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dna vaccine holds an advantage over conventional types which use a target protein as an immunogen in the stability and its relatively low systemic toxicity and has been examined for the efficacy in experimental animal models and moreover in clinical settings [1, 2 ]. previous studies demonstrated that administration of dna potentially induced immune responses to an antigen encoded by the dna and produced protective immunity [3, 4 ]. nevertheless, the low transduction efficacy with dna vaccine administered in vivo hampered extensive clinical application. a possible use of a molecular adjuvant, which can be also administered as dna, can circumvent the inefficient transduction level [5, 6 ]. fas ligand (fasl), type ii transmembrane protein, is a member of tumor necrosis factor family with 40 kda and plays a major role in inducing programmed cell death when it is interacted with fas. the fas / fasl interactions induce apoptosis of immune cells including t, b cells and macrophages and the cell death is often associated with activated stages of immune cells. the activation - induced cell death is a mechanism to inhibit excessive immune responses and to terminate ongoing immunity. naive t cells come to express fasl upon antigen stimulation, and the activated t cells are subjected to apoptosis, which ceases the t cells - mediated responses. moreover, expression of fasl contributed to enhanced antigen uptake in dendritic cells, which indicates that fasl are involved not only in decreasing immunity but in augmenting immune responses. the fas / fasl interactions thus regulate immune responses in multiple ways. in the present study, we examined a role of fasl expression as an adjuvant in vaccination effects on tumor growth. we used -galactosidase (-gal) that was used as a putative tumor antigen in a murine animal model and tested whether administration of fasl dna modulated antitumor responses induced by immunization of -gal - encoding dna. murine colon carcinoma colon 26 cells and packaging cells, 2 and pa317, were maintained with rpmi1640 or dmem medium supplemented with 10% fetal calf serum. balb / c mice were purchased from clea japan slc (tokyo, japan). a.d. miller, fred hutchinson cancer research center, seattle, wa, usa) was used to harbor -gal cdna. the retroviral dna was transfected into ecotropic 2 cells and the cell - free supernatants were further incubated with amphotropic pa317 cells. transduction of colon 26 cells with the -gal gene (colon 26/-gal) was confirmed with 5-bromo-4-chloro-3-indolyl -d - galactoside (x - gal) staining. full - length -gal, mouse fasl cdnas were cloned into expression plasmid vectors, pcdna3 (the transgene is activated by cytomegalovirus promoter) or pcaggs (cag promoter), respectively, and plasmid dna of pcdna3/-gal, pcaggs / fasl was purified with an endotoxin - free dna extraction kit (qiagen, hilden, germany). cardiotoxin (latoxan, valence, france) was injected into thigh muscle of mice 5 days before dna administration. for investigation of -gal expression, dna (10 g or 50 g) was injected in the same area in thigh, and the muscles were fixed with 2% formaldehyde and 0.05% glutaraldehyde and then reacted with x - gal solution. balb / c mice were injected with cardiotoxin (1 mol) and with pcdna3/-gal and/or pcaggs / fasl dna (50 g each) on day 5. they were subcutaneously inoculated with colon 26/-gal cells (1 10) 21 days after dna injections, and the tumor volume was calculated according to the formula (1/2 length width). all the animal experiments were approved by the animal experiment and welfare committee at chiba cancer center research institute. a specific epitope peptide sequence tphparigl of -gal for h-2l haplotype was loaded onto the soluble dimeric h-2l - linked immunoglobulin (ig) complex (dimer x i, bd bioscience, san jose, ca, usa). inguinal lymph node cells were reacted with fluorescence isothiocyanate- (fitc-) conjugated anti - mouse cd8 antibody (ab) (bd bioscience) and with the dimeric h-2l - linked ig complexes loaded with the peptide, followed by phycoerythrin - conjugated anti - mouse igg1 (bd bioscience). the dimeric complex - positive or -negative and cd8 t cells were examined with facscalibur (bd bioscience) and cellquest software (bd bioscience). amounts of anti--gal ab were estimated with enzyme - linked immunosorbent assay (elisa) using purified -gal protein (invitrogen, carlsbad, ca, usa) as a standard and horseradish peroxidase- (hrp-) conjugated anti - mouse igg ab (ge healthcare, buckinghamshire, uk) as previously described. an isotype of anti--gal ab in mice sera was detected with hrp - conjugated anti - mouse igg1 (southernbiotech, birmingham, al, usa), igg2a, igg2b, or igm (invitrogen) ab. the values of respective isotypes were calculated based on optical density at 450 nm since isotype - specific standard anti--gal ab is currently unavailable. we conducted statistical analyses with the one - way analysis of variance (anova) and p values less than 0.05 were judged as significant. we examined expression of the -gal gene in muscles of mice that were injected with pcdna3/-gal plasmid dna and investigated a possible enhancement of the gene expression with a cardiotoxin treatment (figure 1). cardiotoxin destroys muscle tissues and the regeneration process facilitates uptake of dna [13, 14 ]. expression levels of -gal detected with the x - gal staining method depended on amounts of pcdna3/-gal dna used, and the cardiotoxin treatment prior to dna administration augmented the -gal expression. we thereby treated mice with cardiotoxin and 5 days later immunized the mice with 50 g dna in the following experiments. we firstly transduced murine colon 26 cells with the -gal gene and confirmed that the growth of colon 26/-gal cells in vitro and in vivo was not different from parental colon 26 cells. syngeneic balb / c mice were injected with cardiotoxin and then with dna expressing the -gal and/or fasl gene or vector dna as a control. the mice were then inoculated with colon 26/-gal cells and the tumor volumes were monitored. growth of colon 26/-gal cells was not statistically different among mice that were inoculated with vector dna, pcdna3/-gal, or pcaggs / fasl dna (figure 2), and the tumor growth in these mice was not different from that in naive mice (data not shown). in contrast, the tumor growth in mice that received both pcdna3/-gal and pcaggs / fasl dna was retarded compared with that in mice immunized with vector dna, pcdna3/-gal, or pcaggs / fasl dna (p < 0.05). these data indicated that immunization of dna encoding the -gal or the fasl gene alone did not produce antitumor effects but a combinatory use of both dna achieved vaccination effects. we firstly examined induction of antigen - specific cd8 t cells that mediated cytotoxic activities. cells from inguinal lymph nodes that were obtained on days 7, 14, and 21 after dna immunization were stained with antibody against cd8 ab and peptide - loaded class i antigens (figures 3(a) and 3(b)). immunization of both -gal and fasl dna did not increase the antigen - positive cd8 t cells compared with other dna immunizations or naive cases irrespective of days examined. we also calculated total cd8 cell numbers in lymph nodes and found that the numbers in mice which received both -gal and fasl dna did not increase compared with those in other experimental groups (figure 3(c)). these data suggest that cytotoxic t cells were not responsible for the antitumor effects by immunization of -gal and fasl dna. we examined a possible involvement of humoral immunity in the antitumor effects by the immunization of -gal and fasl dna. we firstly measured serum concentrations of anti--gal igg ab produced by dna immunization (figure 4(a)). injection of -gal dna increased anti--gal ab as demonstrated between the group injected with pcdna3/-gal + pcaggs dna and that with pcdna3 + pcaggs dna (p < 0.05), whereas injection of fasl dna did not (pcdna3 + pcaggs / fasl versus pcdna3 + pcaggs, p = 0.48). coinjected fasl dna together with -gal dna however augmented the ab production since the group injected with pcdna3/-gal + pcaggs / fasl dna showed greater responses than that with pcdna3 + pcaggs / fasl or pcdna3/-gal + pcaggs dna (p < 0.01). we then further examined a possible influence of fasl dna injection on differential ig isotype production (figure 4(b)). igg2a amounts were greater in immunization with both -gal and fasl dna than in that with -gal dna alone (p < 0.01), whereas igg2b amounts were rather less in the injection of -gal plus fasl dna than in that of -gal dna alone (p < 0.01). the amounts of igm and igg1 were not different between the mice injected with both -gal and fasl dna and those with -gal dna (igm ; p = 0.29, igg1 ; p = 0.85). the present study demonstrated that administration of fasl dna functioned as an adjuvant and augmented ab production against a tumor antigen. the adjuvant effects by fasl expression generated antitumor immunity which was primed by dna vaccination targeting the tumor antigen. a combinatory use of dna against the tumor antigen and fasl however did not influence the antigen - positive cd8 t cell numbers, suggesting that the antitumor immunity by dna vaccine was not attributable to cell - mediated immunity. in contrast, previous studies showed that vaccination of a tumor antigen with plasmid dna achieved antitumor effects through antigen - positive cytotoxic t cells. moreover, the fas / fasl interactions have negative effects on efficacy of dna vaccine not only by inducing apoptosis of cytotoxic t cells but also by promoting clearance of injected plasmid dna. nevertheless, dharmapuri. demonstrated that downregulation of fas with sirna did not influence the antitumor responses produced by dna encoding a tumor antigen although sirna for bak1 or caspase-8, both of which were involved in apoptotic processes, enhanced the responses in the same experimental settings. a possible role of fasl and fas in the context of dna vaccine in vivo is thus subjected to multiple factors such as immunological microenvironments where tumors develop. the present study did not examine a role of cd4 t cells but the population can be involved in dna vaccine - mediated antitumor responses in which cd8 populations did not play a central role. we however demonstrated that the fasl dna administration augmented production of anti--gal igg ab and igg2a ab specific for a tumor antigen. enhanced anti--gal ab production suggested involvement of ab - dependent cellular cytotoxicity that involved ab binding to fc receptors and/or complement - dependent cellular cytotoxicity that activated complement cascades. the previous study by dharmapuri. also indicated that antitumor responses augmented by coinjected sirna for bak1 or caspase-8 were attributable to class switch from igg1 to igg2a. in fact, igg2a bound to fc receptors better than other isotypes in a murine system. in addition, comparison among immunoglobulin subtypes which, respectively, have a similar affinity to same antigen showed that igg2a activated complement greater than igg2b. nimal. showed increased t helper type 2 rather than t helper type 1 cell responses in vaccination with fasl gene - fused dna and demonstrated that igg2a production was greater than igg1 without generating t cell responses. the data were concordant with the current study although their vaccination targets viral infections. the present data together with the previous studies collectively imply that expressed fasl at local dna injection sites facilitated not only ab production but also class switching, which resulted in augmentation of ab - mediated cytotoxic reactions. nevertheless, a precise mechanism of how the fasl molecules enhanced the humoral immunity is currently unknown. cardiotoxin at the injection sites may also contribute to the humoral immunity since the treatment induces inflammatory reactions with local cytokine productions. proinflammatory cytokines such as interleukin-6, which is produced by cardiotoxin injection, potentiate b cell differentiation. tissue destruction can therefore be crucial not only for integrating plasmid dna but also for conditioning microenvironment for ab production. we demonstrated that administration of fasl dna together with dna encoding a putative tumor antigen gene produced antitumor effects on the antigen - expressing tumor cells in vivo. the antitumor responses were not attributable to antigen - positive cd8 t cells but associated with enhanced ab production in particular igg2a subtype. the present study indicates a role of fasl dna in augmentation of humoral immunity and suggests a potential application of fasl in dna - mediated vaccine. | interaction of fas and fas ligand (fasl) plays an important role in the regulation of immune responses by inducing apoptosis of activated cells ; however, a possible role of fasl in dna vaccination has not been well understood. we examined whether administration of dna encoding fasl gene enhanced antitumor effects in mice that were vaccinated with dna expressing a putative tumor antigen gene, -galactosidase (-gal). growth of -gal - positive colon 26 tumors was retarded in the syngeneic mice immunized with -gal and fasl dna compared with those vaccinated with -gal or fasl dna. we did not detect increased numbers of -gal - specific cd8 + t cells in lymph node of mice that received combination of -gal and fasl dna, but amounts of anti--gal antibody increased with the combination but not with -gal or fasl dna injection alone. subtype analysis of anti--gal antibody produced by the combination of -gal and fasl dna or -gal dna injection showed that igg2a amounts were greater in mice injected with both dna than those with -gal dna alone, but igg2b amounts were lower in both dna - injected than -gal dna - injected mice. these data suggest that fasl is involved in boosting humoral immunity against a gene product encoded by coinjected dna and enhances the vaccination effects. |
although a relatively unspectacular, nonmotile coccoid bacterium, staphylococcus aureus is a dangerous human pathogen in both community - acquired and nosocomial infections. a fundamental biological property of this bacterium is its ability to asymptomatically colonize healthy individuals. s. aureus carriers are at higher risk of infection, and they are presumed to be an important source of the s. aureus strains that spread among individuals. the pathogen can cause a wide variety of infections, which can be divided into three types : (i) superficial lesions such as wound infection, (ii) toxinoses such as food poisoning, scalded skin syndrome and toxic shock syndrome, and (iii) systemic and life - threatening conditions such as endocarditis, osteomyelitis, pneumonia, brain abscesses, meningitis, and bacteremia. s. aureus carries a wealth of pathogenic determinants, which promote tissue colonization, tissue damage, and distant diseases [35 ]. s. aureus is able to survive inside host cells and can invade in vitro a variety of nonprofessional phagocytes, including fibroblasts, osteoblasts, endothelial, and epithelial cells [9, 10 ]. after internalization, s. aureus may either persist, escaping host defenses and antibacterial agents, or multiply and further disseminate. this behavior is orchestrated by global regulators, which sense environmental modifications, such as bacterial density, and may or may not trigger the secretion of proteins that lyse the host cells and allow the bacteria to propagate [1114 ]. thus, invading host cells might not only provide a therapeutic sanctuary, but also be part of a subtle hide - and - seek strategy, as observed with enteric bacteria. to prevent colonization by inhaled microorganisms, the respiratory epithelium maintains an effective antimicrobial environment by mucociliary clearance and by producing antimicrobial peptides, surfactant proteins, complement, chemokines, and cytokines mediating immune cell recruitment and inflammation [1618 ]. all of the innate defense mechanisms of the mammalian airways appear to be directly or indirectly activated by contact of bacterial factors with the epithelial cell surface receptors, which may activate various intracellular signaling pathways. it has long been recognized that s. aureus evokes an intense host response dominated by polymorphonuclear leukocytes (pmns). the induction of genes encoding the proinflammatory cytokines requires activation of mitogen - activated protein kinases (mapks) and the transcription factors activator protein-1 (ap-1) and nuclear factor b (nf-b) [1922 ]. some of them are implicated in lung infection and have been known for several years. however, the information published in the recent past demonstrated a new pathogenic properties related to known virulence determinants of s. aureus. better understanding of functions and mechanisms of action of each virulence factor is important for improving prognosis of individuals suffering from pneumonia. in this paper, we summarize recent advance in our understanding of known virulence factors and their role in the initiation of lung inflammation. over the past 90 years, s. aureus has been increasingly recognized as an important cause of pneumonia in both adult and pediatric populations [2325 ]. along with bacteremia, s. aureus pneumonia is one of the most prevalent methicillin - resistant s. aureus- (mrsa-) related diseases, and the incidence of severe pneumonia caused by mrsa strains rises [26, 27 ]. previously, mrsa infections were largely nosocomial infections and a common cause of ventilator - associated pneumonia (vap), a subtype hospital - acquired pneumonia characterized by high morbidity and mortality [28, 29 ]. however, in the last few years, there was a dramatic increase in the incidence of community - associated mrsa (ca - mrsa) infections in otherwise healthy individuals and in patients who do not establish risk factors for mrsa, and now, ca - mrsa becomes a common and serious health problem. ca - mrsa strains can cause a necrotizing pneumonia, a specific disease entity that often follows an influenza infection. the necrotizing pneumonia is a rapid progressive form of extensive pneumonia leading to acute respiratory distress with pleural effusion, hemoptysis and leucopenia. moreover, pneumonia caused by s. aureus is a serious complication in individuals with cystic fibrosis and patients affected by immunosuppressive therapy [22, 26, 30, 31 ]. a characteristic manifestation of s. aureus - caused pneumonia is the intense host inflammatory response characterized by a rapid and excessive recruitment of neutrophils to the site of infection [32, 33 ]. in fact, accumulating evidence suggests that disease progression in bacterial pneumonia is largely mediated by the dysregulated and exaggerated host inflammatory response to infection that causes lung injury [34, 35 ]. because of the high incidence of pneumonia accompanying with high mortality, it is important to gain more insight into the pathogenesis of this prominent infectious disease. the broad range of infections caused by s. aureus is related to a number of virulence factors that allow it to adhere to surface, invade or avoid the immune system, and cause harmful toxic effects to the host [3, 36 ]. the attachment of s. aureus to the host cell surface initiating the colonization process is mediated by several adhesins. one major class of s. aureus adhesins comprises proteins covalently anchored to cell peptidoglycans (via the threonine residue in the sorting signal motif at their c - terminus), which specifically attach to the plasma or extracellular matrix (ecm) components and collectively are termed the microbial surface component recognizing adhesive matrix molecules (mscramms) [4, 3739 ]. these molecules recognize the most prominent components of the ecm or blood plasma, including fibrinogen, fibronectin, and collagens [3, 4042 ]. typical members of the mscramm family are staphylococcal protein a (spa), fibronectin - binding proteins a and b (fnbpa and fnbpb), collagen - binding protein, and clumping factor (clf) a and b proteins [3, 4 ]. nearly all strains of s. aureus secret a group of exoproteins such as exotoxins and enzymes, including nucleases, proteases, lipases, hyaluronidase, and collagenase. the main function of these proteins may be to convert local host tissue into nutrients required for bacterial growth. cytolytic toxins form -barrel pores in the plasma membrane and cause leakage of the cell 's content and lysis of the target cell. s. aureus secrets several cytolytic toxins, among them -hemolysin, -hemolysin, -hemolysin, leukocidin, and panton - valentine leukocidin (pvl). -hemolysin became inserted into the eukaryotic membrane and oligomerizes into a -barrel that forms a pore which causes osmotic cytolysis and is particularly cytolytic toward human platelets and monocytes. pvl is classified as a bicomponent cytolysin (lukf - pv and luks - pv) that insert itself into the host 's plasma membrane and hetero - oligomerize to form a pore. pvl exhibits a high affinity toward leukocytes, while other bicomponent toxins, -hemolysin and leukocidin, are cytotoxic toward erythrocytes and leukocytes, respectively. s. aureus produces additional group of exotoxins, which include the toxic shock syndrome toxin-1 (tsst-1), the staphylococcal enterotoxins (sea, seb, secn, sed, see, seg, seh, and sei) and the exfoliative toxins (eta and etb). among them, tsst-1 and the staphylococcal enterotoxins belong to the group of toxins known as pyrogenic toxin superantigens (ptsags) [46, 47 ]. the best characterized property of this group is superantigenicity, which refers to the ability of this toxin to stimulate proliferation of t - lymphocytes. the exfoliative toxins have been recognized for long time to possess mitogenic activity toward t lymphocytes, but it remains still controversial, whether they should be implicated as superantigens. s. aureus has also other specific proteins that can have profound impact on the innate and adaptive immune system. examples of such kind of proteins are the staphylococcal complement inhibitor (scin), chemotaxis inhibitory protein of s. aureus (chips), staphylokinase (sak), extracellular fibrinogen binding protein (efb), extracellular adherence protein (eap), and formyl peptide receptor - like-1 inhibitory protein (flipr). scin is a c3 convertase inhibitor, which blocks the formation of c3b on the surface of the bacterium and, thereby, the ability of human neutrophils to phagocytose s. aureus. chips and flipr block neutrophil receptors for chemoattractants [51, 52 ], epa blocks migration of neutrophils from blood vessels into the tissue, sak binding to -defensins abolishes their bactericidal properties, while efb inhibits both classical and alternative pathways of complement activation [55, 56 ]. the virulence of s. aureus is generally considered to be multifactorial and due to the combined action of several virulence determinants. one exception is the toxinoses, such as toxin shock syndrome, ssss, and staphylococcal food poisoning, which are caused by toxic shock syndrome toxin, exfoliative toxins a and b, and different staphylococcal enterotoxins, respectively. in s. aureus - induced vap, multiple virulence factors are implicated. through the action of lta, pepg, mscramms, particularly fnbp and spa, and -toxin, s. aureus is able to adhere to respiratory epithelium, to damage the alveolocapillary barrier, and to attract pmn. in turn, necrotizing pneumonia is associated with an action of spa, -toxin, and -toxin, which cause cell damage and play a role in inflammation and necrosis of the respiratory epithelium [32, 35, 58 ]. the pathogenicity of s. aureus is a complex process involving a diverse array of extracellular and cell wall components that are coordinately expressed during different stages of infection (i.e., colonization, avoidance of host defense, growth and cell division, and bacterial spread) [59, 60 ]. the coordinated expression of diverse virulence factors in response to environmental cues during infections (e.g., expression of adhesins early during colonization versus production of toxins late in infection to facilitate tissue spread) hints at the existence of global regulators in which a single regulatory determinant controls the expression of many unlinked target genes. these regulators help bacteria to adapt to a hostile environment by producing factors enabling the bacteria to survive and subsequently to cause infection at the appropriate time. among the environmental signals, changes in nutrient availability, temperature, ph, osmolarity, and oxygen tension have the greatest potential to influence the expression of virulence factors. production of s. aureus virulence determinants is controlled by several global regulatory loci, such as accessory gene regulator (agr) [62, 63 ], staphylococcal accessory regulator (sara) [64, 65 ], sae, sigb [67, 68 ], arl, and number of sara homologues [70, 71 ]. these regulators are parts of an important network modulating the expression of s. aureus virulence genes. cross talk to ensure that the specific gene is expressed only when conditions are favorable. for instance, in vitro studies have demonstrated that agr negatively regulates the expression of spa, which encodes spa, whereas sars binds to the spa promoter and activates its expression. thus, it has been proposed that agr downregulates spa expression by suppressing the expression of its activator, sars. therefore, virulence gene regulators could affect the expression of target genes directly, by binding to their promoters, or indirectly, via other regulators. the majority of initial inflammatory responses to inhaled bacteria is signaled by mucosal cells lining the respiratory tract. s. aureus has a potential to activate the host inflammatory response in several different ways : through the adherence of intact bacteria to the host epithelial cells, by internalization of the bacteria and by direct interaction of bacterial adhesins and toxins with the mucosal epithelium. the main virulence factors that have potential to cause tissue injury and inflammation in the lung are spa, -toxin, -toxin, and pvl [24, 32, 7375 ]. spa is a good example of one of known and well - characterized s. aureus virulence factors that have recently revealed new properties and play a chief role in the induction of pneumonia. since many years, spa is known to be a 42-kda protein covalently anchored in the bacterial cell wall. it belongs to the mscramm family, because it can bind to the von willebrand factor, a large glycoprotein that mediates platelet adhesion at sites of endothelial damage. spa comprises five repeated domains (e, d, a, b, and c), each of them binding with high affinity to the fc region of immunoglobulin (ig) g and to the fab region of ig of the vh3 subclass [76, 77 ]. the interaction with fc of igg hinders phagocytosis, because bacteria coated with igg in an inappropriate conformation becomes not recognizable by the fc receptor on pmn. an additional consequence of the ability of spa to bind to b lymphocytes displaying igm bearing vh3 heavy chains is the induction of proliferation resulting in depletion of a significant part of the b cells repertoire [78, 79 ]. although the interactions between spa and ig chains have long been recognized, only recent studies reveled the central importance of spa in the pathogenesis of s. aureus - induced pneumonia [32, 80, 81 ]. the absence of spa reduces pneumonia incidents and associated mortality in a mice model of infection. apart of spa interfering with opsonization by binding to the fc portion of immunoglobulins, spa was postulated to have a direct effect on the respiratory epithelial cells even in the absence of igg. in the infection of the airways where serum components are lacking, spa plays a chief role in the pneumonia by induction of interleukin- (il-) 8 expression, and recruitment of pmn into the airway. although several receptors for spa, including von willebrand factor and the platelet protein gc1qr / p33, have been reported, they, however, are not responsible for the accumulation of pmn in the airways. tumor necrosis factor- (tnf-) receptor 1 (tnfr1) is widely expressed at the airway epithelium, and its accessibility on the epithelial surface makes it an attractive candidate for mediating host response induced by spa. an exciting recent study of gmez. showed that spa interacts directly with tnfr1 and mimics tnf- proinflammatory signaling by recruitment of the adaptor molecules the tnfr - associated death domain (tradd), receptor - interacting protein (rip), and tnfr - associated factor (traf) 2 to the receptor and the activation of the mitogen - activated protein kinases (mapks) p38 and c - jun nh2-terminal kinases 1 and 2 (jnk1/2), which induces translocation and activation of transcriptional factor nf-b and mediates il-8 gene expression. moreover, spa - tnfr1 interaction leads to phosphorylation of the activating transcription factor 2 (atf-2), a component of the ap-1 transcription complex that is regulated through phosphorylation by p38 and jnk1/2 mapks (figure 1(a)). additionally, tnfr1-deficiency results in reduced morbidity and mortality in a mouse s. aureus pneumonia model. interestingly, in dominant - negative toll - like receptor (tlr)2 and tlr4 mutants, spa still induces nf-b activation in the airway epithelial cells, suggesting that spa is not tlr2 or tlr4 agonist. the abundance of tnfr1 is controlled by its mobilization from intracellular stores and cleavage from the cell surface [8286 ]. during staphylococcal pneumonia, tnfr1 is specifically mobilized to the apical surface of the airway epithelial cells, providing access to inhaled staphylococci. cleavage of tnfr1 is known to be mediated by the tnf- converting enzyme (tace), a central regulator of tnf- signaling [82, 87, 88 ]. tace (also known as a disintegrin and metalloprotease (adam) 17) is a member of the adam family of proteases involved in release of several cell surface proteins, including receptors for tnf-, the epidermal growth factor (egf) and il-6. tace plays an important role in the regulation of inflammation by its ability to cleave and release the extracellular portion of tnfr1 from the surface of airway epithelial cells and macrophages. shed of tnfr1 from the epithelial surface prevents ongoing signaling and serves to neutralize free tnf- as well as spa in the airway lumen, and, consequently, the loss of the receptor from the cell surface prevents further epithelial activation. activation of tace depends on a discrete interaction between spa and egf receptor (egfr), which in turn induces tace phosphorylation through a c - src - erk1/2-mediated cascade (figure 1(b)). while tace is highly expressed on the apical surface of the airway epithelial cells, the substrate, tnfr1, has to be mobilized to the surface, where it colocalizes with tace. interaction between egfr and bacterial spa and the consequent activation of tace serve to counteract the proinflammatory consequences of tnfr1 signaling, pmn recruitment and activation. thus, activation of the tnfr1 pathway not only stimulates mobilization of pmn, but also provides a mechanism to regulate spa - induced recruitment of neutrophils. therefore, spa is involved in the s. aureus pneumonia by activating tnfr1 and inducing pmn infiltration that is deleterious to the host. the discovery of the new spa- tnfr1 signaling axis highlights additional molecular targets to modulate the host immune response and to treat s. aureus - caused pneumonia. s. aureus -toxin, -toxin, and pvl play an essential role in pneumonia and lung injury. both, -toxin and pvl, are pore - forming toxins, which exaggerate the host inflammatory response by inducing the expression of proinflammatory cytokines and lysing inflammatory cells to release additional inflammatory mediators. thus, these toxins have both direct and indirect means to cause a lung damage [73, 9092 ]. however, little is known about the significance of these toxins in s. aureus - induced pneumonia and lung injury. -toxin is the major cytotoxic agent released by s. aureus, and it was the first bacterial exotoxin to be identified as a pore former. pore formation on susceptible host cell membranes triggers alterations in ion gradients, loss of membrane integrity, activation of stress - signaling pathways, and cell death [93, 94 ]. s. aureus -toxin is known to play an important role in the pathogenesis of staphylococcal diseases, as s. aureus mutants lacking hla display reduced virulence in invasive disease models. interestingly, the dosage of the toxin can result in two different modes of activity. this pore allows the exchange of monovalent ions, resulting in dna fragmentation and, eventually, in apoptosis. high concentrations result in the toxin absorbing nonspecifically to the lipid bilayer [97, 98 ] and forming large, ca - permissive pores. this results in massive necrosis and other secondary cellular reactions triggered by the uncontrolled ca influx. -toxin is secreted as a water - soluble monomer that undergoes a series of conformational changes to generate a heptameric, -barrel structure in host membranes. recently, wilke and wanderburg reported that -toxin binding to eukaryotic cell requires adam 10 expression to initiate the sequence of events (see below). -toxin possesses additional biological functions such as binding to a putative glycoprotein receptor on host cells, activation of intracellular signaling, and modulation of several processes [9193, 96, 100 ]. it was recently described, that -toxin facilitates the secretion of newly synthesized chemokines into the airway and exaggerates neutrophil - mediated inflammatory lung injury through syndecan-1 ectodomain shedding (see below). recently, it has been reported that -toxin - adam 10 interaction identifies adam 10 as the likely proteinaseous cellular receptor for the toxin, which is required for -toxin - mediated cytotoxicity when the toxin is present at low concentrations. multiple lines of evidence confirm the importance of the membrane lipid environment in -toxin - induced injury, because the membrane opposed region of the toxin interacts with phosphatidylcholine, and cholesterol / sphingomyelin - rich membrane domains. it has been shown that clustered phosphocholine head groups serve as the high - affinity binding site for -toxin and provide a mechanistic view of the assembly of -toxin, suggesting that its initial interaction with adam10 and the plasma membrane directs the assembly of the -toxin - adam10 complex in cholesterol / sphingolipid - rich caveolar rafts. this clustering likely increases the local concentration of -toxin, permitting caveolin 1-directed oligomerization of the toxin and providing accessibility to caveolae - associated proteins fak and src, which mediate the biologic effects of -toxin. focal adhesion disruption by the -toxin - adam10 complex provides a mechanism by which the toxin may perturb cellular barriers to cause invasive disease and facilitate superantigen permeation through impenetrable stratified cell layers. among s. aureus toxins, based on literature data, s. aureus -toxin is a mg - dependent neutral sphingomyelinase that hydrolyzes sphingomyelin of the host cell plasma membrane to generate phosphocholine and the bioactive secondary messenger, ceramide [104106 ]. depending on the chain length of their fatty acids or the mode of metabolism, these ceramides may have a number of effects in eukaryotic cells, including stimulation of second messenger systems, activation of mapks, changes in cell shape, and even apoptosis [107, 108 ]. -toxin does not lyse most types of host cells but leaves them susceptible to a number of other lytic agents, such as -toxin and pvl. in fact, the cytotoxic effect of -toxin is cell type - specific and species - specific, suggesting that its primary virulence activity is to modulate host processes that affect pathogenesis, rather than to directly kill host cells. study of hayashida. uncovered a previously unknown in vivo function of -toxin in s. aureus pneumonia. s. aureus -toxin has been shown to maximize lung injury not through its cytotoxic activity, but rather through its capacity to enhance pmn infiltration in a syndecan-1-dependent manner (see below). moreover, this toxin can activate different, as yet unknown, cell signaling pathways involved in the induction of c - fos expression through the nf-b and p38 mapk signaling cascades [94, 109111 ]. ectodomain shedding is a proteolytic mechanism of releasing the extracellular domains of cell surface proteins as soluble ectodomains that can regulate many pathophysiological processes, such as microbial pathogenesis, inflammation, and tissue repair [112, 113 ]. the diverse list of shed proteins includes cytokines, growth factors, and cell adhesion molecules, including tnf-, transforming growth factor- (tgf-), egf, l - selectin, cd44, and syndecans. s. aureus and other bacterial pathogens activate ectodomain shedding of cell surface molecule syndecan-1 to enhance their virulence [35, 58, 100 ]. syndecan-1 is the major heparan sulfate proteoglycan of epithelial cells, which binds and regulates a wide variety of biological molecules through its heparan sulfate chains. both -toxin and -toxin shed syndecan-1 ectodomains through stimulation of the host cells shedding machinery [35, 58, 100 ]. several independent lines of evidence suggest that the primary function of syndecan-1 in - and -toxin - induced inflammation is to facilitate pmn infiltration through the generation of chemotactic signals [35, 58 ]. forming the small discrete pores by -toxin may trigger syndecan-1 shedding [91, 100 ]. -toxin does not directly shed syndecan-1 ectodomains, but rather stimulates an endogenous mechanism which involves protein tyrosine kinases (e.g., syk), but not protein kinase c and mapk signaling pathways, that enhance the cleavage of syndecan-1 ectodomains by host cell metalloproteinase. staphylococcal -toxin enhances syndecan-1 shedding by activating ceramide production in the alveolar epithelial cells and by implicating protein tyrosine kinases syk and jak2, erk - type mapks, and metalloproteinase [115, 116 ]. the mechanism of syndecan-1 shedding was well characterized in a mouse model. in bleomycin - induced acute inflammation and lung injury, shedding of syndecan-1 by metalloproteinase-7 generates a chemokine gradient that attracts pmn into the alveolar compartment. lung injury caused by bleomycin induces the expression of the cxc chemokine kc (cxcl1, mouse functional homologue of human il-8) and metalloproteinase-7. newly synthesized kc binds to the heparan sulfate proteoglycans of syndecan-1, and shedding of the syndecan-1/ectodomain - kc complex by metalloproteinase into the alveolar space generates a chemokine gradient across the alveolar epithelial border. both s. aureus toxins exaggerate lung injury and inflammation through its capacity to enhance neutrophil infiltration [35, 58 ]. thus, the shedding of syndecan-1 mediated by - and -toxins may be a critical mechanism in development of a broad range of acute inflammatory disorders. the toxin was first described by van de velde (1894), but only in 1932 panton and valentine associated the leukotoxin with skin and soft - tissue infection. clinical studies propose the exotoxin pvl being a virulence factor in necrotizing diseases [24, 118 ]. previous studies revealed that human and rabbit neutrophils are highly sensitive to the pore - forming properties of pvl and rapidly undergo cell death. furthermore, it is generally accepted that myeloid cells are the prime target of pvl and that low concentrations of the toxin cause apoptosis, whereas higher amounts induce lysis of neutrophils. pore formation requires the presence of the two components of the toxin, luks - pv and lukf - pv. this pore is an octameric -barrel molecular complex perpendicular to the plane of the cell membrane, similar to that made by s. aureus-toxin [121, 122 ]. sublytic concentrations of purified pvl induce pronounced histamine release from human basophils and stimulate human neutrophils to release enzymes (-glucuronidase and lysozyme), chemotactic components (leukotriene - b4 and il-8), and oxygen metabolites [121, 123, 124 ]. more than 20 years ago, it was suggested that this lytic toxin functions as a virulence factor in cutaneous infection [125, 126 ]. necrotizing pneumonia has long been recognized, but the association with pvl was made by gillet., and numerous cases have been reported worldwide [24, 26, 118, 127131 ]. patients with pvl - positive s. aureus in their lungs develop necrotizing pneumonia and have exceedingly high mortality rates, indicating that pvl might be an important virulence factor. however, several studies that used a diversity of animal models have created conflicting results concerning the role of pvl in pneumonia. in one study applying a mouse acute pneumonia model, labandeira - rey. suggested pvl to be a major virulence factor. using purified toxin or a laboratory strain of s. aureus that overexpressed pvl via a plasmid containing luk - pv operon, it is of interest that when comparing isogenic s. aureus strains lysogenized with either wild - type slt or mutated slt in which the lukpv operon was deleted, no difference in mouse survival was found, indicating that pvl does not exhibit a lethal effect when expressed from a single transgenic copy. labandeira - rey. ascribed to pvl a pronounced global gene regulatory effect, with the regulatory changes reminiscent of disrupting the accessory gene regulator agr. they showed that the expression of pvl induces global changes in transcriptional levels of genes encoding secreted and cell - wall - anchored staphylococcal proteins, including spa. it should be mentioned that this statement is controversial : diep and otto explained that misinterpretation of the data due to the apparent lack of confirmatory experiments might have led to the model in which pvl plays a role in global gene regulation. also, other groups fail to detect any pathogenic function of pvl in murine model of pneumonia. using isogenic pvl mutants in the mw2 and usa300 backgrounds, and when overexpressing pvl in s. aureus strain newman, no significant contribution of pvl to lethal pneumonia was found using mice [75, 134 ]. moreover, it was suggested that hla, but not pvl, was essential for the pathogenesis of staphylococcal pneumonia. passive immunization with anti - pvl immune sera also failed to protect mice against challenge with usa300 in the murine pneumonia model, indicating that pvl is not necessary for the pathogenesis of pulmonary disease. despite the role of pvl as a virulence factor in the lungs is controversial, the pulmonary immune response to pvl, especially responsiveness of alveolar macrophages to this toxin, is known. the recent study of zivkovic. showed that pvl induced a highly specific inflammatory transcriptional response in alveolar macrophages. the alveolar macrophages are considered to represent the first line of defense against pathogens and express receptors, including tlrs, which recognize pathogen - associated molecular patterns. these pathways further modulate proinflammatory gene expression, which is crucial in shaping the innate immune response within the respiratory tract. the idea that tlrs could play an important role in bacterial toxin recognition is not uncommon. other pore - forming toxins have been shown to mediate inflammation via tlrs, particularly via tlr2 and tlr4 [138, 139 ].. demonstrated that pvl directly binds to the extracellular domain of tlr2 and induces immune response via nf-b in a tlr2, cd14, myd88, il-1 receptor - associated kinase 1, and traf6-dependent manner. however, in contrast to data showing that lukf from s. aureus is able to induce inflammation in a tlr4-dependent manner in bone marrow - derived dendritic cells, the study of zivkovic. demonstrated that the active component of the toxin is luks, because the stimulation of macrophages with luks, but not with lukf, resulted in an inflammatory response in vitro and in vivo. furthermore, overexpression of tlr2, but not cd14, is sufficient for luks to induce an inflammatory response, indicating that cd14 can act only as a coreceptor. these data are in line with previous observations, showing that both subunits of pvl are required to perform a pore. interestingly, although single subunits are incapable of forming the pore, luks is capable of inducing tnf- gene expression. furthermore, single submit luks, but not lukf, is able to induce an inflammatory response, suggesting that inflammatory gene expression relies on cellular pathways independent of pore formation. s. aureus deploys a combination of virulence factors, including adhesins, toxins, and immunomodulatory molecules, that facilitate infection of different host tissues [141, 142 ]. the knowledge about host factors, which facilitate eradication of s. aureus in the lungs, is limited. surfactant protein a (sp - a) is the major protein component of pulmonary surfactant. it is involved in organization of large aggregates of surfactant phospholipids lining the alveolar surface and acts as an opsonin for pathogens. previous studies established that sp - a modulates macrophage phagocytosis and a host pro- and anti - inflammatory responses that help in eradication of infection [144148 ]. recent study of sever - chroneos. demonstrated the role of sp - a in opsonization and clearance of s. aureus. macrophage receptor sp - r210 is implicated in the ability of sp - a to coordinate the clearance of pathogens and apoptotic cells, and to participate in temporal control of inflammation in the lungs. sp - r210 mediates also binding of sp - a - opsonized s. aureus by macrophages. phagocytosis of sp - a - opsonized s. aureus via sp - r210 is coordinated with secretion of tnf- and suppression of bacterial growth in macrophages. furthermore, expression of the staphylococcal adhesin eap is necessary for both sp - a binding and enhanced phagocytosis of sp - a - opsonized bacteria by sp - r210. finally, sever - chroneos. revealed previously unknown link between expression of sp - r210 isoforms and the scavenger receptor sr - a. binding of sp - a to sp - r210s induces phagocytosis and release of anti - inflammatory mediators via association with sr - a, leading to an enhanced bacterial killing and resolution of the infection. based on previous findings, sp - r210 and sr - a may coordinate secretion of il-10, tgf-, and hydrogen peroxide in alveolar macrophages. importantly, it is proposed that temporal control of inflammatory responses via sp - r210s and sr - a contributes to the proper recruitment and activation of neutrophils, facilitating eradication of s. aureus infection in the lungs. however, moderate levels of hydrogen peroxide may suppress inflammation through inactivation of nf-b [152, 153 ] and enhance bacterial killing through activation of nadph oxidase during the resolution phase of the disease. the innate defense of the airway epithelial cells against s. aureus includes a regulated secretion of cytokines and chemokines, and involves different signalling pathways. induction of the airway inflammation can be mediated by several staphylococcal determinants and corresponding receptors and is not necessarily dependent on the expression of a particular virulence factor that is crucial for the pathogenesis of s. aureus infection in other body sites. among many virulence factors produced by s. aureus, spa, -, and -toxins play an important role in the of pathogenesis of staphylococcal pneumonia. the shedding of the plasma membrane proteins represents an important mechanism underlying s. aureus properties in the lungs. -toxin and -toxin of s. aureus activate ectodomain shedding of host components to promote bacterial pathogenesis. in addition, the airway epithelial cells regulate their own signaling capabilities by shedding some epithelial receptors (e.g., tnfr1) that serves to bind and neutralize inflammatory cytokines released by immune cells. considerable progress has been made in our understanding of known virulence factors and their implication in pneumonia in the last few years. a detailed analysis of function and mechanisms of action of each virulence factor could open the way to control the proinflammatory response in the lung by using specific inhibitors and may be helpful for the development of novel therapies for s. aureus - caused pulmonary diseases. | airway epithelial cells play a major role in initiating inflammation in response to bacterial pathogens. s. aureus is an important pathogen associated with activation of diverse types of infection characterized by inflammation dominated by polymorphonuclear leukocytes. this bacterium frequently causes lung infection, which is attributed to virulence factors. many of virulence determinants associated with s. aureus - mediated lung infection have been known for several years. in this paper, we discuss recent advances in our understanding of known virulence factors implicated in pneumonia. we anticipate that better understanding of novel functions of known virulence factors could open the way to regulate inflammatory reactions of the epithelium and to develop effective strategies to treat s. aureus - induced airway diseases. |
, recurrent or secondary dental caries has been proven to be one of the most common complications following tooth restoration.1 many investigators have identified microleakage as the primary cause for recurrent or secondary dental caries, pulpal inflammation and necrosis. success in conservative dentistry depends on total removal of the infected dentin and achievement of a good seal ; however, contemporary procedures for treating dental caries do not always eliminate all cariogenic microorganisms in prepared cavities. other studies have shown that microorganisms left in the prepared cavity could survive for a long period of time and this problem may be magnified by microleakage of composite resin at margins not ending on enamel. to solve this problem, the use of an antimicrobial solution has been suggested.5 chlorhexidine solutions has been found to be effective in reducing levels of streptococcus mutans found in occlusal fissures and on exposed root surfaces. the authors of the current study suggest that the use of a 2% chlorhexidine solution to treat the cavity preparation prior to restoration placement could help to reduce residual caries and post - operative sensitivity. its application does not impair the sealing ability and bond strength of adhesive materials, although, in specific situations, some studies showed an interference of chlorhexidine in adhesion.6 in theory, these self - etching systems simultaneously decalcify the inorganic component of dentin and infiltrate the collagen fibers at the same time through the action of acidic primers that minimize the potential for voids. the clinical procedure is less complicated and time - consuming, because there is no need for rinsing.6 different from etch - and - rinse adhesives, self - etch adhesives do not require a separate etching step, as they contain acidic monomers that simultaneously condition and prime the dental substrate. consequently, this approach has been claimed to be user - friendlier (shorter application time, less steps) and less technique - sensitive (no wet - bonding, simple drying), thereby resulting in a reliable clinical performance, though this appeared very product - dependent.2 to provide resin - based materials with antibacterial activity, a new monomer, 12-methacryloyloxydodecylpyridiniumbromide (mdpb), has been developed. mdpb is a compound of an antibacterial agent quaternary ammonium with a methacryloyl group and it exhibits strong antibacterial activity against oral streptococci. the incorporation of mdpb has been reported to be effective in providing dentin bonding systems with antibacterial activity before and after curing. the current study evaluated the influence 2% chlorhexidine gluconate, clearfil protect bond (cpb) (which contains the antibacterial monomer 12-mdpb) on the microleakage of class v composite restorations. standard class v cavity preparations (mesiodistal width of 3 mm, occluso - gingival length of 2 mm and a depth of 2 mm) were prepared on the buccal and lingual surfaces using a high - speed handpiece with air - water spray and a diamond fissure bur. each preparation was designed with the occlusal margin in enamel and the gingival margin in dentin. the teeth were randomly divided into three experimental and one control group, each group containing 10 samples. clearfil se bond (kuraray co. ltd., japan) two step self - etch adhesive system was applied on cavities according to the manufacturer 's instructions. the bonding agent was then applied and light cured for 10 s. each cavity was washed, dried but not desiccated. 2% chlorhexidine gluconate cavity cleanser was applied with a small brush tip, placed for 40 s and then dried with absorbent paper. clearfil se bond (kuraray co. ltd., japan) two step self etch adhesive system similar to group i was applied on cavities according to the manufacturer 's instructions after applying 2% chlorhexidine gluconate. cpb (kuraray co. ltd., japan) self - etching primer was applied to the cavity with a brush and left in place for 20 s. after drying the etched surface with mild air flow, the bonding agent was applied on to the etched primed dentin, gently air dried and light cured for 10 s. each cavity was washed, dried but not desiccated. the self - etching adhesive (xeno iii) was applied to the cavity preparation with an applicator tip, left undisturbed for 20 s and the excess solvent was removed with a gentle stream of air. the cavity was restored incrementally with nano composite filtek z350 using an oblique incremental insertion technique and each increment was light cured for 40 s respectively. finishing and polishing of the restorations was carried out with tungsten carbide finishing bur and a series of polishing discs (3 m sof - lex). the teeth were stored in 37c and 100% humidity for 24 h. the specimens were then thermocycled for 500 cycles with baths conducted in - between (5c and 55c), a dwell time of 30 s and a transfer time of 3 s. after thermocycling, the apices of the teeth were sealed with sticky wax, and all tooth surfaces except a 1 mm wide zone around the margins of each restoration were sealed with nail polish. to minimize dehydration of the restorations, the teeth were then immersed in a 0.5% basic fuchsin solution for 24 h at room temperature. the specimens were then rinsed in tap water, and each specimen was sliced longitudinally using a low - speed diamond disk (isomed buehler, ltd, lake bluff, il, usa) with water coolant and evaluated for marginal leakage. the degree of dye penetration was then graded at 30 original magnification with a stereomicroscope (smz 800, nikon, tokyo, japan) using the following scale according to criteria given by siso. - no marginal leakage1 - basic fuchsin penetrates up to the dentinoenamnel junction (dej) or corresponding length at the dentin wall2 - basic fuchsin penetrates beyond the dej or corresponding length at the dentin wall, surpassing half the cavity depth3 - basic fuchsin penetrates beyond half the cavity depth, without reaching the axial wall4 - basic fuchsin penetrates along the axial wall. 0 - no marginal leakage 1 - basic fuchsin penetrates up to the dentinoenamnel junction (dej) or corresponding length at the dentin wall 2 - basic fuchsin penetrates beyond the dej or corresponding length at the dentin wall, surpassing half the cavity depth 3 - basic fuchsin penetrates beyond half the cavity depth, without reaching the axial wall 4 - basic fuchsin penetrates along the axial wall. none of the procedures tested in the current study completely eliminated microleakagethe scores of microleakage at the enamel margin (occlusal) of the four groups were compared, and no statistical significant differences were found (p > 0.05)the lowest mean microleakage values were obtained from group ii (clearfil se bond + 2% chlorhexidine gluconate group) at the enamel margin (figure 1b) figure 1(a) stereomicroscope photograph of group i (clearfil se bond), (b) stereomicroscope photograph of group ii (clearfil se bond + chlorhexidine), (c) stereomicroscope photograph of group iii (clearfil protect bond), (d) stereomicroscope photograph of group iv (control group), e - enamel margin, d - dentinal margin.the highest values were obtained with group iii (cpb group) and group iv (control group) at the enamel margin (figure 1c)when the scores of microleakage at gingival margin of the four groups were compared, there was no statistical significance (p > 0.05)the lowest mean microleakage values were obtained with group i (clearfil se bond group) (figure 1a) followed by group ii (clearfil se bond + 2% chlorhexidine gluconate group) at the gingival marginthe highest values were obtained from group iii (cpb group) and group iv (control group) (figure 1d) at the gingival margininter - group comparison of mean microleakage between all experimental groups (i, ii, iii) with group iv (control group) using mann test was done both at enamel margin and gingival marginwhen mean microleakage values of group i (clearfil se bond group) were compared with group iv (control group) at the enamel margin, no statistical difference was foundgroup ii (clearfil se bond + 2% chlorhexidine gluconate group) had the lowest mean microleakage values at the enamel margin, but the mean values were not statistically significant when compared with group iv (control group) (p > 0.05)group iii (cpb group) and group iv (control group) had the same mean microleakage values at the enamel margin (graph 1), which were not statistically significant (p > 0.05) when compared graph 1(a) scoring of microleakage at enamel margin, (b) scoring of microleakage at dentine margin.for group i (clearfil se bond group) and group ii (clearfil se bond + 2% chlorhexidine gluconate group) the lowest mean microleakage were obtained at the gingival margin but no statistical significant difference (p > 0.05) was found when each group was compared with group iv (control group)group iii (cpb group) had the highest mean microleakage at the gingival margin but was same as that of group iv (control group)when the mean microleakage of group iii (cpb group) was compared with group iv (control group) there was no statistical significant difference (p > 0.05). none of the procedures tested in the current study completely eliminated microleakage the scores of microleakage at the enamel margin (occlusal) of the four groups were compared, and no statistical significant differences were found (p > 0.05) the lowest mean microleakage values were obtained from group ii (clearfil se bond + 2% chlorhexidine gluconate group) at the enamel margin (figure 1b) figure 1(a) stereomicroscope photograph of group i (clearfil se bond), (b) stereomicroscope photograph of group ii (clearfil se bond + chlorhexidine), (c) stereomicroscope photograph of group iii (clearfil protect bond), (d) stereomicroscope photograph of group iv (control group), e - enamel margin, d - dentinal margin. (a) stereomicroscope photograph of group i (clearfil se bond), (b) stereomicroscope photograph of group ii (clearfil se bond + chlorhexidine), (c) stereomicroscope photograph of group iii (clearfil protect bond), (d) stereomicroscope photograph of group iv (control group), e - enamel margin, d - dentinal margin. the highest values were obtained with group iii (cpb group) and group iv (control group) at the enamel margin (figure 1c) when the scores of microleakage at gingival margin of the four groups were compared, there was no statistical significance (p > 0.05) the lowest mean microleakage values were obtained with group i (clearfil se bond group) (figure 1a) followed by group ii (clearfil se bond + 2% chlorhexidine gluconate group) at the gingival margin the highest values were obtained from group iii (cpb group) and group iv (control group) (figure 1d) at the gingival margin inter - group comparison of mean microleakage between all experimental groups (i, ii, iii) with group iv (control group) using mann whitney u test was done both at enamel margin and gingival margin when mean microleakage values of group i (clearfil se bond group) were compared with group iv (control group) at the enamel margin, no statistical difference was found group ii (clearfil se bond + 2% chlorhexidine gluconate group) had the lowest mean microleakage values at the enamel margin, but the mean values were not statistically significant when compared with group iv (control group) (p > 0.05) group iii (cpb group) and group iv (control group) had the same mean microleakage values at the enamel margin (graph 1), which were not statistically significant (p > 0.05) when compared graph 1(a) scoring of microleakage at enamel margin, (b) scoring of microleakage at dentine margin. (a) scoring of microleakage at enamel margin, (b) scoring of microleakage at dentine margin. for group i (clearfil se bond group) and group ii (clearfil se bond + 2% chlorhexidine gluconate group) the lowest mean microleakage were obtained at the gingival margin but no statistical significant difference (p > 0.05) was found when each group was compared with group iv (control group) group iii (cpb group) had the highest mean microleakage at the gingival margin but was same as that of group iv (control group) when the mean microleakage of group iii (cpb group) was compared with group iv (control group) there was no statistical significant difference (p > 0.05). each cavity was washed, dried but not desiccated. clearfil se bond (kuraray co. ltd., japan) two step self - etch adhesive system was applied on cavities according to the manufacturer 's instructions. primer was applied for 20 s and gently air dried. the bonding agent was then applied and light cured for 10 s. 2% chlorhexidine gluconate cavity cleanser was applied with a small brush tip, placed for 40 s and then dried with absorbent paper. clearfil se bond (kuraray co. ltd., japan) two step self etch adhesive system similar to group i was applied on cavities according to the manufacturer 's instructions after applying 2% chlorhexidine gluconate., japan) self - etching primer was applied to the cavity with a brush and left in place for 20 s. after drying the etched surface with mild air flow, the bonding agent was applied on to the etched primed dentin, gently air dried and light cured for 10 s. the self - etching adhesive (xeno iii) was applied to the cavity preparation with an applicator tip, left undisturbed for 20 s and the excess solvent was removed with a gentle stream of air. the cavity was restored incrementally with nano composite filtek z350 using an oblique incremental insertion technique and each increment was light cured for 40 s respectively. finishing and polishing of the restorations was carried out with tungsten carbide finishing bur and a series of polishing discs (3 m sof - lex). the teeth were stored in 37c and 100% humidity for 24 h. the specimens were then thermocycled for 500 cycles with baths conducted in - between (5c and 55c), a dwell time of 30 s and a transfer time of 3 s. after thermocycling, the apices of the teeth were sealed with sticky wax, and all tooth surfaces except a 1 mm wide zone around the margins of each restoration were sealed with nail polish. to minimize dehydration of the restorations, the teeth were then immersed in a 0.5% basic fuchsin solution for 24 h at room temperature. the specimens were then rinsed in tap water, and each specimen was sliced longitudinally using a low - speed diamond disk (isomed buehler, ltd, lake bluff, il, usa) with water coolant and evaluated for marginal leakage. the degree of dye penetration was then graded at 30 original magnification with a stereomicroscope (smz 800, nikon, tokyo, japan) using the following scale according to criteria given by siso. - no marginal leakage1 - basic fuchsin penetrates up to the dentinoenamnel junction (dej) or corresponding length at the dentin wall2 - basic fuchsin penetrates beyond the dej or corresponding length at the dentin wall, surpassing half the cavity depth3 - basic fuchsin penetrates beyond half the cavity depth, without reaching the axial wall4 - basic fuchsin penetrates along the axial wall. 0 - no marginal leakage 1 - basic fuchsin penetrates up to the dentinoenamnel junction (dej) or corresponding length at the dentin wall 2 - basic fuchsin penetrates beyond the dej or corresponding length at the dentin wall, surpassing half the cavity depth 3 - basic fuchsin penetrates beyond half the cavity depth, without reaching the axial wall 4 - basic fuchsin penetrates along the axial wall. none of the procedures tested in the current study completely eliminated microleakagethe scores of microleakage at the enamel margin (occlusal) of the four groups were compared, and no statistical significant differences were found (p > 0.05)the lowest mean microleakage values were obtained from group ii (clearfil se bond + 2% chlorhexidine gluconate group) at the enamel margin (figure 1b) figure 1(a) stereomicroscope photograph of group i (clearfil se bond), (b) stereomicroscope photograph of group ii (clearfil se bond + chlorhexidine), (c) stereomicroscope photograph of group iii (clearfil protect bond), (d) stereomicroscope photograph of group iv (control group), e - enamel margin, d - dentinal margin.the highest values were obtained with group iii (cpb group) and group iv (control group) at the enamel margin (figure 1c)when the scores of microleakage at gingival margin of the four groups were compared, there was no statistical significance (p > 0.05)the lowest mean microleakage values were obtained with group i (clearfil se bond group) (figure 1a) followed by group ii (clearfil se bond + 2% chlorhexidine gluconate group) at the gingival marginthe highest values were obtained from group iii (cpb group) and group iv (control group) (figure 1d) at the gingival margininter - group comparison of mean microleakage between all experimental groups (i, ii, iii) with group iv (control group) using mann test was done both at enamel margin and gingival marginwhen mean microleakage values of group i (clearfil se bond group) were compared with group iv (control group) at the enamel margin, no statistical difference was foundgroup ii (clearfil se bond + 2% chlorhexidine gluconate group) had the lowest mean microleakage values at the enamel margin, but the mean values were not statistically significant when compared with group iv (control group) (p > 0.05)group iii (cpb group) and group iv (control group) had the same mean microleakage values at the enamel margin (graph 1), which were not statistically significant (p > 0.05) when compared graph 1(a) scoring of microleakage at enamel margin, (b) scoring of microleakage at dentine margin.for group i (clearfil se bond group) and group ii (clearfil se bond + 2% chlorhexidine gluconate group) the lowest mean microleakage were obtained at the gingival margin but no statistical significant difference (p > 0.05) was found when each group was compared with group iv (control group)group iii (cpb group) had the highest mean microleakage at the gingival margin but was same as that of group iv (control group)when the mean microleakage of group iii (cpb group) was compared with group iv (control group) there was no statistical significant difference (p > 0.05). none of the procedures tested in the current study completely eliminated microleakage the scores of microleakage at the enamel margin (occlusal) of the four groups were compared, and no statistical significant differences were found (p > 0.05) the lowest mean microleakage values were obtained from group ii (clearfil se bond + 2% chlorhexidine gluconate group) at the enamel margin (figure 1b) figure 1(a) stereomicroscope photograph of group i (clearfil se bond), (b) stereomicroscope photograph of group ii (clearfil se bond + chlorhexidine), (c) stereomicroscope photograph of group iii (clearfil protect bond), (d) stereomicroscope photograph of group iv (control group), e - enamel margin, d - dentinal margin. (a) stereomicroscope photograph of group i (clearfil se bond), (b) stereomicroscope photograph of group ii (clearfil se bond + chlorhexidine), (c) stereomicroscope photograph of group iii (clearfil protect bond), (d) stereomicroscope photograph of group iv (control group), e - enamel margin, d - dentinal margin. the highest values were obtained with group iii (cpb group) and group iv (control group) at the enamel margin (figure 1c) when the scores of microleakage at gingival margin of the four groups were compared, there was no statistical significance (p > 0.05) the lowest mean microleakage values were obtained with group i (clearfil se bond group) (figure 1a) followed by group ii (clearfil se bond + 2% chlorhexidine gluconate group) at the gingival margin the highest values were obtained from group iii (cpb group) and group iv (control group) (figure 1d) at the gingival margin inter - group comparison of mean microleakage between all experimental groups (i, ii, iii) with group iv (control group) using mann whitney u test was done both at enamel margin and gingival margin when mean microleakage values of group i (clearfil se bond group) were compared with group iv (control group) at the enamel margin, no statistical difference was found group ii (clearfil se bond + 2% chlorhexidine gluconate group) had the lowest mean microleakage values at the enamel margin, but the mean values were not statistically significant when compared with group iv (control group) (p > 0.05) group iii (cpb group) and group iv (control group) had the same mean microleakage values at the enamel margin (graph 1), which were not statistically significant (p > 0.05) when compared graph 1(a) scoring of microleakage at enamel margin, (b) scoring of microleakage at dentine margin. (a) scoring of microleakage at enamel margin, (b) scoring of microleakage at dentine margin. for group i (clearfil se bond group) and group ii (clearfil se bond + 2% chlorhexidine gluconate group) the lowest mean microleakage were obtained at the gingival margin but no statistical significant difference (p > 0.05) was found when each group was compared with group iv (control group) group iii (cpb group) had the highest mean microleakage at the gingival margin but was same as that of group iv (control group) when the mean microleakage of group iii (cpb group) was compared with group iv (control group) there was no statistical significant difference (p > 0.05). microleakage has been defined by sidhu and henderson as the clinically undetectable passage of bacterial fluids, molecules and/or ions between the cavity wall and the restoration material applied to it.9 microleakage at the tooth restoration interface especially with resin restoration is considered, to be a priority issue influencing the retention of dental restorations with no clinical problems. cavosurface margin of restorations, leak more which may lead to recurrent caries at the tooth restoration interface, post - operative sensitivity and pulpal pathosis. the ability of a composite to minimize the extent of microleakage at the tooth / restoration interface is an important factor in predicting clinical success. in the current study, disadvantages include subjective evaluation of the results and low molecular weight of the dye, which is less than that of bacteria. researchers have adopted several methods for estimation of dye leakage under in - vitro condition. the available in - vitro studies include the use of dyes, chemical tracers, radioactive isotopes, air pressure, bacteria, neutron activation analysis, scanning electron microscopy, artificial caries techniques and electrical conductivity. the use of organic dye as tracers is one of the oldest and most common methods of detecting leakage in - vitro. the review of published literature demonstrates that there have been wide variations in choice of dye used either as solutions or particle suspension of different particle size. the concentration of dyes used also ranges between 0.5% and 10% while the time of immersion of specimen in dye varied between 4 h and 72 h or more.8 christen and mitchell (1966) found that different concentration of dyes can vary in penetration time between 5 min and over 1 h. most researchers have used 0.5% basic fuchsin as a standard dye in all in - vitro leakage studies.10 in the present study, we have utilized 0.5% basic fuchsin and the time of immersion of specimen in the dye was 24 h. this included three experimental groups and one control group. in all the three experimental groups self - etching primers were used as preliminary step while in group ii 2% chlorhexidine gluconate was used before application of the self - etching primer. however in group iii cpb which is supposed to have antiseptic properties was utilized, while in the control group however conventional self - etching primer was used. filtek z350 composite resin was utilized according to the manufacturer 's instructions for the restoration of the cavities in all the groups. results of the present study indicated that the scores of microleakage of the four groups at the enamel margin when compared, there was no statistical significant difference found (p > 0.05). the lowest mean microleakage values at the enamel margin were obtained from group ii when 2% chlorhexidine gluconate was used as a coating. the highest mean microleakage was obtained from group iii at enamel margin where cpb was used and group iv the control group. when the scores at gingival margin of four groups were compared there was no statistical significance (p > 0.05). however the lowest mean values at gingival margin were obtained from group i (clearfil se bond) followed by group ii (clearfil se bond + 2% chlorhexidine gluconate). the highest mean value were obtained at gingival margin from group iii (cpb) and group iv (control). when comparing the enamel and gingival margin in the entire three experimental groups, microleakage was higher in the gingival margin compared to the enamel margin. alani and toh (1997) observed that dentin permeability is also an important factor to be considered.8 in the present study both enamel and gingival margin were studied for microleakage and found to be higher in the gingival margin when compared to enamel margin. (2010)3 observed that chlorhexidine gluconate 2% is a well - known disinfectant and has antimicrobial effect when applied prior to placing a restoration. 2% chlorhexidine gluconate functions as a matrix metalloproteinase inhibitor apart from its antibacterial property, which may also prevent collagen degradation and disintegration of the bonding interface over a period of time. (2009) observed in their studies on microleakage in resin composite restorations that the most critical factor affecting the microleakage of resin composite is the polymerization shrinkage. the forces generated by polymerization shrinkage exceeded the bond strength especially in the gingival margin. polymerization shrinkage in this area is not compensated for by acid etching and the application of dentin adhesive.6 clearfil se bond is one of the first self - etching primer in the market and belong to the mild group of self - etch adhesive with a hydrogen ion concentration very close to two clearfil se bond contains functional monomer 10-methacryloxyloxydecyl dihydrogen phosphate (10-mdp), which has two hydroxyl groups that also may bind to calcium. moreover, 10-mdp causes minimal dissolution of smear plugs and the limited opening of the tubules, thus reducing dentin permeability. the functional monomer 10-mdp also facilitates penetration, impregnation, polymerization and the entanglement of monomers with demineralized dentin to form a relatively thick hybrid layer.7 others have reported that mdp tightly adheres to hydroxyapatite and that its calcium salt hardly dissolved in water. same mean microleakage values were obtained with application of 2% chlorhexidine gluconate as a pre application. with the cpb application almost similar microleakage values were obtained without application of 2% chlorhexidine gluconate and there were no statistical significant difference in leakage values among the experimental group. in the control group xeno iii also similar results were obtained without the application of 2% chlorhexidine gluconate on the dentin margin. xeno iii is again a self etching primer with a two bottle system used as a control. cm esteves (2010)4, evaluated the antibacterial properties of some self etching / primer solutions against oral streptococci. they concluded that the self etching adhesives or self etching primers used in the current study demonstrated different levels of inhibition for oral streptococci tested. clearfil protect bond self etching primer exhibited the most effective antibacterial activity against oral streptococci. cpb is a two - step self etch adhesive system composed of a self etching primer containing the antibacterial monomer 12- mdpb and a fluoride releasing adhesive. the antibacterial monomer 12- mdpb is a polymerizable biocide and has a strong bactericidal activity against oral bacteria. although cpb is derived from clearfil se bond with modifications in the components mean microleakage values were higher than clearfil se bond in the current study. (2009)6 however, no statistical difference in microleakage were found among all the groups in gingival surface and when the scores of microleakage at the occlusal surface position of the four groups were compared. cpb contains crystal - like structures, and these are likely to the naf crystals. although the filled adhesive resins have been said to have greater mechanical properties, differences in the filler content and composition of the adhesive may account for variations between them. the non - uniform distribution of the nanometer - sized fillers observed may contribute to a relatively poor mechanical property in some regions.6 hence, microleakage values could be higher than clearfil se bond in the current study. results of the study indicated that the score of microleakage of the four groups at the enamel margin and gingival margin when compared did not have statistical significance (p > 0.05). under the conditions of this in - vitro study the following assessments were made : none of the materials tested in this study completely eliminated microleakage at the enamel and at the gingival marginall of the tested materials provided better sealing at the enamel margin than at the gingival marginthe lowest mean microleakage were obtained from group ii (clearfil se bond + 2% chlorhexidine gluconate) followed by group i (clearfil se bond) at the enamelgroup i (clearfil se bond group) had the lowest mean microleakage values which were same for group ii (clearfil se bond + 2% chlorhexidine gluconate group) at the gingival margin. none of the materials tested in this study completely eliminated microleakage at the enamel and at the gingival margin all of the tested materials provided better sealing at the enamel margin than at the gingival margin the lowest mean microleakage were obtained from group ii (clearfil se bond + 2% chlorhexidine gluconate) followed by group i (clearfil se bond) at the enamel group i (clearfil se bond group) had the lowest mean microleakage values which were same for group ii (clearfil se bond + 2% chlorhexidine gluconate group) at the gingival margin. | aim : to evaluate microleakage in resin composite restorations after antimicrobial pre treatmentsmaterials and methods : forty freshly extracted non carious human premolars were procured. in all forty premolar specimens, class v preparation of standard dimension were prepared and were randomly divided into three experimental and one control group. in all control and experimental groups the class v preparations were restored with filtek z350 composite restorative material. the experimental groups included different self etching primers and 2% chlorhexidine gluconate. the control group included xeno iii and no antimicrobial pre - treatment was done for the control group. thereafter these specimens were thermocycled, dried and sealed with nail varnish, leaving 1 mm around the restoration and immersed in 0.5% basic fuchsin for 24 hours and then the specimens were subjected for microleakage evaluation. the results were statistically analyzed by kruskal wallis test and mann whitney u test.results:results indicate that group ii (2% chlorhexidine gluconate group) had the minimum mean value (15.05) and group iii(clearfil protect bond group) and iv(control group) had the maximum mean microleakage at the enamel margin (23.00). at the gingival margin the lowest mean microleakage values were obtained with group i (clearfil se bond group) and group ii (2% chlorhexidine gluconate) (20.25) and highest with group iii and group iv (20.85). the difference was not statistically significant both at the enamel margin and the dentin margin (p>0.05).interpretation & conclusions : within the limitations of this in - vitro study, we conclude that : none of the materials tested in this study completely eliminated microleakage at the enamel and at the gingival margin.all of the tested materials provided better sealing at the enamel margin than at the gingival margin. |
advances in the ventilatory management of acute lung injury (ali) and ards over the past decade have been dramatic. in particular, the use of a low - tidal volume (6 ml / kg predicted body weight) and plateau pressure - limited strategy has been demonstrated to reduce mortality from 40% to 31%. further, a large, multicenter, randomized, controlled trial, demonstrated the equivalence of higher and lower levels of positive end - expiratory pressure. over this time, a number of nonventilatory therapies for ali / ards have been investigated. many of these have not proven to be effective, while others appear more promising, lipids being one of them. the lipid emulsions generally used in the parenteral nutrition of critically ill patients are rich in long - chain triglycerides (lct), especially linoleic acid (polyunsaturated series 6 fatty acid ; pufa n-6, 18:2 n-6). these fatty acids, aside from producing adverse metabolic effects (transitory hyperlipidemias), can alter pulmonary gas exchange due to their potentially proinflammatory properties. the mct / lct emulsions are oxidized faster and provide fewer amounts of pufas than lct emulsions. thus, mct / lct emulsions have been associated with a lower risk of lipid peroxidation and fewer alterations of membrane structures. polyunsaturated fatty acids of the n-3 series (pufa n-3) are precursors of biologically active substances, e.g., the series 3 and 5 eicosanoids. these molecules use the same metabolic routes and compete for the same elongases and desaturases as linoleic and arachidonic, but ultimately they are mediators that have a much less active biological profile than linoleic acid derivatives. in 146 patients with ards treated with an enteral diet rich in eicosapentaenoic acid, -linolenic, and antioxidants, an improved pao2/fio2 ratio, a reduction in pulmonary inflammatory response, frequency of new organ failures, days of mechanical ventilation and length of stay in icu were observed. in 100 patients with acute pulmonary injury receiving a similar enteral diet, oxygenation was better, pulmonary compliance improved, and there was less need for mechanical ventilation. the administration of lipid emulsions has been associated with changes in pulmonary function that depends on pulmonary disease, dose, duration, speed of administration, and kind of infused lipids. a meta - analysis of 12 randomized controlled trials comparing standard enteral nutrition with antioxidant nutrition found decreased rates of infection but again no effect on mortality. many patients with ards require sedation / analgesia and muscle relaxation, inducing intestinal ileus and making intolerance to enteral nutrition. it has been hypothesized that adding n-3 to a mct / lct mixture used in enteral nutrition, should be beneficial because of the reduced amount of pufa, would be less toxic than a lipid emulsion based on soybean oil, and that the addition of fish oil would be protective because of its anti - inflammatory properties. a study which evaluated the hemodynamic changes and variations in pulmonary gas exchange that occurred in patients with ards given an intravenous lipid emulsion enriched with n-3 fatty acids could not show any benefit. in unselected critically ill medical patients, fish oil supplementation that increased the n-3/n-6 pufa ratio to 1:2 did not affect inflammation or clinical outcome, compared to parenteral lipid nutrition with an mct / lct emulsion. a small study showed that while the lct emulsion induced no deleterious effects on oxygenation in ards patients, the lct / mct emulsion improved the pao2/fio2 ratio and had a further beneficial effect on oxygen delivery. the present study evaluated the effects of an intravenous lipid emulsion enriched with n-3 fatty acids in ventilated patients with acute lung injury. this single - center, placebo - controlled, investigator blind, prospective, randomized clinical trial was approved by the institutional ethics committee. written informed consent all patients admitted to the medical icu of this hospital between july 1 and december 31 2009 were screened for eligibility. we studied 86 consecutive patients with suspected ards in the first 48 hours of admission.the inclusion criteria were : bilateral pulmonary infiltrates of sudden onset in the chest radiograph, pao2/fio2 less than 200, and pulmonary capillary pressure less than 18 mm hg. patients were excluded for age younger than 18 or older than 80 years, pregnancy, liver failure (bilirubin > 3), hiv positivity, leukopenia (2.5 mg / dl) or need for renal dialysis, signs of heart failure, transplantation, multiple blood transfusions, participation in other clinical trials simultaneously or in the last 60 days, treatment with nitrous oxide or corticoids (prednisolone 2 mg / kg / d or equivalent), multiple organ failure, severe dyslipidemia, propofol treatment, head injury, cerebral hemorrhage, receiving immunosuppressive regimen, radiation, allergy to any of the constituents of nutritional products. patients were divided into two groups : group 1 : standard diet (high fat, low carbohydrate kitchen feed) group 2 : standard diet + parenteral omega 3 fatty acids, omegaven (fresenius kabi), an emulsion of 10% fish oil, for 14 days. patients in each stratum were assigned to the intervention or control group by the institutional intensivists, by use of a computer - generated block randomization list inaccessible to the investigators. all participants were started on enteral feeding through a nasogastric tube within 24 hrs of icu admission. conventional modes of ventilation were used in all cases, including assist control ventilation (pressure mode), pressure support ventilation (evita 4, drager, lubeck, germany). the main goals of mechanical ventilation were an oxygen saturation of 90%, peak airway pressure of 35 cm h2o, and tidal volume of 6 ml / kg. levels of peep and fio2 were adjusted to achieve these goals. ventilation settings and decisions regarding readiness for extubation were left to the discretion of the investigators who were blinded to the nutritional prescription. following data were collected : demographic data : age, sex, weight, height, body mass index (bmi), and diagnostic category for icu admission (medical, surgical, trauma)assessment of oxygenation and respiratory function : abg at baseline, 4, 7, and 14 days or discharge from icu. ventilatory settings, tv, peep were also recorded simultaneouslyassessment of metabolic and nutritional variables : harris benedict equation was used to calculate the predicted energy expenditure according to anthropometric variables. albumin levels were assessed at baseline and on days 4, 7, and 14. demographic data : age, sex, weight, height, body mass index (bmi), and diagnostic category for icu admission (medical, surgical, trauma) assessment of oxygenation and respiratory function : abg at baseline, 4, 7, and 14 days or discharge from icu. ventilatory settings, tv, peep were also recorded simultaneously assessment of metabolic and nutritional variables : harris benedict equation was used to calculate the predicted energy expenditure according to anthropometric variables. albumin levels were assessed at baseline and on days 4, 7, and 14. since the study medication was approved for therapeutic use, additional parameters of drug safety were not analyzed as outcome measures. as inhibition of platelet function is an established possible side effect of fish oil, the total quantity of transfused packed red cells / platelets, and bleeding events were recorded. primary outcome measures included changes in oxygenation and breathing patterns assessed at days 4, 7, and 14. secondary outcomes included length of ventilation, length of icu stay, length of hospital stay, and in hospital mortality. data will be presented as mean, standard deviation, median values, and mode as appropriate. demographic data were compared across with standard t tests or one way analysis of variance for all continuous variables. all p values were two sided, significance was assigned at a threshold of 0.05. a t test was used to compare a continuous variable (e.g., pao2/fio2 ratio, length of stay). a contingency table (fishers or chi - square test) of the 86 patients screened, 61 were enrolled [figure 1 ]. of the remainder, 30 patients received the standard diet and 31 patients received the drug in addition to standard diet. pao2/fio2 ratio was better in the control group to start with but still not significantly different from the drug group. albumin levels were low at baseline and did not correlate with patient response over time, reinforcing the poor diagnostic and prognostic value of albumin in critical care illness. consort diagram showing conduct of the study baseline patient values oxygenation shown in table 2 was no different in the two groups at days 4, 7, or 14. however, the observed fall in pao2/fio2 ratio in the control group from baseline to day 14 was significantly higher as compared to the drug group (199 to 95 vs. 145 to 128, p = 0.0004) [figure 2 ]. gas exchange values (pao2/fio2 ratio) trend line showing significant difference (p < 0.0004) in oxygenation (pao2/fio2 ratio) between the intervention and control group at day 14. survivors were more in the drug group (24 vs. 17) at 28 days ; table 4. at 28 days ; lov and los were same in the two groups, both for survivors and non survivors. outcome variables at 28 days overall survival was better in the drug group (22 vs. 16) table 5. length of hospital stay was shorter in the survivors group that received the drug compared to the survivors in the control group (21.5 13.49 vs. 26.63 18.22) but not statistically significant (p = 0.32). oxygenation shown in table 2 was no different in the two groups at days 4, 7, or 14. however, the observed fall in pao2/fio2 ratio in the control group from baseline to day 14 was significantly higher as compared to the drug group (199 to 95 vs. 145 to 128, p = 0.0004) [figure 2 ]. gas exchange values (pao2/fio2 ratio) trend line showing significant difference (p < 0.0004) in oxygenation (pao2/fio2 ratio) between the intervention and control group at day 14. lov and los, table 3, was no different between the two groups. survivors were more in the drug group (24 vs. 17) at 28 days ; table 4. at 28 days ; lov and los were same in the two groups, both for survivors and non survivors. outcome variables at 28 days overall survival was better in the drug group (22 vs. 16) table 5. length of hospital stay was shorter in the survivors group that received the drug compared to the survivors in the control group (21.5 13.49 vs. 26.63 18.22) but not statistically significant (p = 0.32). oxygenation shown in table 2 was no different in the two groups at days 4, 7, or 14. however, the observed fall in pao2/fio2 ratio in the control group from baseline to day 14 was significantly higher as compared to the drug group (199 to 95 vs. 145 to 128, p = 0.0004) [figure 2 ]. gas exchange values (pao2/fio2 ratio) trend line showing significant difference (p < 0.0004) in oxygenation (pao2/fio2 ratio) between the intervention and control group at day 14. survivors were more in the drug group (24 vs. 17) at 28 days ; table 4. at 28 days ; lov and los were same in the two groups, both for survivors and non survivors. outcome variables at 28 days overall survival was better in the drug group (22 vs. 16) table 5. length of hospital stay was shorter in the survivors group that received the drug compared to the survivors in the control group (21.5 13.49 vs. 26.63 18.22) but not statistically significant (p = 0.32). in our opinion, this is the first study to examine the impact of parenteral omega 3 fatty acids on ventilatory parameters and clinical outcome of ards patients who were otherwise fed enterally. supplementation of enteral nutrition with fish oil for 14 days could not be proven to affect the examined ventilatory parameters or measures of clinical outcome in these patients. the hypothesis had been that inflammatory and subsequent anti - inflammatory reactions would be reduced, resulting in clinical benefit. this expectation had been based on results of clinical studies.[1619 ] the majority of such studies showing a benefit from intravenous supplementation of fish oil had been performed in surgical patients. there may be several reasons why the expected effects of fish oil supplementation could not be demonstrated in this study. first, control patients were more severely ill in terms of pao2/fio2 ratio though the apache scoring was comparable. second, the choice of lipids for standard care could partially explain why fish oil supplementation was not efficacious. in most clinical studies showing a benefit for fish oil, pure lct lipids had been used in the control groups. lct lipids, however, have been demonstrated to exert inflammatory and immunosuppressive influence on septic patients. parenteral omega 3 fatty acids have been shown to improve glucose control and decrease inflammatory markers in coad and after major surgery patients. in the present study, standard diet containing 50% mct and 50% lct was used, which reduced the average daily dose of linoleic acid to less than 0.3 g / kg. thus, the expected effects of modifying the lct / mct ratio may not have been detected because the standard nutrition itself had fewer immunological side effects. third, the route of use and specific composition of n-3 pufas could be important for their immunomodulatory effect. enteral application of a mixture of fish oil, canola and borage oil, which in contrast to fish oil alone contains a substantial amount of gamma - linolenic acid, has shown to improve outcome parameters in acute lung injury and acute respiratory distress syndrome. it may also be that this pufa composition and its enteral application carry more benefit than the parenteral application of fish oil. considering their mechanism of action, one could logically expect two principal side effects of n-3-pufas : impaired hemostasis (due to reduced synthesis of thromboxane) and reduced immune function. bleeding events did not occur more frequently in the intervention group. no influence of fish oil supplementation on nutritional status, as assessed by weight and albumin (data not shown), was observed. in effect, the present study produced no evidence that fish oil supplementation is harmful for the critically ill. some limitations of the present study merit consideration. sample - size estimates were based on data from a previous study in a less heterogeneous population. to compensate for greater variability, much larger sample sizes could well have been necessary to detect an effect of fish oil supplementation. however, the intervention in this study was aimed at altering the n-3/n-6 pufa ratio to 1:2. this aim was generally achieved with a daily fish oil dose of 0.1 g / kg. favorable effect of fish oil have been demonstrated with daily doses as low as 0.10.2 g / kg (17). considering their mechanism of action, one could logically expect two principal side effects of n-3-pufas : impaired hemostasis (due to reduced synthesis of thromboxane) and reduced immune function. bleeding events did not occur more frequently in the intervention group no influence of fish oil supplementation on nutritional status, as assessed by weight and albumin (data not shown), was observed. in effect, the present study produced no evidence that fish oil supplementation is harmful for the critically ill. sample - size estimates were based on data from a previous study in a less heterogeneous population. to compensate for greater variability, much larger sample sizes could well have been necessary to detect an effect of fish oil supplementation. however, the intervention in this study was aimed at altering the n-3/n-6 pufa ratio to 1:2. this aim was generally achieved with a daily fish oil dose of 0.1 g / kg. favorable effect of fish oil have been demonstrated with daily doses as low as 0.10.2 g / kg (17). in comparison to standard enteral nutrition, supplementation with parenteral fish oil did not change parameters of ventilation or clinical outcome in selected ards patients. | objective : to determine the effects of parenteral omega 3 fatty acids (10% fatty acids) on respiratory parameters and outcome in ventilated patients with acute lung injury.measurements and main results : patients were randomized into two groups one receiving standard isonitrogenous isocaloric enteral diet and the second receiving standard diet supplemented with parenteral omega 3 fatty acids (omegaven, fresenius kabi) for 14 days. patients demographics, apache iv, nutritional assessment and admission category was noted at the time of admission. no significant difference was found in nutritional variables (bmi, albumin). compared with baseline pao2/fio2 ratio (control vs. drug group : 199 124 vs. 145 100 ; p = 0.06), by days 4, 7, and 14, patients receiving the drug did not show a significant improvement in oxygenation (pao2/fio2 : 151.83 80.19 vs. 177.19 94.05 ; p = 0.26, 145.20 109.5 vs. 159.48 109.89 ; p = 0.61 and 95.97 141.72 vs. 128.97 140.35 ; p = 0.36). however, the change in oxygenation from baseline to day 14 was significantly better in the intervention as compared to control group (145/129 vs. 199/95 ; p < 0.0004). there was no significant difference in the length of ventilation (lov) and length of icu stay (los). there was no difference in survival at 28 days. also, there was no significant difference in the length of ventilation and icu stay in the survivors group as compared to the non survivors group.conclusions:in ventilated patients with acute respiratory distress syndrome, intravenous omega 3 fatty acids alone do not improve ventilation, length of icu stay, or survival. |
evolutionary processes, such as genetic drift and natural selection, can cause organisms developmental processes to evolve. the evolution of such processes can promote morphological and physiological diversity, as well as help an organism adapt to new environmental challenges and ecological niches (hall, 1999). to understand in detail how developmental processes evolve, we first must identify the selection pressures and molecular mechanisms that elicit these changes e.g. this multidisciplinary approach is difficult to achieve for most organisms because the environmental conditions that originally led to their developmental changes and thereby afforded specific selection advantages are often ambiguous. a few recent studies have begun to reveal the advantages associated with developmental traits, including a feeding advantage for animals with certain jaw morphs or different tooth shapes that depend on an animals ' food sources (wainwright., 1995 ; albertson., 2001 ; kocher, 2004 ; alfaro., 2005 ; laffont., 2009 ; parsons., 2009) ; increased mechanosensors to compensate for animals with poor visual cues (yoshizawa., 2010) ; as well as conspicuous (hert, 1989 ; couldridge, 2002) and cryptic pigmentation patterns (klingenberg, 2010 ; linnen., 2013). yet, these scientific accomplishments also emphasize the vast knowledge gap regarding how animals evolve adaptive traits as their genomes and developmental processes shift. if we can identify an animal that has evolved under clear and simple selection pressures, however, it will be possible to more directly test the advantages of the developmental changes they have evolved to undergo. the cave ecosystem is characterized by perpetual darkness, the absence of primary productivity, and sparse food resources (culver, 1982 ; culver., 2009). these conditions exert significant pressure on cavedwelling animals such that caveadapted animals from most major phyla exhibit a remarkable convergence in morphological and physiological changes related to cave life, including features that are both constructive (longer appendages, novel behaviors, elaborate nonvisual sensory systems) and regressive (reduced vision and pigmentation) (culver, 1982 ; culver., 2009). some aquatic cave populations, including teleost, have been isolated from their surfacedwelling relatives for such a long time that they have accumulated cavespecific mutations (for examples, see protas., 2006 ; gross., 2009 ; elipot., 2014). in contrast, it is relatively difficult to identify the original mutations and selection pressures influencing surfacedwelling animals because they frequently hybridize with other populations and live in complex fauna. in this respect, cave animals are valuable models that we can use to analyze adaptive changes that occur under clearly defined selection pressures. behavior is a convenient window through which we can observe the selective advantages associated with morphological and physiological traits. for example, better sensitivity of a sensory organ can improve behavioral responses in predator avoidance, prey hunting, or mating, which lead to enhanced survivorship or fecundity. natural selection then screens animals with the best combination of traits, depending on their fitness (mayr, 1963 ; bateson, 1988 ; wcislo, 1989 ; westeberhard, 1989 ; gittleman., 1996 ; wimberger., 1996 this review discusses recent advances in our understanding of behavioral evolution and related morphological and physiological traits in an animal that lives in a habitat with clear selection pressures : the mexican cavefish, astyanax mexicanus, an established animal model for studying evolution and development (mitchell., 1977 ; wilkens, 1988 ; jeffery, 2001, 2008, 2009 ; protas. 2013). within the past few million years, at least five independent colonizations by two different migrating waves of eyedsurfacefish ancestors have established 29 geographically isolated astyanax cavefish populations in northeastern mexico (ornelasgarca., 2008 ; bradic., 2012, 2013 ; strecker., 2012 ; coghill., 2014 after an initial radiation underground, the founder cavefish populations became isolated and evolved independently. food sources are limited in these caves, and likely consist of small soil crustaceans, microorganisms, and organic matter present in the water that drops from the cave ceiling (culver., 2009). depending on the cave, additional nutrient sources for cavefish may come from the guano dropped by bats living at the cave ceiling and organic matter brought in by seasonal flooding (culver. when the habitat of these fish changed from the surface to the cave, the cavefish ancestor evolved regressed eyes, reduced pigmentation or albinism, enhanced oralpharyngeal morphologies, and expanded nervous systems (wilkens, 1988 ; yamamoto., 2000, 2004, 2009 ; jeffery, 2005 ; protas., 2006 ; alunni., 2007 ; franzodendaal., 2007 ; 2013). despite their 26 million year separation from a. mexicanus surface cohorts, the cavefish and surface fish cohorts remain interfertile, allowing us to study the evolution of behavioral, morphological and physiological traits by genetic analysis. recent work is revealing the genetic architectures that underlie the behavioral evolution of cavefish species, including the examination of the following behaviors : vibrationattraction behavior (vab) (eigenmann, 1909 ; hill, 1969 ; parzefall, 1983 ; yoshizawa., 2010, 2012a, 2012b) ; sleep loss (dubou., 2011, 2012) ; reduced aggression (parzefall, 2001 ; elipot., 2013) ; loss of schooling and shoaling (parzefall, 2001 ; kowalko., 2013a) ; stabilized feeding posture (schemmel, 1980 ; kowalko., 2013b) ; enhanced chemosensory ability (protas., 2008 ; bibliowicz.,, 2011 ; dubou., 2012, 2011 ; beale., 2013). among these behaviors, vab is the moststudied, positively selected behavior regarding how it benefits cavefish in their environment, its sensory and genetic basis, and its developmental process. here, i review the behaviors that have been evolutionarily advantageous and most informative in deciphering how a. mexicanus populations have adapted to their caves. cavefish exhibit vab ; that is, they swim toward an oscillating object, either in a natural cave pool or in a laboratory (fig. vab represents a potential foraging behavior that has evolved repeatedly in at least three different astyanax cavefish populations (fig. 1b) (parzefall, 1983 ; abdellatif., 1990 ; yoshizawa., 2010). considering these populations likely evolved separately under similar ecological conditions (mitchell., 1977 ; borowsky, 2008 ; ornelasgarca., 2008 ; wilkens, 2010 ; bradic., 2012 ; gross, 2012 ; strecker., 2012 ; coghill., 2014), the convergence of these populations towards vab suggests the distinct advantage this behavior would have in the cave environment. yet, the extent of vab is variable within and among populations, and some individuals of one of the oldest cavefish populations, pachn, even lack this behavior (fig. the converse is true for surface fish : while most surface fish lack vab, a few individuals exhibit low levels of this behavior (fig. (a) swimming path (purple lines) of surface fish (left) and pachon cavefish (right) during a 3min assay period. dotted lines represent the 2cm diameter quantification area surrounding the glass rod (dark spot in the center of the chamber). three separated cave populations showed vab (above the threshold level of 4 approaches), but no vab was apparent in either surfacefish population. onetailed mannwhitney tests with bonferroni correction for multiple comparisons were performed between a group of ro choy and ro tampan surface fish, and each cavefish population., p 4 approaches) and without (4 approaches) and without (< 4 approaches) vab using a stimulus of 50 hz. bars show the proportion of strikes at prey between pairs of surface fish (black fish cartoons) and cavefish (orange fish cartoons) with or without vab during a 1min assay period in darkness (left bars) and in light (right bars). a total of eight pairs of cavefish versus surface fish (ba), and five pairs of surface fish with versus without vab (bb) in the dark and light are shown. values are mean ratio of strikes 95% confidence intervals of the mean., p < 0.05 ; p < 0.01. for details about the method, please see yoshizawa. taking advantage of this variation within populations, the adaptive significance of vab was tested in competitive preycapture experiments wherein pairs of fish with and without vab were fed small amount of vibrating prey : brine shrimp (fig. 2b). in darkness, cavefish were significantly better at capturing the brine shrimp than surface fish (fig. the key finding, however, was that surface fish with vab had significantly more brineshrimp captures in the dark than surface fish without vab, a difference that disappeared in the light (fig. thus, an individual 's ability to utilize vab plays a role in foraging and is likely advantageous for survival in dark caves. in wild populations of surface fish, vab is presumably deleterious because fish with vab may swim toward predators, such as the nocturnal prawn, at night (wilson., 2004 in contrast, vab is adaptive in cavefish because it increases foraging in an environment with limited food availability, minimal light, and macroscopic predators (yoshizawa., 2010, 2011). consequently, the vab already present as a standing variation in ancestral surface populations may have been subject to positive selection once the cavefish ancestors started colonizing caves. probing a little deeper reveals hints at how these cave populations have honed vab to survive. cavefish vab occurs at a relatively low frequency range (about 550 hz), with a peak frequency of 35 hz (yoshizawa., these values are within the range that cavefish can detect with their mechanosensory lateral line (2080 hz) (coombs. interestingly, none of the surface fish, even those with vab, displayed such behavioral tuning (yoshizawa. furthermore, because many crustaceans produce water fluctuations at 3040 hz while swimming (lang, 1980), the tuning of cavefish 's vab at 35 hz is a novel trait that is likely adaptive in the cave ecosystem. the cavefish 's optimized frequency detection system suggests that the lateralline sensory organ that cavefish use to detect water flow is likely a vibrationsensory receptor that has been enhanced in cavefish compared to surface fish (schemmel, 1967 ; mnz, 1989). one type of lateralline sensory organ is the superficial neuromast (sn), which is composed of ciliated hair cells and a gelatinous cupular matrix (fig. the sns of cavefish are larger and more numerous than those of surface fish, and are primarily responsible for vab, as determined by sn ablation studies and genetic analysis (schemmel, 1967 ; teyke, 1990 ; yoshizawa., 2010). although sns appear throughout the body and are particularly abundant on the cavefish 's head, those sns located within the orbit of the cavefish 's degenerated eye seem to play a particularly important role in vab (yoshizawa., 2012b). this suggests that the extra cranial space created with the loss of the cavefish 's eyes is an important event that promoted this novel behavior. as there was no difference in the number of eyeorbit sns (eo sns) among surface fish in fact, no eo sn was observed in surface fish their appearance in cavefish did not arise through the selection of a standing phenotypic variation (yoshizawa., 2012b) ; instead, there must be a distinct evolutionary path linking vab and eo sn evolution. surface fish, n = 10 at 2 months postfertilization (mpf), = 9 at 3 mpf, = 10 at 4 mpf, and = 12 at 1yearold ; pachn cavefish, n = 9 at 2 mpf, = 8 at 3 mpf, = 9 at 4 mpf, and = 11 at 1yearold ; tinaja cavefish, n = 7 at 1yearold. pachn cavefish significantly increased vab and eo sn quantity, whereas tinaja cavefish were indistinguishable from surface fish in both vab level and eo sn number at 1 year old (oneway anova followed by plannedcontrast analysis at 1 year old)., p < 0.01 ;, p < 0.001 ; n.s., not significant. (c) example fluorescence images of daspeivital staining of surface fish and two cavefish. thick, white dotted lines indicate the infraorbital canal, and white arrowheads indicate eo sns that were counted in (b). thin, white dotted line encloses the neuromasts in an adjacent region on the cranium, the third infraorbital bone (previously denoted as the third suborbital bone, so3) (yoshizawa. cavefish eyes start degenerating around 36 hr postfertilization, but no significant increases in any sns (including eo sns) are detectible, relative to surface fish, until 2 months postfertilization (yoshizawa., 2010). many aspects of eye degeneration are controlled by increased hedgehog signaling in cavefish (yamamoto., 2004 ; jeffery, 2005), so a surface fish was engineered to overexpress shh by mrna injection so that its eyes would also degenerate to test the relationship between eye retention and eo sn development ; these shhoverexpressing fish did not develop eo sn (yoshizawa., 2012b). therefore, neither eye degeneration alone nor excess hedgehog signaling in early development induces eo sn formation or enlargement ; note that tests that ask if hedgehog signaling plays a role later in development, when eo sns first appear at 2 months old, have not yet been conducted. although there is no shared developmental process between eye degeneration and eo sn formation, ontogenic analysis and a comparison using different cave populations could help establish the relationship between vab and eo sn evolution. the first approach was to determine how the timing of vab onset correlates with the development of eo sns in surface fish versus pachn cavefish by studying fish at 2, 3, and 4 months postfertilization and in the youngadult stage (1yearold). cavefish eo sn quantity gradually increases between 2 months and the first year of age (fig 3) ; this temporally correlates with the appearance and enhancement of vab, which is first detectible between 2 and 3 months postfertilization (fig. interestingly, another cavefish population, tinaja, presents with a weak level of vab, similar to the amount that some individual surface fish display, and does not have eo sns even though their eyes degenerate (fig. this difference further supports the correlation between cavefish vab and eo sn evolution, and also reinforces that eo sns are not directly attributable to eye degeneration. while eo sn ontogeny corresponds with vab cavefish, there is no correlation between vab and the total number of sns in an individual. while pachn and tinaja cavefish have significantly more sns in their infraorbital region than surface fish do (fig. 3c), the development of these organs is not genetically correlated with the level of vab, and their ablation does not detectably affect vab (yoshizawa., 2012b). these observations further emphasize the model that the eo sn, as a minor group of sensory organs, plays an important role in facilitating vab whereas other sns in the infraorbital region may contribute to other sensations, such as hydrodynamic imaging (see below, and also hassan, 1989 ; montgomery., 2001 ; windsor., 2008a, 2010a ; coombs., 2010). development of the eo sn may therefore have evolved as a consequence of positive selection for the enhancement of an adaptive behavior, indicating that studying behavioral traits can help reveal distinct roles for sns residing in different cranial areas. considering the importance of the eo sns in vab, and thus the cavefish 's ability to forage, it is surprising that vab and eo sns emerge so late in development. such timing may occur for two reasons : (1) the developmental process might be constrained or (2) their foraging preference might change with age. to address the first possibility, wada. the sns first form at the edge of a developing intramembranous bone in the cranial region (the operculum, at 45 days postfertilization). in astyanax, however, the second and third infraorbital (suborbital) bones, which underlie the infraorbital sns and eo sns, develop in cavefish around 23 months postfertilization (yamamoto., 2003). therefore, the number of sns in cavefish may only be able to increase after the infraorbital bone forms, meaning that the development of eo sn and thereby vab is developmentally constrained until the infraorbital bone forms at 23 months postfertilization. the second reason for the late development of vab and eo sn that cavefish foraging preferences change over time derives from the hypothesis that cavefish larvae and adults may have different foraging preferences, possibly to reduce competition with one another. cavefish adults quickly respond and move toward a water surface that is disturbed by falling water droplets or bat guano (likely via vab), but they also scavenge for food, perhaps relying on chemosensory inputs originally adapted for bottom feeding. in contrast, relatively small, younger fish scavenge for food exclusively at the bottom, again possibly using chemosensation (parzefall, 1983 ; personal and s. rtaux 's observation at the cave los sabinos). in the laboratory, however, large cavefish occasionally eat smaller cavefish, suggesting that small, younger fish can not compete with mature adults. therefore, the late development of eo sn and vab may offer small, young fish a better chance to survive by avoiding the hazards of larger, hungry conspecifics that are using vab to forage just beneath the water 's surface. while it is difficult to determine which of these possibilities developmental constraints or an ecological advantage is responsible for the late development of vab and eo sns, genetic experiments have helped. by overexpressing or knocking down the gene(s) responsible for eo sn development using available transgenic and/or genome editing methods (e.g., talen and crispr technologies) (gaj., 2013 ; 2014 ; ma., 2015), and performing generegulatorynetwork analysis (gavinsmyth., 2013), we will gain insight to the relationship between eo sn development and the timing of dermalbone formation. deciphering which gene(s) are responsible for vab will through in situ hybridization and/or immunohistochemistry techniques help reveal if the novel sensory tuning occurs at the level of first projection, the octavolateralis nucleus, or higher, such as at the torus semicircularis area. it is unlikely that sensory tuning is achieved at the neuromast level, however, since the sensitivity estimation based on the morphometrics of neuromasts does not peak at 35 hz (yoshizawa. nevertheless, the foraging advantage associated with vab gives us a new way to resolve the driving forces for the evolution of other morphological traits, including cranial sn, intramembranous bone, and also neural connections in the central nervous system. the pleiotropic effects of enhanced hedgehog signaling promote eye degeneration as well as increases in jaw size and number of taste buds in astyanax cavefish (table 1) (yamamoto., 2009, 2004 ; jeffery, 2005). given the necessary redistribution of attention to the nonvisual senses, selection for these other constructive traits, which are fostered by hedgehog signaling, during cavefish evolution no one has tested if these gustatory traits (larger jaws, more taste buds) improve cavefish 's foraging abilities. nevertheless, an enlarged jaw is likely beneficial in the darkness, possibly as an adaptation to bottom feeding, and carrying more taste buds likely improves foraging because they could help navigate to food in the dark. to understand the adaptive significance of these morphological and sensory shifts, however for example, behavioral analyses showing that these gustatory traits are advantageous would support the hypothesis that oralpharyngeal shifts can promote eye reduction through pleiotropy. if having a large jaw and more taste buds does not enhance foraging efficiency, however, these traits would constitute another example of not all evolved traits are adaptive hedgehog signaling is also required for other critical developmental processes, beyond cavefish eye degeneration and a few gustatory traits. during development, enhanced hedgehog signaling increases the number of migratory cells that enter the olfactory bulbs (menuet., 2007) and serotonergic neurons at the hypothalamus (elipot, 2013). while the former may represent a positive behavioral response linked to the chemical stimulus of food (table 1) (bibliowicz., 2013), the latter is an example of a change in behavioral strategy. for instance, having an enhanced serotonergic system in the anterior hypothalamus and the raphe nucleus redirects attacking behavior towards foraging behavior. attacking behavior is frequently used to establish a hierarchical relationship between individuals in surface dwelling fish (magurran, 1993 ; elipot., 2013), but this does not occur in cave populations, possibly because a. mexicanus cavefish lack schooling behavior (elipot., 2013 ; kowalko., 2013b ; see below). indeed, cavefish have higher serotonin levels and more serotonergic neurons at the hypothalamic anterior paraventricular nucleus and hindbrain raphe than their surface counterparts ; these anatomical and endocrine changes are believed to have shifted cavefish behavior from attacking to foraging (elipot., 2013). another study reported that cavefish have higher serotonin levels in the brain because of mutations in the serotonin degradation enzyme, monoamine oxidase (mao)which again promotes foraging behavior (table 1) (elipot., 2014). the cavefish 's heightened foraging is likely adaptive to the cave environment, where there is little food and few predators : fish eat on a first come, first served basis. in fact, enhanced foraging behavior has converged in independentlyevolved cave populations (elipot., 2013). yet even though cavefish have higher numbers of serotonergic neurons in their hypothalamus by 1 week of age (elipot., 2013), there is no obvious difference in attacking behavior between cavefish and surface fish at that age (personal observation). this may be because fish at this young age are too immature to establish hierarchical positions, or because some unknown benefit exists for having an enhanced serotonergic system in the larval stage. overall, if we could better understand the difference in the aggression neural circuits between cavefish and surface fish, and their relationship with the number of serotoninergic cells in 1weekold juveniles, we would gain muchneeded insight to the evolution of cavefish 's foraging behavior. in general, this behavioral shift in cavefish from attacking to foraging provides the first example of a likely behavioral advantage underlying their enhanced hedgehog signaling, whose tradeoff is eye development (c.f. many behaviors have evolved in the company of morphological changes, yet behavior can also evolve without obvious anatomical adaptations e.g., feeding posture. when in the dark, surface fish feed at a steep angle of 90 relative to a substrate. in contrast, multiple cave populations feed at a much shallower angle of 45 (table 1) (schemmel, 1980). although no one has yet determined how lowangle feeding is advantageous in the dark (though this could be demonstrated in a competition assay), three a. mexicanus cave populations (pachn, tinaja, and los sabinos) (kowalko., 2013b and personal observation) and other benthic feeders (for example, see ferrygraham., 2002) all perform lowangle feeding, suggesting that this behavior may be advantageous for foraging at the bottom of caves. since jaw morphology is frequently associated with feeding (wainwright., 1995 ; albertson., 2001 ; kocher, 2004 ; alfaro., 2005 ; parsons., 2009), it was surprising when a set of studies revealed no detectable correlation between feeding angle and eight craniofacial phenotypes in an f2 intercross derived from a surface fish mated to a cavefish (kowalko., 2013b). the genetics therefore suggests that a cavefish 's low feeding angle represents an exclusively behavioral adaptation potentially through changes in motor control that required no accompanying morphological changes. the convergent evolution of a cavefish 's feeding posture has also been supported by genomic evidence (kowalko. using quantitative trait loci (qtl) mapping of feeding posture (a potentially adaptive behavior) and eye degeneration in two independently evolved cavefish populations, kowalko reported distinct qtl results for different genes in different populations, suggesting that many cavefish traits evolved by de novo mutation rather than by selection from standing genetic variation. at the singlefeedingangle qtl detected in one cavefish population (tinaja), the cave allele actually worked to increase the feeding angle, which was unexpected and indicates there are still undetected qtls (table 1) (kowalko., 2013b). importantly, a different cave population (pachn) showed two feedingposture qtls, and at both of them the cave alleles worked to reduce the feeding angle, suggesting that different genes were utilized during the evolution of feeding behavior among independent cave populations (kowalko., 2013a). surface fish readily stabilize their feeding angle at a 45 angle in lighted conditions, so cavefish may have evolved their posture control without visual cues, instead relying on or modifying the vestibular posture control centers. this is supported by observations at the larval stage, when cavefish and surface fish feed similarly. instead of feeding at either 45 or 90, both larval types bob along the bottom of the tank and attack sunken food, trying out different feeding angles during each attempt. since this larval feeding style is somewhat similar to adult surface fish, it would be interesting to investigate the ontogeny of neural wiring in the basal ganglia, cerebellum, pontine, and vestibular systems which together control posture and how this wiring is associated with feeding angle in cavefish. in addition, future competition assays should test if a lower feeding angle is advantageous in the dark, which will provide insight into the selection pressure for this behavior. foraging is not the only behavior to have evolved in cavefish ; they also show differences from surface fish in their social interactions, one of which is schooling. schooling has multiple benefits, including helping fish avoid predators and foraging, but these benefits may not be valid in caves, which have limited food and few predators. indeed, the loss of schooling depends on the loss of visual sensing and on a nonvisuallyrelated genetic factor (kowalko., 2013a). yet, the same genetic factor encoded at this allele actually promotes schooling in cavefish compared to surface fish (table 1) (kowalko., 2013a). such a finding suggests that the absence of cavefish schooling is mainly due to eye loss, which is supported by ablation of the lateralline sensory system. although it was once suggested that the lateral line controls schooling behavior (partridge., 1980), ablating the lateral line did n't affect schooling in either surface fish or cavefish (kowalko., 2013a). therefore loss of schooling behavior may simply be a consequence of eye loss which could be beneficial, as opposed to deleterious, because being solitary may provide a better chance to find the rare food sources. a test of foodfinding ability using eyed, schooling individuals and eyed, nonschooling individuals (2013a) isolated among f2 hybrids from a surface fish cavefish cross should reveal an advantage for the loss of schooling under sparsefood condition. female surface fish prefer to mate with large males ; in dark environments, though, this preference disappears, suggesting that mate preference depends on visual cues (table 1) (plath., 2006). yet even in the dark, two out of six cavefish populations evolved a preference for large males (micos and yerbaniz ; others are molino, pachn, piedras, and curva cavefish), suggesting the evolution of a nonvisual mate preference (plath., 2006). such a preference could be a consequence of adaptations and reliance on the mechanosensory lateralline system, which can sense the vortices produced by fish (bleckmann., 1991). it would therefore be interesting to investigate if the frequency stimulus of vortices produced by large males (100 hz) (bleckmann., 1991) attracts females and stimulates their release of gonadotropin releasing hormone (gnrh) to initiate reproductive behavior (hofmann, 2006). such an investigation could then explore how the reproduction process adapted after cavefish were no longer able to use visual sensory inputs. another behavior cavefish exhibit is wallfollowing, which is thought to function in space recognition and avoiding collisions with cave walls (table 1) (hassan 1989 ; de perera, 2004 ; windsor., 2008, 2010a, 2010b ; coombs., 2010). this behavior has evolved in all astyanax cavefish populations, and likely helps them navigate in the absence of visual cues. as mentioned, the cavefish 's lateralline system senses hydrodynamic changes in the flow field of water caused by objects in the cave (hassan 1989, 1992 ; coombs., 2010). the ontogeny of this behavior is not clear, although it seems to appear by 34 months postfertilization (personal observations). because numbers of infraorbital sns significantly increase after 2 months postfertilization (yoshizawa. furthermore, wallfollowing behavior may be tightly associated with the ability to recognize and navigate spaces in the absence of visual cues (de perera, 2004 ; holbrook., 2009). the part of the nervous system most likely involved in space recognition is at a region homologous to the hippocampus, in the lateral part of the dorsalis of the telencephalon, where spatial memories form (lo., 2002). it would therefore be interesting to find out how cavefish establish spatial memory that is only based on temporal information from hydrodynamic stimuli. for the most part, cave animals adeptly confront the challenges of sparse food and perpetual darkness. consequently, many researchers have associated cave animals ' traits with these selection pressures without actually testing the advantages of these evolved traits, thereby leading to misunderstanding or an overemphasis of the significance of the evolution of these traits. for example, the significant increase of infraorbital sn was first predicted as the receptor for adaptive vab, but the minor sensory population of eo sns turned out to be the major receptors for vab (yoshizawa., 2010, 2012b). this case study reminds us to consider a classic criticism of the adaptive program : not all evolved traits are adaptive (gould., 1979). the a. mexicanus cavefish system makes it easy to avoid such pitfalls by permitting laboratory studies in simulated cave environments, which are easy to replicate with dark and fooddeprived conditions. some interesting, classic evolutionary questions could be answered by surveying behavioral and morphological phenotypes in the a. mexicanus system, such as : do behavioral adaptations emerge before or after morphological changes ? since behavior can be drastically modified by a simple change such as a hormone concentration in the central nervous system (kobayashi., 1999)the evolution of behaviors has been assumed to precede morphological and sensory evolution (reviewed in westeberhard, 1989). yet, multiple studies in the cockroach, moth, silkworm, and mouse suggest that changes in the expression level of some sensory receptors is enough to trigger adaptive shifts of behaviors (jacobs., 2007 ; sakurai., 2011 ; leary., 2012 ; wadakatsumata a few behavioral traits, including vab and stabilized feeding angle, may have preceded morphological or sensory evolution because there are standing phenotypic variations of these behaviors among the cavefish and their related surfacedwelling cohorts. thus, during the initial steps of the adaptation process, individual cavefish ancestors that expressed these caveassociated behaviors were at a selective advantage because they did not require extreme morphological / sensory changes to adjust to the cave enviroment (yoshizawa., 2010, 2011, 2012 ; in contrast, cavefish behaviors that require eye regression, including loss of schooling (kowalko., 2013) and wallfollowing (personal observation of eyeablated surface fish), may have emerged after the morphological loss of the eye. furthermore, loss of pigment, another morphological trait, may have also induced behavioral changes, including higher locomotor activity and/or sleep loss, because a null mutation of the oculocutaneous albinism ii (oca2) gene increases the production of dopamine and noradrenalin, two neurotransmitters that affect sleep and locomotor behaviors (dubou. in addition, wellknown pleiotropic hedgehog signaling controls both morphology / sensory system (eye regression, widening jaw, increase of taste buds) and behavior (behavioral shift from aggression to foraging via increase of serotonergic neurons in the hypothalamus) (yamamoto. thus, traits controlled by pleiotropic genes could have evolved concurrently as a result of the mutation of these genes. in summary, the cavefish serves as an excellent model to study the evolution of multiple morphological and behavioral traits because it has provided evidence that morphological and behavioral traits evolved through complex manners. the latest a. mexicanus behaviors studied include : enhanced prey capture ability in 25dayold cavefish in the dark (espinasa., 2014) ; feeding control via appetiterelated hormones (penney., 2014) ; and loss of circadian rhythm and sleep (dubou., 2011, 2012 ; beale., 2013 ; moran., 2014 the next frontier in cavefish research will be to identify more of the genes and mutations involved in the adaptation to the cave environment, thereby establishing a field where genetics, ontogeny, neuroscience, phylogeny, and ecology are integrated. the recent development of powerful tools has led to a wealth of important information we can use to unravel evolutionary mysteries, including available genome sequences (astmex102 from the ensembl genome browser, at www.ensembl.org) ; available embryology techniques that can modify gene expression (yamamoto., 2000, 2004, 2009 ; gross., 2009 ; bilandija., 2013) ; defined embryonic and larval stages (hinaux., 2011) ; transcriptome datasets (gross., 2013 ; hinaux., 2013) ; and defined evolutionary relationships among populations (ornelasgarca., 2008 ; bradic., 2012 ; gross, 2012 ; bradic., 2013). further advances in transgenic capabilities (elipot., 2014) and genomic engineering methods, such as talen and crispr technologies (gaj., 2013 ; hwang., 2013), allow us to perform more directed genetic studies. such technology is helping to fill the technical gap between the a. mexicanus system with its clear selection pressures and many caveadapted traits and other model animal systems with their historical knowledge base. with new information and better experimental techniques, we can further exploit a. mexicanus as an evolutionary vertebrate model, which will ultimately allow us to comprehensively understand the evolutionary processes through which genomic and developmental shifts produce enhanced or coopted adaptive traits. | summarymany developmental processes have evolved through natural selection, yet in only a few cases do we understand if and how a change of developmental process produces a benefit. for example, many studies in evolutionary biology have investigated the developmental mechanisms that lead to novel structures in an animal, but only a few have addressed if these structures actually benefit the animal at the behavioral level of prey hunting and mating. as such, this review discusses an animal 's behavior as the integrated functional output of its evolved morphological and physiological traits. specifically, we focus on recent findings about the blind mexican cavefish, astyanax mexicanus, for which clear relationships exist between its physical traits and ecosystem. this species includes two morphotypes : an eyed surface dweller versus many conspecific types of blind cave dwellers, some of which evolved independently ; all of the blind subtypes derived from eyed surface dwellers. the blind cavefish evolved under clear selection pressures : food is sparse and darkness is perpetual. simulating the major aspects of a cave ecosystem in the laboratory is relatively easy, so we can use this species to begin resolving the relationships between evolved traits and selection pressures relationships which are more complex for other animals models. this review discusses the recent advances in cavefish research that have helped us establish some key relationships between morphological evolution and environmental shifts. mol. reprod. dev. 82 : 268280, 2015. 2015 wiley periodicals, inc. |
study participants were members of the kaiser permanente northern california (kpnc) diabetes registry in 2005. patients were selected for the study if they had diabetes before 1 january 2005 and were enrolled with an active drug benefit continuously throughout 2005. eligible patients were further assessed for the presence of hypertension and hyperlipidemia using kaiser permanente automated clinical databases. self - reported race / ethnicity data, obtained from kaiser permanente member surveys, study surveys, and hospitalization data, were available for 87.3% of patients. the final study population consisted of 108,555 african american, white, and hispanic adult diabetic patients and 1,750 physicians. asian patients were not included in this analysis because, with current data limitations, ethnically dissimilar asian patients and physicians would be considered race / ethnicity concordant, despite potentially significant cultural and language differences. good a1c risk factor control for diabetes was defined as a patient having an a1c laboratory value of 140 mmhg at any time during the year. this level is higher than that in the seventh report of the joint national committee on prevention, detection, evaluation, and treatment of high blood pressure (22) and kpnc guidelines for diabetic patients of sbp 140 mmhg at any time during the year. this level is higher than that in the seventh report of the joint national committee on prevention, detection, evaluation, and treatment of high blood pressure (22) and kpnc guidelines for diabetic patients of sbp < 130 mmhg but is a conservative target at which a diabetic patient most likely needs therapy modification. good risk factor control for patients with hyperlipidemia was defined as an ldl cholesterol value < 100 mg / dl during the year (23). laboratory and blood pressure values for 2005 were obtained from automated kpnc databases. a binary variable was created to indicate whether pharmacy databases indicated an intensification of pharmacotherapy within 6 months after an instance of poor risk factor control during 2005. a 6-month period (as used in previous studies) was chosen because the high visit rate of diabetic patients within kpnc and the use of primary care teams who can reach out to initiate therapy modification on the physician 's behalf via phone or mail give sufficient opportunity for therapy modification in this setting. intensification was defined as an increase in the number of drug classes, an increase in dosage of at least one drug class, or a switch to a different drug class within 6 months. daily doses were categorized as low (near initial starting doses), medium (maintenance range), or high (high end or above maintenance range) based on package insert recommendations and inspection of actual dosage distributions. patients who were already using insulin were excluded from the treatment intensification for hyperglycemia analysis because treatment intensification for insulin can not be measured in automated pharmacy databases. patient race / ethnicity was the main explanatory variable for multivariable models that assessed predictors of risk factor control and treatment intensification. in these stratified (african american versus white and hispanic versus white) models, separate dummy variables were created for african american and hispanic race / ethnicity, with white patients as the reference group. similar dummy variables were created for african american and hispanic physicians, with white physicians as the reference group. patient - physician interaction terms were included to assess the association of patient - physician race / ethnicity concordance with risk factor control and treatment intensification for african american and hispanic patients. patient race / ethnicity was the main explanatory variable for multivariable models that assessed predictors of risk factor control and treatment intensification. in these stratified (african american versus white and hispanic versus white) models, separate dummy variables were created for african american and hispanic race / ethnicity, with white patients as the reference group. similar dummy variables were created for african american and hispanic physicians, with white physicians as the reference group. patient - physician interaction terms were included to assess the association of patient - physician race / ethnicity concordance with risk factor control and treatment intensification for african american and hispanic patients. stratified probit models assessed the marginal effect of patient race / ethnicity and patient - physician concordance on a1c, ldl cholesterol, and sbp control and intensification. the resulting marginal effects were converted into adjusted percentages of patients in good cvd risk factor control and patients at above - target cvd risk factor levels who received treatment intensification. these models controlled for patient age, sex, preferred language, number of comorbidities, risk factor values (for treatment intensification analysis), number of primary care visits in 2005, medicare status, number of medication classes taken for condition, overall pill burden, geocoded education, and income as fixed effects. physician age, sex, race / ethnicity, language proficiency (which is self - reported by physicians at their onset of employment with the medical group), panel size, and number of diabetic patients in panel were also included as fixed effects. to account for patient clustering at the physician level, all models adjusted for physician as a random effect. this study was developed and approved by the steering committee of the translating research in action for diabetes (triad) study and approved by the kpnc institutional review board. approximately half of the patients in the sample were male (52%) and almost 97% reported speaking at least some english. almost half of the patients (46%) were white, 11% were hispanic, and 10% were african american. spanish was the primary language of almost a quarter (22%) of the hispanic patients. only 10% of african american and 11% of hispanic patients were in race / ethnicity concordant relationships with their providers. physicians were disproportionately white (47%) or asian (40%) ; < 8% of physicians were either african american or hispanic. patients were with their primary care physicians for an average of 5.66.3 years (table 1). patient descriptive statistics after controlling for patient and physician characteristics, patient race / ethnicity was a significant predictor of risk factor control for all three risk factors (table 2). african american patients were also less likely to be at or below target ldl cholesterol (40 vs. 47%, p < 0.001) and sbp (70 vs. 78%, p < 0.001). < 8% (62 vs. 69%, p < 0.001). risk factor control varied little and nonsignificantly by patient - provider race / ethnicity concordance (table 3). percentage of patients with good cvd risk factor control by race / ethnicity data are %, % sem, or ors (95% ci). physician random effect probit models and logistic regression models were adjusted for patient age, sex, preferred language, number of comorbidities, number of primary care visits in 2005, medicare status, number of medication classes taken for condition, pill burden, geocoded education and income, physician age, sex, race / ethnicity, language, panel size, and number of diabetic patients in panel. percentage of patients with good cvd risk factor control by race / ethnicity concordance data are %, % sem, and ors (95% ci). probit random effect and logistic models examined minority patient - physician race / ethnicity interactions. models were adjusted for patient age, sex, preferred language, number of comorbidities, number of primary care visits in 2005, medicare status, number of medication classes taken for condition, pill burden, geocoded education and income, physician age, sex, race / ethnicity, language, panel size, and number of diabetic patients in panel. table 4 shows the proportion of patients at above - target risk factor levels who received treatment intensification within 6 months, by race / ethnicity, after adjustment for patient and physician characteristics. patient race / ethnicity was a significant predictor of treatment intensification for all three risk factors. african american patients were less likely than white patients to have a1c intensification (73 vs. 77%, p < 0.0001) and more likely to receive treatment intensification for sbp above target (78 vs. 71%, p < 0.0001). no significant differences in a1c or sbp intensification were found for hispanic patients compared with white patients. however, hispanic patients were more likely than white patients to have treatment intensification for ldl cholesterol (47 vs. 45%, p < 0.05). treatment intensification was not significantly associated with patient - physician race / ethnicity concordance (table 5). race / ethnicity and race / ethnicity concordance effects on risk factor control and treatment intensification were consistent regardless of whether the patient 's preferred language was included in the model, when cut points of a1c < 7% and sbp < 130 mmhg were used, and when insulin use was adjusted for in models of treatment intensification for hyperglycemia (data not shown). percentage of patients receiving treatment intensification (among patients with elevated risk factor values) by patient race / ethnicity data are %, % sem, and ors (95% ci). physician random effect probit and logistic regression models were adjusted for patient age, sex, preferred language, number of comorbidities, number of primary care visits in 2005, medicare status, number of medication classes taken for condition, laboratory values, pill burden, geocoded education and income, physician age, sex, race / ethnicity, language, panel size, and number of diabetic patients in panel. percentage of patients receiving treatment intensification by patient - physician race / ethnicity concordance data are %, % sem, and ors (95% ci). probit random effects and logistic model examined minority patient - physician race / ethnicity interactions. the model was adjusted for patient age, sex, preferred language, number of comorbidities, number of primary care visits in 2005, medicare status, number of medication classes taken for condition, pill burden, geocoded education and income, physician age, sex, race / ethnicity, language, panel size, and number of diabetic patients in panel. our findings are consistent with previous research showing racial disparities in cvd risk factor control (2). african american patients had worse risk factor control for a1c, ldl cholesterol, and sbp than white patients. after controlling for patient and physician characteristics, hispanic patients were no more likely to have poor risk factor control for ldl cholesterol and sbp than white patients (3). our findings also provide evidence supporting the hypothesis that race / ethnicity is modestly associated with treatment intensification. we found disparities in a1c intensification in comparisons of african american patients with white patients. we also found that, in some cases, minority patients were more likely to receive treatment intensification than whites. one potential explanation for the greater likelihood of treatment intensification for african american patients with elevated sbp and hispanics with elevated ldl cholesterol compared with whites is what is known as the statistical discrimination hypothesis : in instances of uncertainty, physicians may rely on what they know about the prevalence and consequences of the disease for the racial group to which a particular patient belongs (24). aware of high rates of hypertension in african american patients and high cholesterol in hispanic patients, physicians may be more likely to intensify treatment. minority patients are more likely than whites to perceive they would have received better medical care if they belonged to a different racial and ethnic group and that medical staff judged them unfairly or treated them with disrespect based on race / ethnicity (25). these barriers to trust may affect patients ' attitudes toward medicine and may contribute to differential reluctance to intensify therapy. on the other hand, it is also possible that african american patients may be more anxious about elevated sbp than white patients are. our findings do not support the hypothesis that patient - physician race / ethnicity concordance would improve control of intermediate diabetes outcomes. previous research has shown that equal or better care for minority patients does not necessarily close gaps in health outcomes between minority and white patients (13), so it is quite possible that potential benefits of concordance would not translate into (immediate) intermediate outcome improvement. patients and physicians were from a single large, integrated health care delivery system ; it is possible that patients and physicians in this setting may be different from patients and physicians in other settings. however, the patient and physician populations studied were fairly diverse, and the delivery system population is demographically similar to the region it serves (3). omitted variables, such as patient family history of stroke, evidence of end - organ damage, and patient and physician attitudes and beliefs regarding medication, were not captured in these analyses and may influence differential intensification rates. our variables were also limited by the fact that we only had access to socioeconomic status indicators from geocoding ; individual - level data on education and income were not available in this study. another limitation is that we were unable to assess treatment intensification for a1c control in patients who were already taking insulin at the start of the study ; it is possible that this causes underestimation of the level of intensification in this population and potentially biases differences in intensification rates for hyperglycemia by race / ethnicity. we were also unable to measure nonmedication treatment decisions physicians make ; in response to poor control, physicians may provide diet, exercise, and other lifestyle recommendations that we are unable to include in our analysis. another limitation of the study was our inability to assess the impact of race / ethnicity and race / ethnicity concordance on risk factor control and treatment intensification for asians and patients and physicians of other races / ethnicities. finally, another potential reason we did not detect significant race / ethnicity interaction effects is that many diabetic patients also receive care from nutritionists, health educators, and pharmacists and potentially from other physicians and nurses. for example, patients may also have access to diabetes care classes provided by health educators. we were unable to assess patient race / ethnicity concordance with these and other medical care staff, and these relationships may have played a role in predicting risk factor control and intensification. in summary, in this study, minority race / ethnicity was a significant predictor of worse patient risk factor control and better treatment intensification. however, patient - physician race / ethnicity concordance was not associated with either control or treatment intensification for any risk factor. future researchers should try to illuminate the specific barriers to intensification, including possible patient or physician cultural factors that would inform targeted interventions to improve both risk factor control and treatment intensification. | objectivepatient - physician race / ethnicity concordance can improve care for minority patients. however, its effect on cardiovascular disease (cvd) care and prevention is unknown. we examined associations of patient race / ethnicity and patient - physician race / ethnicity concordance on cvd risk factor levels and appropriate modification of treatment in response to high risk factor values (treatment intensification) in a large cohort of diabetic patients.research design and methodsthe study population included 108,555 adult diabetic patients in kaiser permanente northern california in 2005. probit models assessed the effect of patient race / ethnicity on risk factor control and treatment intensification after adjusting for patient and physician - level characteristics.resultsafrican american patients were less likely than whites to have a1c < 8.0% (64 vs. 69%, p < 0.0001), ldl cholesterol < 100 mg / dl (40 vs. 47%, p < 0.0001), and systolic blood pressure (sbp) < 140 mmhg (70 vs. 78%, p < 0.0001). hispanic patients were less likely than whites to have a1c < 8% (62 vs. 69%, p < 0.0001). african american patients were less likely than whites to have a1c treatment intensification (73 vs. 77%, p < 0.0001 ; odds ratio [or ] 0.8 [95% ci 0.70.9 ]) but more likely to receive treatment intensification for sbp (78 vs. 71%, p < 0.0001 ; 1.5 [1.31.7 ]). hispanic patients were more likely to have ldl cholesterol treatment intensification (47 vs. 45%, p < 0.05 ; 1.1 [1.01.2 ]). patient - physician race / ethnicity concordance was not significantly associated with risk factor control or treatment intensification.conclusionspatient race / ethnicity is associated with risk factor control and treatment intensification, but patient - physician race / ethnicity concordance was not. further research should investigate other potential drivers of disparities in cvd care. |
uterovaginal prolapse (uvp) presenting at birth is very rare. to the best of our knowledge, utero - vaginal prolapse is the downward descent and protrusion of the uterus and vagina to the exterior via the introitus. it is as a result of weakness of the cardinal ligaments and uterosacral ligaments which provide support to the uterus. this is a report of a congenital uvp (3 degree) in a 12 h - old - child noticed at delivery. a 12-hour - old female neonate was presented to us with a fleshy mass protruding from the vulva since birth. the baby passed meconium on the 1 day of birth and tolerated breast milk feeding. the mother, a 31-year - old, para 3, seamstress had recurrent febrile conditions throughout the pregnancy and was treated at the primary health center where she booked. there was a fleshy, reddish, and edematous mass protruding from the vulva [figure 1 ]. she has a sacral dimple with tufts of hair in it [figure 2 ]. lower limb motor activity, sensations, respiratory, and cardiovascular systems were all normal. uterovaginal prolapse at birth sacral dimple with tuft of hair under sedation, size 6 foley 's urethral catheter was passed into the bladder. the mass was reduced by gripping it with the right hand and gently pushing it inwards. to prevent re - protrusion on straining, both lower limbs were strapped together using crepe bandage which was applied in a mermaid fashion extending from the buttocks to the lower legs sparing the anus for defecation. both catheter and bandaging were removed after 72 h. following successful reduction, the parents were unwilling to neither subject the child to further investigations nor comply with follow - up schedules. however, through contact tracing, the prolapse has not recurred 6 months after, and the child is growing appropriately. the uterus and the vagina are essentially supported by the muscular pelvic diaphragm and the three condensations of the endopelvic fascia (cardinal ligaments, uterosacral ligaments, and pubocervical fascia). congenital uvp usually results from the weakness of the pelvic muscular support and the ligaments. this may be secondary to either congenital weakness in the pelvic musculature or defects in innervation. in adults the uterovaginal junction is angulated with the uterus lying almost horizontal to the muscular pelvic diaphragm. in the fetus and the neonate there is minimal or no angulation and the orientation is almost vertical, as the true pelvis is not well - formed yet and the pelvic organs are essentially abdominal. hence, raised intra - abdominal pressure as occurs during prolonged breech delivery is transmitted straight down the uterus and out through the vagina and introitus. most of the few reported cases were often associated with some identifiable risk factors [table 1 ]. spina bifida, especially myelomeningocele is the most common risk factor (85% of cases). risk factors associated with congenital uterovaginal prolapse defective innervations of the pelvic floor muscle in meningomyelocele may lead to weakened support of the uterus and vagina. the rise in fetal intra - abdominal pressure during breech delivery stretches the pelvic floor muscle and the ligaments thus increasing the risk of prolapse. this is a genetic disorder characterized by generalized loose and redundant skin with reduced elasticity. early treatment of prolapse is important to prevent injury and metaplasia of the endometrial lining from prolonged exposure. several modalities which can be either conservative or operative have been used in the treatment of genital prolapse in the neonates., conservative management has a success rate of more than 90%, though long - term outcome could not be ascertained yet. the major challenge after the initial digital reduction is the recurrence of prolapse once the intra - abdominal pressure rises during crying or straining. these methods include the use of various forms of pessaries, vaginal tampons, use of a two - way catheter partially inflated in the vagina to prevent recurrence of reduced prolapse, purse string suturing of vaginal wall with 2 - 0 catgut, and even temporary fusion of the labia. baskaran., reported a successful outcome with purse string suturing of the vaginal wall in two rows in a neonate with myelomeningocele. with the resolution of the edema, reduction in the maternal derived estrogen level, surgical procedures like uterine ventrosuspension, sling, sacral cervicopexy, or abdominal sacrocolpopexy have also been used in recalcitrant cases. in our patient, we solved this by strapping the buttocks together by applying crepe bandage from the lower abdomen down to the lower third of the legs in a this method has the advantage of simplicity and easy applicability by even the labor attendants at the primary centers soon after the prolapse is observed, before edema and mucosal ulceration sets in. in conclusion, the possible risk factors present in this patient include breech delivery and spina bifida occulta. successful reduction was achieved by digital reduction and strapping the buttocks together with crepe bandage in a mermaid fashion. | uterovaginal prolapse presenting at birth is very rare. the cause is attributed to conditions that can cause poor innervation or weakness of the pelvic floor muscle and the supporting ligaments. different methods of treatment have been used in the past to reduce and maintain reduction of the prolapse. we report a case of a congenital uvp in a day old child noticed at delivery. he was delivered breech and had a sacral dimple with a tuft of hair. he was successfully managed conservatively with digital reduction and strapping of the buttocks down to the legs with crepe bandage for 72 h. |
spontaneous spinal epidural hematoma (sshe) is a rare entity can have several reasons. for the first time jackson in 1869 reported a case of sshe and after that, it was declared as several hundred cases in literatures. its prevalence in population is 0.1 per 100,000 with the male to female ratio of 1.4:1. sshe is rare among children and no sexual preference was seen from fetus to 14 years - old (1). based on the severity of neurologic deficits two therapeutic approaches, conservative and surgical, have been suggested. here, a case of sshe was reported in a 52-year - old male referred to emergency department following severe low back pain. a 52 year - old male was admitted to the emergency department (ed) with chief complaint of low back pain. the patient was unable to walk because of severe pain ; the severity of pain based on numeric rating scale (nrs) was 10. he stated that his pain has suddenly initiated from two hours before referring and the pain severity in the early was the half of intensity of his current pain. he tried to relieve his pain at home by using drugs like muscle relaxants and non - steroidal anti - inflammatory drugs (nsaids), nevertheless due to increasing the pain he was referred to the ed. the patient did not have a serious trauma history and just mentioned that he jumped from a chair with 40 centimeters height in the previous day, without any hurt in his back or knees. the subject did not have any positive past medical history except a suspicious non - documented history of ischemic heart disease. he has not had any heart problem since that time but has been continuously taking 80 mg aspirin daily. the subject has had the history of smoking and snuff opium since many years ago. on admission to the ed, he had oral temperature of 36.5c, 19/minute respiratory rate, 85/minute pulse rate, and 130/85 mmhg blood pressure ; and 96% oxygen saturation on room air. in physical examination, he did not have tenderness in the spinal column while it presented in left buttock and left inguinal ligament. the sensory and motor examination as well as deep tendon reflexes were normal. on laboratory tests, white blood cell (wbc) = 12000/ l, platelet count = 224000/ l, hemoglobin = 14.7 mg / dl, sodium=139 meq / l, potassium=4.3 meq / l, blood sugar=120 mg / dl, erythrocyte sedimentation rate (esr) = 10 millimeter / hour, prothrombin time (pt) = 13.6 seconds, partial thromboplastin time (ptt) = 33 seconds, and international normalized ratio (inr) = 1.1 were reported. although the treatment of the patient was initiated with an appropriate dose of venous analgesic agent, he did not show an affected response to the treatment and after a short time, the pain began again with the same intensity. in fact, noticing to the history of opium consumption by the patient, it was thought that maybe he wanted to exaggerate his pain to receive more opium. because of tenderness in the inguinal region, the possibility of renal stone was also assumed that rule outed by doing abdomino - pelvic computerized tomography (ct) scan without contrast. the radiologist reported a small epidural hematoma in the l1/l2 regions, as shown in figures 1. immediately a neurosurgical consultant was requested and the patient with sseh diagnosis hospitalized in the neurosurgery ward. the patient s pain was controlled by a pain specialist and then he underwent conservative treatment there. finally, the patient was discharged from the hospital without surgery and any neurologic deficit after six days. lombo - sacrale magnetic resonance imaging (mri) of patients without contrast patients with back pain are the common referrals to the ed and consist of 2.3% of all physician visits. in addition, 84% of adults experience the back pain in their life (2 - 4). some important elements in the history and physical examination can differentiate serious reasons of the back pain. the important point in this case was the presence of severe pain resistant to the treatment, which was not mentioned as red flags. sshe is an idiopathic aggregation of blood in epidural space which can be as acute, chronic, spontaneously, post traumatic, or iatrogenic (5). sseh concludes less than one percent of space occupied lesions ; its pathogenesis is still unknown but believed that bleeding has a venous origin because the lack of valves in the epidural network disposes the intrinsic change in pressure. albeit in terms of recent theories, it is supposed that arterial bleeding causes to traction and mechanic disruptions on nerve roots and in some studies, it was attributed to the spontaneous rupture of an arteriovenous malformation. miyagi. declared that because the pressure of intravenous network is lesser than epidural space, arterial bleeding is more possible for that (6). totally, epidural hematoma is along with a series of underlying diseases and predisposing factors such as organic vascular disease, hemodialysis, coagulation disorders, hemophilia b, thrombolysis for myocardial infarct, factor ix deficiency, long term aspirin using, and vascular malformations as well as vertebral hemangiomas ; liver disease, alcoholism, and thrombocytopenia (7). the pain maybe exacerbated by percussion on the spine or with maneuvers that increase the intraspinal pressure like coughing and sneezing. the sensory and motor findings of the patient depend on the lesion site and hematoma size which can include weakness, paresis, bladder dysfunction, and sensory deficit (8). the spinal epidural hematoma is one of the potentially reversible pressure lesions on the spinal cord and roots, thus its diagnosis and treatment have a vital importance (5). presently magnetic resonance imaging (mri) is considered as the first diagnostic method of choice, which can show a biconvex hematoma in the epidural space with well - defined borders that taper towards up and down. in case of lacking mri, computed tomography scan could be used (9, 10). if neurologic deficit was complete, decompressive surgery should be performed within 36 hours (11). conservative treatment has been suggested just in cases that neurologic deficit improves in the early phase of disease or coagulopathy disorder subjects (12). functional recovery mostly depends on duration of symptoms at presentation and its improving after 72 hours is rare, although some cases improved without surgery has been reported. two cases were showed by hentsched. in 2001, one with complete quadriplegia and another one with complete paraplegia that both of them were cured without surgery and had good neurological function during three months following. another study was performed by kim t and colleagues that 15 patients were evaluated in to two groups. these two groups did not show any difference in initial neurological status after treatment (10). it seems that the hematoma length is a clue in aspect of spontaneous recovery happens in some sseh cases (11, 13). in confronting with a patient complains from the pain, one of the important and priority duties of the physician is relieving the pain. but it is not enough alone and a logical diagnosis is required for justify the pain. of course noticing to the limited time and facilities in the ed, sometimes there is no possibility to the definitive diagnosis. however, at least it should be rule out the critical causes and only in such condition, it is reasonable to discharge the patient to outpatient follow - up. in the case of unknown psychiatric and neurologic manifestations, measuring serum level of thyroid hormones and csf titer of anti - thyroid antibodies could be helpful in limitation of differential diagnosis and timely initiation of proper treatment. all authors passed four criteria for authorship contribution based on recommendations of the international committee of medical journal editors. | spontaneous spinal epidural hematoma (sshe) is a rare entity can have several reasons. its prevalence in population is 0.1 per 100,000 with the male to female ratio of 1/4:1. for the first time jackson in 1869 reported a case of sshe and after that, it was declared as several hundred cases in literatures. here, a case of sshe was reported in a 52-year - old male referred to emergency department following severe low back pain. |
even though it is a benign and self - limiting disease, in many patients it can lead to significant physical, social and psychological morbidity. it has been postulated that bell 's palsy occurs as an inflammatory reaction to viral infection. however, the role of antiviral drugs is controversial. bell 's palsy, usually, occur rapidly, and any pharmacological intervention is difficult. intravenous (iv) corticosteroids are of proven value in similar acute inflammatory neurological illness such as multiple sclerosis, acute disseminated encephalomyelitis and transverse myelitis. hence, it is worthwhile to study the value of iv corticosteroids vis - - vis oral corticosteroids. in this study, we compared the efficacy of a single dose of 500 mg of iv methylprednisolone with a 10 days oral prednisolone regime on recovery of patients with bell 's palsy. patients were enrolled from the outdoor services of the department of neurology, king george 's medical university, lucknow, india. we recruited adult patients with unilateral acute facial palsy of no identifiable cause, within 1-week of onset. patients with any of the following conditions were excluded : pregnancy, diabetes, severe hypertension, renal or hepatic disease, gastric or duodenal ulcer, presence of acute otitis media or ipsilateral chronic otitis, recent head injury, psychiatric disease or any other condition where the use of corticosteroids was contraindicated. the patients were divided into two groups, according to a pre - generated computerized randomization table. patients in group 1 received a single dose of 500 mg of iv infusion of methylprednisolone while those in group 2 received oral prednisolone in a tapering dosage schedule (60 mg daily for initial 5 days, tapered by 10 mg daily over next 5 days). all patients were followed for a minimum of 3 months after inclusion, and the outcome analysis was done at 1-month and at 3 months. cut - off at 1-month and 3 months were used to study the short - term recovery patterns, defined as recovery occurring in < 4 months. brackmann grading system for facial nerve function, which assigns patients to 1 of 6 categories. intermediate severity grades were defined as slight (grade 2), moderate (grade 3), moderately severe (grade 4) and severe (grade 5) depending upon the loss of tone, magnitude of weakness, and presence of synkinesis, contracture or hemifacial spasm. patients were also advised to communicate telephonically or report immediately in case of any serious adverse event. assessment of the patients was carried out on the basis of grade of palsy as well as early (3 days) versus late (between 4 and 7 days) initiation of treatment regimes. final outcome was measured in terms of complete recovery of the facial nerve function (grade 1 of house the chi - square fisher exact test, where ever applicable, was used to compare the dichotomous variables. the chi - square for trend analysis was used to compare categorical variables. the unpaired t - test all the analyses were carried out using spss version 16.0 (spss inc., chicago, il). we recruited adult patients with unilateral acute facial palsy of no identifiable cause, within 1-week of onset. patients with any of the following conditions were excluded : pregnancy, diabetes, severe hypertension, renal or hepatic disease, gastric or duodenal ulcer, presence of acute otitis media or ipsilateral chronic otitis, recent head injury, psychiatric disease or any other condition where the use of corticosteroids was contraindicated. the patients were divided into two groups, according to a pre - generated computerized randomization table. patients in group 1 received a single dose of 500 mg of iv infusion of methylprednisolone while those in group 2 received oral prednisolone in a tapering dosage schedule (60 mg daily for initial 5 days, tapered by 10 mg daily over next 5 days). all patients were followed for a minimum of 3 months after inclusion, and the outcome analysis was done at 1-month and at 3 months. cut - off at 1-month and 3 months were used to study the short - term recovery patterns, defined as recovery occurring in < 4 months. brackmann grading system for facial nerve function, which assigns patients to 1 of 6 categories. intermediate severity grades were defined as slight (grade 2), moderate (grade 3), moderately severe (grade 4) and severe (grade 5) depending upon the loss of tone, magnitude of weakness, and presence of synkinesis, contracture or hemifacial spasm. patients were also advised to communicate telephonically or report immediately in case of any serious adverse event. assessment of the patients was carried out on the basis of grade of palsy as well as early (3 days) versus late (between 4 and 7 days) initiation of treatment regimes. final outcome was measured in terms of complete recovery of the facial nerve function (grade 1 of house the chi - square fisher exact test, where ever applicable, was used to compare the dichotomous variables. the chi - square for trend analysis was used to compare categorical variables. the unpaired t - test all the analyses were carried out using spss version 16.0 (spss inc., chicago, il). we recruited adult patients with unilateral acute facial palsy of no identifiable cause, within 1-week of onset. patients with any of the following conditions were excluded : pregnancy, diabetes, severe hypertension, renal or hepatic disease, gastric or duodenal ulcer, presence of acute otitis media or ipsilateral chronic otitis, recent head injury, psychiatric disease or any other condition where the use of corticosteroids was contraindicated. the patients were divided into two groups, according to a pre - generated computerized randomization table. patients in group 1 received a single dose of 500 mg of iv infusion of methylprednisolone while those in group 2 received oral prednisolone in a tapering dosage schedule (60 mg daily for initial 5 days, tapered by 10 mg daily over next 5 days). all patients were followed for a minimum of 3 months after inclusion, and the outcome analysis was done at 1-month and at 3 months. cut - off at 1-month and 3 months were used to study the short - term recovery patterns, defined as recovery occurring in < 4 months. brackmann grading system for facial nerve function, which assigns patients to 1 of 6 categories. intermediate severity grades were defined as slight (grade 2), moderate (grade 3), moderately severe (grade 4) and severe (grade 5) depending upon the loss of tone, magnitude of weakness, and presence of synkinesis, contracture or hemifacial spasm. patients were also advised to communicate telephonically or report immediately in case of any serious adverse event. assessment of the patients was carried out on the basis of grade of palsy as well as early (3 days) versus late (between 4 and 7 days) initiation of treatment regimes. final outcome was measured in terms of complete recovery of the facial nerve function (grade 1 of house the chi - square fisher exact test, where ever applicable, was used to compare the dichotomous variables. the chi - square for trend analysis was used to compare categorical variables. the unpaired t - test all the analyses were carried out using spss version 16.0 (spss inc., chicago, il). twenty - seven patients were excluded out of whom 14 patients had been initiated on corticosteroids, 9 on antiviral treatment, and 4 patients had a known contraindication to corticosteroids., 7 patients were lost to follow - up and 117 patients were subjected to outcome analysis [figure 1 ]. all the baseline parameters under study were comparable between two groups [table 1 ]. flow chart of the study baseline demographic and clinical characteristics of patients with bell 's palsy after 1-month of treatment, 31 (54.38%) patients from group 1 and 23 (38.33%) from group 2 recovered completely ; a total of 54 (46.16%) patients thus recovered completely. the patients treated with iv methylprednisolone and oral prednisolone, both, showed improvement in the symptoms. after 3 months of treatment, 46 (80.7%) patients from group 1 and 47 (38.33%) from group 2 recovered completely ; a total of 93 (79.48%) patients recovered completely. no statistically significant differences were observed between the two treatment group. according to grade of palsy after 1-month, patients with facial palsy (grade 2 and 3) showed early and complete recovery. all patients of grade 2 and 3 recovered completely while only 55% and 32.2% patients in grade 4 and grade 5, respectively, showed complete recovery. after 3 months, trends of recovery almost converged in terms of the difference in the two groups. in grade 6, only 1 (20%) patient from group 1 showed complete recovery [table 2 ]. outcome assessment (complete recovery to grade 1) at 1-month and 3 months between the two groups in different grades the patients were also analyzed according to early (3 days) versus late (between 4 and 7 days) initiation of treatment based upon their time of arrival for the treatment. in the methylprednisolone group, at 1-month, 58.33% patients (early group) and 47.61% patients (late group) had complete recovery. at 3 months, 83.33% and 76.19% patients in the two respective group had complete recovery. in oral prednisolone group, at 1-month, 40% patients (early group) and 34.78% patients (late group) had complete recovery, while at 3 months 81% and 73.91% patients, respectively, had complete recovery. a trend toward better recovery with early institution of treatment (iv or oral) was observed where iv methylprednisolone was found to be better than oral prednisolone, but the difference was not statistically significant. none of the patients in either treatment groups reported any adverse event during the study period. according to grade of palsy after 1-month, patients with facial palsy (grade 2 and 3) showed early and complete recovery. all patients of grade 2 and 3 recovered completely while only 55% and 32.2% patients in grade 4 and grade 5, respectively, showed complete recovery. after 3 months, trends of recovery almost converged in terms of the difference in the two groups. in grade 6, only 1 (20%) patient from group 1 showed complete recovery [table 2 ]. outcome assessment (complete recovery to grade 1) at 1-month and 3 months between the two groups in different grades the patients were also analyzed according to early (3 days) versus late (between 4 and 7 days) initiation of treatment based upon their time of arrival for the treatment. in the methylprednisolone group, at 1-month, 58.33% patients (early group) and 47.61% patients (late group) had complete recovery. at 3 months, 83.33% and 76.19% patients in the two respective group had complete recovery. in oral prednisolone group, at 1-month, 40% patients (early group) and 34.78% patients (late group) had complete recovery, while at 3 months 81% and 73.91% patients, respectively, had complete recovery. a trend toward better recovery with early institution of treatment (iv or oral) was observed where iv methylprednisolone was found to be better than oral prednisolone, but the difference was not statistically significant. none of the patients in either treatment groups reported any adverse event during the study period. according to grade of palsy after 1-month, patients with facial palsy (grade 2 and 3) showed early and complete recovery. all patients of grade 2 and 3 recovered completely while only 55% and 32.2% patients in grade 4 and grade 5, respectively, showed complete recovery. after 3 months, trends of recovery almost converged in terms of the difference in the two groups. in grade 6, only 1 (20%) patient from group 1 showed complete recovery [table 2 ]. outcome assessment (complete recovery to grade 1) at 1-month and 3 months between the two groups in different grades the patients were also analyzed according to early (3 days) versus late (between 4 and 7 days) initiation of treatment based upon their time of arrival for the treatment. in the methylprednisolone group, at 1-month, 58.33% patients (early group) and 47.61% patients (late group) had complete recovery. at 3 months, 83.33% and 76.19% patients in the two respective group had complete recovery. in oral prednisolone group, at 1-month, 40% patients (early group) and 34.78% patients (late group) had complete recovery, while at 3 months 81% and 73.91% patients, respectively, had complete recovery. a trend toward better recovery with early institution of treatment (iv or oral) was observed where iv methylprednisolone was found to be better than oral prednisolone, but the difference was not statistically significant. none of the patients in either treatment groups reported any adverse event during the study period. we observed that a single dose administration of iv methylprednisolone is as effective as 10 days treatment with oral prednisolone in patients with acute bell 's palsy. treatment options include corticosteroids and antiviral drugs, alone or in combination. even without treatment, a large number of patients (approximately 70%) recover completely within 6 months and approximately 30% of patients may not recover completely. we noted that approximately 80% patients recovered completely at 3 months of treatment with either iv methylprednisolone or oral prednisolone. intravenous methylprednisolone resulted in nonsignificantly better functional recovery rate especially at 1-month when assessed against oral prednisolone and in terms of early institution of treatment (3 days). combined treatment with a corticosteroid and an antiviral agent has been shown to be more effective in treating severe to complete bell 's palsy than corticosteroid treatment alone. physical therapy appears to be effective only in the more severe bell 's palsy, whereas less severe bell 's palsy might result in complete spontaneous recovery, regardless of physical therapy. we showed that grade of bell 's palsy (severity) is the most important prognostic determinant for recovery. our results are similar to those reported by sullivan. who found that patients with facial palsy of lower grades had a better outcome. grade 6 patients infrequently had complete recovery. in patients with bell 's palsy, early treatment with prednisolone significantly improves the chances of complete recovery at 3 and 9 months. consistent with these findings ; our results also suggested that treatment of bell 's palsy should be commenced promptly. in majority number of patients, facial nerve spontaneously and completely recovers. the limitation of our study was the non - inclusion of a control group owing to an ethical concern of depriving patients of a recommended form of treatment. the study was targeted to look for short - term recovery patterns ; an extension to 6 months would have provided more details of patients with incomplete recovery or those in the process of recuperation. a multicentric double - blind study, with a longer follow - up, a 5 days regimen of iv methylprednisolone, for severe bell 's palsy, also needs to be investigated. single dose of 500 mg of iv methylprednisolone may be an equally efficacious alternative to a 10 days course of oral prednisolone. | objectives : corticosteroids have been used in the treatment of bell 's palsy and several other postinfectious neurological conditions. we hypothesized that administration of a single dose of intravenous (iv) methylprednisolone might be an effective alternative to oral prednisolone.materials and methods : in this open label, randomized trial, patients with acute bell 's palsy were randomized into two groups. one group received single dose (500 mg) of iv methylprednisolone while the other group received 10 days of oral prednisone. outcome was assessed at 1 and 3 months with house brackmann scale.results:at 3 months, 93 (79.48%) patients had completely recovered. iv methylprednisolone and oral prednisolone groups had similar recovery rates (80% vs. 78.33%, p > 0.05). patients with grade 2 and 3 recovered completely. in patients with grade 6, the recovery rate was 20%. a better outcome was observed if corticosteroids were administered within 3 days of onset of palsy.conclusion:intravenous methylprednisolone and oral prednisolone showed equivalent benefit in patients with acute bell 's palsy. |
lichen planus (lp) is a chronic inflammatory, autoimmune disease, affecting a wide variety of sites, including skin and mucous membranes.[13 ] in contrast to dermal lp, oral lichen planus (olp) demonstrates clinical variability. the prevalence of olp ranges between 0.5% and 3% and may occur in 70% to 77% of patients with cutaneous lp. most studies suggest that lp is a cd8 + t cell - mediated autoimmune disease. it is believed that the cd8 + t lymphocytes induce keratinocyte apoptosis and cause epithelial basal cell layer damage. possible causes of olp such as an allergy to dental restorative materials (amalgam, gold), local trauma (koebner phenomenon) and hepatitis b or c virus infection have been reported. moreover, it is rare in children, with the youngest case documented in a child aged 3 months. paucity of reported cases of juvenile olp may be due to lack of patient and parent awareness of lesions, lack of recognition by practitioners, low incidence of autoimmune diseases and precipitating factors such as stress. of all the cases of olp reported in children, there is a higher prevalence in the indian population suggesting probable differences in the genetic background and / or environmental triggers. in adults, women some researchers have found an equal sex distribution in children. although olp is usually a sporadic disorder there is also a familial form. olp commonly affects buccal mucosa, although tongue, gingiva and palatal mucosa may also be affected. in both adults and children cutaneous lp is characterised by extremely pruritic eruption of flat - topped, polygonal and violaceous papules with overlying fine reticular striations known as wickham striae. olp presents in mainly three different clinical forms. in order of advancing severity and symptomatology, the plaque form is characterised by slightly elevated, irregular white patch while the erosive form is erythematous and frequently painful. histopathology of olp reveals hyperkeratosis, acanthosis, liquefaction degeneration of basal cells and existence of a band of lymphocytic infiltrate in close proximity to surface epithelium. the objective of treating lichen planus is to control the episodic outbreaks that occurs, given that the lesions are usually not completely cured. reticular and plaque forms usually do not require treatment other than reassurance and follow up. the mild cases of erosive lichen planus are treated with topical corticosteroids combined with antifungal agent. in cases where such treatment proves to be inefficient plaque and erosive lesions do not usually disappear and tend to recur in the majority of the cases. the objective of this article is to highlight the most characteristic histopathological findings of olp in paediatric population and to explore their correlation with the most frequent clinical manifestations and forms in the sample. a retrospective, nonrandomized study was carried out on 22 patients, over a period of 14 years, from 1997 to 2010, at a private oral and maxillofacial pathology clinic in kolkata, india. inclusion criteria were an age younger than 18 years and without any history of drug allergy or contact allergy. investigations for possible signs of extraoral lichen planus, routine blood examination including hepatitis b and c serology were performed. the clinical characteristics such as age, sex, topography, symptoms, clinical pattern of lesion, family history, preexisting medical conditions were documented. histopathologic features were observed at epithelial level, at epithelial connective tissue interface and at connective tissue level. histopathologic criteria for olp consisted of type and degree of keratosis, thickness of epithelium (acanthosis or atrophy), type of rete peg (wavy, saw tooth or bulbous), degree of liquefaction degeneration of basal cells (mild or moderate) and density of lymphocytic infiltration (mild, moderate or severe) at subepithelial lamina propria. patients having reticular, pigmented and hypertrophic form of olp were treated with topical corticosteroid or tacrolimus ointment. 13 cases with erosive form of olp were treated with systemic corticosteroids in tapering dosage. only one patient having extensive erosive olp with repeated recurrence was treated with intralesional corticosteroid. the statistical analysis of the data was performed using the statistical analysis software (sas), version 9.1. fisher 's exact test was employed to explore the association between the clinical pattern and histopathological features. the results obtained are considered significant and highly significant when p values were less than 0.05 and 0.01 respectively. we analysed a total 22 patients, which included 11 males (50%) and 11 females (50%). the average age of the patients was 15.18 years ranging from 8 years to 18 years. the mean duration of olp in our cases prior to seeking consultations was about 14 months. five patients (22.73%) had olp of tongue, three patients (13.64%) had lesions both on the buccal mucosa and tongue, two cases (9.09%) were on both buccal mucosa and lip and only one patient (4.05%) was affected on buccal mucosa, tongue and lip. the most significant finding while comparing the site with age and sex [figure 1 ], we observed that seven male patients(63.63%) and four female patient (36.36%) had olp on buccal mucosa. out of seven male patients, one was from 8 - 9 years age group, four patients in 13 - 15 years age group and two patients in 16 - 18 years age group. out of four females all are from 16 - 18 years age group. in case of tongue lesions comparing site with age and sex of oral lichen planus the most frequent clinical form of olp was erosive type which manifested in 14 patients (63.64%), followed by hypertrophic type, found in five patients (22.73%). reticular type was present in two patients (9.09%) and only one case (4.05%) of pigmented variety was reported. comparing clinical type with site [figure 2 ] it is evident that erosive type [figure 3 ] exclusively occurred in buccal mucosa of nine patients (64.29%), buccal mucosa and tongue in two patients (14.29%), lip and buccal mucosa in two patients (14.29%) and only tongue in one patient (7.14%). the hypertrophic (plaque) type was found mostly on tongue, being reported in eight patients (80%) and concomitantly in lip, tongue and buccal mucosa in one case (20%). the reticular type is evenly distributed over the sites buccal mucosa [figure 4 ] and both buccal mucosa and tongue, being reported in one patient each case. routine hemogram was within normal limits for all the cases and serological testing for hepatitis b and c were not significant. comparing clinical form and site of oral lichen planus clinical photograph showing erosive lichen of right buccal mucosa in a 9 year old male child clinical photograph showing reticular lichen planus of right buccal mucosa in a 14 year old male patient histopathologically [figures 5 and 6 ] we observed that parakeratosis was found in 19 cases (86.36%) and orthokeratosis in three cases (13.64%). acanthotic epithelium was found in 14 cases (63.64%) and atrophic epithelium in eight cases (36.36%). wavy rete peg was most common and was found in 14 cases (63.64%). saw tooth rete peg was present in six patients (27.27%) and bulbous rete peg in two cases (9.09%). moderate basal cell degeneration was found in 14 cases (63.64%) and mild basal cell degeneration in eight cases (36.36%). severe inflammatory infiltrate at subepithelial layer was detected in 13 cases (59.09%), moderate infiltration in three cases (13.64%) and mild in 6 cases (27.27%). direct immunofluoroscence of lesional tissues performed in four equivocal cases were negative for any vesicullobulous lesions. distribution of histological features seen in pediatric oral lichen planus photomicrograph of oral lichen planus showing basal cell degeneration and dense lymphocytic infiltration at subepithelial connective tissue.(h and e stain, 10 magnification) a cross tabulation of histopathological findings and clinical forms was performed [table 1 ]. we observed orthokeratosis was found in a total three patients of which two were of erosive form and one of reticular form. atrophic epithelium was found in eight patients of which five belonged to erosive variety, two were hypertrophic variety and one was reticular variety. acanthotic epithelium was identified in a total 14 patients of which nine were of erosive form, three of hypertrophic form, one of pigmented form and one of reticular form. wavy rete pegs were evident in 14 sections in which ten were erosive type, three hypertrophic form and one reticular form. saw tooth rete peg was present in six sections (27.27%), of which three were of erosive form, one of the hypertrophic form, one of pigmented form and one of the reticular form. mild basal cell degeneration was identified in eight patients (36.36%) of which five were of erosive variant. moderate basal cell degeneration was found in 14 patients (63.64%), nine of which were of erosive form, three hypertrophic form, one pigmented form and one reticular form. regarding subepithelial infiltrate density, severe infiltration was identified in 13 cases (59.09%), mild in six cases (27.27%) and moderate in three cases (13.64%). percentage distribution of histological parameters and in various clinical forms of lichen planus fisher 's exact test was employed to assess the relationship between each of the histopathological findings and clinical forms. the study revealed a significant relationship between the clinical type and site of occurrence (p=0.0112). however no case of hypertrophic olp was found on buccal mucosa of paediatric patients. while comparing clinical form with histopathological findings of olp in pediatric patients we found that parakeratosis was most frequent type of keratosis which occurred in 63.16% of erosive lesion. significant basal cell degeneration and band like subepithelial lymphocytic infiltration were present in all cases. oral lichen planus is extremely rare in childhood with very few cases cited in the literature. out of which only seven patients showed concomitant involvement of the oral mucosa and only one patient had isolated olp. kumar. reported involvement of the oral mucosa in only one of 25 children with cutaneous lesions. however, sharma and maheswari reported 50 children with lp and with concomitant oral lesions in 15 of them. generally the oral mucosa seems to be less commonly involved in children with lp than in adults. in our study, out of 22 cases of olp only two patients had concomitant cutaneous involvement. several studies concluded that patients with lp have a higher risk of being hcv seropositive and similarly, hcv - infected patients had a higher probability of developing lp. according to tilly. there is no increase in hepatitis c among indian childhood lp cases studied to date. serological testing for hepatitis b and c were also not significant in our cases. according to some studies predominance of lp was from five to 15 years of age. in our study kumar. observed more female predominance in his series while male predominated in the studies of sharma and maheswari and woo. some researchers have found an equal sex distribution in children and this observation is consistent with our study. in most of the literature buccal mucosa was the most commonly affected site with the next most common location being tongue. our study was also corroborative to the finding as 50% of the cases had involvement of buccal mucosa and 22.73% of cases had olp in tongue. according to previous studies, most of the paediatric patients had reticular olp. but we found most frequent clinical form of olp was erosive type which is a rare finding in paediatric population. focusing on histological findings our study revealed parakeratosis in 86.36% and acanthosis in 63.64% of cases. we observed wavy rete peg as most common (63.64%) type of rete peg and that was described by other authors as well. liquefaction degeneration of basal layer of epithelium and the band - like subepithelial lymhocytic infiltrate were present in all cases and these were corroborative to observations by other authors.[13 ] most of the authors are of opinion that prognosis and effect of treatment of olp in children seems to be more favourable than olp in adults. in conclusion, oral lichen planus in childhood is rare. it is more prevalent in 15 - 16 years of age with no gender predilection. the most common type is erosive form, primarily manifesting in buccal mucosa. topical corticosteroid therapy and a plaque control regimen in children with symptomatic olp have shown favourable responses. the use of topical calcineurin inhibitors like tacrolimus is not recommended in patients below 2 years. safety of long term continuous use of these drugs in pediatric patients has not been adequately evaluated. although laeijendecker. reported no olp - related malignancies to date in the pediatric population, the schedule of follow up of pediatric olp should be atleast one or two visits per year as long as olp in children persists. paedodontists must be aware of its clinical presentation, diagnosis and management and periodic follow - up visits should be emphasized to control possible recurrence of the disease. the patients were diagnosed following the who diagnostic criteria (1978) of olp both clinically and histologically. the paper also reveals some significant association between the clinical forms and histopathological findings of olp. a better understanding of different clinical forms of oral lichen planus in children would help the pediatric dentists to make an early diagnosis and management of the lesion. | aim : well documented cases of oral lichen planus, a cell mediated immune condition is infrequently reported in paediatric population. this study was undertaken to obtain epidemiological data retrospectively and also to explore the possibility of any association that might exist among the clinical and histopathological features in paediatric patients suffering from oral lichen planus.subjects and methods : a retrospective study was carried out on 22 patients, younger than 18 years with clinical and histopathological diagnosis of oral lichen planus over a period of 14 years. the clinical characteristics and histopathological features were observed. the statistical analysis of the data was performed using statistical analysis software (sas), version 9.1.results:analysis of data of 22 patients revealed that the average age of patient is 15.18 years with equal male and female predilection. the most common site is buccal mucosa (50%) and most frequent clinical form is erosive (63.64%). focusing on the histopathological findings, parakeratosis was found in 86.36% of the cases, acanthosis in 63.64% of cases, moderate basal cell degeneration was identified in 63.64% of cases and dense lymphocytic infiltration at juxtaepithelial connective tissue region was found in 59.09% of cases.conclusions:oral lichen planus in paediatric population is rare and appeared between 8 to 18 years of age. there is no significant gender predominance. the most common clinical form is erosive, manifesting mainly in buccal mucosa. histopathological findings characteristic of oral lichen planus in paediatric patients include parakeratosis, acanthosis, liquefaction degeneration of basal cells and lymphocytic infiltration in the subepithelial layer. |
allergic rhinitis (ar) is a very common disease with a high and still increasing world prevalence.1 ar causes an important medical and social burden,2 which further increases when the disease is associated with allergic asthma.3 in fact, the concurrence of ar and asthma requires more doctor visits and more drugs and worsens patients quality of life.4 the management of respiratory allergy relies on, when possible, allergen avoidance, drug treatment, and allergen - specific immunotherapy (sit).3 sit is the practice of administering gradually increasing doses of the specific causative allergen to reduce the clinical reactivity of allergic subjects. this treatment has pivotal importance because of its ability to modify the natural history of the disease and to extend its effectiveness after treatment withdrawal, provided it is administered with sufficiently hgh doses and for an adequate duration.5 subcutaneous immunotherapy (scit) has for decades been the traditional technique of administration, but it is flawed by the problem of adverse systemic reactions, which, when they are of an anaphylactic type, may be severe and, though very rarely, even fatal.6 in recent years, sublingual immunotherapy (slit) has emerged as an actual treatment option because of its clinical efficacy and safety.7 the first studies on slit used low allergen dosages,8,9 but it soon became apparent that doses much higher than those administered by scit were needed to expect clinical efficacy. in consensus documents, an optimal dose as high as at least 50 times the dose administered by injection was suggested.3 the high number of trials on slit conducted in recent years allows an accurate evidence - based assessment of its efficacy based on several meta - analyses, and the most recent data also give an indication on optimal doses to be used. this review aims to analyze the current role of slit in the treatment of ar using evidence - based demonstrations of its efficacy and safety. the medical literature was searched by means of the medline database for the years 19902010 using the following key words : sublingual immunotherapy, efficacy, safety, tolerability, compliance, meta - analysis, and systematic review. the search was augmented by scanning the references cited in the articles identified. only full - length articles in english that had been published in referenced journals full copies of the articles were retrieved for analysis and the relevant data were extracted. the clinical efficacy of slit in ar, as for sit in general, can be evaluated by a decrease in symptom scores of rhinitis and in the use of symptomatic drugs. most of the placebo - controlled studies usually include small patient populations and thus are exposed to the issue of not having fully reliable statistical significance. an adequate statistical method to achieve statistical reliability is meta - analysis that estimates more powerfully the effect size of a medical treatment by combining the results of several trials and expresses the results as standardized mean difference (smd), comparing the effect of active and placebo treatment on patients. the results from the meta - analyses on slit in ar are summarized in table 1. in 2005, when 22 randomized controlled trials (rcts) were available, wilson published the first meta - analysis on slit,10 which demonstrated a significantly higher efficacy of slit versus placebo, with an smd corresponding to 0.42 for symptom scores (p = 0.002) and to 0.43 for medication scores (p = 0.00003). in the same year, another meta - analysis dealt with seven rcts conducted on children aged up to 14 years and found that slit was significantly effective on asthma symptoms (smd 1.42 ; p = 0.01) and on drug consumption (smd 1.01 ; p = 0.06), but the improvement did not reach significance for nasal and eye symptoms.11 however, a further meta - analysis on slit in children, concerning only efficacy on ar, showed positive results.12 ten rcts with an overall number of 484 patients (245 actively treated and 239 placebo treated) were included, and a significant reduction of both symptoms (smd 0.56, p = 0.02) and medication scores (smd 0.76, p = 0.03) was found. of note, the subanalysis addressing the length of treatment and the kind of allergen administered demonstrated a higher efficacy for durations longer than 18 months and for pollen allergens compared with house dust mites. the most recent meta - analyses highlighted the results according to the allergen used for slit. compalati examined eight rcts for ar (including 194 adults and children).13 a significant reduction in symptoms of ar (smd 0.95 ; p = 0.02) and a significant decrease in antiallergic medication use (smd 1.88 ; p = 0.04) in slit - treated patients compared with placebo was found. di bona performed a meta - analysis on rcts conducted with grass pollen extracts, concluding that slit significantly reduces both symptoms (smd 0.32) and medication use (smd 0.33) compared with placebo, that it is more efficacious in adults than in children, and that prolonging the duration of pre - seasonal treatment for more than 12 weeks improves treatment efficacy.14 a recognized limit of meta - analysis is the usually relevant heterogeneity of the included studies, due to the different dosages, standardization methods, treatment schedules, and patient populations. nieto checked the data reported in the original studies and concluded that the meta - analyses show discrepancies, inconsistencies, and lack of robustness and do not provide enough evidence for the current routine use of slit in patients with allergic asthma or rhinoconjunctivitis.15 by contrast, the overall evaluation of all meta - analyses (five on slit and two on scit) by compalati, despite a significant heterogeneity of studies and one negative meta - analysis, led the authors to conclude that sit can be recommended for the treatment of respiratory allergy because of its efficacy in reducing asthma and rhinitis symptoms.16 the limit of heterogeneity can be overcome by analyzing single studies conducted on large numbers of patients that allow adequate statistical power. the recent preparations for slit in tablets of grass pollen extract were evaluated on large populations, including 855 adults treated by a timothy grass extract,17 628 adults treated using a five - grass pollen extract,18 and 278 children treated using the same five - grass preparation.19 these studies showed a highly significant improvement in symptoms and rescue medication scores in actively treated compared with placebo - treated patients during the grass pollen season. important observations from such studies concern the dose dependence of clinical efficacy : only high doses, corresponding to 75,000 sq in the trial with the timothy grass pollen17 and to 300 ir in the trial with the five - grass extract,18 were effective. these doses correspond to 15 mcg and 20 mcg of the major allergens phl p 5, respectively. calculating the monthly cumulative dose, the world allergy organization position paper on slit suggested the monthly dose of 600 mcg of the grass pollen major allergen phl p 5 as being optimal.20 this is confirmed by the meta - analysis on grass pollen slit by di bona, showing clearly better results (smd 0.47) in patients receiving monthly doses of 275600 mcg than in patients receiving monthly doses lower than 275 mcg (smd 0.16).14 another important aspect of slit efficacy is the identification of patients more prone to respond to the treatment. devillier performed a post hoc analysis of data from the registrative studies with the new grass pollen tablets for slit.18,19 from such analysis it was apparent that the magnitude of efficacy was higher in patients with more severe symptoms during the grass pollen season. in fact, for the adult trial, the differences of the symptom medication score in the active versus placebo groups were 15%, 26%, and 37% for the low-, moderate-, and high - severity tertiles, respectively ; in the pediatric trial, these values were 10%, 33%, and 34%, respectively.21 the meta - analyses including, along with efficacy, the safety and tolerability aspects of slit found that the most common adverse events were local reactions in the oropharynx followed by gastrointestinal reactions (such as vomiting and diarrhea) and that systemic reactions such as asthma, rhinitis, or urticaria were quite rare.10,13 specific reviews on safety are also available that address only children22,23 or patients of any age.24,25 of note, differently from scit, there was not a dose dependence of safety, as the rate of systemic reactions was similar in studies using low doses and in studies using high doses.24 in a subsequent trial evaluating seven groups of patients treated with increasing doses, the treatment - related adverse events, including irritation of the throat, and itching sensations in the mouth and ears, increased with dose.26 in fact, local reactions are generally estimated to concern 20%40% of patients, but they can be easily managed and usually do not result in treatment withdrawal.27 anaphylactic reactions are extremely rare ; a review of published reports showed that in most cases the reaction is associated with mistakes, such as the use of an improper mix of allergens or the consumption of very high allergen doses.28 however, an increased risk is apparent in subjects undergoing slit because of previous systemic reactions to scit,29,30 especially when no updosing regimens are used. this warrants reconsideration of systemic reactions to scit as an admission criterion to slit.31 in any case, starting directly with the maintenance dose is not recommended in any patient, regardless of previous reactions to it. in fact, a phase i study comparing different doses and different updosing or no updosing regimens showed that only the group of patients treated with the highest dose with no updosing had severe local reactions with swelling of throat.32 based on the available studies, the major cause of non - compliance with slit is inconvenience due to visits to allergists offices for the injections.33 slit has different compliance issues from scit, because it is administered at home by patients themselves, and thus should have compliance problems similar to drug treatment. however, the findings from studies specifically designed for compliance and adherence indicate quite satisfactory results, with values between 75% and 95% of treated subjects.33 in a survey on allergists opinions on the factors positively influencing adherence to slit, the issues judged to be most important were the patient s perception of efficacy, reimbursability, and tolerability and the patient s education.34 concerning perception of efficacy, as previously noted in scit studies, it was observed that a lack of compliance to slit may be caused by the erroneous perception that once allergic symptoms are improved, slit is no longer needed.35 however, this aspect also belongs to the field of patient education, which has pivotal importance, as shown in a recent study that found a clear difference in compliance between patients receiving a complete educational course on slit and those receiving only standard instructions.36 a major advantage of scit over drug treatment is that efficacy on allergic symptoms persists after its discontinuation.5 recent studies showed that slit also has such a carryover effect. in a survey on slit using a dust mite extract, 137 patients were divided into two groups, 67 receiving slit for 2 years and 70 receiving slit for 3 years.. a greater improvement of symptoms was found in the patients treated for 3 years.37 in a prospective open controlled study, patients monosensitized to mites were divided into four groups, one receiving only drug treatment and the other three receiving slit for 3, 4, or 5 years. the observation period reached 15 years, and the clinical scores showed that the benefit persisted for 7 years in patients treated for 3 years, whereas the benefit persisted for 8 years in those treated for 45 years.38 moreover, new allergen sensitizations occurred in all patients treated only with drugs and in less than 25% of patients receiving slit. slit has gained ample evidence of efficacy and safety and in some european countries is currently used more frequently than scit. apart from its better safety profile, the advantages of slit over scit are with regard to compliance, which is higher because slit does not need to be administered in a medical setting, and cost - effectiveness, because the cost of the injections is not involved.39 however, it is important to note that such favorable outcomes exist only if slit meets its needs, ie, the administration of high doses is continued regularly for at least 3 consecutive years. in fact, slit efficacy is dose dependent, and a sufficient duration of treatment is necessary to induce the immunologic changes underlying clinical effectiveness. recent studies showed that the mechanism of action of slit is similar to that demonstrated for scit,40 and that when high doses are administered, immunoglobulin g - blocking antibodies, which were not found in slit studies employing low doses, are produced in significant amounts and persist after the discontinuation of treatment.41 | allergic rhinitis is a very common disease affecting about 20% of people. it may be treated by allergen avoidance when possible, by antiallergic drugs such as antihistamines and topical corticosteroids, and by allergen - specific immunotherapy. the latter is the only treatment able to act on the causes and not only on the symptoms of respiratory allergy and is able to maintain its efficacy even after stopping, provided an adequate duration of treatment of 35 years is ensured. sublingual immunotherapy (slit) was introduced in the 1990s as a possible solution to the problem of adverse systemic reactions to subcutaneous immunotherapy and has been demonstrated by more than 50 trials and globally evaluated thus far by five meta - analyses as an effective and safe treatment for allergic rhinitis. life - threatening reactions are extremely rare. however, it is important to note that clinical efficacy occurs only if slit meets its needs, ie, sufficiently high doses are regularly administered for at least 3 consecutive years. this is often overlooked in the current practice and may prevent the same success reported by trials from being achieved. |
hypertension is a powerful independent risk factor for the development of well - known cardiovascular and cerebrovascular morbidities that include myocardial infarction, stroke, atherosclerosis, and death. it is a prevalent disorder that affects close to a quarter of the adult population worldwide. although often considered as a late - onset chronic disorder, hypertension is now recognized to afflict the young population, from neonates to adolescents. while the diagnosis of hypertension in adults is based on empirically determined cut - off values for both systolic and diastolic blood pressure (bp), the diagnosis of pediatric hypertension is not as straightforward since it is based on normative data. hypertension in the young is defined as the average systolic or diastolic bp that is 95th percentile according to age, sex, and height on three or more occasions. there are no target bp values that indicate high blood pressure in children, since bp changes with age ; neonates have low bp that gradually increases with age. the prevalence of pediatric hypertension is estimated at 1 - 2% and seems to have steadily increased during the past decade, which correlates with the increase in the prevalence of obesity. a striking concept on the etiology of hypertension that has emerged during the last few decades is what is known as fetal imprinting, or developmental programming. this concept refers to the observation that adverse environmental insults early in life, particularly during critical periods of development in utero and the early postnatal period, can result in silent yet long - term morphological and physiological alterations that eventually translate into disease in adulthood. the correlation between adverse intrauterine conditions and subsequent cardiovascular disorders was first proposed by barker who reported a link between the mortality rate of coronary artery disease and birth weight [7, 8 ]. barker hypothesized that adaptive responses to an environmental insult during early life may persist and become harmful during adulthood when the insult is no longer present. a compelling body of work from various groups around the world has corroborated these findings and has expanded the concept to include other conditions such as type 2 diabetes, obesity, and chronic kidney disease. the biological and molecular mechanisms involved in fetal imprinting are multiple, though not completely understood, and include low birth weight (small baby syndrome), glucocorticoid excess, uteroplacental deficiency, sexual dimorphism, and epigenetics. low birth weight (lbw), defined as birth weight of < 2500 g by the world health organization, may arise from intrauterine growth restriction (iugr, or birth weight that is < 10th percentile for gestational age) or prematurity, with the former exerting a stronger predisposition for disease to develop and manifest later in life. several animal models have demonstrated the inverse relationship between lbw and hypertension [10, 11 ], which was attributable to a deficit in nephron number. moreover, humans with lbw have significantly higher bps, even after correction for various modifiers such as sex, cigarette smoking, weight, and use of oral contraceptives. two distinct mechanisms have been proposed to explain why lbw antedates the development of adult hypertension. numerous studies indicate that birth weight is a strong predictor of nephron number and mean glomerular volume in several racial groups [12, 13 ]. in humans, nephrons are formed between 2834 weeks of gestation, after which the individual has achieved a finite nephron number endowment for life (an average of 750,000 per kidney), that is, no additional nephrons are formed thereafter [14, 15 ]. postnatally, an increase in tubular length and glomerular size, which varies inversely with nephron number, is achieved. iugr curtails the formation of adequate nephron number in the growing fetus, which compromises the available filtration surface area and leads to hypertension in adults. nephron - deficient rats have been shown to exhibit sodium - dependent hypertension and albuminuria, while postmortem examination of kidneys from hypertensive caucasians reveals significantly fewer glomeruli per kidney and greater glomerular volume. endothelial dysfunction in individuals with lbw is another mechanism that may explain the development of hypertension later in life. the perturbation of endothelial function may be due to impaired angiogenesis during fetal development or an apparent reduction in the production or function of nitric oxide [21, 22 ]. other factors such as increases in inflammatory cytokines, the expression of metabolic genes, and changes in blood vessel formation all of which can contribute to high blood pressure in adults have been detected in fetuses with iugr [2326 ]. additionally, the expression and activity of ion transporters such as nak - atpase and sodium - hydrogen exchanger 3 are increased with iugr, which may affect how electrolyte uptake or excretion is regulated in adulthood. in 310% of pregnancies in the western world, the flow of blood from mother to fetus is inadequate, resulting in utero - placental insufficiency (upi). upi subjects the fetus to stressors which are not normally encountered during development, such as hypoxia, altered hormone concentrations, and decreased nutrition. these unfavorable environmental factors result in the development of conditions such as iugr, which typically begins midway through pregnancy and continues through the end of gestation. in the presence of upi, the fetus must deviate from its normal developmental trajectory during this critical period of growth in order to ensure the formation of vital organs and survival in adverse conditions. though these changes may be beneficial during early life, they often lead to deleterious conditions such as hypertension in adulthood [3234 ]. the trafficking of nutrients, hormones, and other factors essential for the development of the kidneys and vasculature is disrupted in iugr placentas and thus may play a role in the development of hypertension. studies have shown that downregulation of amino acid transporters [3540 ], lowered fetomaternal blood flow [35, 41 ], and structural abnormalities are present in iugr placentas [4244 ]. since the placenta is the medium through which the fetus receives all factors necessary for growth, disruptions in placental structure or function may alter the development of fetal vasculature, organs, or signaling pathways in ways that predispose the fetus to later development of hypertension. as previously mentioned, the process of nephrogenesis in humans is completed by weeks 3436 of gestation [14, 15 ]. iugr that occurs prior to the generation of the full nephron complement may result in reduced renal mass [4547 ], decreased nephron and glomerular endowment, and increased protein excretion [46, 4850 ]. iugr may result in a decrease in the expression of genes involved in nephrogenesis [51, 52 ], as well as a rerouting of nutrients from the kidney to more essential organs such as the brain, adrenal glands, and heart in order to enable survival of the fetus [53, 54 ]. while such changes allow for fetal survival during early development, they may increase the risk of susceptibility to renal injury or malfunction during adulthood [5557 ] as a result of diminished nephron number and renal size [17, 5860 ]. studies of iugr in animal models have demonstrated how highly sensitive the developing kidney is to perturbations of the intrauterine environment, especially during the early stages of nephrogenesis. maternal dietary manipulation is commonly employed to induce iugr during fetal development [46, 47 ] which results in reduced nephrogenesis, suppression of the renin - angiotensin system (ras), and hypertension in the adult animal. significantly, the timing of the induction of fetal iugr is directly related to the outcome in the adult. the greatest impact on adult blood pressure is observed when iugr coincides with the period of nephrogenesis. this indicates the importance of kidney morphology in the programming of blood pressure in the adult. sex hormones may have a regulatory effect on the development of hypertension in adult iugr offspring. while both male and female iugr offspring are hypertensive early in life, only males remain hypertensive into adulthood. many studies have shown that estrogen may play a protective role against the development of hypertension in female iugr offspring. this is supported by the fact that postmenopausal women are at an increased risk for hypertension. in animal models, ovariectomized female iugr offspring become hypertensive like their male counterparts, whereas non - iugr control females do not become hypertensive. it was also found that e2 levels at 16 weeks of age were not significantly different between iugr and control offspring. this seems to suggest that the protective role of estrogen does not play a direct role in regulating blood pressure, but may do so by regulating another mechanism such as the ras. testosterone seems to play a role in the stimulation of the ras as well, which may explain the increased blood pressure in male iugr offspring. testosterone levels were significantly higher in intact male iugr offspring when compared to control offspring. hypertension was abolished in male iugr offspring that underwent castration, and treatment with testosterone raised their blood pressure back to hypertensive levels [62, 63 ]. castration of the male control rats did not have a significant effect on blood pressure [62, 63 ]. the ace inhibitor enalapril abolished hypertension in both male and female iugr offspring, indicating that the ras is involved in the regulation of hypertension. taken together, it seems that testosterone acts as a stimulant of the ras, which increases blood pressure, while estrogen acts as an inhibitor, lowering blood pressure in females. maternal diet has been shown to have a large impact on the fetal programming of hypertension, although this too has a clear sexual dimorphism. males appear to be much more sensitive to insult in utero than their female counterparts. studies show that in cases of moderate fetal insult due to a protein - restricted or global - restricted diet, male iugr offspring developed and remained hypertensive while their female counterparts seemed to be protected [16, 64 ]. female iugr offspring only responded with an increase in blood pressure to severe insult during development. one may be the influence of the sex hormones testosterone and estrogen on the ras. increased testosterone levels in male iugr offspring may explain their increased sensitivity to hypertension, while the effect of estrogen explains the protected status of female offspring. another explanation may be catch - up growth of iugr offspring. while both males and females exhibit low birth weight as a result of reduced uterine perfusion, male iugr offspring undergo catch - up growth [16, 64, 65 ]. this causes male iugr offspring to have an above - average body mass as adults, which may be a factor in the development of hypertension. glucocorticoids are potent regulators of fetal growth and have been associated with long - term effects on the development and morphogenesis of the growing fetus. corticosteroid therapy for neonates at risk of preterm delivery and respiratory distress syndrome (rds) has been the standard of treatment since the landmark paper by liggins and howie in 1972. some pathophysiological effects of glucocorticoid excess are hypertension, impaired sugar metabolism, and abnormalities in neuroendocrine responses [6769 ]. first, suboptimal placental or maternal nutrient supply results in increased glucocorticoid levels, which restrict fetal growth and program permanent changes in the cardiovascular, endocrine, and metabolic systems. second, exposure to exogenous glucocorticoids may occur, such as in neonates treated with glucocorticoids for respiratory - related disorders. antenatal administration of exogenous or increased endogenous glucocorticoids results in lbw and alters the maturation of a variety of organs. fetal glucocorticoid levels are much lower than maternal levels. in order to maintain this difference, the placenta is saturated with the enzyme 11--hydroxysteroid dehydrogenase type 2 (11--hsd2), which rapidly inactivates glucocorticoids to their inert 11-keto forms (cortisone, 11-dehydrocorticosterone). if the fetus is genetically deficient in the 11--hsd2, it may exhibit lbw and metabolic disorders. for example, heterozygous individuals carrying the deleterious 11--hsd2 gene allele had extremely low birth weights with respect to siblings who were homozygous for the wild - type gene and born with normal weights. studies in rats show that a decrease in 11--hsd2 activity results in lbw and hypertension in the adult animal. the exact mechanism of action for glucocorticoids and the pathogenesis of hypertension have been characterized in several physiological systems. increased production of reactive oxygen species, the activation of the ras, and impairment of nephron development are all associated with increased sodium retention in neonates exposed to glucocorticoids [68, 74 ]. research has found a critical period in early fetal development during which glucocorticoid treatment will lead to hypertension ; soon after this time period, most of the organs will have developed, and thus glucocorticoid exposure will not have as profound an effect on their morphogenesis. in one study, offspring of pregnant ewes exposed to glucocorticoids developed impaired renal function and glomerular filtration rate. in two - month - old female offspring of these glucocorticoid - treated ewes, the -, -, -subunits of na / k - atpase were upregulated in the kidney, indicating a potential mechanism of action for development of hypertension. genetic predisposition can also play a role in the alteration of the glucocorticoid receptor function. the 23 k variant of the r23k snp of the glucocorticoid receptor has been shown to protect against neonatal development of insulin resistance and growth failure. this has been shown by a study that evaluated the relative sensitivity of an individual to cortisol in a cohort of 249 19-year - old subjects who were born at less than 32 weeks gestational age. the investigators determined that genomic polymorphisms may play a role in the sensitivity of neonates to glucocorticoid exposure and may have permanent effects as a result of fetal programming. mutations in the 11--hsd2 have been linked with very low birth weights in patients with apparent mineralocorticoid excess, leading to juvenile hypertension. epigenetic modification of dna by methylation occurs primarily at cpg dinucleotides and serves as a mechanism of gene silencing. studies using rodent models of maternal protein restriction during gestation have shown that reduced methylation of genes occurs in the offspring [77, 78 ]. using a similar system, bogdarina. demonstrated decreased methylation at the proximal promoter region of the angiotensin receptor, type b gene (agtr1b), which correlated with increased expression of the receptor in the adrenal gland of the offspring. nevertheless, these results suggest a link between iugr and epigenetic modification of genes related to the regulation of blood pressure. more recently, bogdarina. found that treating pregnant rats with metyrapone, an 11 -hydroxylase inhibitor, during the first two weeks of pregnancy normalized the methylation of the promoter region of the agtr1b, reduced the expression of the agtr1b receptor, and prevented the development of hypertension in the offspring. while few of these types of studies have been conducted, the results are exciting and further investigation is certainly warranted. twenty years ago, hales and barker proposed that events that occur in the early fetal environment may be linked to long - term health and lifespan consequences in the adult. numerous subsequent studies have largely supported their hypothesis and serve to illustrate the overarching complexity of the maternal - fetal - environmental interaction. while this review was necessarily limited in scope to only the effect of iugr on hypertension, the reader should be made aware that this is but one component of a constellation of metabolic disorders that may arise as a result of a severe perturbation of the fetal environment. the focus of most of the studies reviewed has been the impact of iugr on the adult animal. we would suggest that additional studies be devoted to elucidating the mechanisms employed by the fetus in order to rapidly adapt to its altered environment. it is by better understanding these mechanisms that we may be able to develop interventional strategies that may prevent the onset of disease in the adult. | events that occur in the early fetal environment have been linked to long - term health and lifespan consequences in the adult. intrauterine growth restriction (iugr), which may occur as a result of nutrient insufficiency, exposure to hormones, or disruptions in placental structure or function, may induce the fetus to alter its developmental program in order to adapt to the new conditions. iugr may result in a decrease in the expression of genes that are responsible for nephrogenesis as nutrients are rerouted to the development of more essential organs. fetal survival under these conditions often results in low birth weight and a deficit in nephron endowment, which are associated with hypertension in adults. interestingly, male iugr offspring appear to be more severely affected than females, suggesting that sex hormones may be involved. the processes of fetal programming of hypertension are complex, and we are only beginning to understand the underlying mechanisms. |
urinary tract infection (uti) is one of the most common diseases in children, and vesicoureteral reflux (vur) is estimated to occur in 25 to 50% of children with febrile uti. approximately 30 to 40% of children with vur have renal scarring at the time of diagnosis. recurrent uti related with renal scarring and vur are risk factors for progressive renal damage, which may lead to severe sequelae such as hypertension and permanent renal failure. however, vcug is an invasive procedure and has many side - effects, such as pain, irradiation, and uti. the dimercaptosuccinic acid (dmsa) renal scan is a noninvasive test that allows for cortical imaging ; this approach is currently recommended for evaluation of first - time febrile uti because of its high sensitivity for detection of renal parenchymal injury. however, it is not clearly known whether dmsa scintigraphy performed during febrile uti has any prognostic value for outcome assessment in these children. camacho. advocated that children with an abnormal dmsa scan result have a higher frequency of vur than do children with a normal dmsa scan result (48% vs. 12%) and that children with normal dmsa scan results during acute uti have a low risk of renal damage. in the present study, we evaluated the usefulness of dmsa scans performed during febrile uti for prediction of the severity and persistency of vur that may lead to progressive renal damage. a total of 142 consecutive children (57 boys and 85 girls ; mean age, 6.72.6 years) who presented with an episode of acute febrile uti from january 2004 to december 2006 and who were followed up for more than 1 year were included in this retrospective study. dmsa and renal ultrasound scans were performed during the first 7 days of the acute episode, and vcug was done 1 month after the acute phase of disease to determine the presence of vur. children with detected vur underwent a follow - up vcug study within 1 year after the first episode of acute uti. vur was graded according to the recommendations of the international reflex study and then categorized as low grade (grade children with bilateral reflux, voiding difficulty caused by neurogenic bladder, dysfunctional elimination syndrome, or duplicated kidney were excluded from this study. appropriate antimicrobial prophylaxis was continued in children with a high grade of vur and a recurrent uti episode. for patients with breakthrough uti and aggravated vur, endoscopic subureteral injection of deflux (dextranomer hyaluronic acid copolymer) was considered. dmsa scintigraphy was performed by using a standard protocol applied by the calculated dose of dmsa according to the body surface area performed within 7 days of diagnosis. dmsa scintigraphy was interpreted according to the recommendations of the european association of nuclear medicine pediatric task group. a focal reduction or absence of uptake in more than one area of the kidney vur was found in 47 children (33.1%) with febrile uti, of whom the majority had low - grade vur (76.6% vs. 27.1%). there were no significant differences in basal characteristics according to gender or in vur incidence between boys and girls (p=0.2) (table 1). at the initial examination, abnormal dmsa scintigraphy results were found in the majority (72.5%) of children with febrile uti. the frequency of vur with an abnormal dmsa scan result during the acute febrile uti episode was significantly higher than that of vur with a normal dmsa scan result (38.8% vs. 25.8%, p=0.004). in all children with a normal renal scan result, the reflux grade was low - grade ; high - grade vur was not found in this group (table 2). none of the 13 patients with high - grade vur had a normal dmsa scan result. at the follow - up evaluation, the rate of vur resolution in children with an abnormal dmsa scan result during the acute febrile uti episode (7/40) was significantly lower than the rate in children with a normal dmsa scan result (6/8 ; 17.5% vs. 75.0%, respectively, p<0.05) (table 3). most of the children with vur with an abnormal dmsa scan result at the initial visit had persistent vur (33/40, or 82.5%) at the follow - up evaluation. in the present study, vur was found in 33.8% of children with febrile uti, and abnormal dmsa scintigraphy results were found in 72.5% of children with febrile uti. the purpose of this study was to assess the usefulness of dmsa study performed during acute febrile uti in identifying children at risk of vur or of resolution of vur. in the present study, vur had a high rate of association with abnormal dmsa scan results compared with normal dmsa scan results. furthermore, the frequency of vur with an abnormal dmsa scan result during acute uti was significantly higher than that of vur with a normal dmsa scan result. the present findings are consistent with the view that the dmsa scan obtained in children with febrile uti is useful in the formulation of a proper management plan for those children in the clinical setting. those with abnormal dmsa results and renal scarring are more likely to display vur and are at increased risk of persistent vur. this information is useful in counseling children and parents about the natural course of vur and in prediction of vur resolution during the follow - up period. vcug is an unpleasant and invasive procedure that involves radiation exposure and introduces the risk of catheter - induced uti. these risks can dissuade clinicians from routinely performing vcug in children after their first febrile uti. instead of vcug dmsa scintigraphy has high sensitivity for detection of renal abnormalities and is considered the gold standard for diagnosis of renal damage. in the present study, 103 of 142 children (72.5%) presented with abnormal findings on the dmsa scintigraphy performed during the acute period of uti. reported that dmsa abnormalities are found in 51% of children with febrile uti when dmsa is performed early in the course of uti. the relatively higher incidence of abnormal dmsa studies found in our study may be attributed to our including recurrent uti in our data. vur is one of the most frequent risk factors for the development of renal damage. in the present study, vur was found in 33.8% of the children with acute febrile uti, whereas other studies have reported an incidence of vur of 24 to 25% of children with febrile uti. an abnormality in a dmsa scan could occur in the absence of vur, and in the present study, as many as 63 of 103 children with an abnormal dmsa scan result showed no reflux. this finding suggests that more than reflux itself is involved in the pathogenesis of parenchymal defects and renal scarring ; additional factors could include bacterial virulence factors or host immunity. in an earlier retrospective study, temiz. showed that a strategy to perform vcug only in patients with abnormalities on the dmsa scan to identify patients with high - grade vur would have reduced the number of vcug procedures by half. a recent study by tseng. retrospectively analyzed the medical records of 142 children with vur and reported that all the children with grades iii to v vur (21 patients) had an abnormal dmsa scan result. those authors further stated that if vcug had been performed only in children with an abnormal scan result, 41 of 142 (29%) investigations could have been omitted. in the present study, the incidence of vur with a normal dmsa scan result was very low (8/39, or 20.5%), and the degree of vur in those children was low - grade (grade i, n=4 ; grade ii, n=3 ; grade iii, n=1) and clinically insignificant. many studies have reported a spontaneous vur resolution rate of 53 to 68% during follow - up, with the rate differing according to the grade of vur. a recent study reported that low - grade vur (grades i to iii) diagnosed after uti resolves significantly more rapidly than does high - grade vur (grades iv to v). camacho. evaluated the prognostic value of dmsa renal scans performed during febrile uti and reported that the frequency of vur was higher by as much as 48% when the dmsa scan result was abnormal than the frequency of vur when the dmsa scan result was normal in children (12%). another study reported that the rates of 2-year reflux resolution in abnormal and normal renal scan groups were 13% and 53%, respectively. in our study, all the subjects with high - grade reflux showed an abnormal renal scan, and the vur of those children did not show spontaneous resolution on follow - up evaluation. the rate of vur resolution differed depending on the dmsa scan result during the acute uti. at the follow - up evaluation, the rate of vur resolution in children with an abnormal dmsa scan result during the acute febrile uti was significantly lower than in children with a normal dmsa scan result. most of the children with vur with an abnormal dmsa scan result at the initial visit were found to have persistent vur at the follow - up evaluation. we suggest that in children with an abnormal result on a dmsa renal scan during acute febrile uti, most of the reflux does not resolve after follow - up evaluation, in contrast with the case in children with a normal result on a dmsa renal scan, in whom reflux tends to resolve. furthermore, in the present study, in children with a normal renal scan, most of the reflux was low - grade, whereas none of the patients with high - grade vur had a normal dmsa scan result. these results concur with previous reports that higher grades of reflux are associated with an increased incidence of an abnormal result on a dmsa renal scan and a strong association between reflux grade and the incidence of renal scarring. however, goldman. reported that renal scarring was not related in children with pyelonephritis and low - grade reflux and that there was no correlation between parenchymal defects and recurrent uti. concerning the reversible defect of renal damage, it is important to consider the timing of dmsa renal scintigraphy. we made every effort to conduct the dmsa scintigraphy within the acute period of febrile uti within 7 days of diagnosis. vcug was performed within 1 month after the diagnosis, but with small variation among the subjects. we excluded children with bilateral vur and could not evaluate the dmsa renal scan results according to the frequency of febrile uti or differences in gender or age. another limitation of our study is that we did not distinguish between first uti and recurrent uti. in our study, the mean age of the children with febrile uti was relatively older than in other similar studies. renal scars may have developed in older children after previously unrecognized utis, and these scars can not be differentiated from acute photon defects on dmsa renal scintigraphy. to avoid such a limitation, the current study limited the age to younger than 1 year. abnormal dmsa scan results during febrile uti were associated with high - grade and persistent vur. dmsa scanning performed during febrile uti was useful as an indicator of reflux resolution in childhood. a dmsa scan should be considered when counseling families about the prognosis of febrile uti with respect to vur and renal damage. | purposethis study assessed whether 99mtechnetium dimercaptosuccinic acid (dmsa) scintigraphy used for the assessment of renal sequelae after febrile urinary tract infection (uti) has any prognostic value for outcome measurement of vesicoureteral reflux (vur) by retrospectively evaluating the correlation between abnormal dmsa scintigraphy results and persistence of vur in children with febrile uti.materials and methodsthe medical records of 142 children (57 boys, 85 girls) admitted with febrile uti from january 2004 to december 2006 and who were followed up for more than 1 year were retrospectively reviewed. at the initial and follow - up visits, renal ultrasound and dmsa scans were performed within 7 days from the diagnosis and voiding cystourethrography (vcug) was performed within 1 month in all case and follow - up evaluations.resultsthe children 's mean age was 4.83.6 years (range, 0.3 to 14 years). the mean follow - up was 28.24.8 months. at the initial examination, vur was more often associated with an abnormal dmsa scan result (83.3%) than with a normal dmsa scan result (16.7%, p=0.02). the frequency of vur with an abnormal dmsa scan during acute uti was significantly higher than the frequency of vur with a normal dmsa scan (38.8% vs, 25.8%, respectively, p=0.004). also, high - grade vur was associated with an abnormal dmsa scan result (32.5%) more often than with a normal dmsa scan result (0%, p=0.01). children with an abnormal dmsa scan had a lower resolution rate of vur (17.5%) than did children with a normal dmsa scan (75.0%) at the follow - up vcug (p=0.02).conclusionsan abnormal result on a dmsa scan during febrile uti is associated with high - grade and persistent vur. dmsa scans performed during febrile uti are useful in reflux resolution in childhood. |
winey and colleagues developed an mps1-as1 mutant in budding yeast, which can be specifically inactivated by an atp - analog inhibitor 1nm - pp1. they arrested this mps1 mutant after duplication of spindle pole bodies (spbs ; microtubule - organizing centers in yeast) but before dna replication and spb separation, by using a cdc34 - 2 temperature - sensitive (ts) mutant. upon release from cdc34 - 2 arrest by lowering the temperature, they inactivated mps1-as1 by means of its inhibitor ; in this way, the mps1 requirement for spb duplication was bypassed in order to study its function in a later phase of the cell cycle. the majority of chromosomes showed missegregation when mps1 was inactivated, and relevant defects were already detected prior to anaphase entry, suggesting that mps1 plays very crucial roles in chromosome segregation, independently of spb duplication and the spindle - assembly checkpoint. to address how mps1 regulates chromosome segregation, we also used cdc34 - 2 and mps1-as1 and analyzed behavior of centromeres when mps1 was inactivated. cen3 was visualized by adjacent insertion of a tet operator array that was bound by tet repressors fused with green fluorescent protein (gfp), and therefore cen3 was visualized as a small gfp dot (figure 1a, top). microtubules were also visualized by expression of -tubulin (tub1) fused with yellow fluorescent protein (yfp). after release from cdc34 - 2 arrest and inactivation of mps1, we depleted cdc20, an activator of anaphase promoting complex, to arrest cells in metaphase (cdc20 was under control of the met3 promoter, which can be turned off in the presence of methionine). as a control, a wild - type mps1 strain was treated in the same way as the mps1-as1 strain. mps1 and mps1-as1 strains formed a bipolar spindle with similar timing (figures s1a and s1b in the supplemental data available online). upon formation of bipolar spindle, mps1 cells frequently showed separated cen3 dots on the metaphase spindle, indicative of sister - kinetochore bi - orientation [1215 ] (figure 1a). in mps1-as1 cells, cen3 was also located on the spindle ; but in contrast to mps1 cells, they rarely showed separation of cen3 dots on the bipolar spindle. most of nonseparated cen3 signals stayed in the vicinity of the same spindle pole during time - lapse observation (data not shown) ; they were therefore mono - oriented on the spindle (supplemental results and discussion, note 1). the mono - orientation of cen3 signals in mps1-as1 was not due to failure of dna replication (figure s2). we concluded that mps1 kinase is required for establishing sister - kinetochore bi - orientation on the metaphase spindle. this is consistent with a previous report that bilobular kinetochore distribution in metaphase is disturbed by mps1 inactivation. next we addressed whether a similar defect in mps1-as1 could also be found without using cdc34 - 2 (supplemental results and discussion, note 2 ; figures s3 and s4). the bi - orientation defect in mps1-as1 mutant was not an artifact due to use of cdc34 - 2, as shown by the fact that it was also found in other assays. considering that mps1 is important for a spindle - assembly checkpoint, we studied the behavior of gfp - marked cen3 in mad2-deleted cells, where the spindle - assembly checkpoint is defective, with the same protocol as in figure 1a. in contrast to mps1-as1, mad2-deleted cells showed sister cen3 separation as frequently as in mps1 mad2 cells on the metaphase spindle (data not shown). the bi - orientation defect with inactive mps1 was therefore not due to an impaired spindle - assembly checkpoint. when mps1 was inactive, the majority of centromeres showed mono - orientation on the bipolar spindle, and in addition, the spindle became longer than normal and was sometimes discontinuous in the middle (figures s1b and s1c). we next addressed whether such elongated and discontinuous spindles played any causative roles in centromere mono - orientation upon mps1 inactivation (supplemental results and discussion, note 3 ; figure s5). we concluded that mps1-as1 cells showed defects in bi - orientation before appearance of elongated and discontinuous spindles, which therefore can not be the sole reason for centromere mono - orientation. rather, we suspect that the abnormal spindle is the outcome of extensive centromere mono - orientation in mps1-as1 cells. kinetochores are initially captured by the lateral side of a microtubule and subsequently transported by that microtubule toward a spindle pole, followed by bi - orientation on the spindle [17, 18 ]. frequent mono - orientation in mps1-as1 suggests that one or some of these steps are defective. to identify a defective step, we used an assay system in which we can visualize the initial kinetochore - microtubule interaction in a stepwise manner (figure 1b, top). in this assay, we regulated the activity of a particular centromere (cen3) ; cen3 was displaced from the spindle and other centromeres by conditional inactivation via transcription from the adjacently inserted gal1 - 10 promoter. then, during metaphase arrest by cdc20 depletion, we reactivated cen3 by turning off the gal1 - 10 promoter. after reactivation, the gfp - marked cen3 was captured within 1015 min (figure 1b, blue line) by the lateral surface of yfp - labeled microtubules and was immediately transported toward a spindle pole (figure 1b, pink line : time - lapse microscopy, not shown) in the majority of both mps1 and mps1-as1 cells. subsequently, sister cen3 dots were separated on the spindle in mps1, but such separation was rare in mps1-as1 (figure 1b, red line). thus, when mps1 is inactive, kinetochores are captured and transported pole - ward normally by microtubules, but it is the subsequent establishment of bi - orientation that is extensively defective. subsequently we addressed, once bi - orientation is established, whether mps1 is still required for its maintenance (supplemental results and discussion, note 4 ; figure s6). sister cen3 separation was maintained after mps1 was inactivated, making a sharp contrast with the extensive defect in establishing sister cen3 bi - orientation, shown in figure 1. thus, mps1 might not be required for maintenance of bi - orientation once it is established. one explanation is that one of two spbs is defective in organizing microtubules that extend to capture kinetochores. in particular, because mps1 is required for spb duplication, the new spb might be partly defective even after this requirement is bypassed with cdc34 - 2. to address this, we analyzed the behavior of two pairs of sister centromeres on different chromosomes in the same cells (figure 2a) ; if mono - orientation is caused by a defect of one spb, both pairs of sister centromeres will mono - orient always at the same pole, i.e., the more functional one. we marked cen5 by the adjacent insertion of a tet operator array bound by tetr-3cfp (cyan fluorescent protein) and cen15 by a lac operator array bound by gfp - laci. spbs were also marked by expression of spc42 fused with red fluorescent protein (rfp). after release from cdc34 - 2 arrest and addition of 1nm - pp1, mps1-as1 cells showed extensive mono - orientation of both pairs of sister cen5s and cen15s (figure 2a), as expected. importantly, when both cen5 and cen15 mono - oriented in the same mps1-as1 cells, they mono - oriented at the same pole or at opposite poles with similar frequency. therefore mono - orientation can not be explained by a defect of one spb in organizing microtubules. the two spbs are probably equally functional in mps1-as1 cells, at least under these experimental conditions. another explanation for sister centromere mono - orientation with inactive mps1 is that only a single kinetochore from each pair of sister centromeres is functional. for example, after centromere dna replication, a kinetochore may be formed on one sister chromatid but not on the other. if this explanation is the case, abolition of sister - chromatid cohesion should permit the sister centromere with an active kinetochore to be drawn to a spindle pole, but should leave the other sister centromere lacking an active kinetochore within the middle of the nucleus. when we depleted a cohesin subunit scc1 (also called mcd1 ; by means of a strain whose sole scc1 gene is under control of the galactose - inducible promoter) to abolish cohesion in mps1-as1 cells treated with 1nm - pp1, both sister centromeres moved to the vicinity of spbs in more than 90% of cells, sometimes to the same pole but often to the opposite poles (figure 2b). a similar result was obtained when we depleted scc1 in mps1 cells (figure 2b) ; frequent mono - orientation under this condition confirmed that scc1 was successfully depleted with this procedure. in contrast to the result in mps1-as1 cells, when scc1 was depleted in ndc10 - 1 cells that lack functional kinetochores, centromeres often did not stay in the vicinity of either spb. thus, mono - orientation with inactive mps1 is not caused by a failure to establish functional kinetochores after dna replication. taken together, failure in bi - orientation upon mps1 inactivation is not due to absence or loss of kinetochore - spindle pole connections but is due to defects in achieving proper orientation of these connections. to facilitate sister - kinetochore bi - orientation on the metaphase spindle this error correction stems from stabilization of kinetochore - spindle pole connections by tension, arising from bi - orientation but not syntelic attachment [6, 23 ]. to investigate error - correction mechanism, we previously developed an unreplicated circular minichromosome harboring two centromeres (supplemental results and discussion, note 5 ; figure 3a). in wild - type cells, the two centromeres on this unreplicated dicentric minichromosome always bi - oriented efficiently, suggesting that a tension - dependent mechanism suffices for their bi - orientation. to address whether bi - orientation of the unreplicated dicentric minichromosome, like that of authentic replicated sister centromeres, is dependent on the mps1 kinase, we compared the behavior of this gfp - marked minichromosome in mps1 and mps1-as1 cells after release from cdc34 - 2 arrest and addition of 1nm - pp1 (figures 3b and 3c). in 89% (32/36) of mps1 cells, gfp signals were found halfway between two yfp - labeled spbs and often stretched, indicative of bi - orientation. on the other hand, in 92% (33/36) of mps1-as1 cells, gfp signals remained in the vicinity of one spb, having made connections to a single pole, i.e., mono - oriented. thus, inactivation of mps1 greatly reduced the incidence of bi - orientation of this minichromosome. a corollary is that mps1 is able to facilitate bi - orientation of sister kinetochores through a tension - dependent error - correction mechanism. if mps1 is indeed involved in the tension - dependent error correction, we may see the role of mps1 in detaching a centromere from one spb and attaching it to another spb when tension is not applied on this centromere - spb connection by microtubules. to increase the chance of detecting this re - orientation between different spbs, we created cells containing four spbs, instead of two in normal metaphase, as we did previously. in these cells, we observed the movement of an unreplicated minichromosome with one centromere, on which no tension is applied (supplemental results and discussion, note 6 ; figure 4a). by using time - lapse microscopy, we compared frequency of re - orientation of gfp - marked unreplicated monocentrics between different yfp - labeled spbs in mps1 and mps1-as1 cells (figures 4b and 4c). during observation, 70% (7/10) of mps1 cells showed re - orientation whereas only 23% (3/13) of mps1-as1 cells showed it (p < 0.04). these data suggest that the mps1 kinase does indeed have an important role in eliminating kinetochore - spindle pole connections when they can not come under tension normally generated by bi - orientation. given that mps1 and ipl1 kinases play similar roles in promoting sister - kinetochore bi - orientation [3, 6, 24 ], we addressed whether one may regulate function of the other. first we studied whether mps1 and ipl1 localization is altered in ipl1 and mps1 mutants, respectively (supplemental results and discussion, note 7 ; figures s7 and s8). however, we did not find any significant change in their localization in the mutants. next we investigated a possible change in mps1 kinase activity in an ipl1 mutant and vice versa. mps1 was immunoprecipitated from ipl1 and ipl1 - 321 cells (ipl1 - 321 shows no detectable kinase activity in vitro), whereas ipl1 was immunoprecipitated from mps1 and mps1-as1 cells. the kinase activity was measured in vitro with gst - fused dam1 as a substrate [26, 27 ] (supplemental results and discussion, note 8 ; figure s9). we did not detect any significant change in either kinase activity in cells in which the other kinase was mutated. these results are consistent with mps1 and ipl1 facilitating bi - orientation through similar mechanisms but in parallel pathways. however, it is still possible that one critically regulates the other but we can not detect this regulation. for example, one kinase may change the kinase activity of a small population of the other at kinetochores, in such a way that this small change is sufficient to promote bi - orientation. we have shown that inactivation of mps1 kinase leads to extensive defects in sister kinetochore bi - orientation on metaphase spindle in budding yeast. the role of mps1 in bi - orientation is not secondary to its function in spb duplication or in the spindle - assembly checkpoint (supplemental results and discussion, notes 9 and 10). mps1 facilitates bi - orientation in a tension - dependent mechanism and eliminates kinetochore - spindle pole connections that do not generate tension ; this function is similar to that of ipl1 (supplemental results and discussion, note 11 ; figure s10). this similarity would be explained if one kinase regulates the other ; however, we did not find such evidence. given that both kinases are required for bi - orientation, they may target different substrates at kinetochores (or mps1 may do so at spindle poles ; supplemental results and discussion, note 12) or different sites of the same substrates, to promote re - orientation of kinetochore - spindle pole connections. intriguingly, both mps1 and ipl1 phosphorylate the same dam1 protein (a kinetochore component in metaphase) at different sites [26, 27 ] ; at least ipl1-dependent phosphorylation is important for bi - orientation (supplemental results and discussion, note 13). it is also interesting whether the role of mps1 in promoting bi - orientation is evolutionarily conserved from yeast to humans (supplemental results and discussion, note 14). | summarysegregation of sister chromatids to opposite spindle poles during anaphase is dependent on the prior capture of sister kinetochores by microtubules extending from opposite spindle poles (bi - orientation). if sister kinetochores attach to microtubules from the same pole (syntelic attachment), the kinetochore - spindle pole connections must be re - oriented to be converted to proper bi - orientation [1, 2 ]. this re - orientation is facilitated by aurora b kinase (ipl1 in budding yeast), which eliminates kinetochore - spindle pole connections that do not generate tension [36 ]. mps1 is another evolutionarily conserved protein kinase, required for spindle - assembly checkpoint and, in some organisms, for duplication of microtubule - organizing centers [7 ]. separately from these functions, however, mps1 has an important role in chromosome segregation [8 ]. here we show that, in budding yeast, mps1 has a crucial role in establishing sister - kinetochore bi - orientation on the mitotic spindle. failure in bi - orientation with inactive mps1 is not due to a lack of kinetochore - spindle pole connections by microtubules, but due to a defect in properly orienting the connections. mps1 promotes re - orientation of kinetochore - spindle pole connections and eliminates those that do not generate tension between sister kinetochores. we did not find evidence that ipl1 regulates mps1 or vice versa ; therefore, they play similar, but possibly independent, roles in facilitating bi - orientation. |
aging is the greatest risk factor for neurodegenerative disorders in general, but specifically for alzheimer 's disease (ad). with the increasing life expectancy in developed countries, the incidence of ad, and consequently its socioeconomic impact, ad currently affects about 4.5 million americans, which costs the usa economy more than $ 100 billion each year. the number of ad patients is projected to increase to 1116 millions by 2050, with a cost exceeding $ 380 billion per year [1, 2 ]. to identify ad and monitor disease progression, neuropsychological tests such as the mini - mental state exam (mmse) and the cognitive subscale of the alzheimers disease assessment (adas cog) are currently the most commonly used strategies. however, these tests have several limitations, as follows. mmse is criticized by its marginal or absent assessment of some cognitive abilities that are affected early in the course of alzheimer 's disease or other dementing disorders (e.g., limited memory and verbal fluency items and no problem solving or judgment items), and its relative insensitivity to very mild cognitive decline, particularly in highly educated individuals. adas cog is limited by its relatively poor test - retest reliability, which likely reflects the influence of other factors on the patients ' performance (e.g., the patients ' mood). furthermore, these tests are not able to distinguish the risk for ad in preclinical groups (cognitively normal elderly adults) or predict the conversion to ad from preclinical and mild cognitive impairment (mci) groups. the national institute of aging (nia) has recently announced the revised clinical diagnostic criteria for ad dementia for the first time in 27 years (http://www.nih.gov/news/health/apr2011/nia-19.htm). instead of addressing the disease and describing only later stages when symptoms of dementia are already evident, the updated guidelines cover the full spectrum of the disease as it gradually changes over many years. they describe (i) the earliest preclinical stages of the disease, (ii) mci, and (iii) dementia due to alzheimer 's pathology. importantly, the guidelines now address the use of imaging and biomarkers in blood and spinal fluid that may help determine whether changes in the brain and those in body fluids are due to ad. in this paper, we will focus on the imaging biomarkers as addressed in the new criteria. specifically, we will discuss the surrogate biomarkers developed by the multimodal magnetic resonance imaging (mri) methods for identifying the risk for ad in normal cognitive (nc) adults ; brain anatomical and functional alterations in amnestic mci (amci) and ad patients. the high spatial resolution, sensitivity, and specificity of mri (e.g., resolution : 0.8 mm isotropic ; sensitivity : 8094% ; specificity : 60100%) have made it a powerful tool to identify structural alterations and brain atrophy using volumetric measurements of the entire brain [4, 5 ]. with advanced computer software, the neocortex of the brain on the mri scans can be automatically subdivided into 32 gyral - based region of interests (rois) per hemisphere, including gray matter (gm), white matter (wm), and hippocampus volumes [68 ]. gm loss can also be determined by measuring cortical gray matter thickness (gmt). gmt is determined by calculating the three - dimensional distance from the outer cortical surface to the inner cortical gm - wm boundary using cortical modeling from the high - resolution mri structural images (figure 1). wm integrity can be assessed with diffusion tensor imaging (dti). in brain tissues, highly structured tissue such as wm, this motion is highly anisotropic and dti provides directional information about it. loss in wm structure results in the loss in anisotropy, which can be easily detected by dti [9, 10 ]. functional - based mri can detect alterations and monitor disease progression related to brain metabolism, hemodynamics, and connectivity. functional connectivity mri (fcmri) has been developed as a technique to determine the resting state brain connectivity as measured by the basal blood oxygenation - level - dependent (bold) signal. in its simplest form, functionally connected networks can be identified using a seed - based correlational approach, in which the average resting state time series from a region of interest is correlated with all other voxels in the brain [2, 1114 ]. in contrast to this correlational method, independent component analysis (ica) is a more advanced multivariate analysis method that allows resting state fmri data to be decomposed into sets of independent, intrinsic brain networks [1517 ]. in either approach, each functional network 's neuronal activity is associated with a hemodynamic response, which consists of an increase in cerebral blood flow (cbf) and oxyhemoglobin and a relative decrease in deoxyhemoglobin. the changes in the oxyhemoglobin - deoxyhemoglobin ratio result in changes in bold signal. neuronal activity is tightly coupled with cbf (as mentioned above) ; therefore, another approach to assess the disease progression of ad is to measure cbf (in the units of ml/100 g / min). mr - based cbf measurements have been developed to investigate hemodynamic alteration in ad, including arterial spin labeling (asl) and dynamic contrast techniques. compared with the traditional cbf measurements using single - photon emission computed tomography (spect) with tc-99 m radioactive tracers [20, 21 ], the absence of ionizing radiation or injection and the ability to obtain high quality anatomical images within the same scanning session make mri - based cbf techniques attractive methods for the study of ad, especially when repeated scans are needed for monitoring disease progression or assessing treatment effect. changes in neuronal activity during the progression of ad may be associated with the changes in brain metabolism. using proton (h) mrs, numerous metabolites related to brain functions can be determined, including n - acetyl aspartate (naa), myoinositol (mi), creatine, choline, glutamate, glutamine and lactate. naa is present only within neural cell body, axons and dendrites, it is thus considered to be a marker of neuronal viability and function. mi, on the other hand, has considerably higher concentration in glial cells and thus is often taken as a glial marker. beyond age, family history is the most significant risk factor for ad, with maternal transmission being significantly more frequent than paternal transmission. biomarkers for ad - associated pathological changes, including metabolic deficits and amyloid beta (a) load, have been observed in cognitively normal individuals who have maternal history of late - onset ad (fhm) [25, 26 ]. structural mri has been used to assess brain volume changes for cognitively intact elderly individuals with fhm, a paternal history of ad (fhp) and no parental history of ad (fh-) [2729 ]. compared with fh individuals, cognitively healthy subjects with a family history of late - onset ad had significantly decreased gray matter volume (gmv) in the precuneus, middle frontal, and superior frontal gyri (figure 2 ;). fhm subjects had even significantly smaller inferior frontal, middle frontal, precuneus, and lingual gyri compared with fh and fhp individuals (figures 2 and 3) [27, 28 ]. another chief known genetic factor for ad is 4 allele apolipoprotein e gene (apoe 4). increasing age and carrying apoe 4 are well - established risk factors for ad. healthy older apoe 4 carriers, particularly 4 homozygotes, have demonstrated brain structure changes related to noncarriers. in a recent study with a longitudinal cohort of 1186 healthy elderly persons (6589 years), crivello. found that an annual rate of gray matter volume loss was seen in 4 homozygotes, whereas no age effect was seen in 4 heterozygotes and in noncarriers (figure 4(a)). similarly, 4 homozygotes had a significant larger rate of hippocampal volume loss than heterozygotes or noncarriers. in another anatomical study, filippini. observed white matter atrophy, including corpus callosum (cc) volume and all subregions, in both apoe 4 carriers and noncarriers. however, the slope has been steeper in the apoe 4 carriers compared with the noncarriers particularly in the prefrontal region (p = 0.02) (figure 4(b)). in addition to structural changes, apoe 4 has also shown great impact on brain function. memory is the first cognitive domain to be affected by ad, and impairments have been found in apoe 4 carriers relative to noncarriers [43, 44 ]. using functional mri (fmri), bookheimer. observed that during a memory task in a group of healthy subjects (aged 4782), apoe 4 carriers demonstrated significant increases in the left parahippocampal region, the left dorsal prefrontal cortex, the inferior - superior parietal lobes, and the anterior cingulate gyrus (figure 5 ;). in addition, the extent and the intensity of activation for the apoe 4 carriers were greater in the left inferior frontal region, the right prefrontal cortex, the transverse temporal gyri bilaterally, the left posterior temporal, and inferior parietal regions relative to the noncarriers (carriers of the apoe 3 allele). direct comparisons of apoe 4 carriers and noncarriers further demonstrated the greater extent and magnitude of activity in the left prefrontal, bilateral orbitofrontal, and superior temporal regions. in carriers of the apoe 4 allele, it has been demonstrated in inferior and superior parietal regions. in a younger group (mean age = 2130), apoe 4 carriers demonstrated increased task - induced brain activation in hippocampus relative to the noncarriers [45, 46 ]. overactivity of brain function has also been found in young apoe 4 carriers but disproportionately reduced with advancing age even before the onset of measurable memory impairment (figure 6 ;). in both age groups, a significant interaction has been found between age and apoe 4 status in the hippocampi, frontal pole, subcortical nuclei, middle temporal gyri, and cerebellum. these results have suggested that apoe genotype determines age - related changes in brain function, and greater activation reflects greater cognitive effort by apoe 4 carriers to obtain the same level of performance as the noncarriers, and/or reflect neuronal mechanism to compensate for processes, such as reduced synaptic plasticity, neuronal growth, or altered long - term potentiation in the carriers. apoe 4 carriers have also shown disrupted resting state brain activity in the absence of a or decreased csf in cognitively normal elderly (mean age = 62) using functional connectivity mri method [1113, 47 ]. similarly, young apoe 4 carriers (mean age = 2130), although had no difference in cognition and gm volume compared to their age - mated controls, showed increase in default mode network (involving medial temporal, medial prefrontal, and retrosplenial cortical areas) coactivation, suggesting that the function of these areas subject to the disease process in ad is modulated by apoe 4 allele at very early stage. taken together, these results provide evidence that influence of the genetic effect (familial and apoe 4 allele) on neurophysiological characteristics and the risk for ad can be detected using mri decades prior to any clinical or neuropathological expression of neurodegenerative process. mci is a diagnosis given to individuals who experience memory problems greater than normally expected with typical aging, but who do not show other symptoms of dementia, such as impaired judgment or reasoning. mci has various clinical subtypes, including amnestic single domain (amci - s), amnestic multiple domain (amci - m), nonamnestic single domain (namci - s), and nonamnestic multiple domain (namci - m). nonamnestic forms of mci (namci, i.e., namci - s and namci - m) have had findings suggestive of vascular disease, whereas amnestic forms of mci (amci, i.e., amci - s and amci - m) have appeared to have demographic, genetic, and mri findings suggestive of ad pathology [48, 49 ]. although amci can be defined using neuropsychiatric criteria, brain imaging studies have aimed to develop measures that are sensitive enough to distinguish amci from normal aging with high specificity. many other studies attempt to differentiate between amci subjects who will convert to ad, over a specific followup interval versus those who remain stable or ever recover. in this section, although age - related regional volume loss is apparent and widespread in nondemented individuals [5, 52 ], amci is associated with a unique pattern of structural vulnerability reflected in differential volume loss in specific regions. in a cross - sectional study, amci patients were observed with a significant wm abnormality in the region of crossing fibers in the centrum semiovale in comparison to nc (figure 7). in a ten - consecutive - year longitudinal study, 18 participants (among 138) who converted from normal to mci showed accelerated changes (compared to normal controls) on whole brain volume, ventricular csf (vcsf), temporal gray matter, and orbitofrontal and temporal association cortices, including the hippocampus (p 0.04) (figure 8). similar findings of vcsf increases in amci patients compared to normal controls have been reported by vemuri.. in this study, vemuri and colleagues further demonstrated that changes in serial structural mri differed by apoe 4 status overall among amci, with higher brain atrophy rates in apoe 4 carriers. in addition, mr - based structural biomarkers, compared with other biomarkers (e.g., csf), showed higher correlation with concurrent change on general cognitive and functional indices in impaired subjects. compared with healthy controls, amci patients had a regional pattern of brain disconnection between the posterior cingulate cortex (pcc) and the medial prefrontal cortex and the rest of the brain. these disconnections could be observed even in the absence of gm atrophy (figure 9). cognitively normal elderly subjects convert to ad at a rate of only 1 - 2% per year, whereas amci subjects convert to ad at a rate of 1215% per year. studying the similarities and differences between amci and ad would provide valuable information of the disease mechanism and progression. multimodal mri offers noninvasive methods for detection and possibly prediction of the conversion from amci to ad [68 ]. in a 3-year followup of 118 amci individuals who progressed to a diagnosis of ad, desikan. reported that atrophy in the medial temporal cortex (as measured by hippocampal volume, entorhinal cortex thickness, amygdala volume, temporal pole thickness, and parahippocampal gyrus thickness) can accurately and reliably predict time to disease progression [6, 7 ]. they demonstrated that amci individuals with significant atrophy of the medial temporal factor regions are three times as likely to progress to ad, compared with amci individuals with preserved medial temporal factor regions. their results also demonstrated that amygdala and temporal pole may be additional important structures for predicting the conversion from amci to ad. similar observations were found in a meta - analysis involving 40 studies of imaging data from 1351 patients, suggesting that atrophy in the (trans) entorhinal area and hippocampus most reliably predict the progression from amci to ad. these data provide strong evidence that ad - related volume losses are most readily detected in the medial temporal lobe in amci. the reduction in medial temporal lobe volume is therefore an important indicator in predicting the transition of amci to ad. mr - based asl techniques provide a functional biomarker (perfusion) to predict the progression from mri to ad. in a longitudinal study (2.7 1.0 years), chao. reported that the mci individuals who converted to dementia displayed hypoperfusion in the right precuneus, right inferior parietal cortex, and right middle frontal cortex. meta - analytic study (involving 1351 patients) in which hypoperfusion in the inferior parietal lobules was found to most reliably predict the progression from amci to ad. furthermore, baseline perfusion from the right precuneus predicted subsequent declines in clinical dementia rating sum of boxes, functional activates questionnaire and selective attention (stroop switching), and baseline perfusion from the right middle frontal cortex predicted subsequent episodic memory decline. these results suggest that hypoperfusion as detected by asl mri can predict progression from mci to ad. ad is histopathologically characterized by the formation of -amyloid (a) plaques and neurofibrillary tangles (nft). progressions of the a plaques and nft pathology of ad correlate closely with loss of neurons and synapses. these losses further result in gross atrophy, including cortical gray matter loss, reduced subcortical gray, and white matter volumes, as well as expanding ventricular and sulcal cerebrospinal fluid (csf) spaces [37, 38 ] (figure 10 ; similar regions of a/ntf deposition and brain atrophy in ad). in ad, this brain atrophy is localized to the medial temporal limbic cortex during its earliest states. at later stages of disease since memory impairment is the earliest symptom of ad, the temporal limbic cortex (including entorhinal cortex and hippocampus) has been an attractive target for structural neuroimaging studies [5862 ]. using brain volumetric measurements, patients with mild ad showed significantly smaller brain regions of hippocampus (25%) and entorhinal cortex (37%) than healthy elderly controls [5862 ]. in a number of longitudinal studies, significantly higher rates of brain atrophy were observed in ad [6366 ]. the global atrophy rate in normal aging typically increases from 0.3% to 0.5% per year at age 7080 but increases from 2% to 3% per year in ad [6769 ]. similar regional observations have also been found in hippocampus (controls, 1.0% to 1.7% per year ; ad, 3.0% to 5.9% per year) and in entorhinal cortex (controls, 1.4% to 2.9% per year ; ad, 7.15 to 8.4% per year) [2, 70, 71 ]. mr - based volume measures, particularly for the hippocampus, have been shown to be a strong structural biomarker for ad, as follows [7274 ]. first, it has been demonstrated that a significant correlation exists between mri and histological - based hippocampal volumes (r = 0.97, p 1.25 millimeters of cortical thickness [75, 76 ]. in a followup study 1.5 years later, patients with ad lost significant gm (p 98%) of energy production in mammals, yielding atp through oxidative phosphorylation of glucose. in the brain, oxidative metabolism (o2 consumption) is the predominant source of energy (atp generation), supporting baseline demands and maintaining viability, as well as responding rapidly and in a highly regional manner to changes in neuronal activity induced by task performance. failure to maintain adequate levels of tissue oxygenation rapidly results in tissue death as observed of brain atrophy in ad patients. to identify the integrity of the mitochondrial function, cerebral metabolic rate of glucose (cmrglc) and oxygen (cmro2) are the most - well known indicators. cmrglc measurements in ad research have been well established with positron emission tomography (pet) methods. for instance, significant decreases of glucose metabolism have been found in young apoe 4 carriers (30 years old) in brain areas associated with ad pathology. in contrast, cmro2 measurements are not feasible using pet methods, especially for ad patients, due to the difficulties of obtaining arterial blood samples. in addition, the radioactive nature of less repetitive scans, which limits the monitoring of the disease progress. therefore, considerable efforts have been made to develop mri - based, noninvasive, cmro2 measurements. baseline cmro2 and task - induced changes in cmro2 determinations have been proposed by several methods, including t2-relaxation - under - spin - tagging (trust) and quantitative bold (qbold) techniques [8993 ]. these mr - based metabolic imaging methods, in addition to mrs, are expected to be very useful as diagnostic and prognostic biomarkers in ad. however, future studies allowing for rigorous assessment of test - retest reliability and power calculation compared to the established imaging techniques are necessary before these cmro2 methods can be accepted as other complementary and/or established functional neuroimaging biomarkers for ad. the development and validation of structural and functional biomarkers will enable mri to be utilized as a powerful tool for evaluation of therapeutic efficacy in ad in large - scale clinical trials. for example, jack. estimated that in each arm of a therapeutic trail with conventional volumetric measures for hippocampal volume, only 21 subjects would be required to detect 50% reduction in the rate of decline. this compares 241 subjects if mmse scores were used ; 320 subjects if adas cog scores were used. combining with other biomarkers (e.g., cmrglc by pet and a/nft csf), surrogate markers for ad progress can be identified and used for clinical / cognitive tests in clinical trials. nonetheless, these surrogate markers must be validated to be reproducible in the treatment setting, across various types of treatments, across imaging centers, and across time. a multicenter ad research project, known as the alzheimer 's disease neuroimaging initiative (andi) (http://adni.loni.ucla.edu/), was launched in 2004 to meet this goal. the incidence of alzheimer 's disease is increasing with the extended lifespan in developed countries. the development of neuroimaging biomarkers is a pressing need to detect the early risk of ad (from nc), predict and monitor disease progression (from amci). multimodal mri methods have been developed to meet this demand by providing useful and important structural and functional biomarkers in ad. the validation of the surrogate biomarkers will have profound implications in ad clinical trials, including the prevention and deceleration of ad onset, as well as the evaluation of treatment efficacy. | multimodal magnetic resonance imaging (mri) techniques have been developed to noninvasively measure structural, metabolic, hemodynamic and functional changes of the brain. these advantages have made mri an important tool to investigate neurodegenerative disorders, including diagnosis, disease progression monitoring, and treatment efficacy evaluation. this paper discusses recent findings of the multimodal mri in the context of surrogate biomarkers for identifying the risk for ad in normal cognitive (nc) adults, brain anatomical and functional alterations in amnestic mild cognitive impairment (amci), and alzheimer 's disease (ad) patients. further developments of these techniques and the establishment of promising neuroimaging biomarkers will enhance our ability to diagnose amci and ad in their early stages and improve the assessment of therapeutic efficacy in these diseases in future clinical trials. |
the main method of atp production in the central nervous system is oxidative phosphorylation, which is carried out by billions of specialized organelles, mitochondria. mitochondria harvest the energy available in their transmembrane proton gradient to form atp, which is then exported from the mitochondria for use by energy demanding processes in the cell. the capacitance - reducing sheath surrounding axons can carry out the same atp - producing process. we assess the plausibility of this idea, after outlining both the known mechanism of atp synthesis in mitochondria and the proposed mechanism of atp synthesis in myelin. mitochondria are composed of two compartments : an inner mitochondrial matrix bounded by an inner membrane, and an intermembrane space between the inner and outer membranes (figure 1a). within the matrix, the citric acid cycle produces nadh and fadh2, which are passed to the respiratory chain in the inner membrane. chain complexes catalyze nadh and fadh2 oxidation and o2 reduction, and the energy released from these reactions is used to pump protons from the matrix to the intermembrane space. this makes the intermembrane space acidic (ph 6.9), leaving the matrix alkaline (ph 7.8) and negatively charged (200 mv). both the electrical and concentration gradients drive protons from the intermembrane space into the matrix. as they flow down this gradient through the fo segment of the atp synthase, the energy released is used to power adp phosphorylation by the f1 segment of the atp synthase. one atp molecule is generated for every 2.7 protons that enter the matrix this way. the atp is transported out of the mitochondrial matrix in exchange for a cytoplasmic adp, and an extra proton is cotransported into the mitochondrial matrix with a pi molecule (it is critical to keep the ratio of atp concentration to adp concentration in the mitochondrial matrix low, and the level of pi high, so that atp production rather than hydrolysis is favored). based on evidence from biochemical assays, western blot analysis, and immunocytochemistry, panfoli 's group have put forward the hypothesis that myelin is able to consume oxygen and produce atp through the operation of an atp synthase driven by a proton gradient across the membranes of the sheath. they suggest that mitochondrial fusion with the myelin membrane during formation of the sheath leads to ectopic expression of the respiratory chain complexes and f1fo - atp synthase in the myelin membranes. the respiratory complexes are proposed to pump protons into the cytoplasmic space of the myelin sheath, generating a proton motive force that powers the extracellular production of atp via the ectopically expressed atp synthase (with the f1 segment facing outward ; figure 1b). atp is then proposed to be passed from extracellular compartment to extracellular compartment, through a series of gap junctions, until it reaches the axon (although it is unclear how gap junctions which normally allow diffusion between adjacent intracellular compartments could mediate atp movement between the extracellular spaces of myelin, and unclear how atp would enter the axon). ravera suggest that atp generated in this way is a significant fraction of all the atp generated in the brain. they also argue that the multilamellar structure of myelin is due to a need for a large area for atp generation, rather than being due to a need to reduce axonal capacitance. one potential problem with this arrangement is the possibility that cytochrome c expressed on the inner surface of the plasma membrane could be released into the cytoplasm where it would trigger caspase - mediated apoptosis of the oligodendrocytes (reviewed in jiang and wang). this is not normally a danger for mitochondrial cytochrome c, because it is confined to the mitochondrial intermembrane space by the outer mitochondrial membrane. furthermore, whether axons, in fact, require metabolic support from ensheathing oligodendrocytes is debated. several lines of evidence suggest that myelinating oligodendrocytes provide energetic substrates, such as lactate, to the axons they ensheath, and that this trophic support is essential for axonal integrity. in contrast, harris and attwell have calculated that the glucose supply at nodes of ranvier and the density of axonal mitochondria could make the need for glial metabolic support of central nervous system axons unnecessary, although it is possible that peripheral nervous system axons, with significantly longer internodes, do require such support. although it has been suggested that mitochondria routinely contaminate purified myelin preparations, which would provide an alternative explanation for the presence of mitochondrial proteins in myelin, we can evaluate the theory put forward by ravera on the basis of the requirements it dictates. first, energetic substrates in particular, nadh and fadh2, the electron donors in oxidative phosphorylation must be provided extracellularly. their production (either by the citric acid cycle or glycolysis) must therefore be either extracellular between the sheets of myelin or intracellular with a mechanism for their export. second, there must be sufficient provision of adp and pi to the extracellular space. third, there must be sufficient clearing of atp from the extracellular space. whether any of these critical requirements are met in vivo is unknown. however, two features of the oligodendrocyte that are known its ph and membrane potential allow us to assess perhaps the most critical prerequisite for the hypothesis : that enough proton motive force can be produced across the myelin membrane to generate atp. this analysis is presented below in two stages : first, we assess whether protons will tend to move through the atp synthase in the correct direction to provide energy for atp synthesis, and then we calculate whether the energy thus produced is sufficient to generate atp. mitochondria are composed of two compartments : an inner mitochondrial matrix bounded by an inner membrane, and an intermembrane space between the inner and outer membranes (figure 1a). within the matrix, the citric acid cycle produces nadh and fadh2, which are passed to the respiratory chain in the inner membrane. chain complexes catalyze nadh and fadh2 oxidation and o2 reduction, and the energy released from these reactions is used to pump protons from the matrix to the intermembrane space. this makes the intermembrane space acidic (ph 6.9), leaving the matrix alkaline (ph 7.8) and negatively charged (200 mv). both the electrical and concentration gradients drive protons from the intermembrane space into the matrix. as they flow down this gradient through the fo segment of the atp synthase, the energy released is used to power adp phosphorylation by the f1 segment of the atp synthase. one atp molecule is generated for every 2.7 protons that enter the matrix this way. the atp is transported out of the mitochondrial matrix in exchange for a cytoplasmic adp, and an extra proton is cotransported into the mitochondrial matrix with a pi molecule (it is critical to keep the ratio of atp concentration to adp concentration in the mitochondrial matrix low, and the level of pi high, so that atp production rather than hydrolysis is favored). based on evidence from biochemical assays, western blot analysis, and immunocytochemistry, panfoli 's group have put forward the hypothesis that myelin is able to consume oxygen and produce atp through the operation of an atp synthase driven by a proton gradient across the membranes of the sheath. they suggest that mitochondrial fusion with the myelin membrane during formation of the sheath leads to ectopic expression of the respiratory chain complexes and f1fo - atp synthase in the myelin membranes. the respiratory complexes are proposed to pump protons into the cytoplasmic space of the myelin sheath, generating a proton motive force that powers the extracellular production of atp via the ectopically expressed atp synthase (with the f1 segment facing outward ; figure 1b). atp is then proposed to be passed from extracellular compartment to extracellular compartment, through a series of gap junctions, until it reaches the axon (although it is unclear how gap junctions which normally allow diffusion between adjacent intracellular compartments could mediate atp movement between the extracellular spaces of myelin, and unclear how atp would enter the axon). ravera suggest that atp generated in this way is a significant fraction of all the atp generated in the brain. they also argue that the multilamellar structure of myelin is due to a need for a large area for atp generation, rather than being due to a need to reduce axonal capacitance. one potential problem with this arrangement is the possibility that cytochrome c expressed on the inner surface of the plasma membrane could be released into the cytoplasm where it would trigger caspase - mediated apoptosis of the oligodendrocytes (reviewed in jiang and wang). this is not normally a danger for mitochondrial cytochrome c, because it is confined to the mitochondrial intermembrane space by the outer mitochondrial membrane. furthermore, whether axons, in fact, require metabolic support from ensheathing oligodendrocytes is debated. several lines of evidence suggest that myelinating oligodendrocytes provide energetic substrates, such as lactate, to the axons they ensheath, and that this trophic support is essential for axonal integrity. in contrast, harris and attwell have calculated that the glucose supply at nodes of ranvier and the density of axonal mitochondria could make the need for glial metabolic support of central nervous system axons unnecessary, although it is possible that peripheral nervous system axons, with significantly longer internodes, do require such support. although it has been suggested that mitochondria routinely contaminate purified myelin preparations, which would provide an alternative explanation for the presence of mitochondrial proteins in myelin, we can evaluate the theory put forward by ravera on the basis of the requirements it dictates. first, energetic substrates in particular, nadh and fadh2, the electron donors in oxidative phosphorylation must be provided extracellularly. their production (either by the citric acid cycle or glycolysis) must therefore be either extracellular between the sheets of myelin or intracellular with a mechanism for their export. second, there must be sufficient provision of adp and pi to the extracellular space. third, there must be sufficient clearing of atp from the extracellular space. whether any of these critical requirements are met in vivo however, two features of the oligodendrocyte that are known its ph and membrane potential allow us to assess perhaps the most critical prerequisite for the hypothesis : that enough proton motive force can be produced across the myelin membrane to generate atp. this analysis is presented below in two stages : first, we assess whether protons will tend to move through the atp synthase in the correct direction to provide energy for atp synthesis, and then we calculate whether the energy thus produced is sufficient to generate atp. for the f1fo - atp synthase to generate atp, protons must flow from a state of high potential energy on the side of the membrane expressing the fo segment to a state of low potential energy on the side of the membrane expressing the f1 segment (where atp is generated). both electrical and concentration gradients contribute to the potential energy for proton flow across the membrane. calculating the change in gibbs - free energy for proton flow (gp) tells us whether protons will spontaneously flow across the membrane in the direction that is required for atp generation. in the mitochondrion, the f1 segment of the atp synthase is in the matrix, so, to synthesize atp, protons must flow from the intermembrane space to the matrix. both electrical and chemical gradients across the inner membrane are large and in the right direction, resulting in a negative gp (table 1a). this indicates that protons will spontaneously flow across the membrane in the direction required for atp generation. in the oligodendrocyte, the f1 segment of the atp synthase is proposed to be in the extracellular space, so protons must flow from the intracellular to the extracellular space. with an intracellular ph of 7.0 (ref. 13) and a standard extracellular ph of 7.4, the concentration gradient is in the right direction. however, oligodendrocytes have a resting membrane potential of around 70 mv, implying that the electrical gradient is in the wrong direction. these combine to give a positive gp (table 1b ; the reversal potential for h is 24 mv, more positive than the resting potential), meaning that protons would not tend to flow across the membrane in the right direction (in fact, passively, they would flow in the opposite direction, into the cell) and can not provide energy for atp synthesis. in this scenario, the atp synthase would operate in reverse, hydrolyzing atp rather than phosphorylating adp, while h ions enter the cell. thus, if myelin expressed respiratory chain proteins in the manner suggested by morelli, atp would be broken down, not generated. ectopic expression of the f1fo - atp synthase with the f1 segment on the extracellular side of the membrane has been proposed for several cells (reviewed in champagne, panfoli, and chi and pizzo), often based on the observation that the f1 segment can be antibody - labeled without permeabilizing the membrane (for example, in rat hepatocytes). in myelin, however, it is theoretically possible that the atp synthase and complex proteins could face the other way, so that the f1 segment is expressed intracellularly rather than extracellularly. this arrangement would require a different account of how the proteins come to be expressed in the myelin membrane (it could not be by mitochondrial fusion), but perhaps eases some of the difficulties with the original theory, for example, substrate supply and the danger of cytochrome c - triggered apoptosis. most importantly, this arrangement would now require the flow of protons to be from the extacellular to the intracellular space, resulting in a negative gp and therefore a tendency for passive proton flow in the correct direction (table 1c). thus, for mitochondria, and for myelin with the f1 segment of the atp synthase oriented intracellularly, protons would tend to flow in the right direction for atp generation. but, for atp to actually be generated, the energy released by this proton flow must overcome the positive g required to phosphorylate adp (gatp), which is set by the ratio of substrate (adp and pi) to product (atp) present. knowing the cytoplasmic and matrix concentrations of adp, atp, and pi, we can evaluate gatp in mitochondria and myelin (tables 1a and 1c). for mitochondria, the energy provided by the proton motive force is sufficient to overcome the energy required for atp synthesis if > 1.93 protons per atp flow through the atp synthase. in fact, 8/3=2.7 protons enter per atp formed (since movement of 8 h is required to turn the fo part of the synthase through one rotation which generates 3 atp molecules). for the same proton flow (2.7 protons / atp) across the myelin membrane, on the other hand, even with the f1 segment of the atp synthase facing intracellularly so that protons would tend to passively flow in the right direction, the proton motive force is not large enough to provide sufficient energy for atp synthesis (since gatp+2.7 gp>0 ; table 1c). in this arrangement, as with the f1 segment facing extracellularly, therefore, the atp synthase would run backwards, hydrolyzing instead of generating atp and pumping h out of the cell. it would theoretically be possible for the atp synthase to generate atp using the same proton motive force if the energy provided by at least 14 protons entering the cytoplasm could be harvested for the synthesis reaction. however, this is far more than the 2.7 protons per atp molecule normally used and, even if not mechanistically impossible, indicates that powering atp synthesis by the proton motive force across the myelin membrane would, at the very least, be extremely inefficient. we have assessed the plausibility of an atp - generating function for myelin at the energetic level. it does not seem possible that enough energy could be provided by the electrochemical gradient for protons across the myelin membrane no matter which direction the atp synthase is oriented in to power the synthesis of atp. in fact, if the atp synthase were present in the myelin membrane, then it would function in reverse, hydrolyzing instead of synthesizing atp. this analysis is based on known values for the mature oligodendrocyte 's resting potential and ph. it is possible that these values, measured at the soma (for the resting potential) and in the absence of proper myelin wraps (for the ph), are drastically different from those of the in vivo sheath. it is not clear whether ravera envisage that atp is generated within the compact portions of the sheath (where the water content of the intracellular space is almost negligible and discussion of ion concentrations is of dubious significance) or within the inner and outer tongues or schmidt - lanterman incisures of the myelin (where pockets of cytoplasm are present). in the latter case, if such cytoplasmic pockets are much lower in ph and more depolarized than the oligodendrocyte cell body, it is theoretically possible that enough proton motive force could be generated across the myelin membrane to synthesize atp in the manner suggested by ravera. experimentally, however, evidence for this is completely lacking : voltage and ph differences between the intracellular cytoplasmic pockets and the extracellular spaces of myelin have not been measured (and there is no obvious reason, at least for the outer myelin tongue which is well connected to the soma, why such differences should exist). demonstrating an anomalously positive voltage and acid ph intracellularly in myelin would be essential for the hypothesis that myelin can carry out proton motive force - powered atp synthesis since, to be taken seriously, its most basic prerequisite a sufficient proton motive force must be met. we therefore suggest that previous data indicating the presence of the f1fo - atp synthase in myelin membranes reflect contamination of myelin preparations by mitochondrial membrane (possibly from mitochondria located in the schmidt - lanterman incisures or inner / outer myelin tongues) and nonspecificity of antibody labeling. furthermore, apparent extracellular synthesis of atp may, in fact, reflect regulated release of intracellular atp (reviewed in corriden and insel). although here we have focused on the hypothesis of atp production by myelin, the same line of reasoning is worth considering for similar theories of extracellular atp production in other cells, e.g., endothelial cells and hepatocytes. finally, it is possible that the f1fo - atp synthase is expressed ectopically in myelin, but not in an energy - producing capacity. in hepatocytes, for example, ectopic f1fo - atp synthase expression has a role in regulating the extracellular adp concentration by hydrolyzing but not by synthesizing atp. | it has been hypothesized that myelin acts like a mitochondrion, generating atp across the membranes of its sheath. by calculating the proton motive force across the myelin membrane based on known values for the ph and membrane potential of the oligodendrocyte, we find that insufficient energy could be harvested from proton flow across the myelin membrane to synthesize atp. in fact, if the respiratory chain were present in the myelin membrane, then the atp synthase would function in reverse, hydrolyzing rather than synthesizing atp. this calculation places the hypothesis of an energy - producing role for myelin in considerable doubt. |
the international type 1 diabetes genetics consortium (t1dgc) comprised groups of investigators from many countries throughout the world, with a common goal of identifying genes predisposing to type 1 diabetes. affected sibling pair (asp) families, as well as a large series of case - control collections for genetic studies. the t1dgc organized four recruitment networks (asia - pacific, europe, north america, and u.k.) for collection of data and samples (1,2), and, within these networks, laboratories were chosen for processing biological samples (3). three laboratories were selected to serve the t1dgc, initially to measure islet autoantigens from the baseline samples (4). these autoantigens were gad65 and the intracellular portion of protein tyrosine phosphatase (ia-2a). the t1dgc developed a masked split - pair duplicate sample program that allowed assessment of intra- and interassay reproducibility over time for each of the assays. these assessments included evaluation of different methods of computing results reported in world health organization (who) units per milliliter for sera yielding signals above the highest who standard. the results of the diabetes autoantibody standardization program (dasp) for the three laboratories have previously been described (4) for islet autoantibodies. the dasp workshops have provided insights for the improvement and standardization of autoantibody measurement associated with type 1 diabetes across numerous laboratories, and performance in dasp was used as a criterion for selecting the laboratories and for monitoring their performance. autoantibodies against gad and ia-2a were measured in samples from participants with type 1 diabetes from t1dgc asp families. although the measurement was not used as an entry criterion for participation in the study, quantifying results in standardized who units per milliliter allowed more detailed phenotyping and increased the research value of t1dgc study samples. with recognition of the clustering of multiple autoimmune diseases and overlap among genes contributing to multiple autoimmune diseases, additional organ - specific antibodies were measured in t1dgc samples. these additional assays were for antibodies to thyroid peroxidase (tpo) for hashimoto disease, to transglutaminase (tg) for celiac disease, to 21-hydroxylase (21-oh) for addison disease, and to h+/k+-atpase (atpase) for pernicious anemia (5). the t1dgc autoantibody workshop was designed to distribute data for analyses to discover genes associated with autoantibodies in participants with type 1 diabetes. this article describes the design of the t1dgc autoantibody workshop and the quality - control (qc) procedures to maintain and monitor the performance of each laboratory with respect to nonislet autoantibodies. the design of the t1dgc autoantibody workshop consisted of the t1dgc making data available to analytic teams to apply methods to detect genetic variants that contribute to the variation observed in autoantibody measurements. the primary data sources are 1) the clinical phenotypic data associated with the collected samples from participants with type 1 diabetes, 2) the genetic data from multiple t1dgc experiments, 3) previously generated islet autoantibody measurements, and 4) newly generated nonislet autoantibody data. an announcement was made to the research community of the t1dgc autoantibody workshop and availability of data. cleaned data sets were made available to the research teams (investigators interested in the same topic were encouraged to form teams) and results of the analyses presented at an in - person meeting. each of the first three sources of data (clinical phenotypes, genotypes, and existing islet autoantibodies) had undergone extensive qc procedures. details of the generation, qc, and analyses of the assembled genetic data can be found in previous reports (610). the methods for the autoantibody assays are described in greater detail in the articles in this supplement as well as in other summaries (4,5). measurements of the organ - specific autoantibodies (tpo, tg, 21-oh, and atpase) were performed in the t1dgc north american laboratory (barbara davis center for childhood diabetes, aurora, co), which was designated as the autoantibody workshop laboratory. this article describes the qc components for the nonislet autoantibody measurements used in the t1dgc autoantibody workshop. the details on tracking assay performance for the measurement of islet autoantibodies have previously been reported (4). measurements of nonislet autoantibodies were performed at a single t1dgc autoantibody workshop laboratory at the barbara davis center. similar to the qc assessment procedure for islet autoantibodies, a two - pronged qc system was implemented to assess nonislet autoantibodies. first, univariate analyses were conducted by the coordinating center (wake forest university) on the monthly data results from the t1dgc autoantibody workshop laboratory. based on these analyses, summary statistics (e.g., means, variances) and out - of - range values were obtained and, if necessary, investigated further. second, duplicate autoantibody measures were performed on a random sample of 5% of participants with type 1 diabetes. duplicate sera were collected and labeled with a separate, unique identifier and were sent to the laboratory in the normal sample shipments. these samples were often measured in the same assay and are representative of intra - assay variation. interassay variation was evaluated by a second split - duplicate protocol in which previously measured duplicate pairs were resubmitted to the laboratory. a total of 632 intra - assay split pairs were tested for each autoantibody evaluated in the t1dgc autoantibody workshop. reliability was assessed using intraclass correlations and the technical error measurement for autoantibody measures, in addition to graphical inspection of the data. the technical error is defined as the square root of the pooled between - measures variance as a percentage of the sample mean. the technical error was compared with the laboratory s internal coefficient of variation (cv). if there was evidence of high technical error, the laboratory was contacted and the cause determined. the results of split - duplicate determinations were expressed as antibody positive or negative (as defined within the laboratory) and as who units per milliliter or as indices, depending on the autoantibody. for the autoantibody workshop, autoantibody measurements were certified and provided by the t1dgc coordinating center. the available genetic data in the mhc on chromosome 6p21.3 were derived from the t1dgc mhc fine mapping project (11). these genetic data consisted of a dense single nucleotide polymorphism (snp) map (2 1,536 snp panels), a polymorphic microsatellite map (66 variable number tandem repeats), and classic hla typing to ensure comprehensive coverage of the 4-mb mhc core region (ensembl positions 29.533.5 mb). quality checks, including examination of plate - to - panel yields, snp performance, hardy - weinberg equilibrium expectations, mismatch error rates, mendelian error rates (within families), and allele distribution patterns, were conducted for all data. similar qc methods were implemented for candidate genes (t1dgc rapid response workshop) (12), genome - wide linkage analyses (68), and the immunochip analysis (conducted at the university of virginia). the dna samples used for these studies were derived from t1dgc asp families, representing a total of 9,992 individuals from 2,325 asp families ascertained from nine t1dgc recruitment cohorts (11). the families consisted primarily of nuclear families with an asp, often with two parents and an unaffected sibling. after exclusions for poor - quality dna, there were 9,979 samples and 339 qc samples sent for genotyping for all genetic marker sets. the details on tracking assay performance for the measurement of islet autoantibodies have previously been reported (4). measurements of nonislet autoantibodies were performed at a single t1dgc autoantibody workshop laboratory at the barbara davis center. similar to the qc assessment procedure for islet autoantibodies, a two - pronged qc system was implemented to assess nonislet autoantibodies. first, univariate analyses were conducted by the coordinating center (wake forest university) on the monthly data results from the t1dgc autoantibody workshop laboratory. based on these analyses, summary statistics (e.g., means, variances) and out - of - range values were obtained and, if necessary, investigated further. second, duplicate autoantibody measures were performed on a random sample of 5% of participants with type 1 diabetes. duplicate sera were collected and labeled with a separate, unique identifier and were sent to the laboratory in the normal sample shipments. these samples were often measured in the same assay and are representative of intra - assay variation. interassay variation was evaluated by a second split - duplicate protocol in which previously measured duplicate pairs were resubmitted to the laboratory. a total of 632 intra - assay split pairs were tested for each autoantibody evaluated in the t1dgc autoantibody workshop. reliability was assessed using intraclass correlations and the technical error measurement for autoantibody measures, in addition to graphical inspection of the data. the technical error is defined as the square root of the pooled between - measures variance as a percentage of the sample mean. the technical error was compared with the laboratory s internal coefficient of variation (cv). if there was evidence of high technical error, the laboratory was contacted and the cause determined. the results of split - duplicate determinations were expressed as antibody positive or negative (as defined within the laboratory) and as who units per milliliter or as indices, depending on the autoantibody. for the autoantibody workshop, autoantibody measurements were certified and provided by the t1dgc coordinating center. the available genetic data in the mhc on chromosome 6p21.3 were derived from the t1dgc mhc fine mapping project (11). these genetic data consisted of a dense single nucleotide polymorphism (snp) map (2 1,536 snp panels), a polymorphic microsatellite map (66 variable number tandem repeats), and classic hla typing to ensure comprehensive coverage of the 4-mb mhc core region (ensembl positions 29.533.5 mb). quality checks, including examination of plate - to - panel yields, snp performance, hardy - weinberg equilibrium expectations, mismatch error rates, mendelian error rates (within families), and allele distribution patterns, were conducted for all data. similar qc methods were implemented for candidate genes (t1dgc rapid response workshop) (12), genome - wide linkage analyses (68), and the immunochip analysis (conducted at the university of virginia). the dna samples used for these studies were derived from t1dgc asp families, representing a total of 9,992 individuals from 2,325 asp families ascertained from nine t1dgc recruitment cohorts (11). the families consisted primarily of nuclear families with an asp, often with two parents and an unaffected sibling. after exclusions for poor - quality dna, there were 9,979 samples and 339 qc samples sent for genotyping for all genetic marker sets. the data based on the islet autoimmune markers used in the t1dgc autoantibody workshop are provided elsewhere (4,10,11). for the autoantibody workshop, t1dgc recognized the opportunity to investigate genetic associations between type 1 diabetes and other autoimmune diseases for which autoantibodies could be measured. nonislet autoantibodies were measured in samples from participants with type 1 diabetes ; the nonislet antibodies are focused on the thyroid, adrenal, and parietal cell (gastric) systems. the results of qc that produced the analytic data for nonislet antibody intra - assay reproducibility are provided here. qc split - pair results for tpo, tg, 21-oh, and atpase autoantibodies tpo is an enzyme found in the thyroid gland that contributes to the production of thyroid hormones. the presence of tpo antibodies suggests the presence or development of autoimmune thyroid disease (hashimoto thyroiditis or graves disease). the internal cv at low tpo levels was 6.1% and at high levels was 8.7%. the mean (sd) difference between the pairs was 0.82 (10.92) who units / ml with 95.9% concordance in positive / negative calls within the split - pair samples. tg antibodies bind tg, a major thyroid - specific protein that is important in thyroid hormone synthesis, storage, and release. tg antibodies and tpo antibodies are used in concert to identify likely cases of autoimmune thyroiditis (hashimoto and graves disease). the technical error for samples was 0.01%, and the internal cv was 5.9%. a total of 632 intra - assay split pairs have been tested for tg. the mean (sd) difference between the pairs was 0.00 (0.02) with 98.7% concordance in positive / negative calls within the split - pair samples. the microsomal 21-oh (55 kd) has been shown to be the primary autoantigen associated with autoimmune addison disease. the 21-oh autoantibodies are markers of autoimmune destruction of the adrenal cortex. in the t1dgc autoantibody workshop, the values are expressed as an index, with a technical error for samples of 0.01%. the internal cv at low 21-oh autoantigen levels was 6.1%, and the internal cv at high levels was 8.7%. a total of 632 intra - assay split - pair samples were tested for 21-oh. the mean (sd) difference between the pairs was 0.00 (0.02) with 100% concordance in positive / negative calls within the split pairs. pernicious anemia is characterized by atrophic body gastritis leading to autoimmune chronic atrophic gastritis with destruction of gastric parietal cells and an inability to absorb vitamin b12. diagnosis of pernicious anemia includes the presence of parietal cell antibodies that bind to the atpase. for atpase autoantibodies in the t1dgc samples, every plate of samples in the assay had one well derived from a pool of samples that historically had the very high titers as well as a series of five historically low titer samples as negative controls. the mean of intra - assay cvs was used to represent the typical cv for this assay : 2.6% (high titers), 11.1% (controls), and 4.5% (midrange values). the mean (sd) difference between the pairs was 0.00 (0.03) with 97.9% concordance in positive / negative calls within the split pairs. the purpose of the t1dgc autoantibody workshop was to provide data sets for investigators to be able to determine the contribution of genes to variation in the presence of autoantibodies in a large collection of families enriched for type 1 diabetes (t1dgc asp families). the t1dgc asp families had previously been characterized with islet autoantibodies. given the co - occurrence of other organ - specific autoimmune diseases with type 1 diabetes, there was interest in determining the presence of nonislet autoantibodies in family members with type 1 diabetes. with the extensive genetic data for analysis, a critical component of the workshop was to provide the antibody phenotypes to the working groups with robust measurements and qc. measurements were made of nonislet autoantibodies focused on the thyroid (tpo and tg), adrenal (21-oh), and parietal cell (gastric atpase) systems in t1dgc participants with type 1 diabetes. we evaluated intra - assay precision, using masked sera, with a split - duplicate sample program. with more than 632 masked duplicate samples sent directly from the clinics and mixed with other samples, we determined the intra - assay percentage concordance of original positive / negative calls for selected autoantibodies. for the assays used in the t1dgc autoantibody workshop, there was high concordance in positive / negative calls within the duplicate split pairs, ensuring high - quality data for the genetic analyses of the samples. | the type 1 diabetes genetics consortium (t1dgc) comprised groups of investigators from many countries throughout the world, with a common goal of identifying genes predisposing to type 1 diabetes. the t1dgc ascertained and collected samples from families with two or more affected siblings with type 1 diabetes and generated a broad array of clinical, genetic, and immunologic data. the t1dgc autoantibody workshop was designed to distribute data for analyses to discover genes associated with autoantibodies in those with type 1 diabetes. in the t1dgc - affected sibling pair families, three t1dgc network laboratories measured antibodies to the islet autoantigens gad65 and the intracellular portion of protein tyrosine phosphatase (ia-2a). the availability of extensive genetic data provided an opportunity to investigate the associations between type 1 diabetes and other autoimmune diseases for which autoantibodies could be measured. measurements of additional nonislet autoantibodies, including thyroid peroxidase, tissue transglutaminase, 21-hydroxylase, and the potassium / hydrogen ion transporter h+/k+-atpase, were performed by the t1dgc laboratory at the barbara davis center for childhood diabetes, aurora, co. measurements of all autoantibodies were transmitted to the t1dgc coordinating center, and the data were made available to members of the t1dgc autoantibody working groups for analysis in conjunction with existing t1dgc genetic data. this article describes the design of the t1dgc autoantibody workshop and the quality - control procedures to maintain and monitor the performance of each laboratory and provides the quality - control results for the nonislet autoantibody measurements. |
under feeding conditions, dietary carbohydrates are digested and processed by various glucosidases in the digestive tract, and the resultant monosaccharides, mainly hexose glucose, are transported into various tissues as a primary fuel for atp generation. in most mammalian tissues, the catabolism of glucose into pyruvate, termed glycolysis, is preserved as a major pathway in eliciting atp. in tissues with abundant mitochondria, cytosolic pyruvate is transported into the mitochondrial matrix, converted to acetyl - coa by pyruvate dehydrogenase complex, and incorporated into the tricarboxylic acid cycle in conjunction with oxaloacetate. the cycle generates energy equivalent to atp (that is, gtp) as well as both nadh and fadh2, which serve as important electron carriers for electron transport chain - oxidative phosphorylation, resulting in the generation of atp. in some cases, such as red blood cells lacking mitochondria or cells under ischemic conditions, pyruvate is converted into lactate in the cytosol to regenerate the nad that is necessary for the continued generation of atp by substrate - level phosphorylation via anaerobic glycolysis. excessive carbohydrates in the liver are first converted into glycogen, a storage form of glucose in animals, by glycogenesis. in addition, in a carbohydrate - rich diet, the excessive carbohydrates are also converted into fatty acids via lipogenesis using the acetyl - coa generated from glycolysis - driven pyruvate, which is incorporated into very low density lipoproteins for transport to white adipose tissue for the storage. the regulation of glycogen metabolism is examined in detail in this section, and the transcriptional control of glycolysis and lipogenesis is delineated in the following section. under fasting conditions, the liver has a major role in generating glucose as a fuel for other tissues, such as the brain, red blood cells and muscles. initially, an increase in the pancreatic hormone glucagon initiates the cascade of kinase action (stated below in detail) that releases glucose from the stored glycogen via glycogenolysis. normally, stored glycogen is critical for maintaining glucose homeostasis in mammals during an overnight fasting period. during a longer term fast or starvation, essentially all of the stored glycogen in the liver is depleted (after ~30 h of fasting), and de novo glucose synthesis or gluconeogenesis is responsible for the generation of glucose as a fuel for other tissues. major non - carbohydrate precursors for gluconeogenesis are lactate, which is transported from peripheral tissues such as skeletal muscles or red blood cells, and glycerol, which is released from the adipose tissues via enhanced lipolysis during fasting. most of the initial precursors for gluconeogenesis are generated in the mitochondria (except glycerol 3-phosphate via glycerol kinase activity), but the majority of the reaction occurs in the cytosolic part of the cell. the complex regulatory mechanism is delineated in detail in the following section, with an emphasis on the transcriptional control of key regulatory enzyme genes. the accumulation of glycogen in the liver during feeding conditions provides a storage form of glucose that can be used in times of reduced food intake (figure 1). multiple layers of regulation are required for this process for both the activation of glycogen synthase, which is a key enzyme of glycogenesis (glycogen synthesis), and the inhibition of glycogen phosphorylase, which is a key enzyme of glycogenolysis (glycogen breakdown) in the liver. glycogen synthase is a major enzyme that facilitates the elongation of glycogen chains by catalyzing the transfer of the glucose residue of udp - glucose to the non - reducing end of a pre - existing glycogen branch to produce a new 14 glycosidic linkage. the regulation of glycogen synthase has been mostly studied using a muscle - specific isoform. in the muscle, glycogen synthase is inactivated via phosphorylation on multiple serine residues by various serine / threonine kinases such as casein kinase-1, protein kinase a (pka), and glycogen synthase kinase-3 (gsk-3). most notably, the phosphorylation of glycogen synthase by gsk-3 at serine residues 640, 644 and 648 has been linked to the most important inhibitory post - translational modification for its catalytic activity. under fasting conditions, dephosphorylated and active gsk-3 phosphorylate and inactivate glycogen synthase, leading to the inhibition of hepatic glycogen synthesis. on feeding, increased insulin signaling activates akt in the cell, which in turn phosphorylates and inactivates gsk-3, thus resulting in the activation of glycogen synthase. in addition, increased concentrations of glucose 6-phosphate allosterically activate this enzyme, thus potentiating its catalytic activity under feeding conditions. one recent publication argues against the role of gsk-3 in the regulation of the liver - specific isoform of glycogen synthase. in that study, guinovart. mutated the corresponding serine residues that are shown to be regulated by gsk-3 in the liver - isoform of glycogen synthase. they found that the mutation of those residues did not affect the overall enzyme activity but that the mutation of serine 7 to alanine, a site that is recognized and regulated by pka, led to the increased activity of this enzyme. further study is necessary to determine whether these results can be verified in vivo using animal models such as liver - specific knock - in mice for s7a liver glycogen synthase. the protein phosphatase 1 (pp1) may be responsible for the dephosphorylation and activation of glycogen synthase. accordingly, both glucose and insulin have been shown to activate pp1 activity, whereas glucagon and epinephrine have been linked to the inhibition of its activity. this enzyme catalyzes the reaction of the removal of a glucose residue from the non - reducing end of a glycogen chain, leading to the generation of glucose 1-phosphate. glucose 1-phosphate can be converted into glucose 6-phosphate by phosphoglucomutase, and glucose 6-phosphate can be incorporated into glycolysis or further converted into glucose by glucose 6-phosphatase, depending on the energy status of the organism. the phosphorylation of glycogen phosphorylase requires a cascade mechanism of epinephrine and glucagon in the liver. on the activation of gs by the binding of hormones to cell surface g protein - coupled receptors (beta adrenergic receptor or glucagon receptor), the intracellular cyclic amp (camp) levels increase via adenylate cyclase, leading to the activation of pka. pka is then responsible for the phosphorylation and activation of glycogen phosphorylase kinase, which in turn phosphorylates and activates glycogen phosphorylase to enhance glycogen breakdown. under feeding conditions, this kinase cascade is inactive due to the lack of secretion of catabolic hormones. in addition in essence, the anabolic hormone insulin promotes glycogenesis and inhibits glycogenolysis via the activation of pp1, leading to the dephosphorylation of glycogen phosphorylase (inactivation) and glycogen synthase (activation), and via the activation of akt, leading to the phosphorylation of gsk-3 (inactivation) that is unable to phosphorylate and inactivate glycogen synthase. as stated above, glycolysis is critical to the catabolism of glucose in most cells to generate energy. the key rate - limiting enzymes for this pathway include glucokinase (gk, also termed hexokinase iv), which converts glucose into glucose 6-phosphate ; phosphofructokinase-1 (pfk-1), which converts fructose 6-bisphosphate into fructose 1,6-bisphosphate ; and liver - type pyruvate kinase (l - pk), which converts phosphoenolpyruvate (pep) into pyruvate in the liver. these enzymes are tightly regulated by allosteric mediators that generally promote the catabolism of glucose in the cell. gk is a high km hexokinase that is present in the liver and the pancreatic beta cells, thus functioning as a glucose sensor for each cell type. unlike the other hexokinase isotypes, gk activity is not allosterically inhibited by its catalytic product, glucose 6-phosphate in the cell, thus enabling the liver to continuously utilize glucose for glycolysis during conditions of increased glucose availability, such as during feeding conditions. gk is regulated via its interaction with glucokinase regulatory protein (gkrp). in the low intracellular glucose concentration during fasting, the binding of gk and gkrp is enhanced by fructose 6-phosphate, leading to the nuclear localization of this protein complex. higher concentrations of glucose during feeding compete with fructose 6-phosphate to bind this complex, which promotes the cytosolic localization of gk that is released from gkrp, thus causing the increased production of glucose 6-phosphate in this setting. this enzyme activity is allosterically inhibited by atp and citrate, which generally indicate a sign of energy abundance. reciprocally, it is allosterically activated by adp or amp, making it more efficient to bring about glycolysis to produce more atp in the cell. in addition, pfk-1 activity is allosterically activated by fructose 2,6-bisphosphate (f26bp), a non - glycolytic metabolite that is critical for the regulation of glucose metabolism in the liver. f26bp is generated from fructose 6-phosphate by the kinase portion of a bifunctional enzyme that contains both a kinase domain (phosphofructokinase-2, pfk-2) and a phosphatase domain (fructose 2,6-bisphosphatase, f-2,6-pase). pfk-2 is activated by the insulin - dependent dephosphorylation of a bifunctional enzyme that activates pfk-2 activity and simultaneously inhibits f-2,6-pase activity to promote the increased f26bp concentration. glucagon - mediated activation of pka is shown to be responsible for the phosphorylation and inactivation of the kinase portion of this enzyme. unlike its muscle counterpart, l - pk is also a critical regulatory step in the control of glycolysis in the liver. as in the case of other glycolytic enzymes, l - pk activity is regulated by both allosteric mediators and post - translational modifications. l - pk activity is allosterically activated by fructose 1,6-bisphosphate, an indicator for the active glycolysis. by contrast, its activity is allosterically inhibited by atp, acetyl - coa, and long - chain fatty acids, all of which signal an abundant energy supply. additionally, the amino acid alanine inhibits its activity, as it can be readily converted to pyruvate by a transamination reaction. l - pk is inhibited by pka following a glucagon - mediated increase in intracellular camp during fasting and is activated by insulin - mediated dephosphorylation under feeding conditions. in addition to the acute regulation of key regulatory enzymes, glycolysis is regulated by a transcriptional mechanism that is activated during feeding conditions. two major transcription factors, sterol regulatory element binding protein 1c (srebp-1c) and carbohydrate response element binding protein (chrebp), are responsible for the transcriptional activation of not only glycolytic enzyme genes but also the genes involved in fatty acid biosynthesis (such as fatty acid synthase (fas), acetyl - coa carboxylase (acc), and stearoyl - coa desaturase 1 (scd1)) and triacylglycerol formation (such as glycerol 3-phosphate acyltransferase (gpat) and diacylglycerol acyltransferase 2 (dgat2)), a process that is normally activated by a carbohydrate - rich diet (figure 2). because these processes are often coordinately regulated, that is activated during feeding and inhibited by fasting, they are sometimes collectively called lipogenesis. they are members of the basic helix - loop - helix leucine zipper (b / hlh / lz) type transcription factor families comprising srebp-1a, srebp-1c, and srebp-2. srebp is translated as an endoplasmic reticulum (er)-bound precursor form that contains the n - terminal transcription factor domain and the c - terminal regulatory domain linked with the central transmembrane domain. within this family of transcription factors, srebp-2 is linked to the control of cholesterol uptake or biosynthesis in the liver by the transcriptional activation of the genes involved in the pathway including low density lipoprotein receptor (ldlr), 3-hydroxy-3-methylglutaryl - coenzyme a reductase (hmgcr), hydroxy-3-methylglutaryl - coenzyme a synthase 1 (hmgcs1), and farnesyl diphosphate synthase (fdps). srebp-1c, however, activates the genes encoding the enzymes for lipogenesis (fas, acc, scd1, and dgat2) as well as gk, which is a first enzyme in the commitment step of glucose utilization in the liver. indeed, liver - specific srebp-1c knockout mice showed an impaired activation of lipogenic genes in a high carbohydrate diet, thus confirming the importance of this transcription factor in the regulation of hepatic glycolysis and fatty acid biosynthesis. srebp-1a is not highly expressed in the liver but was shown to be involved in the formation of inflammasomes in response to lipopolysaccharide (lps) treatment in macrophages by transcriptional activation of nlrp1. the expression of srebp-2 is not controlled by sterols, but its proteolytic processing is tightly regulated by intracellular concentrations of cholesterol. it is normally bound in the er via the interaction of srebp - cleavage - activating protein (scap) and insulin - induced gene protein (insig) in the presence of high intracellular cholesterol levels, and the reduction in the cholesterol concentration releases the interaction of scap and srebp-2/insig complex, resulting in the translocation of the latter complex into the golgi apparatus and the liberation of the active srebp-2 factor by sequential proteolytic cleavages. unlike srebp-2, srebp-1c is mainly regulated at the transcription level by insulin. the exact transcription factor that mediates this insulin - dependent signal is not yet clear, although srebp-1c itself might be involved in the process as part of an auto - regulatory loop. interestingly, the oxysterol - sensing transcription factor liver x receptor (lxr) is shown to control the transcription of srebp-1c, suggesting that srebp-1c and srebp-2 could be regulated differently in response to cellular cholesterol levels. recent studies have revealed the involvement of various kinases in the control of srebp-1c activity. in hepg2 cells, pka was shown to reduce the dna binding ability of srebp-1a by the phosphorylation of serine 338 (equivalent of serine 265 for srebp-1c). a report by bengoechea and ericsson suggested that gsk-3, a kinase known to reduce glycogen synthesis by targeting glycogen synthase, downregulates srebp-1 activity via the phosphorylation of the c - terminal residue that promotes the ubiquitin ligase fbw7-dependent degradation of srebp-1 proteins. in addition, both amp activated protein kinase (ampk) and its related kinase salt - inducible kinase (sik) 1 are involved in the down - regulation of its activity through inhibitory phosphorylation (serine 372 for ampk, which blocks proteolysis and nuclear localization of srebp-1c, and serine 329 for sik1, which directly reduces its transcriptional activity). these data suggest that the fine - tuning of srebp-1c activity is critical to the maintenance of glucose and lipid homeostasis in the liver. the other prominent transcription factor for controlling glycolysis and fatty acid biosynthesis in the liver is chrebp. chrebp was initially known as williams - beuren syndrome critical region 14 (wbscr14) and was considered one of the potential genes that instigate williams - beuren syndrome. later, by using a carbohydrate response element (chore) from l - pk, chrebp was isolated as a bona fide transcription factor for binding chore of glycolytic promoters. indeed, chrebp is highly expressed in tissues that are active in lipogenesis such as the liver, brown adipocytes, white adipocytes, small intestine, and kidney. as in the case for srebp, chrebp belongs to the b / hlh / lz transcription factor family and forms a heterodimer with another b / hlh / lz transcription factor max - like protein x (mlx) on the glycolytic promoter. as in the case for the srebp-1c, the expression of chrebp is increased in the liver as a result of a high carbohydrate diet, and the effect was recapitulated in primary hepatocytes with high glucose treatment. a recent report indeed suggested a role for lxr in the transcriptional activation of chrebp in response to glucose, although the study needs to be further verified because the transcriptional response is shown not only by the treatment of d - glucose, a natural form of glucose present in animals, but also by the treatment of unnatural l - glucose, a form of glucose that is not known to activate lipogenesis in the liver. moreover, studies performed in lxr knockout mice revealed no changes in chrebp expression in the liver, arguing against the role of lxr in the control of chrebp. there are three prominent serine / threonine residues that are targeted by serine / threonine kinases. pka is shown to phosphorylate serine 196, which is critical for cellular localization, and threonine 666, which is critical for its dna binding ability, whereas ampk phosphorylate serine 568 dictates its dna binding ability. all three sites are phosphorylated under fasting conditions by these kinases and are dephosphorylated under feeding by xylulose 5-phosphate (x5p)-mediated activity of protein phosphatase 2a (pp2a). however, the current model needs to be further verified due to the contrasting data that have been published regarding the role of these phosphorylations on chrebp activity. first, high glucose concentrations in primary hepatocytes do not result in decreased camp levels or pka activity, suggesting that other signals might be necessary to mediate the high glucose - dependent nuclear translocation of chrebp. in addition, a serine to alanine mutant of chrebp still requires high glucose for its full activity, suggesting that additional actions are necessary to recapitulate the high glucose - mediated activation / nuclear localization of chrebp in the liver. the physiological role of chrebp in liver glucose metabolism was verified by in vivo studies. the knockout animals showed reduced liver triacylglycerol levels together with a reduction in lipogenic gene expression, thus confirming the role of chrebp in the control of hepatic glycolysis and fatty acid synthesis. interestingly, the compensatory increase in glycogen was observed in the livers of these mice, suggesting that these mice adapted to store more glycogen as a storage form of fuel as opposed to triacylglycerol. in ob / ob mouse liver, increased accumulation of nuclear chrebp was shown, suggesting that this phenomenon might be causal to the fatty liver phenotype in these mice. indeed, knockdown of chrebp in ob / ob mice reduced the rate of lipogenesis with decreased expression of most lipogenic genes. furthermore, the depletion of hepatic chrebp in ob / ob mice improved hyperglycemia, hyperlipidemia, and hyperinsulinemia, suggesting that regulation of chrebp might be critical in the control of metabolic disorders in the presence of obesity and insulin resistance. prolonged fasting or starvation induces de novo glucose synthesis from non - carbohydrate precursors, termed hepatic gluconeogenesis. this process initiates from the conversion of pyruvate to oxaloacetate by pyruvate carboxylase (pc) in the mitochondria and eventually concludes in the conversion into glucose via several enzymatic processes in the cytosol. among the substrates for gluconeogenesis are amino acids, which can be converted into either pyruvate or intermediates of the tricarboxylic acid cycle ; lactate, which can be converted into pyruvate by lactate dehydrogenase ; and glycerol (from increased lipolysis in the white adipocytes under fasting), which can be converted into dihydroxyacetone phosphate, a gluconeogenic intermediate (a two - step process that is catalyzed by glycerol kinase and glycerol 3-phosphate dehydrogenase). key regulatory enzymes in that pathway, including glucose 6-phosphatase (g6pase), fructose 1,6-bisphosphatase (fbpase1), pc, and phosphoenolpyruvate carboxykinase (pepck), are activated under fasting conditions to enhance gluconeogenic flux in that setting. mitochondrial acetyl - coa (derived from the increased fatty acid oxidation under fasting) functions as a key allosteric activator of pc, leading to the increased production of oxaloacetate for the gluconeogenesis. in addition, f26bp, which is a key allosteric regulator for glycolysis by activating pfk-1, was shown to inhibit gluconeogenesis via the allosteric inhibition of fbpase1, which helps reciprocally control gluconeogenesis and glycolysis under different dietary statuses. because fbpase2 is activated but pfk-2 is inhibited under fasting, the lack of f26bp enables the activation of fbpase1 and the increased production of fructose 6-phosphate in gluconeogenesis. major transcriptional factors that are shown to induce gluconeogenic genes include creb, foxo1, and several nuclear receptors (figure 3). under fasting conditions, glucagon and epinephrine can increase the camp concentration in the liver via the activation of adenylate cyclase, leading to the activation of pka and the subsequent induction of creb via its serine 133 phosphorylation. the phosphorylation event is crucial in the recruitment of histone acetyltransferases (hat) cbp / p300, leading to the histone h3 and h4 acetylation and the transcriptional activation of target genes. creb - dependent transcription is further enhanced by association with additional transcriptional coactivators creb regulated transcription coactivators (crtcs), which are a target for cbp / p300-mediated acetylation, which in turn promotes a tighter association of creb, cbp / p300, and crtc on the promoter. the role of creb in the control of hepatic gluconeogenesis has been confirmed by in vivo studies by utilizing albumin promoter - driven acreb (creb inhibitor) transgenic mice and sirna - mediated creb knockdown mouse models. in both mouse models, the inactivation of creb reduced blood glucose levels and reduced the expression of gluconeogenic genes in mice, showing that creb is a bona fide physiological transcriptional regulator of hepatic gluconeogenesis in vivo. disruption of creb - cbp interaction does not appear to affect glucose homeostasis because mice exhibiting a stable expression of mutant cbp that was unable to bind creb showed normal glycemia. furthermore, mutant mice producing ch1 null products (ch1-a domain that is critical for insulin - mediated depression of cbp activity) displayed normal fasting gluconeogenesis. thus, further studies are required to describe the potential role of hats in the transcriptional control of creb activity in this setting. the crtc family of transcriptional coactivators consists of crtc1, crtc2 and crtc3, which were isolated by the expression library screening as activaters of creb - dependent transcription. crtc activity is regulated by cellular localization, and the ampk family of serine / threonine kinases, such as ampk, sik1 or sik2, was shown to be involved in the inhibitory phosphorylation of this factor (serine 171 for crtc2). in addition, the phosphorylation status of crtc is regulated by a pair of serine / threonine phosphatases (pp2b or pp4) in response to camp signaling or calcium concentration in the cell. crtc activity is also further enhanced by o - glcnacylation on serine 171 and arginine methylation by protein arginine methyltransferase (prmt) 6. among the family members, recent studies have delineated the role of crtc2 in the regulation of hepatic gluconeogenesis in vivo. knockdown of crtc2 in mice by rnai reduced blood glucose levels and led to a concomitant repression of gluconeogenic gene expression. in addition, crtc2 knockout mice displayed lower plasma glucose levels and improved glucose tolerance, indeed showing that crtc2 is crucial in controlling hepatic glucose metabolism in vivo. a recent study indicated that crtc2 could also coactivate other bzip transcription factors that are implicated in the regulation of glucose homeostasis. further study is required to delineate the potential contributions from other bzip factors in the control of hepatic gluconeogenesis by using tissue - specific knockout mouse models. the forkhead box o (foxos) belongs to a class of forkhead families of transcription, which recognize the at - rich insulin response element on the promoter. of the four major isoforms in mammals (foxo1, foxo3, foxo4, and foxo6), foxo1 is the predominant isoform in the liver. the activity of this protein is also regulated by phosphorylation - dependent subcellular localization, and three major serine and threonine residues (threonine 24, serine 253 and serine 316 for murine foxo1) are targeted by the insulin / akt pathway. following phosphorylation, foxo1 moves to the cytosol via an association with 14 - 3 - 3, where it is degraded by the ubiquitin / proteasome - dependent pathway. in addition to phosphorylation, foxo1 was shown to be regulated by the hat - dependent acetylation of specific lysine residues (lysine 242, 245 and 262 for murine foxo1), which also inhibit its transcriptional activity. in the liver, foxo1 regulates hepatic gluconeogenesis via the transcriptional regulation of key genes in the pathway such as pepck and g6pase and is considered a major regulatory point for the insulin - mediated repression of hepatic gluconeogenesis. indeed, mice with liver - specific knockout of foxo1 showed lower plasma glucose levels that those associated with reduced hepatic glucose output, thus underscoring the physiological significance of this factor in the control of glucose homeostasis in vivo. as in the case for creb, foxo1 requires transcriptional coactivators for optimal transcriptional activity. peroxisome proliferator - activated receptor gamma coactivator 1 alpha (pgc-1), a known coactivator for nuclear receptors, functions as a key transcriptional coactivator for foxo1 in hepatic gluconeogenesis. pgc-1 was originally isolated in brown adipocytes and was shown to control adaptive thermogenesis in response to cold shock in that setting. in the liver, the expression of pgc-1 is upregulated under fasting conditions via a crtc2-creb - dependent mechanism and is critical in maintaining prolonged gluconeogenesis under starvation by enhancing the transcriptional activity of foxo1 as a coactivator. indeed, the depletion of hepatic pgc-1 in mice results in lower fasting glucose levels with a concomitant reduction in hepatic gluconeogenesis, thus showing the physiological relevance of this coactivator in the control of glucose homeostasis. as is the case for crtc2, foxo1 activity is enhanced by arginine methylation by prmt. in this case, prmt1 promotes the asymmetric dimethylation of arginine 248 and 250 in foxo1, which blocks the binding of akt and the subsequent akt - mediated phosphorylation of the adjacent serine residue (serine 253), thus enhancing the nuclear localization of foxo1. consequently, the chromatin occupancy of foxo1 onto the gluconeogenic promoter and gluconeogenesis itself are increased due to the prmt1-dependent arginine methylation. acute knockdown of hepatic prmt1 in mice reduces foxo1-mediated glucose production, confirming that prmt1 is crucial in modulating foxo1 activity and subsequent gluconeogenesis in the physiological context. nuclear receptors belong to the superfamily of transcription factors that possess two cys2-his2 type zinc finger motifs as a dna binding domain as well as both ligand - independent and ligand - dependent transactivation domains. nuclear receptors can be classified into one of three subgroups based on their dimer - forming potential. homodimeric nuclear receptors are also called cytosolic receptors because they reside in the cytosol and associate with molecular chaperones such as heat - shock proteins. on binding to the ligand, they form homodimers and translocate to the nucleus to bind a specific response element termed the hormone response element to elicit the ligand - dependent transcriptional response. most of the steroid hormone receptors, such as the glucocorticoid receptor (gr), estrogen receptor (er), and progesterone receptor (pr), belong to this subfamily. by contrast, heterodimeric nuclear receptors reside in the nucleus and are bound to their cognate binding sites together with the universal binding partner retinoid x receptor (rxr). in the absence of the ligands, these factors repress the transcription of target genes in association with transcriptional corepressors such as histone deacetylase or nuclear receptor corepressor (ncor)/silencing mediator of retinoid and thyroid hormone receptors (smrt). ligand binding initiates the conformational changes of these heterodimeric nuclear receptors, which promotes the dissociation of corepressors and the association of coactivators such as cbp / p300, p160 steroid receptor coactivator family, and pgc-1. examples of this class of nuclear receptors include members of peroxisome proliferator - activated receptors, lxrs, vitamin d receptors and thyroid hormone receptors. some of them also lack dna binding domain and thus function as transcriptional repressors of various transcription factors, including members of nuclear receptors. gr is activated by cortisol, which is released from the adrenal cortex in response to chronic stresses such as prolonged fasting. gr was shown to directly bind to the cognate binding sites found in the promoters of gluconeogenic genes such as pepck and g6pase and to enhance transcription of these genes under fasting conditions. the same response elements were also shown to be recognized and regulated by hepatocyte nuclear factor 4 (hnf4), a member of heterodimeric nuclear receptors, which suggests that these nuclear receptors could coordinately function to control hepatic gluconeogenesis in response to fasting. in accordance with this idea, the activity of these nuclear receptors can be effectively integrated by the function of transcriptional co - activator pgc-1. recently, estrogen - related receptor gamma (err), a member of monomeric nuclear receptors, was shown to be involved in the regulation of hepatic gluconeogenesis. in the liver, err expression is increased under fasting or by insulin resistance in a crtc2-creb - dependent manner. this factor regulates hepatic gluconeogenesis by binding to unique response elements that are distinct from the known nuclear receptor - binding sites in the promoters of pepck and g6pase. inhibition of err activity by injecting either rnai or the inverse agonist gsk5182 effectively reduced hyperglycemia in diabetic mice, suggesting that the control of this factor might potentially be beneficial in the treatment of patients with metabolic diseases. as is the case for other nuclear receptors that control hepatic gluconeogenesis, err activity is further enhanced by interaction with the transcriptional coactivator pgc-1, showing that this coactivator functions as a master regulator for the hepatic glucose metabolism. three members of atypical orphan nuclear receptors, the small heterodimer partner (shp, also known as nr0b2) ; the dosage - sensitive sex reversal, adrenal hypoplasia critical region, on chromosome x (dax-1, also known as nr0b1) ; and the shp - interacting leucine zipper protein (smile) are implicated in the transcriptional repression of hepatic gluconeogenesis. shp is ubiquitously expressed in mammalian tissues, with the highest expression occurring in the liver. interestingly, metformin directly activates the transcription of shp via an ampk - mediated pathway. shp directly inhibits camp - dependent transcription by binding to creb, resulting in the reduced association of creb with crtc2. the adenovirus - mediated overexpression of shp could effectively reduce blood glucose levels in diabetic mice, thus showing the importance of this pathway in the control of hepatic glucose metabolism. these results provide a dual mechanism for a metformin - ampk dependent pathway to inhibit hepatic gluconeogenesis at the transcriptional level ; an acute regulation of crtc2 phosphorylation to inhibit the crtc2-creb - dependent transcriptional circuit ; and a longer - term regulation of gluconeogenic transcription by enhanced shp expression. both dax-1 and smile were shown to repress hepatic gluconeogenesis by inhibiting hnf4-dependent transcriptional events. sik1, a member of the ampk - related kinases, was shown to enhance dax-1 expression in the liver, whereas akt was shown to activate the transcription of smile to target the hnf4 pathway under feeding conditions. interestingly, smile was shown to directly replace pgc-1 from hnf4 and the gluconeogenic promoters, suggesting that this factor could potentially function as a major transcriptional repressor of hepatic gluconeogenesis in response to insulin signaling. further study is necessary to fully understand the relative contribution of these nuclear receptors in the control of glucose homeostasis in both physiological conditions and pathological settings. in this review, we attempted to describe the current understanding of the regulation of glucose metabolism in the mammalian liver. under feeding conditions, glucose, a major hexose monomer of dietary carbohydrate, is taken up in the liver and oxidized via glycolysis. the excess glucose that is not utilized as an immediate fuel for energy glycogenesis is activated via the insulin - akt - mediated inactivation of gsk-3, leading to the activation of glycogen synthase and the increased glycogen stores in the liver. insulin is also critical in the activation of pp1, which functions to dephosphorylate and activate glycogen synthase. glycolysis is controlled by the regulation of three rate - limiting enzymes : gk, pfk-1 and l - pk. the activities of these enzymes are acutely regulated by allosteric regulators such as atp, amp, and f26bp but are also controlled at the transcription level. two prominent transcription factors are srebp-1c and chrebp, which regulate not only the aforementioned glycolytic enzyme genes but also the genes encoding enzymes for fatty acid biosynthesis and triacylglycerol synthesis (collectively termed as lipogenesis). the importance of these transcription factors in the control of glycolysis and fatty acid biosynthesis has been verified by knockout mouse studies, as described in the main text. initially, insulin counterregulatory hormones such as glucagon and epinephrine are critical in activating the pka - driven kinase cascades that promote glycogen phosphorylase and glycogenolysis in the liver, thus enabling this tissue to provide enough fuel for peripheral tissues such as the brain, red blood cells and muscles. subsequently, these hormones together with adrenal cortisol are crucial in initiating the transcriptional activation of gluconeogenesis such as pc, pepck and g6pase. the major transcription factors involved in the pathway include creb, foxo1 and members of nuclear receptors, with aid from transcriptional coactivators such as crtc, pgc-1 and prmts. these adaptive responses are critical for maintaining glucose homeostasis in times of starvation in mammals. further study is necessary by using liver - specific knockout mice for each regulator of hepatic glucose metabolism to provide better insights into the intricate control mechanisms of glucose homeostasis in mammals. | glucose homeostasis is tightly regulated to meet the energy requirements of the vital organs and maintain an individual 's health. the liver has a major role in the control of glucose homeostasis by controlling various pathways of glucose metabolism, including glycogenesis, glycogenolysis, glycolysis and gluconeogenesis. both the acute and chronic regulation of the enzymes involved in the pathways are required for the proper functioning of these complex interwoven systems. allosteric control by various metabolic intermediates, as well as post - translational modifications of these metabolic enzymes constitute the acute control of these pathways, and the controlled expression of the genes encoding these enzymes is critical in mediating the longer - term regulation of these metabolic pathways. notably, several key transcription factors are shown to be involved in the control of glucose metabolism including glycolysis and gluconeogenesis in the liver. in this review, we would like to illustrate the current understanding of glucose metabolism, with an emphasis on the transcription factors and their regulators that are involved in the chronic control of glucose homeostasis. |
the purpose of this presentation is to detect influencing factors regarding the interface between ambulatory care and a geriatric day hospital. the coordination between different professions in integrated care is a critical success factor for the quality of care. communication skills, time for information exchange and continuity of therapy and medication. we sent a standardized questionnaire to 476 practitioners and asked about their experiences in cooperation with the local geriatric day hospital in 2006. overall most of the practitioners are satisfied with the cooperation and 90% will continue the interaction. eighty - six percent share the opinion that the interaction is very good concerning time. difficulties exist in the case of an emergency, in evening care and at night. challenges in the interface are therapy and medication. because of the different budget systems practitioners discontinue therapy and medication, which was introduced in the day hospital. | purposeintegrated care for older people is becoming more and more important. the purpose of this presentation is to detect influencing factors regarding the interface between ambulatory care and a geriatric day hospital.quality of carethe coordination between different professions in integrated care is a critical success factor for the quality of care. we assume that this quality is, besides other factors, influenced by communication skills, time for information exchange and continuity of therapy and medication.methodwe sent a standardized questionnaire to 476 practitioners and asked about their experiences in cooperation with the local geriatric day hospital in 2006. eighty - six of the professionals responded (18.1%). statistical analysis was conducted using spss and excel.results and discussionoverall most of the practitioners are satisfied with the cooperation and 90% will continue the interaction. eighty - six percent share the opinion that the interaction is very good concerning time. a weak point is communication between professionals and access by phone. difficulties exist in the case of an emergency, in evening care and at night. challenges in the interface are therapy and medication. because of the different budget systems practitioners discontinue therapy and medication, which was introduced in the day hospital.the results underline the relevance of coordinating interface activities. communication is a prominent success factor between partners in integrated care arrangements. |
visceral leishmanisis (vl) is a protozoal disease manifested as prolonged fever, hepatospleenomegaly, anorexia, and weight loss. it shows a spectrum of epidemiological diversity : leishmania donovani is the causative agent of kala - azar in east africa and indian subcontinent, whereas l. infantum is found in latin america and the mediterranean basin (haldhar. direct detection of amastigote forms in splenic or bone marrow aspirate is taken as the gold standard for diagnosis of vl, but these methods are invasive, carry high degree of risk for the patient and require technical expertise to detect. recently, rk39 dip stick test has gained popularity because of high sensitivity, specificity, rapidity, and easy to use (sundar. this can be used as field test for diagnosis of vl cases in kala - azar endemic areas (khan. but, the major limitation of the test is false positive result in cured patients due to persistence of antibody. urine and sputum samples from kala - azar patients proved as useful as serum for the diagnosis of vl by variety of diagnostic tests (singh. however, the role of saliva is not established in the diagnosis of vl patients. in this study, we compared serum, urine, and saliva samples of clinically diagnosed vl cases by rk39 dip stick test for the diagnosis of vl and also followed up to evaluate their utility in the prognosis of patients. as per who, a vl case is defined as a person from an endemic region with fever for more than 2 weeks, spleenomegaly and confirmed positive by either rapid diagnostic test based on rk39 antigen or biopsy (huda mm. a total of 72 clinically suspected vl patients were enrolled in the study from 2011 to 2012 within a period of 2 year. ethical clearance for the study was not required as the samples were referred to the microbiology laboratory for the diagnostic purposes as a public health measure. sixty one out of these 72 patients were diagnosed as vl as per the above definition and included in the test group. rest 11 cases were included in the control group (n= 11), which consisted of pyrexia of unknown origin (puo) (n= 1), post kala - azar dermal leishmaniasis (pkdl) (n= 3), past history of kala - azar (n= 2), mycosis fungoidosis (n= 1), malaria (n= 3), and filaria (n= 2). serum, urine and saliva samples were collected from all patients (cases and controls) and tested for rk39dip stick test. detailed clinical history of the patients such as fever, hepatospleenomegaly, residence, weight loss, loss of appetite, complete blood count, albumin : globulin ratio, liver function test, and past history of kala - azar were obtained from all patients. serum sample was tested for rk39 test (kalaazar detect rapid test, inbios international, usa) as per the instruction of the kit. urine and saliva samples were tested with rk39 strip test following the similar procedure. only 11 patients out of 61 vl confirmed cases in the test group could be followed up to 34 months after completion of treatment and rk39 test with serum, urine and saliva samples were repeated. all the rk39 test strips were graded from 3 + (equal to or more than control) to 1 + (faint) depending on the intensity of test band in comparison with control band. all patients in the test group (n= 61) presented with fever for more than 2 weeks and hepatospleenomegaly during the first visit to the hospital. on examination, features like pancytopenia, anorexia, weight loss, hypoproteinemia, and reversal of albumin : globulin ratio was observed less frequently in comparison to the previous one. rk39 dip stick test with serum sample was positive in all the patients (100%) of test group (table 1). urine showed a complete concordance with serum exhibiting 100 % positivity in all the patients of the test group, whereas only 51 patients (83.6%) were found positive with saliva sample. eleven patients from the test group were followed up after the completion of treatment and tested with serum, urine and saliva samples to observe the response of treatment. all the cases in the follow up group showed positive result with serum and urine samples in comparison to 10 cases with saliva. comparison of rk39 test with serum, urine, and saliva samples in kalaazar patients and the control group we graded the intensity of the test band of the rk39 strip of these 11 patients before and after treatment as it indirectly correlates the disease status (table 2). the test band intensity with serum sample before and after therapy obtained to be same among these patients. the band intensity found to be reduced with the urine and saliva samples in 4 cases after completion of therapy (table 2). among the control patients (n= 11), the rk39 test with serum, urine and saliva sample found positive in 7 (63.6%), 2 (18%), and 1 (9%) case respectively (table 3). gradation of test band intensity rk39 strips in the kalaazar patients before and after treatment 1 + and 2 + : band intensity weaker than control, 3 + : band intensity matches with control, 4 + : band intensity stronger than control comparison of rk39 test with serum, urine, and saliva samples in control group this study was designed to detect the performance of non - invasive samples for the diagnosis and prognosis of vl patients. it is known that the test band intensity of the ict strip correlates with the course of many diseases (moody 2002, sako. the intensity is maximum during the active phase of the disease and gradually fades after treatment. in our study, urine was more useful in comparison to saliva for the diagnosis of kala - azar in the active stage of disease. during the first visit, the result of rk39 test with urine sample showed complete concordance with that of the serum sample. however, saliva was positive in 84 % cases. the urine and saliva samples showed similar results as with serum samples in follow up patients. it shows that, urine and saliva do not have much role in the determination of prognosis of vl patients. but, in the control group, the rk39 test with the saliva sample was found negative in majority (10/11, 91%) of cases in comparison to serum (4/11, 36.4 %). the test was also observed to be negative in majority of cases with urine sample. conditions such as past history of kalaazar, post kala - azar dermal leishmaniasis showed false positive result with serum sample in the control group. singh. found sputum samples to be highly specific for vl patients (2009). many literatures discussed about the false positive result of urine sample for the diagnosis of vl (khan. but, in this study the false positive results by urine sample was much less. the limitation of the study is that, it is comprised of less number of patients in the control group and also did not include the samples of healthy persons from endemic area. non - invasive samples in endemic area can be used as an adjunct to the serum samples to decrease the rate of false positivity. urine and saliva samples can also be used as part of surveillance in countries where animals behave as reservoir and help to control vl (taran. the role of non - invasive samples in the prognosis of the patients is also difficult to establish because of small sample size in the follow up group. to overcome these limitations and to determine their role, further studies in field with a large sample size the present data highlights the usefulness of non - invasive samples such as urine and saliva as the diagnostic tools for the patients of kala - azar particularly in endemic regions. hence, rk39dip stick test with noninvasive samples such as urine and saliva may be adapted in routine diagnostics along with serum samples in endemic areas for the diagnosis of vl conditions. | background : immunochromatographic based rk39 antibody detection test became popular for the diagnosis of visceral leishmaiasis (vl) because of high sensitivity, rapidity, easy to interpret, and cost effectiveness. however, false positive result after complete cure of the patients is the major limitation with this test. the aim of the study to access the usefulness of non - invasive samples i.e. urine and saliva by rk39 test for the diagnosis of visceral leishmaniasis in comparison to serum.methods:seventy two clinically suspected vl patients were enrolled in the study among which 61 cases were confirmed as vl and 11 cases were included in the control group. serum, urine, and saliva samples of all the cases were tested for rk39 dip stick test.results:urine and saliva both were equally sensitive as serum for the diagnosis kala - azar. in the control group, rk39 antibody test was negative in 10 cases out 11 (91%) with saliva in comparison to 4 cases with serum (36%), thereby found to be more specific.conclusion:saliva sample found to be highly reliable for the diagnosis of vl cases by rk39 test. the test with saliva sample showed less false positive result in comparison to serum sample, thereby can be used an adjunct with serum sample for the diagnosis of kala - azar in endemic areas. |
traditional chinese medicines (tcms), also known as botanical medicines or phytomedicines, have been used to promote health and treat diseases for over a millennium. differing from synthetic drugs, medicinal herbs and their preparations because of low toxicity and effective therapeutic performance, a rapid growth in worldwide demand for tcms has appeared in the last few decades, especially in asia, europe, and north america. as demand grows, the requirement for product quality is also improving. each batch of tcms should meet certain product specifications including both the time of production and over its shelf life. however, the quality control for tcms is more difficult than that for synthetic drugs, because even a simple preparation consisting of only a few herbs may comprise hundreds of mostly unique or species - specific compounds. complete characterization of all the compounds in a preparation is indeed a challenge, while selection of several compounds for determining either efficacy or quality is not in line with the principle of tcms. in recent years, significant efforts have been made to develop new analytic methods for quality control of tcms. among all quality control methods, chromatographic fingerprint, as a unique pattern indicating the information on multiple chemical components within a sample, could provide more informative and accurate assessment of complex matrix, such as tcm. in order to promote the valid fingerprint method for the quality control of herbal medicines, the china food and drug administration (cfda) suggested that all of herbal chromatograms should be evaluated by similarity, which is based on the calculation of correlative coefficient and/or cosine of vectorial angle (i.e., congruence coefficient) between fingerprint data. now similarity evaluation is widely used as a close measure for assessment of chromatographic fingerprints in practice. however, similarity based on correlative coefficient or cosine of vectorial angle is the overall evaluation of fingerprints ; it is ambiguous and insensitive to the changes in content of components. that is, the similarity between chromatographic fingerprints could not sufficiently indicate the closeness in the contents of components, especially for low - content components. recently, hplc fingerprinting combined with quantitative determination of some characteristic compounds has been developed and validated for quality control of herbal preparations. although sample fingerprints shared a satisfactory similarity, the contents of some compounds between samples may still appear quite different (i.e., similar fingerprints do not mean similar components in content). this may indicate that the slight difference in similarity between fingerprints could suggest the large difference in amounts of components. it is unclear whether the difference in fingerprint similarity is significant or not. how to assess the difference in fingerprint similarity is worthy of investigating. to ensure the quality of tcms but for now it is unrealistic to identify and quantify all the chemical components in such a complex system as tcm. the xin - ke - shu (xks) tablet is one of the most commonly used tablets in tcms for treatment of coronary heart and cerebrovascular disease. it is a really complex matrix, which comprises five medicinal materials or extracts thereof, including salviae miltiorrhizae radix et rhizoma, pueraria lobatae radix, crataegi fructus, notoginseng radix et rhizoma, and aucklandiae radix. due to a high complexity in chemical composition, we developed a high performance liquid chromatography - diode array detector (hplc - dad) method for quantification of multiconstituent in xks tablet and found that the component contents in different batches of xks tablet had large differences. with the development of chemometric methods, a number of data processing tools were introduced and applied to process chromatographic fingerprints [1013 ]. in our present study, chromatographic fingerprint combined with chemometric methods was developed for batch consistency evaluation of xks tablet. firstly, four types of fingerprints characterized by different modes were used and compared by similarity analysis. according to the results of comparison, then, hierarchical cluster analysis was applied to observe whether class difference occurred in different batch samples. if high fingerprint similarity was achieved, but apparent class difference existed in samples, then mahalanobis distance analysis was employed to estimate the probability level of obviously different samples. subsequently, for the significantly different fingerprints of batches determined by mahalanobis distance, t - test was used to find the peaks with significant difference in areas. although these peaks found by t - test were based on peak areas, the results were theoretically identical to those based on concentration. the peaks having significant differences were identified by hplc / it - ms as well. finally, a useful strategy based on the combination of hplc fingerprint and chemometric methods was proposed for rational evaluation of batch - to - batch consistency of xks tablet and could be also useful for assessing the batch consistency of other tcms. hplc - grade acetonitrile and methanol (meoh) were purchased from merck company (merck, darmstadt, germany). ultrapure water was purified with a milli - q system (millipore, bedford, usa). (tedia way, fairfield, usa). reagent grade meoh was purchased from tianjin kemiou chemical limited company (tianjin, china). in order to include as many sources of sample variability as possible, seventy - one batches of xks tablet that were produced in a long time span of three years were provided by shandong wohua pharmaceutical limited company in china. all the xks tablet samples were kept in a cool room at 4c and 30% relative humidity with packages ; storage stability of xks tablet, both the physical and chemical properties, was validated. in the cool room, the chemical profiles and the concentration of the major compounds (we determined 6 of the major compounds in xks tablet, details not shown here) of xks tablet were consistent for 3 years. the pulverized powder of 100 mg for each sample was accurately weighed and ultrasonically extracted with 5 ml 70% methanol for 20 min in a conical flask at room temperature. this sample solution (4 ml) was transferred into 10 ml centrifuge tube and centrifuged at 3000 rpm for 10 min. the supernatant was further filtered through a 0.45 m membrane filter before hplc injection., santa clara, ca, usa) with a diode array detector (dad) was used to acquire chromatograms and uv spectra. all of the chromatographic analysis was performed on a phenomenex c18 column (250 mm 4.6 mm i.d. 5 m) protected by a precolumn (12.5 mm 4.6 mm i.d. 5 m) of the same material. the mobile phase was composed of acetonitrile (a) and 0.1% formic acid in water (b). the conditions of solvent gradient elution were 812% (a) in 021 min, 1217% (a) in 2131 min, 1738% (a) in 3155 min, 3890% (a) in 5565 min, and 90% (a) in 6575 min, at a flow rate of 1.0 mlmin. the uv detector was set at 278 nm with full spectral scanning from 190 nm to 400 nm at 2 nm step. the column temperature was maintained at 30c, and all the injection volumes of sample solutions were fixed at 10 l. santa clara, ca, usa) was connected to hplc system via an esi interface. ultrapure helium (he) was used as the collision gas, and pure nitrogen (n2) was employed as the nebulizing gas. the instrumentation and chromatographic condition of hplc for lc - ms were the same as those used for hplc - dad analysis. for identification of the interesting constituents in the fingerprints of xks tablet, the following conditions of ms analysis were used : negative ion mode ; capillary voltage 3.5 kv ; drying gas flow rate 12 lmin ; drying gas temperature 350c ; nebulizer 35 psi ; mass analyzer range was from m / z 50 to 1250 amu. all the data were processed using data analysis for 6300 series ion trap lc / ms (version 3.4). in order to investigate whether different types of fingerprints characterized by different modes have notable differences in characterization of xks tablet samples, four types of fingerprints were applied, including single wavelength fingerprint, 3d fingerprint, maximum absorption wavelength fingerprint, and multiwavelength fingerprint. the four types of fingerprints were developed as described in additional file 1 in supplementary material available online at http://dx.doi.org/10.1155/2015/589654. hplc - dad data of the 71 batches of samples were obtained from agilent chemstation software. the software similarity evaluation system for chromatographic fingerprint of traditional chinese medicine (version 2004a) recommended by cfda (the china food and drug administration) was used to align the fingerprints of different batches and to simultaneously calculate similarity between samples. natick, ma, usa) was used for handling chromatographic data matrices, calculating the mahalanobis distance and carrying out hierarchical clustering analysis (hca) principle component analysis (pca) and statistical test (i.e., t - test and f test). about similarity calculation, one is cosine of vector angle (rcos) that is often used ; the other is correlation coefficient (r). both rcos and r were calculated for similarity evaluation of chromatographic fingerprints among different batches of samples. ward 's method was used for hca due to relatively powerful performance compared with other methods. mahalanobis distance is a distance measure in statistics and takes into account the correlated variables in data set, which can be written as(1)di2=xixs1xix,where xi, x-, and s are the ith sample, the mean vector (i.e., the centroid of class), and the variance - covariance matrix of a class, respectively. according to mahalanobis distance, the unknown samples can be classified as acceptable or unacceptable for the training set at a certain probability level [17, 18 ]. critical values at a certain probability level () are obtained by f distribution. the mahalanobis distance of a sample was compared with critical value to determine whether the difference of the sample relative to the mean of the training set was significant or not. the sample having a probability level in the range from 1.0 to 0.05 is classified as a member of training set. the sample having a probability level in the range from 0.05 to 0 is classified as an outlier. since the single wavelength fingerprint was often used in practice, the validation of hplc fingerprint method for xks tablet was performed on the basis of this type of fingerprint. the relative standard deviation (rsd) values of common peak areas and relative retention time of common peaks were in the range of 0.63.5% and 0.31.2%, respectively. the repeatability was estimated by running five independently prepared xks tablet samples, which were from the same batch, and the corresponding rsd values of common peak areas and relative retention time of common peaks were lower than 1.8% and 1.4%, respectively. a sample solution placed over 3 days in room temperature was employed to carry out the stability test. the corresponding rsd values of common peak areas and relative retention time of common peaks were less than 3.5% and 2.3%, respectively. the results of validation suggested that the developed hplc fingerprint method was satisfactory and applicable to sample analysis. the typical fingerprints (single wavelength fingerprints) of 71 batches of xks tablet samples after peak alignment were displayed in figure 1. the similarity between the fingerprint of each sample and the standard fingerprint (i.e., the mean fingerprint of 71 batches of samples) was shown in additional file 2 (supplementary figures 1 and 2). there are no apparent differences found in similarity trend along with the sample distribution for different types of fingerprints. although fingerprints are based on different characterization modes, the fingerprints of different batches of samples have a high similarity to the standard fingerprint (all above 0.94 based on r or rcos). according to the literature, in addition to the maximum wavelength fingerprint, the other three types of fingerprints are considered to have good similarity for different batches of samples. the range and the degree of dispersion of similarities based on four types of fingerprints were summarized in table 1. according to table 1, the maximum wavelength fingerprint has the largest range and the dispersion degree of similarity as compared with the other three types of fingerprints. because this type of fingerprint is characterized with the data at maximum absorbance wavelength, the sensitivity is relatively high. the 3d fingerprint is very close to the single wavelength fingerprinting in the range and the dispersion degree of similarity. among four types of fingerprints, multiwavelength fingerprint exhibits the minimum range and dispersion degree of similarity due to the quite close distribution of multiactive ingredients in different batches of samples. up to now, the single wavelength fingerprint is the most used in practice. in this type of fingerprint, the particular wavelength the 3d fingerprint for characterizing each sample contained rich information on components ; however, redundant information was also present. additionally, poor resolution and unstable baseline appeared in some wavelengths, making the subsequent data processing (such as integration and peak alignment) time - consuming, difficult, and complicated. the multiple - wavelength fingerprint could have good reflection on characteristic components but lost some useful information on other constituents. the maximum wavelength fingerprint was a satisfactory type of fingerprint and could better reflect the differences in chemical composition between samples. however, it was not easy to get the maximum absorbance fingerprint, because some complicated and time - consuming data handling methods or special chemstations (such as waters empower chemstation) should be necessary. according to the comparison mentioned above, the single wavelength fingerprint seems to be still the most practical, popular, and easy - handling fingerprint pattern for quality evaluation of herbal medicine product. considering fingerprint applicability and representativeness, overall, the dendrogram revealed two clear clusters, suggesting that there were apparent class differences in different batches of xks tablet samples. although high similarity between samples was acquired (supplementary figure 2), the class differences seemed to be significant. in order to determine whether or not the differences between batches of xks tablet samples were statistically significant, mahalanobis distance method was employed. for calculation of mahalanobis distance good quality samples should be candidates of the reference class. in our study, a sample having larger peak areas in its fingerprint was selected as the member of the reference class due to large peak area generally indicating high content of component. sample 5 was first selected as a reference sample for development of the reference class, because most of peak areas in sample 5 fingerprint are larger as compared to other batches of samples. the euclidian distances between other batches of samples and sample 5 were then calculated for evaluation of sample closeness. the reference class was developed by choosing the closest 10 batches of samples plus sample 5 ; therefore, 11 samples were involved in the reference class according to the requirement of the documents on the development of fingerprint. the mean fingerprint of the samples in the reference class was also calculated as the reference fingerprint. with respect to the centroid of the reference class, the mahalanobis distance and its probability level of each sample in the reference class the results reveal that all sample fingerprints in the reference class have nonsignificant differences as compared to the reference fingerprint. the mahalanobis distances between the fingerprints of other batches of samples and the reference fingerprint were also calculated and shown in figure 3. there are 19 batches of samples having significant differences with respect to the centroid of the reference class determined by mahalanobis distances, indicating that their fingerprints may have large differences in peak areas relative to the reference fingerprint. to explore the relationship between similarity (both cosine of vector angle and correlation coefficient) and mahalanobis distance, similarity, mahalanobis distance, and corresponding probability of each batch fingerprint relative to the reference fingerprint however, although the fingerprints of some samples (such as samples 9, 15, and 20, and their r and rcos are both above 0.99) have high similarity to the reference fingerprint, their corresponding mahalanobis distances are still larger, which result in significant differences (< 0.05). notice that higher similarity does not exactly indicate small mahalanobis distance, while lower similarity indicates larger difference in mahalanobis distance. according to (1), the mahalanobis distance is actually based on the differences in peak areas between fingerprints. for r or rcos, although the chromatographic peak areas are used in similarity calculation, similarity indicates a measure of the closeness of the overall shapes of the compared fingerprint profiles. when the shapes of the two hplc fingerprints are very close, high similarity can be obtained even if the peak areas in the compared fingerprints are quite different (e.g., the peak areas vary greatly with different injection volumes in different fingerprints, but the similarity is still high owing to close shapes of fingerprint profiles). if the difference in peak area is very great and can significantly affect the shape of the compared fingerprints, the similarity between the fingerprints will change accordingly. therefore, the mahalanobis distance takes the difference of peak area into account, while similarity emphasizes the overall shape of the fingerprint profile. in some cases, although the fingerprints of some batches showed high similarity to the reference fingerprint, their differences in peak areas were still significant based on mahalanobis distances. therefore, the mahalanobis distance could reflect the inconsistency between batches of samples relatively better than similarity, and the corresponding probability level was also estimated for assessing whether the inconsistency between fingerprints was significant or not. if peak area has large difference in various batches of samples, quality consistency will be strongly affected. therefore, it is quite necessary to find the significant different peaks across the fingerprints of batches. the mahalanobis distance was used to detect whether there are significant differences between the sample fingerprints and the reference fingerprint. however, it is not easy to find the significantly different peaks in a sample fingerprint by mahalanobis distance. in our study, because common peaks are common features of different batches, differences in areas of common peaks will have great impact on quality consistency. moreover, for each sample fingerprint, the common peak areas also accounted for above 81% of the total peak areas. therefore, only common peaks were identified by t - test for significantly different batches determined by mahalanobis distance. more attention should be paid to the significantly different common peaks for the quality control of xks tablet. the reference class could be regarded as the training set. to carry out the t - test on the peaks in a sample fingerprint, the variable zi was calculated according to(2)zi = xixisi, where xi referred to the ith chromatographic peak area in a fingerprint of sample, and x - i and si were the average peak areas and the corresponding standard deviations of peak i in the fingerprints of the samples in the training set (i.e., the reference class), respectively. n was the number of samples in the training set (n = 11 in the present study). the ti was compared with the tcrit given by student 's t distribution to determine whether the difference between xi and x - i was significant or not. although, t - test was calculated with chromatographic peak area, the result indirectly denoted the differences in component contents of different batches of samples, because, in the linear range, a chromatographic peak area was expressed by(4)xi = fici+bi, where xi and ci referred to the chromatographic peak area of peak i and its corresponding concentration (or content), respectively. fi and bi denoted the response factor and the intercept of the calibration curve, individually. then, (2) combined with (4) could be written as(5)zixixisi = ficicifi1/n1i=1ncici21/2=cicisci, where c - i and sci represented the average content and the standard deviation of ith component concentrations in the samples of the reference class. so, according to (5), the value of zi based on peak area was identical to that based on component content for peak i. figure 5 shows the z values of common peaks in 19 significantly different batches of samples determined by mahalanobis distance analysis. among the 19 batches of samples, most of common peak areas with significant differences identified by t - test are the small ones in peak areas, which indicate that the amounts of these components may be lower than the average contents of the corresponding components in the samples of the reference class. figure 6 shows the frequency proportion of significantly different common peaks based on t - test versus 19 batches of samples. the results clearly reveal the common peaks 13 and 19 having the highest frequency percentage that occurred in the 19 batches of samples. the components corresponding to the common peaks with high frequency percentage may have large differences in amount. hplc / it - ms was employed to identify seven of them (peaks 13, 15, 16, 19, 20, 26, and 29) which displayed high frequency proportion in figure 6 and are marked in figure 1. in the esi - ms experiment, the molecular weight of each peak and some fragments could be obtained. in negative ion esi mode, the peaks were detected according to the deprotonated molecules ([m - h ] and/or [m - h + hcooh ]). except peak 26, which was identified as lithospermic acid (characterized based on the retention time, uv spectrum, and its ms data compared with literature), the other six interesting peaks were tentatively identified as isoflavonoids. the diagnostic ions of isoflavonoids are the loss of 162 da and 120 da in molecular weight, which are related to the o- and c - glycoside isoflavones [21, 22 ], respectively. the possible structures of peaks 13, 15, 16, 19, 20, 26, and 29 were characterized based on their retention times, uv spectra, and their ms data compared with literatures. using peak 15 as an example, the fragmentation of [m - h ] ion at m / z of 445 gives product ions at m / z 325 [m - h-120 ], 297 [m - h-120-co ], and 282 [m - h-120-co - ch3 ], which indicate that peak 15 has a -och3 group in the aglycone skeleton and the neutral loss of 120 da characteristic of c - glycosides. more information about these seven peaks and their structures are shown in table 3 and figure 7, respectively. according to ms results, lithospermic acid and six isoflavonoids as characteristic markers may have strong contribution to the inconsistency of batches of xks tablet samples. the isoflavonoids were the main active ingredients of pueraria lobatae radix, suggesting that the pueraria lobatae radix could have large fluctuations in the production of xks tablet. consequently, in order to improve the consistency of different batches of xks tablet, more attention should be paid to the quality of pueraria lobatae radix, including selection of good quality of raw materials, improvement of manufacturing technologies, and enhancement of process control level. to find a rational way to evaluate the consistency of different batches of xks tablet samples, the useful strategy was proposed for the solution of the routine issue existing in the consistency evaluation of chromatographic fingerprints of xks tablet samples. although high similarity occurred in some batches of samples, the significant differences between samples could still be found by mahalanobis distance. the chromatographic peaks with significant differences in peak areas determined by t - test could suggest that the corresponding constituents would have significant differences in content, which resulted in affecting the consistency of quality. our study demonstrated that the proposed strategy based on the combination of hplc fingerprints and chemometric methods would provide a useful way to assess the quality consistency of xks tablet. | evaluation of the batch consistency of traditional chinese medicines (tcms) is essential for the promotion of the development and quality control of tcms. the aim of the present work was to develop a useful strategy via liquid chromatography and chemometrics to evaluate the batch consistency of tcm preparations. xin - ke - shu (xks) tablet was chosen as a model for this method development. four types of chromatographic fingerprint approaches were compared by using similarity analysis based on cosine of angel or correlation coefficient. differences in the fingerprints of 71 batches of xks tablet were illustrated by hierarchical cluster analysis. then, mahalanobis distance was employed for estimating the probability level (p < 0.05) of the differences mentioned above. additionally, t - test was applied to find out the chromatographic peaks which had significant differences. for xks tablet, the maximum wavelength fingerprint had the largest range and dispersion degree of similarity as compared with the other three ones. there were two clear clusters in all the batches of samples. and we clearly arrived at the conclusion that higher similarity does not exactly indicate small mahalanobis distance, while lower similarity indicated larger mahalanobis distance. finally, a useful strategy was proposed for evaluation of the batch consistency of xks tablet. |
first reported in western medical literature in 1958, fmt remained a fringe medical practice over the next 50 years. however, advances in molecular microbiology and the rising incidence and severity of c. difficile infection prompted a resurgence of interest in the therapy, especially as investigations consistently found that 90 per cent of recurrent c. difficile cases treated with fmt achieve clinical resolution. fmt refers to the practice of transplanting a filtered stool preparation from a healthy donor into the lower gastrointestinal tract, typically via colonoscopy or enema, or the upper gastrointestinal tract, for instance by nasojejunal tube. published protocols guide the screening of stool donors for transmissible diseases including hepatitis and hiv, and the preparation of stool either for immediate use or for cryopreservation for later use. these protocols call for the stool to be mixed with a saline solution, filtered to remove fibrous material, and either administered to the patient immediately or mixed with glycerol and frozen until the time of administration. as mentioned above, systematic reviews and meta - analyses reviewing case series of fmt for recurrent c. difficile infection found that 90 per cent of patients were cured by the therapy. by comparison, as noted above, first - line antibiotics treatments cure c. difficile infection 82 per cent of the time, and after two or more recurrences, the cure rate of standard antibiotic therapies drops to below 40 per cent. in the first clinical trial evaluating the therapy, fmt proved so superior to standard antibiotics that the study 's data and safety monitoring board stopped enrollment early, concluding that it was unethical to withhold the treatment from the members of the control group. neither the systematic reviews and randomized trial nor a one- to two - year follow - up investigation found a link between fmt and any adverse events, though colonoscopy and upper routes of administration carry procedural risks. however, even though studies concluding a year or two post - fmt (a timeline which is typical of many clinical trials) have reported no adverse events, diseases that have been linked to the microbiome may surface years after fmt. as such, there remains a need for more investigation of the safety profile of fmt in the extreme long term. the mechanism by which the transplanted microbiota out - compete the c. difficile infection also requires further investigation. analyses of microbiota communities before and after transplantation indicate that fmt addresses the lack of bacterial diversity in recurrent c. difficile patients and restores the composition of a normal microbiota community. it was even demonstrated that the presence of a single bacterial strain of c. scindens could enhance resistance to c. difficile infection in mice. these findings and others suggest the possibility of more targeted synthetic bacterial treatments of the infection, which companies including seres therapeutics are pursuing. while there is a paucity of data on the scale at which fmt is practiced today, openbiome, a public stool bank that supplies fecal microbiota preparations for clinical use, has provided material for more than 4,000 treatments of recurrent c. difficile infection to 300 clinical sites across the usa since it first began operations in 2013. openbiome is also sponsoring a multicenter study of the long - term safety profile of fmt in recurrent c. difficile patients, under an investigational new drug (ind) application. beyond c. difficile, studies of the microbiome have revealed important relationships between intestinal bacteria and human health. fmt for the treatment of recurrent c. difficile infection represents a first foray into engineering the human microbiome to yield positive clinical results. investigations into other applications of this therapy are underway, including testing fmt for the treatment of irritable bowel syndrome, inflammatory bowel disease, and a variety of metabolic diseases. companies such as vedanta biosciences are also pursuing targeted synthetic bacterial treatments of subsets of these indications. in may 2013, the fda held a public workshop on fmt at which they first confirmed that fecal microbiota would be regulated as a drug. the fda had written that while fmt may be an effective therapy for the management of refractory c. difficile infection, its efficacy ha[d ] not yet been demonstrated in controlled clinical trials. all uses of fmt would therefore need to be part of an ind application. in other words, patients who wished to be treated with a fecal transplant for recurrent c. difficile or other indications would need to participate in a clinical trial to do so. physicians and scientists, including representatives of the centers for disease control and members of professional medical societies, quickly responded with concern to the fda 's decision. they argued that the available evidence supporting fmt 's effectiveness as a therapy for refractory c. difficile infection was too compelling for regulators to restrict its availability to the treatment groups of clinical trials. as such, just two months later, the fda revised its decision and announced that it would exercise enforcement discretion when fmt was used to treat patients with c. difficile infection not responding to standard therapies. so long as the treating physician obtained adequate informed consent, the fda would not require recurrent c. difficile patients to receive treatment through an fda - reviewed clinical trial. the fda 's decision not to enforce the ind requirement for recurrent c. difficile infection has significantly expanded access to the therapy, in no small part by creating a window in which public stool banks like openbiome could operate. as a result a recent study of the cost - effectiveness of fecal transplantation has determined that it saves a conservative estimate of \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ { \$ } $ \end{document}17,000 per patient. public stool banks have developed protocols for screening and processing stool that are even more rigorous than previously published guidelines for directed donor fecal transplants, and that can represent even more cost savings by spreading the costs associated with stool screening and preparation over many treatments. however, the long - term regulatory outcome for fmt remains unclear, and as will be explained infra, if the fda proceeds along its current pathway, the most likely outcome is one in which a single provider receives a license from the fda to produce fecal microbiota for transplantation, at which time the fda will likely no longer exercise its enforcement discretion. there are several problems with this outcome, each of which we will address in further detail in the sections to follow. first, to effectively regulate stool as a drug, regulators will need to address the challenge of characterizing active ingredients and guaranteeing safety and quality in spite of the variability of stool. second, this outcome will likely impose significant burdens on the health care system, burdens that the fda has not yet faced with other therapies. as an initial matter, it is probable that a license to produce fecal microbiota for transplantation will have the effect of granting market exclusivity to a single provider for the provision of ubiquitous human stool. yet, in this case, like only a very few others, doing so would allow that provider to capture value from knowledge that already exists in the public domain, and is already being used by multiple providers to meet an urgent and growing public health need. this outcome would work against the public interest by inhibiting treatment choices for patients and raising prices across the health care system. when combined with the do - it - yourself potential of fmt, the decision to regulate fecal microbiota as a drug may impose safety risks that are unique to this therapy and do not arise in the context of other, more traditional drugs. third, given the relative freedom with which clinicians may prescribe drugs off - label, regulating stool under the traditional drug paradigm may also discourage investment into explorations of fecal therapies for other indications, if off - label uses of fecal microbiota can meet demand from patients with those conditions to which researchers have made tentative links to microbiome health. this concern is not unique to fmt, but is worth attention because in this case the fda has the ability to select an alternative regulatory paradigm that would mitigate, if not eliminate, this result. from a scientific perspective, the regulation of stool as a drug is complicated by the material 's complexity and inconsistency across samples. the microbial and metabolic contents of human stool are known to vary enormously across individuals and over time within individuals. unless the active components are identified, purified, and tested, it will not be possible to guarantee that the product is consistent across batches. this characteristic suggests that the regulation of stool should be tied to the process by which it is prepared for transplantation, rather than to the variable contents of the product. process not only includes preparation methods following stool collection ; it is also driven by the complex and very specific life history of the individual donors, and might arguably be defined by the donors themselves. more practically, the process associated with preparing fecal microbiota for transplantation could be narrowly defined by the very particular methods used to filter and prepare stool from donors who are selected in accordance with a consistent protocol. thus, under the drug regulation paradigm, given the characteristic variance of stool, the fda would be underregulating stool from both a safety and an efficacy perspective should it simply provide traditional drug licenses to stool - based products. stool preparations have distinct compositions and resultant therapeutic properties, and they also carry distinct pathogenic threats, and thus warrant ongoing safety monitoring and evaluation beyond that which typically accompanies an approved drug. perhaps more importantly, it would be inappropriate to provide licenses that bear little relationship to the specific donor screening or stool preparation methods used by the stool provider. instead, an approach that narrowly defines the process by which stool material is assessed and prepared for fecal transplantation would be more consistent with our understanding of the extreme complexity of the microbial system. however, the standard drug paradigm does not clearly allow the fda to specify who may donate stool and which pathogens must be screened for. it is important that these complicated issues, many of which are still not well understood by science and medicine, be considered in a public regulatory context, rather than in the fda 's private negotiations with a single company. other problems with the fda 's decision to regulate fmt as a drug stem from one of the fda 's often - overlooked powers : its statutory requirement to award periods of exclusivity for approved drugs under a variety of conditions. as one example, the orphan drug act awards seven years of marketing exclusivity to fda - designated orphan drugs, during which time the fda may not approve a new or generic drug application for the same product and indication. the recently enacted biologics price competition and innovation act goes further, providing 12 years of data exclusivity for reference biologics, during which the fda may not approve an application for a biosimilar product that relies on the innovator company 's clinical trial data. although the appropriate lengths of exclusivity periods like these have been hotly contested, there is general agreement that in many cases an exclusivity period helps provide innovative drug manufacturers with sufficient incentives to carry new products through the long, expensive, development process. the orphan drug act is the paradigmatic example : where the market for a drug is by definition small, a long exclusivity period helps assure pharmaceutical companies that they can recoup their investment into a drug for treating an orphan disease. exclusivity then functions to raise the price of the drug above what it would be in an otherwise free market, but this is a social bargain that has been made for purposes of allowing companies to capture more of the value that they generate through innovation, incentivizing them to engage in innovative drug development activities that would not otherwise have occurred. but sometimes this bargain breaks down. in some cases, the fda has approved a drug when that very same drug was already widely, cheaply available on the market. the fda 's approval and grant of an exclusivity period in such cases may function to clear the market of existing therapies, permitting the new applicant to charge monopoly prices without fulfilling their end of the social bargain and bringing a truly new product to market. scholars have been concerned about two such cases that have occurred recently, with disparate outcomes. the story of colchicine, a drug used to treat acute gout flares, has likely been the most widely reported. colchicine had been widely and cheaply available in generic form since the 1800s, long before the fda began regulating the safety and efficacy of drugs. as such, even after the fda acquired such authority in 1962, many drugs that were already on the market remained publicly available, although they were never formally evaluated by the fda. a pharmaceutical company decided to invest in conducting a series of trials involving colchicine, to demonstrate its safety and its efficacy for gout as well as for an orphan disease known as familial mediterranean fever. in 2009, the fda approved the use of colchicine for these indications, and under the orphan drug act the manufacturer received seven years of marketing exclusivity for a product that was already on the market. the manufacturer and the fda subsequently took legal action to force all other makers of colchicine to exit the market, and the manufacturer then raised the price per pill from about \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ { \$ } $ \end{document}0.09 to \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ { \$ } $ \end{document}4.85. the grant of exclusivity resulted in significantly increased costs not only for patients but also for medicare and medicaid, common payers for the drug. scholars have persuasively argued that the rewards granted to colchicine 's now monopoly supplier vastly exceed the value of the information provided to the public, and that public health is likely to suffer as a result. another recent example involves a synthetic hormone used to reduce the risk of preterm births in pregnant women with a history of preterm births. the hormone had been available to women in compounded form for many years, and it was relatively inexpensive, at \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ { \$ } $ \end{document}15 per injection or \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ { \$ } $ \end{document}300 per pregnancy. but in february 2011, the fda approved makena, a branded form of the hormone, for this orphan indication. the marketing company first set a price roughly 100 times higher than that of the compounded therapy, for a total cost of almost \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ { \$ } $ \end{document}30,000 per pregnancy. although they later cut that price in half, the cost still vastly exceeded the cost of the compounded form. in this case, though, makena 's maker could not secure a monopoly over the distribution of the hormone. although the fda could not have approved another manufacturer 's application under the orphan drug act, the fda stated that it would decline to take enforcement action against pharmacies that compounded the drug for patients unless safety problems were reported. its regulatory authority in this area is unclear, and the fda 's exercise of its enforcement discretion in this case avoided a legal battle with the compounding pharmacies. however, the fda was also likely motivated by concerns about patient access, having observed the results of the colchicine case. if the fda continues in its efforts to regulate fecal microbiota as a drug, it may soon be required to make a similar choice in the case of fmt. responding to an application from a company called rebiotix, the fda has recently designated fecal microbiota as an orphan product for the treatment of recurrent c. difficile infection. rebiotix is currently conducting clinical trials in an effort to gain fda approval, and if it is successful, the orphan drug act would prevent the fda from approving another manufacturer 's application to process and distribute whole stool material for fmt, barring a showing of clinical superiority. the question then becomes whether other stool banks would be able to stay in business, as in the case of makena, or whether they would be forced out of the market, as in the case of colchicine. in our view, the fda ought to continue exercising its enforcement discretion and permit other stool banks to remain as market participants, analogous to its behavior in the makena case. perhaps most obviously, the fda has prioritized the removal of unapproved drugs from the market. the fda would also likely be sued for this behavior, as it was in the makena case. also, given the fda 's past attempts to curtail its use of enforcement discretion in the fmt context, it is difficult to think that it would be likely to continue exercising its enforcement discretion here. even if the fda did continue to exercise its enforcement discretion after granting orphan drug exclusivity to one company, the award of exclusivity itself would likely spell trouble for other stool banking operations. in the case of colchicine, recall that before the fda took action, the approved company independently brought suit against other colchicine manufacturers, seeking to force them to exit the market. should the approved fmt company do the same, even if it would not be likely to win such a suit, the time and expense required by litigation would likely have an adverse effect on the ability of existing stool banks to continue providing care, especially given their non - profit approach. importantly, the possibility of having a single stool provider on the market in itself is not a problem. the survival of any particular fmt company is incidental to the primary goal : to ensure patients have access to safe, effective treatments. first, to the extent that the prospect of granting exclusivity in the colchicine and makena cases has troubled scholars because the reward received by companies would be disproportionate to the social contribution made, exclusivity for fmt raises the same concerns. but second, and even more troubling, is a factor unique to fmt : the potential for do - it - yourself treatments. since monopoly power leads to monopoly prices, if rebiotix chooses to sell patients stool for thousands or tens of thousands of dollars, depending on health insurance coverage, patients may resort to essentially free at - home transplantations, using friends or family members, screened at the patient 's discretion, as donors. the problem is a simple one of trading off access, cost, and safety. at a low price but at a high price, the prospect of a monopoly fmt system poses significant safety concerns. as we have explained, fmt as a therapy is unique for the difficulty of its characterization and the simplicity of its production, and each of these characteristics raises special safety concerns. first, the complexity of the microbial community in stool and the variability across stool samples makes it nearly impossible to guarantee the contents from batch to batch. as such, ongoing monitoring for the presence of possible pathogens is necessary for maintaining a safe product and should either be considered part of the approved manufacturing process or a condition imposed on manufacturers. second, although there is little chance that patients will manufacture traditional small molecule therapies in their bathrooms, processing stool for transplantation at a basic level requires very little training or equipment. instructional guides produced by non - professionals and posted on youtube have received tens of thousands of views. patient support groups and online forums include lengthy step - by - step instructions along with discussions of best practices for mixing stool in a low - cost blender and administering it via enema. the risk with any regulatory strategy that restricts access to this therapy, whether by raising barriers to clinical practice or by supporting monopolistic pricing, is that it will motivate desperate patients to pursue self - treatment. unsupervised, do - it - yourself treatments carry considerable risk of the transmission of pathogens from improperly screened and handled stool. only six per cent of prospective donors to openbiome pass the full screening process, which includes a 109-item clinical assessment administered by a nurse or physician, and 30 stool and blood screens. with a lack of long - term safety data on patient outcomes post - fmt, it is prudent to be overly cautious about screening for diseases that are potentially mediated by the microbiome ; for instance, researchers have notably linked the microbiome to obesity, metabolic syndrome, and behavior. similarly, it is just as important to collect longitudinal safety data to identify any conditions that may be transmitted via stool of which we are unaware. thus, given the known and unknown risks that come with improper donor screening and inadequate patient follow - up, the ease with which patients may prepare and administer fecal transplants themselves without medical supervision, any regulatory outcome that results in restricted access by either limiting supply or significantly increasing the cost of therapy should be adopted extremely cautiously. as we have explained, while fmt may fall within the broad statutory definition of drug, there are reasons to be concerned about regulating it wholesale under that paradigm. in the past, when similar concerns have arisen for other technologies involving human cellular or tissue - based products of various types, the fda has adopted or developed alternative regulatory paradigms. drug under the fdca, the fda has promulgated regulations establishing an entirely separate system for regulating blood and blood products as biologic products under the phs act. this regulatory system is largely focused on ensuring the safety of the blood supply, with the fda restricting the possible donor pool and requiring extensive testing and quarantine of donated blood before it can be transfused. the fda also regulates blood banks themselves, with highly specified registration and facility requirements. yet, generic licenses are issued to banks meeting requirements, rather than being awarded exclusively to one or a small number of institutions. in many ways, the fda 's regulation of whole blood and blood products is in some ways an artifact of history, however. blood transfusions were routinely performed in the 1800s, and the discovery of blood typing in the early 1900s greatly improved the safety and efficacy of the treatment. by the time the fda officially acquired the authority to regulate blood in 1970, the safety and efficacy of transfusions was beyond question. a far greater concern, in the fda 's view, was the potential for scarcity. it relies on donors, and the possibility of granting exclusive licenses to certain banks would be unthinkable, in light of the potential human cost. the fda 's commitment of resources to create blood 's existing regulatory structure must be understood in the context of this history. blood and fecal microbiota share many scientific characteristics that distinguish them both from traditional drugs. further, in both cases we ought to be concerned about the potential for scarcity, due to the need at present to procure both blood and stool from human donors. and just as the efficacy of blood and blood products was known and appreciated prior to the fda 's involvement in this area, fmt 's efficacy for at least one indication blood and fecal microbiota are distinct in at least one important way : unlike blood, scientists believe that fmt may be useful for a wide range of potential indications that implicate the human microbiome. some of these, such as ulcerative colitis or crohn 's disease, may naturally be suspected due to fmt 's locus of action. yet others, like parkinson 's disease, multiple sclerosis, and childhood regressive autism, are also potential indications. research into these indications but also into the microbiome more broadly is desperately needed, and the current paradigm for regulating blood is not set up to oversee such clinical examination. instead, then, we might look to another regulatory paradigm whose history more closely resembles that of fmt. specifically, fmt 's development quite strongly resembles the history of the use of cord blood, whose potential to treat a range of diseases was not discovered until the 1980s. cord blood, which is regulated by the fda under its related oversight system for human cells and tissues, is a second potential model for fmt. blood is not the only therapeutic that the fda has carved out of its system for regulating traditional drugs. the fda has also issued special regulations for human cells, tissues, and cellular and tissue - based products or hct / ps, which are human cells or tissues that are intended for implantation, transplantation, infusion, or transfer into a human recipient, a group that includes bones, ligaments, skin, and cord blood. like blood, these human tissues are regulated as biological products under the phs act, but for hct / ps and specifically for cord blood, the fda has chosen to regulate different uses in different ways. specifically, where banked cord blood is intended for use by its donor or that donor 's close relatives, that cord blood is regulated like general whole blood, as a biological product, in a way that prioritizes the safety of the process. accordingly, private cord blood banks must be registered and licensed much like typical blood banks, and they must comply with all tissue regulations. cord blood stored for personal use may be used for any indication. where cord blood is intended for use by a patient unrelated to the donor, however, it is regulated both as a biological product and as a drug. this means that cord blood when sourced from an unrelated donor must be approved for use under an ind or bla for the requested indication, a process that might involve extensive clinical trials. to put it succinctly, for cord blood, fda regulation is concerned with both the intended recipient and the intended use, toggling the type and level of regulation along both dimensions. a key virtue of adopting the hct / p approach in the fmt context is that it would permit the fda to regulate different uses of fmt differently. there is already strong evidence to suggest that fmt can be safe and effective for the treatment of recurrent c. difficile infection. however, whether fmt can be safe and effective for the treatment of other indications is not known. as such, the fda might use their hybrid approach to regulating hct / ps like cord blood as a guide. in effect, choosing to regulate fmt as a biological tissue product would allow the fda to permit its use in treating recurrent c. difficile infection, while still requiring additional proof before it could be prescribed for other indications. adopting the hct / p regulatory paradigm wholesale would, however, permit individuals to bank and use their own stool or stool from any first- or second - degree relatives for any purpose. as such, regulators ought to be concerned about the potential for this carve - out to reduce the benefits of adopting the hct / p approach in terms of regulating different indications in different ways. arguably, though, it is preferable for individuals who are determined to use fmt for unapproved indications to do so through a process that at the very least minimizes the safety concerns associated with the therapy. if no such exemption existed, the reality is that patients would undergo fmt in an unregulated, do - it - yourself fashion, not that they would be unable to undergo fmt at all. the fda has faced this same tradeoff in the context of semen, which has similar potential for at - home administration. the fda therefore imposes fewer and less strict requirements on directed sperm donors than on anonymous donors. in order to regulate the safety of fmt along the same lines as either blood or tissue, though, the fda would need to either interpret or amend its existing regulations. at present, although fecal material qualifies as a biological product, the fda 's tissue reference group has recommended that it does not meet the statutory definition of an hct / p and therefore could not at present be subsumed within the relevant regulations. the reference group did not publicly explain their reasons, but a close reading of the regulations suggests two primary reasons the reference group may have reached this conclusion, each of which can be surmounted. first, the group members may have reasoned that although fecal microbiota preparations are at least partly composed of human cells or tissues that are intended for implantation, transplantation, infusion, or transfer into a human recipient, fecal microbiota falls within the explicit regulatory carve - out for secreted or extracted human products, which include milk and collagen. these products are not considered to be hct / ps, and therefore are not regulated under the tissue paradigm their collection and storage is not subject to the same rigorous safety requirements that apply to cord blood products. the carve - out for secreted products goes onto state that semen is considered an hct / p, suggesting that otherwise semen would be considered a secreted product as well. if this limitation on what constitutes an hct / p is the basis for the reference group 's conclusion, the fda would likely need to conduct an official notice - and - comment rulemaking to clarify that fecal microbiota is also considered an hct / p. a simpler approach would be to attempt to issue an interpretive rule or guidance document indicating that fecal matter simply does not qualify as a secreted or extracted human product. however, this approach might be viewed by courts to purport [] to impose legally binding obligations or prohibitions on regulated parties, and therefore might be rejected as an effort to circumvent the administrative procedure act. second, the reference group may have reasoned that fmt does not require the transplantation of human cells or tissue, but that it rather requires the transplantation of microbial cells or tissue. the simplest option would be for the fda to clarify that fmt as it is currently practiced simply does fall into the category of articles containing human cells or tissues. although it is possible that in the future the material for fmt will not need to be sourced from humans and therefore will not necessarily contain human cells, at present the raw material for fmt is inexorably sourced from humans and inevitably contains human cells. yet, either of these approaches would require a significant expenditure of resources, which the fda might not be willing to allocate without political pressure of the kind they faced in the cord blood context. given the potential for publicly reported adverse events that may be attributed to the underregulation of processing and storage of stool samples, the fda might be willing to take such actions, but it is by no means clear at this time. if they are not willing to devote the resources, a third option as discussed above, the fda has currently chosen to use its enforcement discretion to de facto permit physicians to provide fmt for the treatment of recurrent c. difficile infections. if the fda continued to exercise this discretion, it would be able to achieve a similar outcome as it could achieve in the hct / p context. that is, physicians would be free to provide fmt to treat c. difficile infections not responding to standard therapies, but a company hoping to market fmt for other indications would still need to complete clinical trials. importantly, if the fda chose to continue exercising its enforcement discretion for fmt for recurrent c. difficile, health care providers would not be without expert advice on the subject. professional organizations like the american gastroenterological association have issued best practice guidelines for fmt, including restrictions on donor selection, sample processing, and facilities management., private standard - setting organizations like the american association of blood banks had already begun to regulate the blood supply before the fda was involved in the process, a role which the professional association continues to fulfill today. although these guidelines lack the legal force of the fda 's regulatory scheme, they are not toothless. physicians subject to malpractice actions who have failed to adhere to guidelines that represent the standard of care may be subject to professional discipline. by extension, stool banks would face pressure to demonstrate compliance with best practice guidelines from the clinical sites and insurers using their services. a key virtue of this approach is that it permits the fda to postpone the off - label prescription problems that might arise if it does approve rebiotix 's fmt application. essentially, although manufacturers are not permitted to advertise their products for off - label uses, doctors are free to prescribe therapies and devices for off - label indications, with one study suggesting that about 20 per cent of all drugs are prescribed off label. too often there is no scientific support for off - label prescriptions, and with a grant of exclusivity, the manufacturer has little financial incentive to conduct further validating studies. because approving fmt for the treatment of recurrent c. difficile infections would permit physicians to prescribe it for all other unproven indications, it would perversely delay the fda 's acquisition of the very information it wants most data on the use of fmt for other, non - c. when compared with the possibility of constructing an entirely new regulatory system, as the fda has done in the context of blood, or even completing a notice - and - comment rulemaking to bring fmt within the scope of the hct / p regulatory scheme, the possibility that the fda could achieve a somewhat similar result by simply doing nothing is undoubtedly attractive. further, it is useful to recall that the use of stool for fmt is only a bridge technology, at least in the c. difficile context. companies seeking to understand the microbiome and use it directly to treat the disease will (perhaps soon) obviate the need to use donated stool for these purposes. as such, any regulatory action taken by the fda may be obsolete within a decade. the possibility that enforcement discretion may be withdrawn at any time places stool processing companies in a precarious, uncertain position regarding their legality. it also does not avoid the off - label prescribing problem, but simply delays it until such time as the fda approves fmt for a different indication. the possibilities above must be considered in light of decisions that the agency has already made in this area that may inhibit its ability to adopt a more flexible paradigm in the near term. to briefly summarize what has been described above, in 2012, the fda 's tissue reference group recommended that fecal microbiota did not meet the statutory definition of an hct / p. in 2013, the fda explained that it considered fecal microbiota for transplantation a drug, and would regulate it as such. a few months later, the agency permitted rebiotix to pursue licensure for its fecal microbiota preparation under the approval program for orphan drugs and subsequently awarded it an orphan drug designation for treating recurrent c. difficile infection. in light of the scientific, legal, and public health arguments against regulating fecal microbiota preparations for c. difficile therapies as drugs, what options do the rest of these decisions leave the fda ? above, we considered the option of surmounting the reference group 's recommendation through regulation, and indeed, the fda could conceivably regulate fmt as an hct / p in addition to regulating it as a drug, though this route may prove costly. we also considered the fda 's ability to continue exercising its enforcement discretion in an attempt to promote access to fmt for recurrent c. difficile, but this option would indeed be jeopardized in light of its prior decisions in this area. beyond regulation, though, there are other ways in which the fda could use various legal tools to minimize the social harms that granting an exclusive license to provide stool for transplantation would cause. one option would be for the fda to narrowly construe its orphan drug designation, defining the drug itself narrowly by the very specific processing methodology to which it is subject. as a result, other companies seeking to provide stool for transplantation would still need to complete clinical trials, but companies would be able to invent around rebiotix 's method. or alternatively, the fda might approve another company 's fmt preparation on the grounds of clinical superiority. as a third option, since the existing designation is limited to recurrent c. difficile infections, a product could be tested and approved for initial occurrences of c. difficile. there are still other more promising ways for the fda to regulate this new and rapidly evolving field with flexibility. perhaps the most novel would be to carefully monitor any approved company with exclusivity for the presence of a drug shortage. defined broadly as a situation in which the demand or projected demand for the drug within the united states exceeds the supply of the drug, in such an event the fda is empowered to take a range of actions, including obtaining the product from other sources. and here it is again critical to remember the ways in which fmt is not a typical drug because a company can not simply openbiome, with which one of the authors of this essay is affiliated, has curated the raw material for just 7400 treatments in its two years of existence. the fda might reasonably decide that in the event of a shortage, whether produced by an unanticipated surge in demand or barrier to supply, it would take any single corporation too long to recruit enough healthy donors and de - risk them through multiple rounds of screens for it to provide coverage alone. indeed, any shortage, whether now or in the future, would be made worse if a single company held a monopoly over stool procurement and processing. the question of how the fda should regulate fmt to prioritize the safety of the procedure while at the same time ensuring its effectiveness is a complicated one. as we have explained, trying to shoehorn fmt into the traditional drug regulatory paradigm is problematic, with the potential to both underregulate safety and overregulate access. regulating fmt as a tissue would more fully address our concerns, since so much is still unknown about fmt 's potential efficacy across a wide range of indications. a model much like the one the fda has adopted in the case of hct / ps like cord blood would seem to be the most appropriate solution. under this scenario, the fda would enforce a suite of regulations that control for the safety of fecal microbiota preparations so long as the material is only being used to treat recurrent c. difficile infection. for all other uses, the fda would require demonstrations of safety and efficacy following the path for investigational new drugs. this paradigm would grant c. difficile patients safe access to ubiquitous human stool, the data for which is available in the public domain, while at the same time encouraging scientists to study new applications for the therapy. fmt presents the fda with a new regulatory challenge, and it provides the agency with a chance for thinking critically and creatively about the most appropriate ways to oversee this new technology. the fda should seize the opportunity, as other emerging technologies like 3-d printing and mobile health may soon pose similar regulatory challenges for allowing access under medical supervision while restricting that access to evidence - based practices. we hope the considerations presented in this essay will help the fda in its endeavors to do so. | scientists, policymakers, and medical professionals alike have become increasingly worried about the rise of antibiotic resistance, and the growing number of infections due to bacteria like clostridium difficile, which cause a significant number of deaths and are imposing increasing costs on our health care system. however, in the last few years, fecal microbiota transplantation (fmt), the transplantation of stool from a healthy donor into the bowel of a patient, has emerged as a startlingly effective means to treat recurrent c. difficile infections. at present, the fda is proposing to regulate fmt as a biologic drug. however, this proposed classification is both underregulatory and overregulatory. the fda 's primary goal is to ensure that patients have access to safe, effective treatments and as such they should regulate some aspects of fmt more stringently than they propose to, and others less so. this essay will examine the nature of the regulatory challenges the fda will face in deciding to regulate fmt as a biologic drug, and will then evaluate available policy alternatives for the fda to pursue, ultimately concluding that the fda ought to consider adopting a hybrid regulatory model as it has done in the case of cord blood. |
liver cirrhosis is an ultimate outcome of irreversible damage of liver parenchyma by variety of pathological agents such as environmental toxins metabolic derangements and viral infection particularly hepatitis c virus (hcv) and hepatitis b virus (hbv) (1). liver transplantation is increasingly done as a curative treatment worldwide (2). despite this, 10% of those listed for transplantation will die while awaiting a donor liver (3). in addition, the risk of invasive surgical procedure, use of immune suppression, risk of disease recurrence and the extremely heavy cost for the remainder of life, calls for other therapeutic modalities (4). stem cells (scs) are unspecialized cells that have two defining properties : the ability to differentiate into other cells and the ability to self regenerate (5). they are thought to act as progenitors with the ability to differentiate to hepatocytes (6). bone marrow contains at least two kinds of stem cells, hematopoietic stem cells (hscs) and mesenchymal stem cells or marrow stromal cells (mscs) (7). this movement is critical for hematopoietic homeostasis and is hypothesized to contribute to homeostasis and repair in solid organs. the use of pluripotent stem cells capable of differentiation into different cell lineages provides an optimistic solution for the problems associated with transplantation. injection of bone marrow stem cells (bmscs) directly into the liver was proved to be one of the preferred routes of administration (9). this study was done to evaluate the safety, feasibility and outcome of stem cell transplantation (sct) using autologous hscs in egyptian patients with hcv induced liver cirrhosis. this study was conducted on 20 patients with hcv induced liver cirrhosis, diagnosed by clinical, laboratory and ultrasound features of liver cirrhosis. they were collected from internal medicine department and hepatology out - patient clinic, ain shams university hospitals over the whole period of a year. they were selected as late child b and child c with scores ranging from 9 to 12 according to child - pugh grading (10). one week after last attack of encephalopathy, haematemesis or infection had to pass before beginning of stem cell mobilization. these patients were on medical treatment including silymarin, diuretics and vitamin k support and continued on the same treatment after the stem cell transplantation (sct). changes in the doses of the diuretics were done according to the clinical assessment during the follow up period. they were included as control group matching group i in age, sex and child score. they continued on the conventional therapy they were on including silymarin, diuretics and vitamin k, with no stem cell transplantation done for them. exclusion criteria : patients with liver tumors, heterogenous liver ; history of other cancer ; renal impairment (creatinine > 1.5 mg / dl) ; other viral co - infection such as human immunodeficiency virus (hiv) or hbv ; life expectancy less than 3 months ; evidence of extrahepatic biliary diseases (e.g. presence of primary sclerosing cholangitis, or dilated common bile duct on ultrasound) ; presence of hepatic, portal, or splenic vein thrombosis on doppler ultrasound ; presence of severe co morbid diseases (e.g. severe renal, respiratory, or cardiac disease) and inability to give informed consent were excluded from the study. detailed history taking and full clinical examination including : body weight, body mass index (bmi), abdominal girth, vital data, general and abdominal examination. laboratory investigations including : hepatic profile including total proteins, serum albumin, total bilirubin, direct bilirubin, aspartate transaminase (ast), alanine transaminase alt, alkaline phosphatase, gamma glutamyltransferase (gt) ; bleeding profile including prothrombin time (pt), international normalized ratio (inr), partial thromboplastin time (ptt) ; complete blood picture and platelet count ; renal functions and electrolytes including creatinine, blood urea nitrogen (bun), sodium (na) and potassium (k) ; viral markers and serology including hepatitis b surface antigen (hbsag), hiv antibody, hcv antibody by enzyme - linked immunosorbent assay (elisa), and hcv ribonucleic acid (rna) by polymerase chain reaction (pcr), and -fetoprotein level (afp), done by standard laboratory tests. abdominal ultrasound with assessment of liver parenchymal echogenicity, focal lesions, liver size, spleen size, portal vein diameter and the presence and severity of ascites (mild, moderate and massive). the above investigations were done for the patients of both groups at beginning of the study and were followed up after 1, 3 and 6 months for both groups. follow up after 15 days after sct was additionally done for patients of group i. upper gastrointestinal endoscopy : for detection of esophageal varices and assessment of their grade was done at the beginning of the study and was followed after 3 months for patients of both groups. statistical analysis of the results was done using : wilcoxon signed ranks test to compare the results of patients of group i before and after the sct, and mann whitney test to compare between the results of the two groups. stem cells mobilization : the peripheral blood hsc count in normal subjects under un - stimulated conditions is very low (< 0.05% of the white blood cells). granulocyte - colony stimulating factor (g - csf) can mobilize a large number of hsc from the bone marrow (bm) into the circulation (15- to 35 fold following 4~5 days of administration). stem cells mobilization for patients of group i was done by giving them g - csf. we used gene - leukimsubcutaneous injections (filgrastim 300 g / ml / vial), in the dose of 10 g / kg / day for 4 consecutive days. another dose of 300~600 g was given to the patients on the 5 day 3 hours before the session of leukapheresis. leukapheresis : separation and collection of the peripheral blood stem cells was done in the bone marrow transplantation unit (bmt unit) (ain shams university hospital), on the day of g - csf administration using special mononuclear cell (mnc) processing machine separator (cobe spectra apheresis system, gambro bct). processing of two times the patient s blood volume was done over a period of 3~4 hours at a rate of 40~80 ml / min. in all patients, placement of a double lumen central venous apheresis catheter was necessary. blood withdrawn from the patient through the access line is mixed with anticoagulant acid - citrate - dextrose - a(acd - a) and pumped into the centrifuge channel. acd - a was administered in a ratio of 1 cm acd : 12 cm blood for prevention of extracorporeal clotting. this required oral and parentral calcium bicarbonate administration to avoid development of citrate reactions in the form of muscle cramps due to hypocalcemia. blood pressure and heart rate monitoring were done before, during and after the session of leukapheresis. assessment of stem cells : upon completion of the stem cell collection, an aliquot is taken from the apheresis bag. the total nucleated cells in this aliquot are counted on an electronic blood cell counter and the percentage of g - csf mobilized peripheral blood mononuclear cells (g - csf pb - mncs) is obtained by flow cytometric analysis. the total number of stem cells in the harvest is calculated by multiplying the percentage of g - csf pb - mncs obtained by flow cytometry the number of nucleated cells obtained by electronic cell counting and the final volume in the apheresis bag which ranged from 140~200 cc in our study. the number of stem cells in the harvests of the patients in our study ranged from 251 to 1911. follow up of platelets count and splenic size by ultrasound after stem cells mobilization by g - csf was done. stem cells injection : was performed into the hepatic artery in the following steps : after local anesthesia, puncture of right femoral artery was performed. simon catheter advanced into the descending aorta, and catheterization of celiac axis and then hepatic artery was performed. then the stem cells in the apheresis bag were transfused to the hepatic artery as equal aliquots of 50 ml, taking an average time of 20 minutes. after that, the catheter was flushed with 10 cc of normal saline and the procedure was finished. after the stem cell infusion the catheter was removed. the procedure was done in the interventional radiology department, ain shams university. all the patients received fresh frozen plasma transfusion before and after the scs injection due to prolonged prothrombin time. the patients were kept in hospital under medical observation for 48 hours after which they were discharged. following of their clinical, biochemical, radiological outcomes was performed after 15 days, 1 month, 3 months and 6 months after the sct procedure. parallel follow up of patients of group after 1 month, 3 months and 6 months was done. the follow up of patients of both groups involved assessment of the quality of life, observation of the changes in hepatic biochemical profile, child score, liver size or amount of ascites by ultrasound and body weight, development of any complications in relation to the procedure and occurrence of major side effects such as : renal failure, worsening hepatic decompensation, progressive elevation in the serum afp, or development of liver mass in the ultrasound. written consent was granted in all cases and the study was approved by the ethical committee of ain shams university. detailed history taking and full clinical examination including : body weight, body mass index (bmi), abdominal girth, vital data, general and abdominal examination. laboratory investigations including : hepatic profile including total proteins, serum albumin, total bilirubin, direct bilirubin, aspartate transaminase (ast), alanine transaminase alt, alkaline phosphatase, gamma glutamyltransferase (gt) ; bleeding profile including prothrombin time (pt), international normalized ratio (inr), partial thromboplastin time (ptt) ; complete blood picture and platelet count ; renal functions and electrolytes including creatinine, blood urea nitrogen (bun), sodium (na) and potassium (k) ; viral markers and serology including hepatitis b surface antigen (hbsag), hiv antibody, hcv antibody by enzyme - linked immunosorbent assay (elisa), and hcv ribonucleic acid (rna) by polymerase chain reaction (pcr), and -fetoprotein level (afp), done by standard laboratory tests. abdominal ultrasound with assessment of liver parenchymal echogenicity, focal lesions, liver size, spleen size, portal vein diameter and the presence and severity of ascites (mild, moderate and massive). the above investigations were done for the patients of both groups at beginning of the study and were followed up after 1, 3 and 6 months for both groups. follow up after 15 days after sct was additionally done for patients of group i. upper gastrointestinal endoscopy : for detection of esophageal varices and assessment of their grade was done at the beginning of the study and was followed after 3 months for patients of both groups. statistical analysis of the results was done using : wilcoxon signed ranks test to compare the results of patients of group i before and after the sct, and mann whitney test to compare between the results of the two groups. stem cells mobilization : the peripheral blood hsc count in normal subjects under un - stimulated conditions is very low (< 0.05% of the white blood cells). granulocyte - colony stimulating factor (g - csf) can mobilize a large number of hsc from the bone marrow (bm) into the circulation (15- to 35 fold following 4~5 days of administration). stem cells mobilization for patients of group i was done by giving them g - csf. we used gene - leukimsubcutaneous injections (filgrastim 300 g / ml / vial), in the dose of 10 g / kg / day for 4 consecutive days. another dose of 300~600 g was given to the patients on the 5 day 3 hours before the session of leukapheresis. leukapheresis : separation and collection of the peripheral blood stem cells was done in the bone marrow transplantation unit (bmt unit) (ain shams university hospital), on the day of g - csf administration using special mononuclear cell (mnc) processing machine separator (cobe spectra apheresis system, gambro bct). processing of two times the patient s blood volume was done over a period of 3~4 hours at a rate of 40~80 ml / min. in all patients, blood withdrawn from the patient through the access line is mixed with anticoagulant acid - citrate - dextrose - a(acd - a) and pumped into the centrifuge channel. acd - a was administered in a ratio of 1 cm acd : 12 cm blood for prevention of extracorporeal clotting. this required oral and parentral calcium bicarbonate administration to avoid development of citrate reactions in the form of muscle cramps due to hypocalcemia. blood pressure and heart rate monitoring were done before, during and after the session of leukapheresis. assessment of stem cells : upon completion of the stem cell collection, an aliquot is taken from the apheresis bag. the total nucleated cells in this aliquot are counted on an electronic blood cell counter and the percentage of g - csf mobilized peripheral blood mononuclear cells (g - csf pb - mncs) is obtained by flow cytometric analysis. the total number of stem cells in the harvest is calculated by multiplying the percentage of g - csf pb - mncs obtained by flow cytometry the number of nucleated cells obtained by electronic cell counting and the final volume in the apheresis bag which ranged from 140~200 cc in our study. the number of stem cells in the harvests of the patients in our study ranged from 251 to 1911. follow up of platelets count and splenic size by ultrasound after stem cells mobilization by g - csf was done. stem cells injection : was performed into the hepatic artery in the following steps : after local anesthesia, puncture of right femoral artery was performed. simon catheter advanced into the descending aorta, and catheterization of celiac axis and then hepatic artery was performed. then the stem cells in the apheresis bag were transfused to the hepatic artery as equal aliquots of 50 ml, taking an average time of 20 minutes. after that, the catheter was flushed with 10 cc of normal saline and the procedure was finished. after the stem cell infusion the catheter was removed. all the patients received fresh frozen plasma transfusion before and after the scs injection due to prolonged prothrombin time. the patients were kept in hospital under medical observation for 48 hours after which they were discharged. following of their clinical, biochemical, radiological outcomes was performed after 15 days, 1 month, 3 months and 6 months after the sct procedure. parallel follow up of patients of group after 1 month, 3 months and 6 months was done. the follow up of patients of both groups involved assessment of the quality of life, observation of the changes in hepatic biochemical profile, child score, liver size or amount of ascites by ultrasound and body weight, development of any complications in relation to the procedure and occurrence of major side effects such as : renal failure, worsening hepatic decompensation, progressive elevation in the serum afp, or development of liver mass in the ultrasound. written consent was granted in all cases and the study was approved by the ethical committee of ain shams university. this study was conducted on 20 patients with hcv induced liver cirrhosis who were divided into 2 groups. group ii : included another 10 patients of liver cirrhosis as a control group. patients of this group were male patients (100%). there was no statistical significant differences between the two groups regarding the age, sex, bmi or child score. regarding the safety and feasibility of the sct procedure, all the patients tolerated the treatment protocol well without any major complications or side effects related to the procedure. in particular, there was no bleeding, hemodynamic instability, infection or significant deterioration in liver function after the procedure. there were no cases of hepatorenal syndrome, and injection of the stem cells into the hepatic artery did not result in any thrombotic episode or bleeding. the main adverse reactions related to g - csf use noticed in the patients were the low grade fever, musculoskeletal pain, fatigue and itching for which oral paracetamol was given that improved the fever and the musculoskeletal pain. the other symptoms were mild and well tolerated and disappeared with discontinuation of the drug. although, one patient developed mild hepatic pre - coma in the form of minimal flapping tremors on the 3 day of mobilization, he improved on anticoma measures (enema/6 houres, metronidazol & lactulose) that did nt require stoppage of the sc transplantation. all the patients underwent successful leukapheresis sessions with no complications except for mild circumoral numbness to mild to moderate muscle cramps only in one patient which were handled by increasing the rate of calcium infusion and decreasing the rate of blood processing. on comparing the clinical, laboratory and sonographic parameters of group i patients before and after the sct there were statistically significant differences regarding : the serum albumin after 15 days and 1 month after the sct with p - value<0.05 (table 1). the total bilirubin after 1 month after the sct with p - values<0.05 (table 2) the gt after 1 and 3 months of the sct with p - values<0.05 (table 3) the size of the spleen after 3 months of the sct with p - values<0.05 (table 4). the child score after 1 month after the sct (p - values < 0.05), with decrease in the mean of child score from (10.51.05) to (9.71.4) after 1 month, and maintenance of the mean child score around 10 over the follow up period (table 5). on the other hand there were none significant differences regarding the body weight, abdominal girth, total proteins, prothrombin time, partial thromboplastin time, direct bilirubin, ast (table 6), alt, alkaline phosphatase, creatinine, bun, na, k, total leukocyte count (tlc), hemoglobin (hg), platelets, liver size, portal vein diameter, amount of the ascites, afp and grades of esophageal varices. comparison between the two groups regarding the different parameters showed statistically significant differences regarding : the ast after 1 month of the follow up period with lower mean and standard deviation in the patients of group i and p value<0.05 (table 6) the alt after 1 and 3 months of the follow up period with lower mean and standard deviation in the patients of group i and p value<0.05 (table 7). gt level after 6 month of the follow up period with p value<0.05 (table 3). none significant differences between the two groups were noted regarding total proteins, serum albumin, total bilirubin, direct bilirubin, alkaline phosphatase, pt, ptt, creatinine, bun, na, k, tlc, hg, platelets count, afp, liver size, portal vein diameter, size of the spleen, amount of the ascites, grades of esophageal varices and death rate (table 8). although significant difference regarding the child score between both groups was not found statistically, there was difference in the mean of child score being higher in the control group over the follow up period. as regard the sequelae of liver cirrhosis, there were no significant differences between the two groups regarding the number of attacks of hematemesis, spontaneous bacterial peritonitis (sbp), hepatic encephalopathy, development of hepatocellular carcinoma (hcc) over the follow up period. one patient (10%) who was subjected to the sct developed hepatic focal lesion consistent with hcc after 4 months of the sct for which conservative therapy was given (child c) and the patient died 6 months later. another patient (10%) who was subjected to the sct developed suprarenal mass after 6 months after the sct. on the other hand two patients (20%) of the control group developed hcc over the follow up period. much has been written and rewritten on stem cells (scs), their potentials, their properties, their possible uses and their risks (5). sc - based therapies could be used to cure inherited or genetic degenerative alterations associated with the loss of adult sc functions, such as cancers, immune system and hematopoietic disorders, cardiovascular, muscular and neurological diseases, gastrointestinal pathologies, diabetes and chronic hepatopathies. the manipulation of adult stem cells (ascs) seems to be particularly promising, as it could improve the endogenous regenerative potential without risk of rejection and overcome the ethical and political issues related to embryonic stem cell research (11). preliminary experience with clinical hepatocyte transplantation during the past decade has provided proof of concept that cell therapy can be effective for the treatment of some liver diseases. recent progress in cell biology resulting in the isolation and characterization of bone marrow stem cells and progenitor cells further increases the expectation for a new approach to the treatment of genetic and chronic liver disease (12). we hypothesized that infusion of hscs may help to reverse liver failure in patients with decompensated cirrhosis manifested by improvement of the child score. thus, we conducted a human trial to evaluate safety and feasibility of autologous hsc transplantation in egyptian patients with decompensated cirrhosis and to evaluate the outcome of hsc transplantation by assessing changes in, hepatic biochemical profile, child score, liver size by abdominal ultrasonography and quality of life of the patients over the follow up period. it is important to note that the patients of liver cirrhosis could respond to g - csf treatment and that their white blood cell counts were increased in all cases, because this is a prerequisite for the remainder of the treatment protocol. also the patients tolerated well the sessions of leukapheresis with minimal side effects as have been mentioned in the results. this goes with agreement with yannaki., who initiated a pilot study to assess the safety and efficacy of boost infusions of mobilized peripheral blood stem cells (pbscs) in patients with advanced - stage alcoholic liver cirrhosis. each of the two patients included in their study, safely underwent three rounds of mobilization, which were well tolerated in general (13). in this study, there was improvement in the quality of life of all the studied patients especially over the first two months after the procedure., in which there was improvement in the quality of life of three patients of the four patients included in their study at the end of follow up (14). in the current study, there was significant improvement in the mean serum albumin noticed after 15 days and 1 month after sct in group i patients with improvement of the serum albumin level of about 0.5 gm / dl. this goes with results of the trial of gordon., in which 4 of the 5 patients included in their study showed improvement in the serum albumin of an average of 0.4~0.5 gm / dl (15). although there was none significant statistical difference between the two groups regarding the serum albumin, the results of control groups were lower than the group i patients on the individual level reflecting the stem cells transplantation potential to improve the synthetic function of the diseased liver when compared to conventional treatment. at this stage, the mechanism of the effect on liver function is not fully understood but may reflect activation of genes corresponding to hepatocyte differentiation program in the transplanted stem cells upon exposure to the injured liver environment. as regard the serum total bilirubin, there was statistically significant improvement in its level in patients of group i after 1 month after sct. although the direct bilirubin, showed none significant differences between its level before and after the sct in patients of group i, analysis on the individual level showed decrease in its levels in two patients of about 0.6~1 mg / dl over the first month and in another two patients over the six months of the follow up period., in which four of the five patients showed improvement in serum bilirubin which was maintained for up to 6 months (15). as regard the liver enzymes (ast and alt) there was statistically none significant difference in their levels before and after sct in patients of group i, but there were statistically significant differences between the two groups regarding the alt level with significant lower levels in the patients of group i after 1 and 3 months after the stem cells transplantation. also there was statistically significant difference as regard the ast level between the two groups after 1 month of the follow up period. the decrease in the liver enzymes levels parallel the decrease in the inflammatory process and can be related to the regenerative properties of the stem cells. -gt showed statistically significant decrease in its level in patients of group i after 1 and 3 months after the sct. also there was statistically significant difference between the two groups regarding the -gt after 6 months after sct with lower levels in the patients of group i which can be explained by the improvement of hepatic inflammation. the pt and inr which are important liver function tests that reflect the functional status of the liver showed none significant changes in their levels before and after the sct in patients of group i over the follow up period. analyzing the results on the individual levels showed minimal and transient decrease in the level of the pt and inr for less than 1 month, which may be due to plasma transfusion and intravenous vitamin k injections, the patients received before and after the procedure for fear of bleeding tendency. comparison between the two groups regarding the pt and inr showed none significant difference in their levels. our results regarding the pt and inr are different from those of mohamadnejad., who published results showing improvement in the pt in 2 of 3 patients in their study (14). ultrasound examination was done over the follow up period and revealed none significant differences regarding the liver size before and after the sct. also, there were none significant differences between the two groups regarding the liver size. one of our expectations was the increase in the liver size due to the regenerative properties of the stem cells that was not proved in our study. this may be due to the patients advanced stage of liver cirrhosis (child c stage) and suggests the possibility that sct in earlier stages may have better outcome. the undetected changes in the liver size may also be due to lower potentiality of the abdominal ultrasound to detect the minor differences in the liver size as compared to the computerized tomography (ct) scan and magnetic resonant imaging (mri) which are more accurate but are more expensive modalities with radiation and contrast risks of the ct. g - csf can induce spleen enlargement in peripheral blood cell donors and patients with hematologic malignancies (16), but this increase in spleen size very rarely may result in splenic rupture (17). theoretically, the administration of g - csf in patients with splenomegaly, as in cirrhotic patients, could increase the risk of splenic rupture. the splenic longitudinal diameters of patients of group i ranged from 16 cm to 22 cm before the g - csf administration. following up the size of the spleen after 15 days after the procedure in those patients showed statistically none significant difference. fear of rupture spleen related to the use of g - csf in patients of liver cirrhosis and splenomegaly was excluded in our study. this goes with the results of gaia., who observed a significant reversible increase of splenic longitudinal diameter with no adverse event occurred in relation to g - csf (18). they reported that splenomegaly up to 17 cm does not prevent safe bone marrow cell (bmc) mobilization following g - csf in patients with end stage liver disease. the same conclusion was reached in the current study even in larger splenic sizes reaching 22.4 cm. in a study done by yannaki., g - csf caused an increase in the spleen volume by 49% and 19.5% as compared to baseline value, in two of the three mobilization rounds in one patient, whereas the other patient had a 27% increase during the second round (13). it is possible that in conditions of chronic splenomegaly, the spleen becomes more flexible so that g - csf priming may not result in abrupt changes in splenic size. in contrast, in a previously normal - sized spleen, g - csf could cause striking enlargement over a short period of time, thus increasing the rupture potential. consistent with this hypothesis, it has been shown that the median increase in spleen volume of mobilized healthy donors was significantly greater than mobilized patients with hematologic malignancies (85% versus 57%) (16). there were none significant differences regarding the amount of the ascites of patients of group i before and after the sct, as assessed clinically by abdominal circumference measurement, body weight and abdominal sonar. this may be due to the complex pathophysiological mechanisms involved in the formation of the ascites, which were not reversed by the minimal improvement of the serum albumin level in the patients of group 1 after sct. on the other hand, terai., found that ascites improved after autologous bone marrow infusion (abmi) therapy in their study which included 9 patients (19). as regard the child - pugh score, there was statistically significant decrease in the score. one patient showed sustained decrease in the child - pugh score from 10 to 9 over the whole follow up period. the same occurred with another patient whose score decreased from 9 to 8 for more than 3 months. although there were statistically none significant differences between the two groups regarding the child - pugh score, the mean values of the child score were lower in patients of group i and continued to be inner the last range of disease state. in the study of mohamednejad, one of the 4 patients showed improvement in the model for end stage liver disease (meld) score(14). another patient showed worsening of the meld score at the end of the follow up period. in a study made by terai., nine liver cirrhosis (lc) patients who received autologous bone marrow cell infusion (abmi) from the peripheral vein showed significantly improved child - pugh scores at 4 and 24 weeks (19). we found that sct has not significantly affected the incidence of encephalopathy, hematemesis and sbp on comparing the two groups. upper endoscopy showed none significant differences in the esophageal varices grades after 3 months after the sct. one of the patients who received sct developed hcc after 4 months of the sct, while another patient developed suprarenal mass after 6 months after the sct. it is suggested that embryonic stem cells (escs) may give rise to tumors, while cancers derived from adult stem cell (asc) therapies havent been reported. nonetheless, the long - term safety of asc infusion has not been adequately tested. preclinical studies and clinical trials with longer follow up periods should be recommended prior to large - scale clinical applications of such cell - based therapies (5). five patients of group i died within the first year of sct on top of intractable attacks of hematemesis or hepatic coma. on the other hand, six patients of the control groups died within the first year of the follow up with none significant difference between the two groups regarding the death rate. hematopoietic stem cell transplantation for patients of liver cirrhosis on top of hepatitis c virus infection is a safe and feasible procedure. it can improve the quality of life of cirrhotic patients, the hepatic functions such as serum albumin, bilirubin and also the child score. this improvement is transient and lasts for about 3 to 6 months, or longer periods up to 12 months in other published studies. it did nt affect the incidence of occurrence of complications related to the liver cirrhosis. this means that sct can be used as a bridging therapy till liver transplantation, which is the curative treatment, is available. | background and objectivesuse of pluripotent stem cells is an ideal solution for liver insufficiencies. this work aims is to evaluate the safety and feasibility of autologous stem cells transplantation (sct) in egyptian patients of liver cirrhosis on top of hepatitis c virus (hcv).subjects and results20 patients with hcv induced liver cirrhosis were divided into 2 groups. group i : included 10 patients with liver cirrhosis child score 9, for whom autologous stem cell transplantation was done using granulocyte colony stimulating factor (g - csf) for stem cells mobilization. separation and collection of the peripheral blood stem cells was done by leukapheresis. g - csf mobilized peripheral blood mononuclear cells (g - csf pb - mncs) were counted by flow cytometry. stem cell injection into the hepatic artery was done. group ii : included 10 patients with hcv induced liver cirrhosis as a control group. follow up and comparison between both groups were done over a follow up period of 6 months. the procedure was well tolerated. mobilization was successful and the total number of g - csf pb - mncs in the harvests ranged from 25106 to 191106. there was improvement in the quality of life, serum albumin, total bilirubin, liver enzymes and the child - pugh score of group i over the first two - three months after the procedure.conclusionsct in hcv induced liver cirrhosis is a safe procedure. it can improve the quality of life and hepatic functions transiently with no effect on the life expectancy or the fate of the liver cirrhosis. |
neurofibromatosis type 1 (nf1) is an autosomal dominant disorder caused by germline mutation of the nf1 gene. the nf1 gene encodes the tumor suppressor gene neurofibromin, and patients with nf1 are at an increased risk of developing various types of tumors, either benign or malignant. however, there are few reports of nf1 associated with colorectal cancer. we present the case of nf1 in a 67-year - old patient with rectal cancer. a 67-year - old man with a history of nf1 presented with hematochezia for 2 months. he had been diagnosed as having nf1 40 years earlier and had no family history of nf1. his skin showed numerous caf au lait spots and cutaneous neurofibromas over the whole body (fig. an ulcerofungating mass was palpated at 5 cm above the anal verge on the posterior side of the rectum on manual rectal examination and was observed at colonoscopy (fig. the tumor was confirmed as an adenocarcinoma by colonoscopic biopsy. the abdomino - pelvic computed tomography (ct) scan revealed a 4.5-cm - long, irregular rectal wall thickening at the distal rectum, with a few small perirectal lymph nodes. the pathologic stage of the tumor was stage iii (t3n1), and he received concurrent chemoradiation (6 cycles of 5-fu / leucovorin chemotherapy and 5,040 rad of radiation). after completion of chemotherapy, he was followed up on 3 months interval. at 12 months after the operation, the positron emission tomography (pet)-ct scan showed increased fluorine-18 2-fluoro-2-deoxy - d - glucose uptake (suvmax = 5.1) at the anastomotic site (fig. 3). a 2-cm polypoid mass was found by colonoscopy and was excised transanally (fig. the tumor was composed of spindle cell fascicles of moderately atypical cells intermixed with a scanty amount of lymphoplasma cells (fig. the tumor cells were positive to anaplastic lymphoma kinase and vimentin and were negative to cd34, cd68, s-100, sma, and desmin (fig. nf1 also known as von recklinghausen disease, is an autosomal dominant disorder that affects one in 3,000 live births. the most frequent clinical manifestations are skin pigmentation, iris lisch nodules, and multiple cutaneous neurofibromas. nf1 reduces the average life expectancy by 10 to 15 years, and malignant tumors are the most common cause of death in individuals suffering from this syndrome. malignant neoplasms in nf1 occur with an incidence of 3 to 15% and can arise in the nervous and the non - nervous systems in either childhood or adulthood. malignant peripheral nerve sheath tumors and central nervous system tumors, such as the optic nerve glioma or astrocytomas are the most common tumors associated with nf1.. gastrointestinal stromal tumors, somatostatinomas, breast cancer, and pheochromocytomas are more common in adults with nf1 than in those without the syndrome. development of epithelial malignancies is very rare, and the ontogenic relationship is not apparent. adenocarcinomas in nf1 patients have been reported in the stomach, duodenum, small bowel, colon, and pancreas. in a 12-year follow up study in the swedish nf1 population, 12 adenocarcinomas out of 70 patients (18%) were developed. the authors stated that the incidence of adenocarcinomas seems to be high and that an adenocarcinoma is an important risk factor in nf1 patients. however, estimates of the frequencies of specific tumors in nf1 patients are difficult, and the incidence might be overestimated because most cohort studies are based on hospital data. in the literature, there are only six case reports that indicate an association of nf1 with an adenocarcinoma of the colon [4, 5 ]. nevertheless, considering that both nf1 and colon cancer are relatively common diseases, their concomitant occurrence is probably the result of a chance association. in a mortality study using us death certificates, colon cancer was not listed as often on the death certificates of persons with nf1 as on those of the general population. although malignancy constitutes an important cause of both morbidity and mortality in nf1, the mechanism underlying tumorigenesis remains obscure. neurofibromin, the protein encoded by the nf1 gene, functions as a gtpase - activating protein for ras by catalyzing the hydrolysis of active ras - gtp to inactive ras - gdp. the nf1 gene may, thus, be considered as a tumor suppressor gene, and the high incidence of some types of neoplasms in nf1 patients may be related with mutation of the nf1 gene. it has been suggested that mutation of the nf1 gene can also occur in somatic cells and that loss of heterozygosity of the nf1 gene can be found in some sporadic colorectal cancers. one might expect that the tumor type found occasionally to harbor somatic mutations in the nf1 gene would have an increased incidence in nf1 patients. however, colon cancer does not seem to have an increased incidence among nf1 patients. there is a precedent for this paradox among hereditary neoplastic syndromes. somatic mutations of the rb gene are found frequently in sporadic small cell lung cancer, yet this tumor does not have an increased incidence among individuals with hereditary retinoblastoma. these considerations suggest that the nf1 mutation might promote growth of both the nf1-associated tumor type and other types of tumors by a somewhat different mechanism. in addition to the uncommon concurrence of the two diseases, nf1 and cancer, there are some concerns about managing nf1 patients with cancer. some nf1 patients have coexisting clinical problems that may complicate the clinical presentation, possibly delaying diagnosis and resulting in consequent presentation of the disease at a late stage. multiple primary tumors, synchronous or metachronous, are more common in nf1 patients with cancer. the initial cancer staging and the postoperative surveillance of nf1 patients may also be complicated by the presence of other benign or malignant lesions, visualized on imaging scans or identified on physical examination. in this case, an imft was mistaken as an anastomotic recurrence of the rectal cancer on both pet - ct and colonoscopy. therefore, one should consider the possibility for synchronous or metachronous development of other types of tumors, besides tumor recurrence, when some tumors are found during follow up of nf1 patients with cancer. in summary, we report an unusual case of rectal cancer in a patient with neurofibromatosis type 1. this case shows that a slightly different interpretation is needed for findings of initial staging and postoperative surveillance because multiple primary tumors are common in nf1 patients. | a rectal cancer was found in a 67-year - old man with a history of neurofibromatosis type 1. a low anterior resection was performed, and he received concurrent chemoradiation for 6 months. twelve months after the surgery, a tumor was found at the anastomotic site by positron emission tomography - computed tomography and colonoscopy and was mistaken as anastomotic site recurrence. the tumor was confirmed as an inflammatory myofibroblastic tumor through transanal excision. |
frostbite represents a spectrum of tissue injury, ranging from superficial insult to deep tissue freezing. although it is not usually associated with mortality, there is a high level of morbidity that can occur with severe frostbite with potential loss of digits or limbs. historically, frostbite has affected military personnel ; however, the incidence of frostbite in the military population is declining, as shown in a 2010 study by hall., who found only 19 reported cold injuries, two of which were frostbite, in a retrospective review of over 18,000 patients identified by trauma registry, compared to 6,300 cases of cold weather injury seen previously in the korean war. the epidemiology of this condition has changed over the past twenty years with greater involvement of the civilian population. there is no formal reporting system for frostbite cases and the incidence of frostbite in the general population has not been widely studied. a 2009 cross - sectional study performed in finland sent out a cold questionnaire to the general population. subjects were randomly chosen from a pre - existing nationwide health survey system from all over finland in the years 1997 and 2002. of 13,713 participants ranging from ages 25 - 74 years, 697, or 5.1% sustained some type of frostbite. in another cross - sectional finnish study of male military recruits aged 17 - 30, the prevalence of frostbite was 2555/5839, or 44% with an annual incidence of 2.2%. the incidence of frostbite was also examined in a high risk population, mountaineers, and found to be 366/1000 mountaineers per year. frostbite or damage to tissues from exposure to temperatures below their freezing point, can occur at -2c (28f). risk factors for frostbite injury in the civilian population include prolonged exposure, decreased ambient temperature, inadequate clothing, smoking, peripheral vascular disease, diabetes, raynaud 's disease, sepsis, previous cold injury, concomitant use of alcohol or drugs, psychiatric illness, dementia, trauma, vehicular breakdown, and homelessness.[68 ] until the early 1990s the goal of frostbite treatment was to preserve viable tissue and prevent infection, with rapid thawing, daily wound care, antibiotics as indicated, and watchful waiting being the mainstays of therapy. the treatment of frostbite had not progressed until the introduction of tpa for limb and digit salvage in the 1990s. the following case report outlines a case of frostbite treated with tpa at our institution. a 59-year - old woman was found outside her home by a neighbor. the average temperature that day was -7f. the patient was confused initially, and gave various stories to different providers about how she came to be outside and exposed for a prolonged period of time. she was unable to provide a complete medical history ; however, she was able to state she had a history of depression, anxiety, and migraines, and medications included alprazolam and sumatriptan as needed. examination revealed mild hypothermia (temp 34.2c) and cold, mottling and woody induration of all ten digits of the hands. after re - warming, her hands became purple and swollen [figures 1 and 2 ]. left hand after rewarming right hand after rewarming multiple imaging and laboratory studies were obtained in the ed. pertinent studies included a normal head ct, a wbc count of 17.5k, and a ck of 441. her mental status improved, and although unable to recall details of her exposure, she thought she had been outside longer than 2 hours. the surgery team was consulted regarding the patient 's frostbite and she was admitted to the burn service. a bone scan was urgently obtained which revealed : (1) no perfusion identified within the left third through 5 digits and right 4 and 5 digits, and (2) small amount of uptake at the base of the right first 3 digits and left first and second digits [figure 3 ]. intravenous (iv) tpa was given as a 0.15 mg / kg bolus followed by an infusion of 100 mg at 0.15 mg / kg / hr. after tpa, a heparin drip at 12 units / kg / hr was administered for 36 hours. a repeat bone scan was performed two days after admission, showing increased perfusion in the first through 4 digits bilaterally to at least the proximal interphalangeal joints, and in the 5 digits bilaterally, more modestly improved flow to the mid proximal phalanx level [figures 4a and b ] there is no perfusion identified within the left 3 through 5 digits and right 4 and 5 digits, and a small amount of uptake at the base of the right first 3 digits and left 1 and 2 digits (a - b) : bone scan after thrombolytic therapy showing increased perfusion in the first through 4 digits bilaterally to at least the proximal interphalangeal joints, and in the 5 digits bilaterally, more modestly improved flow to the mid proximal phalanx level after a twelve day hospital stay, the patient was discharged to a nursing facility to allow demarcation of her injuries. the patient returned to the hospital approximately one month after her initial presentation for amputation of mummified tissue. all ten fingers had amputation : bilaterally, her index, middle and ring fingers were amputated to the level of the proximal phalanx. the treatment of frostbite injury had been stagnant for many years prior to the introduction of thrombolytic therapy. the mainstays of treatment had been re - warming, local wound care, watchful waiting, and then progression to either recovery or amputation. other investigations into anti - thrombotics, dextran, and hyperbaric oxygen therapy have not been fruitful.[911 ] in frostbite, two elements, tissue freezing and reperfusion, contribute to the damage seen in frostbite patients. parts of the mechanism of injury are not clearly understood.first, tissue freezing leads to crystallization of the extracellular space with increase of extracellular oncotic pressure, causing fluid shifts out of the cells. this causes cellular dehydration and disruption of intracellular metabolism and instability of the cell membrane. vasoconstriction, ice crystal formation in the plasma, and increased blood viscosity with subsequent microvascular damage leads to an inflammatory response. this begins a cascade of events that ends with thrombosis, tissue ischemia, edema, and endothelial injury. early thrombolytic therapy could limit microvascular thrombosis and help prevent reperfusion injury. with thawing, further endothelial damage and lysis of cells occur, with release of inflammatory cytokines and further development of microvascular clot and edema. the initial examination of the frostbitten extremity may reveal a range of severity of injury from superficial (erythematous area around a white center) to deep (hemorrhagic blisters or full thickness tissue necrosis). the final demarcation of viable tissue is hard to determine at this stage. at this time, some supportive measures can be started, including addressing hypothermia, pain management, iv hydration, and consideration of transfer to a higher - level facility. rapid transport to a facility capable of giving tpa is of utmost importance ; it may be the re - warming needs to be done there. the decision to proceed with thrombolysis of frostbitten extremities is typically based on results of technetium-99 m triple - phase bone scanning or angiography. findings on physical exam that should prompt further evaluation by one of these two methods include absent capillary refill, absent doppler pulses, purple discoloration and hemorrhagic blisters. bruen,. published a 2007 retrospective review of patients at their institution who received ia therapy for severe frostbite. they compared patients treated with ia tpa starting in 2001, (when they implemented their protocol), to historical controls from 1995 - 2001 as well as more recent patients presenting greater than 24 hours after injury. patients were given ia tpa if they had evidence of significant perfusion defect on tc - scintigraphy or distal angiography and had no contraindications for tpa. they reported a 10% amputation rate among those treated with ia tpa within 24 hours of injury (vs. 41% in those not receiving tpa). the authors reported one complication in the patients who received ia tpa, with one patient developing a retroperitoneal hematoma that was managed non operatively and resolved without further issue. bruen recommends dosing as follows : (1) ia catheter(s) are placed, (2) an initial bolus of 2 - 4 mg is given, (3) a maximum infusion of 1 mg / hr is started ; this infusion dose is divided amongst the catheters (e.g., two extremities involved = 0.5 mg / hr per extremity, etc.). tpa is continued until there is evidence of tissue reperfusion, 48 hours has passed, or the attending surgeon and interventional radiologist feel there is no further therapeutic gain by continuing the infusion. more recently, adding a vasodilator to ia tpa has been suggested. in 2005, twomey. published a non - randomized prospective trial with historical controls to assess the safety and efficacy of tpa for severe frostbite. patients received either ia tpa (6 patients) or iv tpa (13 patients), and were included in the study if they had no improvement with re - warming, absent doppler pulses in limbs and/or digits, and no perfusion on technetium 99 m three - phase bone scan, and no contraindications to tpa. the authors reported of 174 digits at risk in 19 patients treated with tpa ; part or all of 33 digits were amputated (10 fingers and bilateral bkas in one patient with 60 hours of cold exposure with repeat freeze - thaw cycles). no complications were reported in the iv tpa group. in the ia group, two patients had reported complications, where one patient had bleeding at arterial puncture sites requiring premature cessation of therapy and the other developed hematuria that required cessation of therapy. overall, this study showed safety and efficacy of tpa in a small number of patients, with equivalent results in iv and ia patients. two patients who received ia tpa patients had complications. ultimately the authors recommend tpa at dosing of 0.15 mg / kg bolus with a 0.15 mg / kg / hr infusion over 6 hours up to a maximum of 100 mg and heparin therapy (defined as ptt twice control values) for 3 - 5 days, and aspirin therapy for several weeks after discharge. contraindications to use of tpa in the bruen study included superficial frostbite, involvement of the tips of the distal phalanges, concurrent trauma, neurological impairment, recent surgery or hemorrhage, or bleeding diasthesis. contraindications to the use of tpa in the twomey study included : severe hypertension, recent trauma, stroke, or bleeding disorder, pregnancy, mental incapacity, drug or alcohol intoxication, repeated freeze - thaw cycles, or more than 48 hours of cold exposure. however, the outer range of tpa administration after exposure is not clear. in the bruen study, one patient received tpa treatment approximately 48 hours after exposure with no improvement in blood flow. the remainder of their patients treated with tpa were treated within 24 hours after exposure. in the twomey study, the authors found that patients that did not respond to tpa were those with greater than warm ischemia time greater than 6 hours, more than 24 hours of cold exposure, or those with multiple freeze - thaw cycles. based on these two studies, most of the literature now recommends tpa be given within 24 hours of exposure. although our patient 's bone scan did show improvement after tpa and heparin infusion, her digits were ultimately unable to be salvaged. further discussion with family revealed that she had likely been exposed for a much longer period of time, possibly 7 or 8 hours. iv tpa shows promise for a condition that has had no advances in treatment for decades. evidence is limited but suggests iv tpa is an effective treatment, and possibly the only opportunity for digit and limb salvage. in the ed, re - warming the affected digits in a 40c water bath along with concomitant treatment of hypothermia or co - morbid trauma and medical conditions access to a facility with bone scanning or angiography and the ability to monitor a tpa infusion is important and should be considered early in the disposition decision. the risk / benefit ratio must be considered before starting tpa empirically prior to any bone scan or angiography. consultation with a plastic / burn or general surgeon is encouraged prior to transfer in questionable cases. our facility recommends transfer to a burn center for new severe frostbite, danger of skin or limb loss, or if the transferring facility feels uncomfortable caring for the patient. based on limited existing literature, patients who are eligible for tpa should have less than 24 hours of cold exposure, no evidence of multiple freeze / thaw cycles, show no improvement with rapid re - warming in tepid water, absent doppler pulses in limbs and/or digits, and absence of perfusion on angiography or technetium-99 m triple - phase bone scanning, with no indications to tpa treatment. patients should be able to consent for treatment or have a representative able to consent for them. iv tpa appears to be equally as effective as ia tpa with a lower complication rate. | until recently, the treatment of frostbite injuries has been limited to supportive care only, with mediocre outcomes. the use of thrombolytic therapy has been presented in a limited fashion in the literature since 2005. this case study describes the work - up and treatment of a patient with severe frostbite injury who received tpa. we then discuss thrombolytic therapy in more detail, with particular attention to the two studies outlining different treatment regimens. |
the use of gene targeting with homologous recombination in murine embryonic stem (es) cells has led to many mechanistic insights about human diseases. however, global gene disruption has two major limitations that may prevent the identification of gene function in a target tissue or in adults. second, disruption of a ubiquitously expressed gene may not yield mechanistic insights regarding the function of a protein of interest in a particular cell type [2, 3 ]. in these scenarios, temporal or / and spatial gene disruption is far more advantageous. the seminal work on the utilization of bacteriophage p1 site - specific recombination system in mammals by dr. brian sauer and his coworkers [4, 5 ] established a firm foundation for the cre / lox - based gene targeting, which is the most widely used conditional gene targeting approach to date. cre recombinase is a 38 kda protein and belongs to the integrase family of recombinases. biochemically cre catalyzes site - specific dna recombination, both intra- and intermolecularly, between the 34 base pair loxp sites. cre carries a eukaryotic nuclear targeting sequence and is efficient in performing site - specific dna recombination in mammals. therefore, cre / lox system has become the primary choice for the site - specific dna recombination - based manipulation of the mouse genome. efficient cre - mediated excision of dna between directly repeated loxp sites has been widely used in gene activation and deletion of small or large segment of chromosomal dna [911 ]. cre - mediated recombination also permits the translocation of large dna fragments on chromosomes and integration (knock - in) or replacement of a gene or dna segment [1315 ]. conditional gene knockout is by far the most widely used application of cre - mediated site - specific recombination. the use of this strategy in retinal degeneration studies will be the focus of this paper. in addition to the general strategy of cre / lox gene targeting, this review will address various factors influencing the outcomes of conditional gene targeting studies, limitations of current technologies, availability of cre - drive lines for various retinal cells, and issues related to the generation of cre - drive lines. finally, this paper will update the current status on the use of cre / lox - based gene targeting approach in mechanistic studies for retinal degeneration, including the two most advanced areas, rod photoreceptor survival under photo - oxidative stress and protein trafficking in photoreceptors. cre / lox conditional gene targeting requires a mouse that has been pre - engineered with a loxp - flanked gene (or gene segment), generated with homologous recombination in murine es cells (figure 1). as the loxp sites are placed in introns, this engineered mouse is phenotypically wild type. a conditional gene knockout mouse is generated by breeding this mouse with a mouse that expresses cre under the control of a tissue - specific promoter for two generations (figure 1). in the conditional gene knockout mouse only cells / tissues that express cre carry the deleted gene, and thus they are phenotypically mutants (figure 1). in this way, one can analyze the gene function in cre - expressing tissues without affecting the gene expression in nontargeted tissues. one concern regarding the use of conditional gene targeting in vivo is that the cre - mediated excisive recombination is usually not 100 percent. it is important to understand that there is a fundamental difference between cre - mediated gene disruption and conventional gene knockdown. as only four cre molecules are required for a productive cre - mediated recombination, cre - mediated gene disruption occurs usually in an all - or - none fashion in a particular cell. a most likely scenario for a 20 percent efficiency of cre - mediated recombination is that approximately 20 percent of targeted cells have 100 percent gene knockout. this characteristic has made cre / lox - based gene targeting a useful approach in gene function analysis, even though it is rare that transgenic cre mice express the recombinase in all targeted cells / tissues. since most gene function studies are targeting the effect of gene inside the cells, a fraction of targeted cells with gene deletion could produce stable phenotypic changes in animals [44, 45 ]. however, in a scenario that no phenotypic change is observed in animals that have a small portion of targeted cells carrying cre - mediated gene disruption, the interpretation of data needs to be cautious. another misconception in designing conditional gene targeting studies is that a complete cre - mediated excision is more desirable. this is not always true, particularly, in a situation that cre may have toxic effect to the cells or phenotypic changes are too strong to be characterized. in a previous study, we intentionally used a rod - expressing cre line with a lower efficiency of cre - mediated recombination to avoid unnecessary complication derived from potential cre toxicity in rods, as observed by others. in a scenario that conditional gene targeting results in a massive or / and rapid phenotypic change that hampers the understanding of the biology and diseases, a lower level of cre expression in targeted tissues / cells may produce a genetic mosaic that attenuates the development of pathological changes in animal models. although cre can be exogenously delivered to a targeted tissue, it is usually expressed under the control of tissue / cell specific promoters. a critical factor for a successful conditional gene inactivation study table 1 includes a list of published cre - expressing drive lines for various retinal cells. since most retinal degeneration studies are related to the photoreceptors and rpe, all published rod-, cone-, and rpe - expressing cre mouse lines are listed in table 1. retinal mller glia is the major supporting cell and plays a critically role in maintaining structural and functional integrity in the retina under stress conditions. as most cre - drive lines for mller glia were usually developed for brain and cre expression occurred outside ocular tissues in these mice, table 1 only lists a few that either have been characterized more thoroughly or have been shown to be successful in conditional gene targeting in the retina [3, 47, 48 ]. degeneration of retinal ganglion cells (gcs) is becoming a focused research area for their role in glaucoma and for the relevance to the safety of treating amd patient with anti - vegf strategies. a number of characterized gc - expressing cre - drive lines are thus listed in table 1. while inner nuclear layer (inl) neurons are not often investigated for retinal degeneration, they are retinal neurons. the cre - drive lines for inl neurons can be used for studies related to retinal neurobiology and are listed in table 1. finally, cre - drive lines that are expressed in almost all retinal neurons are also listed in table 1. it is worth noting that some of the listed cre - expressing mouse lines were originally designed to trace cell lineage and had strong developmental cre expression. although some promoters employed for cre expression are useful in circumventing embryonic lethality, due to their ubiquitous expression they can not be utilized to study a tissue / cell type - specific gene function. for most retinal cell - types, it is important to know that these seemly redundant cre - drive lines are necessary. as most published cre - drive lines derived from the same or similar promoters are not identical, it is ideal to have several usable cre - drive lines for a particular cell - type due to the following considerations. first, a range of cre expression levels provide choice to achieve a suitable degree of gene inactivation for a particular study. second, variable ecotopic expression patterns between the cre - expressing lines may produce unintended phenotypes that may be beneficial. there is a 5 percent of possibility that cre may be residing on the same chromosome where a loxp - flanked gene is localized. having more than one cre - drive line for a targeted tissue / cell - type is likely to provide a choice for the successful generation of a conditional gene knockout mouse therefore, publishing a cre - drive line for a particular cell - type with already established drive lines should be encouraged. since there have not been many side - by - side studies comparing different cre - drive lines as performed by ivanova. recently, it is not possible to give an accurate account of the differences among cre - drive lines that target a particular cell - type. this review only provides a roadmap about the available resources. to select the most desirable cre - drive line, end users should perform side - by - side comparison, if necessary. while the traditional transgenic approaches have proved to be useful for generating cre - drive lines, the inherent problems associated with this approach may cause variability in mutant phenotypes among animals. this variability sometimes may result in unintended expression pattern that may or may not be useful for other studies. the use of knock - in or bacterial artificial chromosome based transgenic approaches is likely to produce cre - drive lines with the expression patterns that more closely resemble the characteristics of the promoters. in addition, the variability in cre expression among animals can be reduced using these transgenic approaches. for these reasons, the cre - drive lines referenced in table 1 also provide information on how these cre - expressing mice were generated. it is important to keep in mind that a cre - drive line generated with a knock - in approach may affect the expression of the native gene and careful phenotyping of cre - expressing mice are necessary. table 1 also includes information about whether cre - expressing lines are generated using an inducible promoter system such as tetracycline- or tamoxifen - inducible systems [51, 52 ]. while inducible tissue-/cell - specific gene knockout approach is more advantageous, there are inherent problems associated with these systems, such as leakiness [53, 54 ]. efficient delivery of inducing agents to the targeted retinal cells at the peak of promoter activity is the key to the success of inducible cre expression. although inducing gene expression in a tetracycline - inducible system with doxycycline for a short period of time may not be harmful to the retina, one should always keep in mind that tamoxifen may be toxic to the retina. one distinctive advantage of using inducible systems is their ability to turn off / down the expression of cre, which may be toxic to the targeted cells [19, 21 ]. cre is a dna recombinase and may cause unintended chromosomal rearrangement at cryptic sites [57, 58 ]. proper control of cre expression is required for cre - drive lines and a careful phenotypic analysis of cre - drive lines is a prerequisite for conditional gene targeting. however, the cre toxicity may not be the only contributing factor that caused retinal denegation in cre - expressing rod - specific cre mice [19, 21 ]. as expression of human rhodorpsin - gfp fusion, a nontoxic protein, also caused progressive rod photoreceptor degeneration, it is likely that a high level of expression of an exogenous protein may be toxic to the host protein transcription / translation / maturation system in rods. for the past decade or so, many laboratories have contributed considerable effort in establishing various cre - drive lines. while cre - expressing mice have been used successfully in conditional gene targeting, there are not sufficient cre - drive lines, even for the most advanced field, photoreceptor biology. due to a high level of cre expression causes rod degeneration, it would be ideal to have at least one inducible cre - drive line for rods. as there are at least fifty types of retinal neurons, the current list (table 1) is far from completion. however, for most retinal cell - types, a major shortcoming of most currently available cre - drive lines is a lack of temporal or spatial specificities and desired efficiencies. a major challenge for cre / lox - based conditional gene targeting is the difficulties to obtain cre - drive lines with desired tissue - specificities. therefore, it is worthwhile to invest some effort on studying the expression pattern of potential promoters that drive cre expression before making a mouse. as many of the survival factors are essential for development, global disruption of these essential genes often causes embryonic lethality. using cre / lox - based conditional gene targeting approach, haruta. demonstrated that rac1, a component of nadph oxidase that produces reactive oxygen species, was required for the rod photoreceptor protection from photo - oxidative stress. to determine photoreceptor survival mechanisms under photo - oxidative stress, ueki. used rod - specific gp130 knockout mice and showed that preconditioning of mice with a sublethal photo - oxidative stress activated an autonomous protective mechanism in rods through gp130, an il6 cytokine receptor, and, its downstream target stat3. to determine further whether mller cells, major retinal supporting cells often played a role in photoreceptor protection by releasing survival factors, were involved in this process, they demonstrated that gp130 activation in mller cells had no additional effect for rod survival under photo - oxidative stress. while this study demonstrates the neuroprotective role of gp130-stat3 pathway in the rod photoreceptors under the chronic photo - oxidative stress, another series of studies showed that the pi-3 kinase / akt pathway could protect rod photoreceptors under the acute photo - oxidative stress. using a conditional gene knockout approach, rajala. showed that insulin receptor, a pi-3 kinase upstream regulator, had a protective effect to rod photoreceptors under the acute photo - oxidative stress. in another study using a conventional gene targeting approach, disruption of akt2, a pi-3 kinase downstream target, accelerated the acute photo - oxidative stress - induced rod photoreceptor degeneration. finally, zheng. demonstrated that bcl - xl, a downstream target of akt, was a rod survival factor under acute photo - oxidative stress. these studies clearly mapped the significance of pi-3 kinase / akt pathway in stress - induced rod photoreceptor survival in vivo. kinesin - ii is a molecular motor localized to the inner segment, connecting cilium, and axoneme of mammalian photoreceptors. the involvement of kinesin - ii in protein trafficking through the mammalian photoreceptor cilium was initially probed with cre / lox - based conditional gene targeting. loss of kinesin - ii in rods caused significant accumulations of opsin, arrestin, and membrane proteins within the photoreceptor inner segment, which ultimately led to the death of photoreceptors, a phenotype that is commonly observed in retinitis pigmentosa. further experiments also suggested that ectopic accumulation of opsin was a primary result of rod - specific kinesin - ii deletion. using a conditional gene targeting approach, avasthi. recently demonstrated that heterotrimeric kinesin - ii acted as a molecular motor for proper trafficking of membrane proteins within the cone photoreceptors. these conditional gene targeting studies established an unequivocal role of kinesin - ii as a molecular motor that facilitates protein membrane trafficking in the photoreceptors. rpe is the gatekeeper of the retina and plays a pivotal role in the maintenance of retinal neurons. abnormal rpe function is associated with both the wet and dry - forms of age - related macular denegation (amd) (for review see [65, 66 ]). although the pathogenic mechanisms for dry - amd is unclear, clinical evidence suggests that photoreceptor degeneration is a consequence of impaired rpe functions [67, 68 ]. rpe - specific gene targeting will be a powerful approach for functional analysis of the rpe - expressed genes in the pathogenesis of dry - amd. whereas the use of conditional gene targeting in the pre is still at its infancy, investigating the role of vascular endothelial growth factor (vegf or vegf - a), a potent angiogenic factor whose polymorphisms are associated with amd [69, 70 ], in choroidal vascular development has yield some information related to the relationship between the rpe - derived vegf and choroidal vasculature [2, 71 ]. as abnormal choroidal vasculature is clearly associated with both the dry- and wet - amd [7275 ], the genetic systems established in these studies may have some utility for amd research. while the conditional gene targeting approach has yet to reach its full potential in amd research, lewin. recently demonstrated that disruption of mitochondrial manganese superoxide dismutase (sod) in the rpe produced a geographic atrophy - like phenotypes in mice. here again, tissue / celltype - specific disruption of widely expressed genes, such as vegf and sod, circumvents the interference of nontargeting tissues / cells and is likely a direction for generating animal models used for mechanistic, diagnostic, and therapeutic investigations in the years to come. remarkable progress has been made since the publication of the first study on the retinal denegation using a conditional gene targeting approach a decade ago. it is also important to realize that, except in protein trafficking and photoreceptor survival, progress in other areas of retinal biology is not keeping the pace. at present, cellular mechanisms of many trophic factors and their signaling pathways in the retina remains unclear. although the rpe and mller cells are two major retinal supporting cell - types, the post - developmental functions of rpe and retinal mller cell - derived trophic factors and their signaling mechanisms have remained largely uninvestigated. substantial effort is necessary to establish a framework for cellular mechanisms of inherited retinal degeneration, amd, and diabetes - induced retinal neuron degeneration. many of these investigations will require the use of conditional gene targeting approach. with the improved cre - drive lines and effort in investigating cell - specific function of trophic factors and their signaling, significant progress in our understanding of retinal degeneration will be achieved in the near future. ultimately, these findings will help to design therapeutic approaches for the treatment of the retinal degenerative diseases. | retinal neuron degeneration and survival are often regulated by the same trophic factors that are required for embryonic development and are usually expressed in multiple cell - types. therefore, the conditional gene targeting approach is necessary to investigate the cell - specific function of widely expressed and developmentally regulated genes in retinal degeneration. the discussion in this review will be focused on the use of cre / lox - based conditional gene targeting approach in mechanistic studies for retinal degeneration. in addition to the basic experimental designs, this article addresses various factors influencing the outcomes of conditional gene targeting studies, limitations of current technologies, availability of cre - drive lines for various retinal cells, and issues related to the generation of cre - expressing mice. finally, this review will update the current status on the use of cre / lox - based gene targeting approach in mechanistic studies for retinal degeneration, which includes rod photoreceptor survival under photo - oxidative stress and protein trafficking in photoreceptors. |
malignant ascites may occur in the evolution of digestive or gynecologic cancers when disseminated to the intra - abdominal cavity [1, 2 ]. disseminated intra - abdominal tumor induces peritoneal neovascularization and increases microvascular permeability and production of peritoneal fluid. besides, intraperitoneal fluid accumulation is also related to intra - abdominal and diaphragmatic lymph vessel obstruction due to tumor invasion. it can be due to vascular extension of peritoneal carcinomatosis or bleeding from the primary tumor. patients quality of life is often severely impaired due to symptoms including chronic anemia, and even worsened by repeated paracentesis and blood transfusions. iterative paracentesis, diuretics and albumin perfusion are used to treat cirrhotic ascites and might be useful to treat malignant ascites at its beginning, but lose effectiveness over time because of different physiopathology. as peritoneal dissemination is a major poor prognosis factor in patients with intra - abdominal malignancy, it represents a classical contraindication for aggressive therapeutic strategies such as surgical resection. palliative laparoscopic hyperthermic intraperitoneal chemotherapy (hipec) has been proposed to treat debilitating malignant ascites [1, 3, 4, 5, 6, 7 ]. the goal of this procedure is palliative symptom relief with reduced invasiveness and morbidity. to our knowledge, no treatment has been reported to show effectiveness in carcinomatosis - associated hemoperitoneum, and patients are usually treated with iterative paracentesis, repeated blood transfusions and eventual palliative systemic chemotherapy. two patients with peritoneal carcinomatosis causing malignant ascites and peritoneal bleeding requiring iterative blood transfusion treated by laparoscopic hipec are reported. endoscopic examination of the upper gastrointestinal tract showed a thickened and stiff appearance of the cardial region. abdominal computed tomography (ct) scan was suggestive of gastric linitis with synchronous peritoneal carcinomatosis. the patient was treated by chemotherapy in the avagast randomized study (6 cycles of capecitabine and cisplatin + / after 15 months of chemotherapy the disease was uncontrolled, as ascites relapsed more often and became hemorrhagic. a second - line chemotherapy using 5-fluorouracil and oxaliplatin did not have an effect on hemorrhagic ascites. anemia was severe (mean hemoglobin 5.28.7 g / dl) and needed iterative blood transfusions (1 unit of blood every 7 days), erythropoietin and iron intravenous injection. as blood transfusions were more and more frequent, an explorative laparoscopy followed by hipec was proposed after discussion in multidisciplinary care team meeting. explorative laparoscopy confirmed hemorrhagic ascites in which hemoglobin was measured at 0.6 g / dl. the primary tumor was ill - bounded and completely sheathed the submesocolic region, and particularly the left lobe of the liver. the epiploon was impaired by carcinomatosis, but the small intestine and pelvis were not involved. the hipec procedure consisted in intraperitoneal perfusion of mitomycin c (75 mg) and cisplatin (250 mg) during 30 min with an inflow temperature of 43c under laparoscopy. the postoperative course was unremarkable. during the following months the ascites did not relapse and hemoglobin became stable (11.2 g / dl) and no more transfusion support was needed. five months after laparoscopy, the ascites relapsed, and scanography showed progression of the disease. the patient then received everolimus or placebo in the granit study, but died 2 months later, 7 months after surgery and 25 months after diagnosis. a diagnostic laparoscopy was performed and found a pelvic infiltrative tumor with diffuse infracentimetric peritoneal carcinomatosis nodes. no primary tumor could be clearly identified, although ovarian origin was strongly suspected. despite systemic chemotherapy with cisplatin- (8 cycles) and paclitaxel - based regimen (2 cycles), the patient suffered from abundant bloody ascites with severe anemia (mean hemoglobin 6.19.5 g / dl) that required iterative paracenteses (one paracentesis every 4 days) and transfusion support. preoperative exploration confirmed the ill - bounded pelvic bulk, with retro- and subperitoneal development. the procedure consisted in intraperitoneal perfusion of mitomycin c (75 mg) and cisplatin (184 mg) for 20 min with an inflow temperature of 43c. this therapeutic procedure resulted in regression of ascites and complete resolution of hemoperitoneum, leading to cessation of blood transfusions and improvement of quality of life. three months later, as the patient complained of asthenia and weight loss. a ct scan was performed and showed resurgence of ascites and progression of the abdominal lesions. malignant ascites may complicate digestive or gynecologic cancers in their terminal phase, especially ovarian or gastric cancer. the prognosis is poor as median survival rarely exceeds 6 months. in their retrospective analysis of 209 patients with malignant ascites, ayantunde and this can be due to vascular extension of peritoneal carcinomatosis or bleeding from the primary tumor. hemorrhagic malignant ascites might be an even poorer prognostic factor due to anemia and repeated blood transfusions. as malignant ascites is a poor prognostic factor, the aim of therapy is palliation and improvement of quality of life. iterative paracentesis and intravenous chemotherapy may be initially effective, but classically lose their effectiveness over time. use of palliative laparoscopic hipec has been proposed to treat debilitating malignant ascites [1, 4, 5, 6, 7, 8 ]. by destroying the peritoneal carcinomatosis, hipec may induce hemostasis and progressive fibrosis of the peritoneum, preventing ascites and bleeding [4, 5 ]. however, hipec is linked to important morbidity and mortality, mostly determined by the extent of cytoreduction and peritoneal resection, the number of digestive anastomoses, the dose of chemotherapy and the operative time. in patients with malignant ascites and eventually related hemoperitoneum,, no extended resection is required that might result in a relative decrease of the morbidity impact related to hipec. the laparoscopic approach has been considered in the last decade in an attempt to reduce the invasiveness of the procedure in patients undergoing hipec with a palliative intent [5, 9 ]. indeed, laparoscopic hipec may integrate the benefits of a definitive palliation of ascites with a minimal invasion, leading to fewer postoperative complications. retrospective studies related complete clinical regression of ascites in most patients, resulting in symptom relief, and significant improvement in quality of life [4, 5, 6, 7, 8 ]. in the larger studies, valle. reported a definitive regression of ascites in 94% of 52 patients. although laparoscopic hipec has been evaluated in patients with malignant ascites, we have presented here the originality of its efficacy in malignant hemoperitoneum [4, 7 ]. we have reported two cases of peritoneal carcinomatosis causing peritoneal bleeding requiring iterative blood transfusion, treated by laparoscopic hipec, resulting in the reduction of debilitating ascites and cessation of peritoneal bleeding without postoperative complication. this laparoscopic procedure could be considered as a palliative success as far as it improved quality of life and reduced symptoms, especially anemia.. reported that 2 of their 16 patients presented bloody ascites that turned clear after laparoscopic hipec, but there is no information about the severity of peritoneal bleeding in these patients. however, this supports that laparoscopic hipec might be effective to dry up carcinomatosis - associated peritoneal bleeding. although there are not enough reported cases of laparoscopic hipec to treat malignant bleeding ascites, we may consider it as a therapeutic option. recent studies reported the potential benefit of using new targeted agents by intraperitoneal route in malignant ascites. demonstrated clinically relevant benefits of catumaxomab in patients with recurrent malignant ascites due to carcinomas of different origin, with frequent but acceptable chemo - induced toxicity. additionally, bevacizumab is currently under evaluation in clinical studies to treat and prevent malignant ascites. combination of laparoscopic hipec with these targeted drugs might be an option to consider, and its efficacy and toxicity should be evaluated. in conclusion, laparoscopic hipec might be an interesting therapeutic option in selected patients with debilitating malignant ascites associated with hemoperitoneum. this technique appears to be interesting because of improvement of quality of life with a minimally invasive approach. | malignant hemorrhagic ascites may complicate the terminal evolution of digestive cancers with peritoneal carcinomatosis. it has a bad influence on prognosis and may severely impair patients quality of life. palliative laparoscopic hyperthermic intraperitoneal chemotherapy (hipec) has been proposed to treat debilitating malignant ascites. two cases of peritoneal carcinomatosis causing hemorrhagic ascites and severe anemia that needed iterative blood transfusions are reported. these patients were treated by laparoscopic hipec (mitomycin c and cisplatin with an inflow temperature of 43c), resulting in cessation of peritoneal bleeding. no postoperative complication or relapse of ascites occurred during the following months. no more blood transfusion was needed. laparoscopic hipec might be an effective and safe therapeutic option to consider in patients with malignant hemorrhagic ascites. |
overall, breast cancer five - year age - standardized survival rates are around 80%. survival varies with age and stage of disease from 8869% (i - ii) to 4312% (iii - iv). although newer markers of oncogene expression show promise with respect to treatment of disease, the nodal status still remains the primary prognostic discriminant and is important for tailoring treatment. axillary nodes are the most common sites of expansion outside the breast, occurring in approximately 41% of cases, and prognosis is better when there is no lymphatic invasion. additional large, well - conducted studies are required to obtain more accurate data on sensitivity and specificity of imaging techniques, in addition to the accuracy and costs of the different diagnostic methods. where metastases are present, surgical removal of axillary nodes is indicated in order to ensure staging accuracy and local disease control. furthermore, even a combination modality of three noninvasive diagnostic imaging techniques (us, pet - tc, and mri) can not substitute for an invasive method to make decisions for appropriate systemic interventions. traditional staging requires dissection in level i and ii axillary lymph nodes (alnd) with 10 or more removed nodes. alnd is very accurate in establishing the presence of axillary disease and has the therapeutic advantage of being associated with a high long - term local disease control rate. however, alnd is associated with significant complications (e.g., 21% of arm lymphedema, 22% of seromas, and 14% of infection rate). in recent years, however, sentinel lymph node biopsy (slnb) has been proven to be a feasible, accurate, and suitable method for staging the axilla, while avoiding the morbidity of alnd, in patients with clinically node - negative breast cancer on clinical examination, ultrasound, or fine needle aspiration cytology. as a minimally invasive approach, slnb has become a standard surgical technique in the management of early breast cancer patients whereby prevalence is assumed fixed at 41.2%. a reduction in morbidity is an obvious goal but the more challenging metric is demonstrating that survival is not adversely affected. sentinel node surgery represents the next major step in reducing the extent of surgical procedures, but, despite the revolution of quality conserving care, recent data collection and analyses using anatomical techniques suggest that the exact lymphatic drainage of the breast continues to be debatable. different lymphatic patterns may help to explain some important unresolved clinical problems, including different detection rates in different studies and high false - negative rates of about 10% in multicenter randomized controlled trials. further anatomical investigation and knowledge of the exact sentinel lymphatic channels (slcs) will provide more underlying information about patterns of breast cancer in order to improve surgical strategy, locoregional recurrence, and survival. in the era of slnb, the exclusion of nodal involvement using noninvasive methods could reduce the rate of axillary surgery, thereby preventing patients without lymph node (ln) metastases from suffering complications. therefore, the sensitivity, specificity, and accuracy of diagnostic imaging techniques (us, pet - tc, and mri) have been appropriately examined (table 1). ultrasound (us) is currently recommended prior to surgical assessment of the axilla for all patients with early - stage breast cancer. a systematic review estimated the average sensitivity at 4461% in patients with nonpalpable axillary nodes and the specificity at 7586% in all patients. furthermore, if clinical and us scans suggest nodal metastases on the basis of size or abnormal morphology, us - guided biopsy (fnac or core biopsy) of abnormal nodes is undertaken, which detects 45% of metastases. preoperative axillary us is a widely accepted diagnostic method that provides additional value in detecting pathological spread but on its own is insufficiently specific to obviate the need for slnb because of the substantial number of fn results, particularly in n1 disease, although it may almost exclude n2 and n3 disease. a large number of prospective randomized studies must be completed for validation of performance results with additional relevant costs. positron emission tomography (pet - tc) is a nuclear medicine imaging technique that produces a three - dimensional map of functional processes in the body. across 26 studies (n = 2591 patients) evaluating pet or pet - ct or pet only for assessment of axillary metastases, the mean sensitivity was 5666% and the mean specificity was 9396%. however, there was a trend for lower sensitivities where metastatic lymph nodes were smaller or less in number. micrometastases (> 2 mm) were associated with a mean sensitivity of 11% based on data from five studies (n = 63), while macrometastases (> 2 mm) were associated with a mean sensitivity of 57% based on data from four studies (n = 111). a further meta - analysis of 25 studies including 2460 patients reported that pet - ct provided lower sensitivity (37% to 85%) and high specificity (84% to 100%). compared with the combination of breast sonography and mammography, pet - ct was less sensitive and had less accuracy in detecting node disease. consequently, it is not a reliable noninvasive modality to assess node involvement to replace alnd or slnb before decisions are made on appropriate systemic interventions. mri scanning may provide information on whether a lesion is suspicious for metastasis based on criteria such as size, morphology, and enhancement characteristics following administration of a contrast agent. several mri studies reported more than one set of diagnostic accuracy results, according to different criteria, but the contrast of use and uptake pattern was the main requirement for defining a node as metastatic with a better combined sensitivity (90%) and specificity (90%) [11, 12 ]. the morphological features of lymph node enhancement defects and dilated lymphatic vessels show potential for differentiating metastatic nodes through magnetic resonance lymphangiography (mrl). the physiology of mrl makes the identification of slns readily apparent due to gadolinium - based contrast uptake within lymphatic vessels and lymph nodes following intradermal injection, but further investigations would have to be performed to identify the accuracy of new biomarkers on these pathological findings. in order to evaluate the effects on patient outcomes and cost - effectiveness of enhanced imaging techniques compared with the standard assessment, cooper. modified the standard pathway. key findings from diagnostic results suggested that the most cost - effective strategy might be mri or pet rather than slnb or 4-ns, reducing costs, and increasing quality of life years due to fewer adverse events for the majority of patients. however, the two strategies of replacing axillary sampling with imaging techniques may be considered unacceptable on clinical grounds due to the higher numbers of fn cases (leading to higher risk of recurrence) and fp cases (leading to unnecessary alnd). in the mri replacement strategy, the number of fp cases increased significantly from 0.2% to 6.3% and the number of fn cases increased to a lesser extent, from around 1.0% to 1.9%. in the pet replacement strategy the numbers of both fp and fn cases increased significantly, from 0.2 to 3.6% for fp cases and from around 1.0% to 7.2% for fn cases. positron emission tomography and mri are assumed to be associated with neither short nor long term adverse events. however, due to the lower accuracy of the imaging techniques, more fp and fn cases will be produced, which will lead to increased costs, poorer quality of life due to adverse events, and, in some cases, a higher probability of recurrence and subsequent death from breast cancer. evaluation of slnb in 323 patients with breast cancer suggests that no imaging techniques like us, mri, and pet - ct can replace surgical staging and histologic confirmation of nodal status. the presence of axillary ln metastasis on preoperative imaging carried 82.1% sensitivity, 45.9% specificity, 33.8% positive predictive value, and 86.1% negative predictive value for determining axillary metastasis on final pathology. the aim of this data review was also to determine whether it is safe and feasible to perform slnb in patients with clinically suspicious axillary nodes in preoperative imaging studies, which showed that the sentinel procedure works well in a wide range of practice settings. furthermore, alternative imaging addition diagnostic pathway demonstrated that the sensitivity and specificity of both pet and mri before sampling methods vary significantly between studies. the advantage of adding strategy before sampling results in fewer fn cases (reduced from around 1.0% to 0.1% for mri and to around 0.5% if pet is placed before sampling) due to the use of two sequential tests and fewer sampling procedures performed because sampling methods are avoided if mri or pet results are positive. the disadvantages of these strategies are that there are more fp cases because the specificities of mri and pet are lower than those of slnb and 4-ns. fp increases from 0.2 to 6.3% for mri prior to slnb, which is the same as for the mri replacement strategy. in conclusion, in order to have similar levels of fp and fn cases for the sampling methods, the specificity of mri and pet needs to be improved by close to 100%, which by definition is the specificity of 4-ns and slnb. however, availability of pet and mri scanning facilities would need to be considered if pet or mri was recommended as part of the routine screening pathway for all patients with early breast cancer. axillary involvement is found in 1030% of patients with t1, depending on size, reaching 45% for small t2 tumors (2.13 cm) and 5570% for larger tumors. randomized trials in which the primary aim was to assess morbidity conclusively demonstrated a marked diminution of complications associated with the slnb strategy when compared with routine alnd. in the almanac outcome measures, the twelve - month risks of lymphedema and sensory loss after surgery were, respectively, 13% and 31% in the alnd group compared to 5% and 11% in the slnb group. furthermore, specific results concerning the impact of sentinel strategy on recurrence and survival have confirmed the oncologic safety of healthy lymph nodes preservation. the eligibility criteria for a procedure are the main elements of a prognostic management classification system, but new quality indicators still need to be incorporated into clinical practice to evaluate the applicability and relevance for surgeons. when the authors excluded patients with tumors larger than 2 cm or multicentric, with prior excisional biopsy, younger than 40 years, or when a sentinel node was not found at lymphoscintigraphy and preoperative probe - guided inspection, the axillary relapse rate in the slnb group was as low as 1.2% per 10-year follow - up. although this data is fully reassuring, the adopted wide exclusion criteria might limit the generalization of these conclusions in order to develop a more practical approach to quality assessment. all studies on sln biopsy, according to established breast cancer quality indicators (qis), report a variable false - negative rate whose prognostic consequences are still unclear [1725 ] (table 2). at a median follow - up of 56 months, zavagno. reported positive non - slns rate of 16.7% and there were more locoregional recurrences in the sln arm than in patients randomly assigned to receive axillary lymph node dissection. it is highly debated which patients can be offered sentinel surgery but the clinical research could benefit from new operational perspectives since it represents the next major step in reducing the extent of surgical procedures to treat breast cancer. false - negative slnb results might impair patient outcomes for several reasons since missed nodes might lead to axillary recurrence that is difficult to treat and understaging could affect decisions about systemic and radiation therapy. systematic review, results from large multi - institutional series showed that all have achieved excellent identification rates, ranging from 93.5% to 97.2%, but none achieved an fn rate lower than 5%, with a weighted average of 9.2%. identification rate may thus serve as a reasonable quality indicator for sentinel biopsy because the lowest fn rates were obtained in the studies in which preoperative lymphoscintigraphy and dual mapping during surgery were required. dual mapping with radiotracer and blue dye, combining 2 different injection sites, and routinely using lymphoscintigraphy may improve accuracy. by providing the surgeon with a map of sentinel lymphatic channels, the new anatomical and functional acquisitions could validate the resection of all hot or blue spread, thereby showing the influence of the quantity of removed nodes on the fn rate. the different techniques used to identify the sentinel node form the basis for defining a concept which goes beyond any definition : the lymphatic drainage. the slnb procedure uses radiotracers, dyes, or nanoparticles, and at the same time, produces different results including the node closest to the primary lesion, the first node visualised at lymphoscintigraphy, the hottest node, all radioactive nodes, all blue nodes, or all nodes with a count rate that is a certain factor higher than that of the background or compared to other nodes. the administration of radiopharmaceuticals may result in acceptable accuracy according to the introduction of a threshold of percentual activity in order to distinguish sns from nsns. however, the amount of tracer that is accumulated by a node depends not only on its positions in the drainage order but also on the number of lymphatic channels that enter the node and on parameters such as lymph flow rate. the exact tracer patterns of a tumor site can simultaneously drain to more than one sentinel node, and differentiating a true sn from a secondary echelon node can be difficult. goyal. reported that the false - negative rate in 842 clinically node - negative patients was 10.1% in those who had one sln harvested compared to 1.1% in those with multiple slns (three or more) removed (p = 0.010). in the nsabp b-32, the fn rates were 17.7% if only one node was resected, 10% if two, 6.9% if three, 5.5% if four, and 1% if five or more. these results should not translate into routine removal of multiple nodes but rather pose a question about the sln procedure optimization. moreover, variations in lymphatic channels may exist and thus may influence identification of positive sentinel nodes according to different procedures and modalities. to determine the effect of different injection sites for radiotracer and blue dye on fn rate the fransenode trial strongly validates the periareolar injection technique giving the high detection rate (99.1%) of slnb and high concordance (95.6%), thereby improving probe detection. however, identification rate could also represent a false reassurance in the accurate analysis of the tumor lymphatic network if considering the different drainage patterns compared to the optimal injection path. according to anan., no matter which mapping agents are used, a two - site injection method based on subareolar (sa) and peritumoral (pt) technique may be superior to a one - site injection in limiting the false - negative rate of slnb for early breast cancer. the success of sentinel channels mapping is optimized not only by using dye and isotope but also by a combination of pt and sa injections, which is useful for identifying the potential discordance between the hot and the blue nodes found in the 8.5% of patients by noguchi.. based on available data, the intraparenchymal or peritumoral technique is necessary to evidence cases of extra - axillary drainage (internal mammary or infra- or supraclavicular) that is present in about 20% of patients and focus the discussion on several points that are still open to debate. therefore, surgical results are optimized when preoperative lymphoscintigraphy mapping is obtained in addition to preoperative probe detection. failure to visualize a sentinel node is predictive of difficult intervention, and negative scintigraphy also heralds a higher risk of axillary involvement. in brenot - rossi 's. experience, positive nodes were identified in 28.5% of patients with successful axillary drainage and in 63.3% with unsuccessful axillary drainage. more than four invaded axillary nodes (p < 0.0001) and the presence of lymphovascular invasion in the breast tumor (p = 0.004) were the only significant variables of univariate analysis, although multivariate analysis showed that only the increased number of invaded nodes was statistically significant. then, these results seem to indicate a prognostic predictive value for this event and, in cases of nonvisualization, some authors superficially reinject after peritumoral injection. however, sentinel nodes that appeared with rescue injections were associated with a significantly higher false - negative rate (23.8%) in patients for whom deep and peritumoral injections had failed. thus, when scintigraphy is negative after an adequate delay, one should check for the presence of macrometastases by ultrasound before surgery. when no sentinel node is identified at surgery, the new criteria produced by clinical trials might form the basis for designing a target node sampling according to the patient 's risk of nodal involvement. the exact route of breast lymphatic drainage to the axilla continues to be debated, although recent studies provide more knowledge on the anatomical network. different drainage patterns may help to investigate some important unresolved clinical problems, including different detection rates in different studies and high false - negative rates. reasons for this have been technical, related to personal surgeon experience or the site of the radioactive tracer, which may not reach the lymph node especially if sited in a peritumoral position. suami. suggested that anatomical studies of the breast and anterior upper torso might help explain the percentages of false - negative slnbs and identify an appropriate injection site for sln detection. the cross sectioned specimens, radiographed to provide three dimensional images of lymph collecting vessels, showed that although the majority of the breast drains to one sentinel node, every breast area is drained by more than one first - tier node in each study (figure 1). although the intradermal injection technique has attractive practical features, the relationship between the superficial lymphatics and the adult cancerous breast tissues has not been adequately described. there is currently insufficient certainty that drainage of tracer injected anywhere in or underneath the skin of the breast reflects drainage from the cancer, and some authors state that there is no constant route via the subareolar plexus. also, evidence that some of the torso vessels (perforating lymphatic system) pass from the periphery through the breast tissue on their way towards the axilla questioned the concrete evidence of a centripetal anatomical lymphatic pathway towards the subareolar plexus. turner - warwick revealed a direct pathway from the tumor injection site to the axillary lymph node, concluding that the collecting vessels through the breast contribute to the drainage pattern and lymphoscintigraphy examinations. the perforating system is connected to the deep lymphatic system and these collecting vessels have the same appearance as the superficial lymphatics as they course with the internal thoracic blood vessels. the color - coded diagrams of dissection, to simulate various injection sites for lymphatic mapping and sentinel node biopsy in breast cancer, suggest a mechanism for false - negative slnb since more than one sentinel node drains every glandular tissue area. as reviewed by wang., six lymphatic drainage patterns based on three types of sentinel lymphatic channels (slcs) were found in 107 early stage breast cancer patients. the drainage pattern was a significant risk factor for unsuccessful identification of sentinel lymph node (p < 0.001) and false - negative slnb ; whereas, patient age, tumor location, tumor size, pathology, and tumor grade do not affect the sentinel detection rate. this helps explain the fact that in all quadrants of the breast, cancer has the potential to spread via the internal mammary lymphatics, especially if the tumor is medial or deep in the breast parenchyma. the variable contribution of perforating lymphatics along the branches of the internal mammary artery to lymphatic drainage can not be clinically predicted, thus suggesting that accurate mapping for additional information requires peritumoral injection and alternative operating pathways in surgical management. moreover variations in axillary clearance put the arm lymphatics at risk for disruption during axillary lymph node surgery. the recent data collected on successful identification and protection of the arm lymphatics, in addition to further investigations of lymphedema occurrence, define any crossover between a hot breast node and a blue arm node. the goal of the axillary reverse mapping (arm) pilot study was to develop a technique to identify and preserve arm lymphatic drainage, thereby decreasing the likelihood of disruption during alnd and, to a lesser degree, slnb. in a series of 220 patients undergoing sentinel biopsy with or without axillary dissection, boneti. revealed a rare arm - sln crossover rate (2.8%) but a more frequent anatomical juxtaposition in 40.6% of patients placing at risk for disruption during lymphadenectomy (figure 2). disruption of the blue arm node due to proximity to the hot sln may also explain the high rate of lymphedema seen after slnb. metastasis of the arm node develops in 043% of patients according to several topics [3436 ]. early reports have suggested that arm lymph nodes rarely contain metastatic disease and that their preservation may translate into a lower incidence of postoperative lymphedema, especially in cases of completion alnd. other authors have used the technique during alnd in node - positive patients and found nonnegligible prevalence (10%20%) of disease involving arm nodes. bedrosian. reported a phase i trial conducted in 30 patients with cytologically documented axillary metastasis, 23 (77%) of whom received preoperative therapy. of 11 patients who had axillary metastasis and at least one arm lymph node identified, 18% had metastasis to the arm node. the small number of patients enrolled in this pilot trial may have resulted in an overestimate of the risk of metastasis to the arm node. furthermore, even if axillary metastases were noted in 60% of cases, the disease clinical stage was ii (a - b) and iii (a - b - c) in 36.6% and 53.3%, respectively. reported that arm nodes showed metastatic involvement in 3 of 21 patients with n1 - 3 (14%). similarly, in a study by noguchi., the metastatic rate for arm nodes was 3 of 7 patients (43%) with a clinically positive node or positive slnb, and all were pn3, highlighting a potential contraindication for high n stage. therefore, additional prospective studies in this patient population are warranted to provide a more comprehensive understanding regarding the relation between arm lymph nodes with oncologic and functional endpoints. it is also important to point out that the concordance between the radioactive sentinel node and the blue arm node needs to be better defined on the basis of clinical and pathological prognostic factors, varying in clinical trials from 3.9% to 18.9% [38, 39 ]. if more patients are included, statistical analysis would be possible and the relationship between the location and metastasis of arm nodes would be identified. the novel findings on anatomical mapping knowledge could produce further advances in surgical techniques, thus leading to optimal information for axillary staging and lymphedema microsurgical preventive healing approaches. there are clearly unfinished areas of research in the field of axillary lymph node surgery but the nodal status still remains the primary prognostic discriminant in breast cancer patients. the aims of new alternative diagnostic pathways were to evaluate the effects on patient outcomes and cost - effectiveness of enhanced imaging (mri and pet) compared with standard techniques in the assessment of axillary lymph node metastases. lymphatic mapping with sentinel node approach is one of the most interesting recent developments in surgical oncology, allowing patients with metastasis to be treated in early phase without submitting other patients to unnecessary regional dissection. the technique assumes orderly progression of tumour spread to the regional node and biopsy of the first nodes in the lymphatic chain at risk for metastasis should therefore reflect involvement of the remaining nodes. however, the lymphatic interconnections variability makes the diagnostic procedure complex and with different results. lymphatic mapping for early breast cancer has become the standard of care but there is as yet no single study that demonstrates conclusively which particular sentinel node protocol is best for a specific patient. it may also be useful for future studies to report diagnostic accuracy according to subgroups of patients with different stages and molecular subtypes of primary breast tumors in order to inform management decisions for these categories. the false - negative rate is one of the safety parameters of slnb and this result might impair patient outcomes for several reasons. optimization of procedure could be implemented by dual mapping injection site skills, resection of all hot or blue nodes through tracer combination, and improvement in atypical drainage patterns mapping. variations in lymphatic channels may exist and thus influence detection of positive sentinel nodes. frequently, more than one sentinel node drained the breast and the risk of missed axillary disease after negative slnb would also progressively increase depending on tumor size. this anatomical analysis suggests safety measures in patients with high probability of node involvement through a renewed interest in surgical management. the perspective of a guided axillary sampling (gas) could represent a potential development of recent anatomical and functional acquisitions, offering a dynamic technique shared according to clinical and anatomical disease parameters. evolutions in lymphatic drainage mapping and its interconnections could explore the concept of disease progression with a new therapeutic value for the axillary staging procedures. furthermore, lymph node dissection may adopt a conservative approach through the evaluation of upper arm lymphatics during axillary surgery, thus defining a functional model aimed at the reduction of short- and long - term adverse events. quality results in breast cancer surgery need to generate oncological safety devoid of complications through renewed clinical experience. | even in the era of gene - expression profiling, the nodal status still remains the primary prognostic discriminant in breast cancer patients. the exclusion of node involvement using noninvasive methods could reduce the rate of axillary surgery, thereby preventing from suffering complications. however, lymphatic mapping with sentinel node biopsy (snb) is one of the most interesting recent developments in surgical oncology. optimization of procedure could be implemented by dual mapping injection site skills, resection of all hot or blue nodes through tracer combination, and improvement in atypical drainage patterns mapping. this anatomical analysis suggests safety measures in patients with high probability of node metastasis through a renewed interest in surgical management. the perspective of a guided axillary sampling (gas) could represent a potential development of recent anatomical and functional acquisitions, offering a dynamic technique shared according to clinical and anatomical disease parameters. furthermore, the surgical staging procedures may adopt a conservative approach through the evaluation of upper arm lymphatics, thus defining a functional model aimed at the reduction of short- and long - term adverse events. quality results in breast cancer surgery need to generate oncological safety devoid of complications through renewed clinical experience. |
many potential pain generators exist in the thoracic spine, and variable clinical symptoms and the close proximity of related anatomical structures lead to poor localization of the exact pain source. therefore, the diagnosis and treatment of thoracic back pain have been historically considered difficult for the pain physician. thoracic facet joints and intervertebral disks are considered to be the most typical pain sources in the thoracic spine ; therefore, clinical practice has focused on the assessment and treatment of these structures. the costovertebral and costotransverse (ctrv) joints are the articulations that connect the ribs with the bodies of the thoracic vertebrae and transverse processes (fig. the costovertebral and ctrv joints also significantly contribute to the stability and movement of the thoracic spine, which is not limited to motion of the ribs. although the ctrv joints are relatively less studied compared to the thoracic facet joints and intervertebral disks, it has been well established that the ctrv joints may produce clinically significant thoracic back pain. therefore, we hypothesized that the ctrv joints might be a source of pain and that this treatment could lead to clinical improvements. the aim of this study is to evaluate the clinical effectiveness of the ctrv joint injection in thoracic back pain patients with suspected ctrv joint problems. this study is a retrospective observational audit of patients who received a ctrv joint injection for pain treatment. we reviewed clinical records and interviewed patients individually at a follow - up visit or via telephone. the study protocol was approved by the institutional review board, and written informed consent was obtained from all patients on an outpatient basis. the study population was defined as thoracic back pain patients with localized paraspinal tenderness 2 - 3 cm lateral to the midline where the transverse process meets the rib, at anticipated points that correspond to the ctrv joint on physical examination. the patients were placed in the prone position during prescanning for identification of the 12th rib to investigate the thoracic level of the target ctrv joint using an ultrasound machine with a 7.5-mhz linear probe (sonosite s - nerve ultrasound system, sonosite, bothell, wa, usa). all ultrasound - guided ctrv joint injections were performed by one pain physician (k.b.y.) as previously described by deimel.. for image optimization, we identified the transverse process, ctrv joint, rib, and lung in the same plane (fig. as the needle passed under the transverse process of the target ctrv joint, it was advanced until the tip penetrated the capsule (fig. 0.5 ml of triamcinolone acetonide 2.5 mg and 0.75% ropivacaine mixture were injected at each ctrv joint. clinical and demographic data were collected for analysis, including age, gender, coexisting pain sites, duration of pain, previous pain intervention history, characteristics of thoracic back pain, and ctrv joint injection sites. before and two weeks after ctrv joint injection, patients were asked to rate their pain score using a 10-point numeric rating scale (nrs ; 0 = no pain, 10 = worst possible pain). we also assessed the degree of patient satisfaction as excellent, good, fair, or bad two weeks after injection. continuous data are reported as mean and standard deviation (sd) unless otherwise indicated. a paired t - test was used to compare mean pain scores before and two weeks after ctrv joint injection. statistical analyses were performed with spss statistical software, version 18.0 (spss inc., twenty consecutive patients who were treated with ctrv joint injections were enrolled in this study. patients enrolled in this study previously experienced a variety of interventional procedures for thoracic back pain treatment. in particular, all patients had previously experienced thoracic medial branch blocks, but they reported no lasting improvement after any of these interventions. twenty symptomatic thoracic back pain patients received a combined total of 32 ctrv injections at the t3-t10 spine levels. table 3 shows the change in pain scores before and after the ctrv joint injection treatment, and the degree of patient satisfaction two weeks after the treatment. the mean pre - procedure pain score decreased by 37.9% (7.2 1.5 to 4.5 1.7, p < 0.001) two weeks after ctrv joint injection. although most patients reported reduced pain two weeks after injection, two patients reported the same pain score. in addition, 70% of patients (14/20) reported excellent or good satisfaction levels during the two weeks after the injection. this study reports the clinical outcomes of ctrv joint injection for treating ctrv joint generated pain. we demonstrated that an ultrasound guided ctrv joint injection with local anesthetic and steroid led to reduced pain scores and a high level of satisfaction at short - term follow - ups of thoracic back pain patients with suspected ctrv joint problems. the provoked pain patterns of thoracic facet joints overlap considerably in the thoracic area, and no referral zone can be attributed solely to one joint. in contrast to the thoracic facet joint, the ctrv joint pain pattern is very localized and presents with mainly unilateral symptoms which are limited to the affected ctrv joint. this pain pattern is also distinguished from referred pain caused by an active trigger point on the rhomboid or trapezius muscles. the medial branches of the thoracic dorsal rami innervate the thoracic facet joints, whereas innervation of the ctrv joints comes from the lateral branch of the thoracic dorsal rami. therefore, conventional thoracic medial branch interventions may not fully relieve pain originating from the ctrv joints. costovertebral joints, which receive sympathetic innervation, have also been known to cause thoracic back pain. however, this joint pain pattern sometimes radiates to the anterior chest area, similar to visceral - origin chest pain, which is called atypical chest pain and pseudo- angina. some studies have demonstrated that intercostal nerve blocks effectively reduced this costovertebral joint - generated pain. although no patients reported radiating pain to the anterior chest wall in this study, we can not fully exclude costovertebral joint problems in some patients because the costovertebral and ctrv joints have a very close biomechanical relationship that is associated with rib movements. it is therefore difficult to differentiate which of the costovertebral or costotransverse joints is problematic with any kind of specificity on manual testing. moreover, ultrasound guided - costovertebral joint injection is technically very difficult due to its location. the ctrv joint is the joint formed between the facet of the tubercle of the rib and the adjacent transverse process of a thoracic vertebra. the ctrv joint is a plane - type synovial joint which allows both gliding and rotation of the connected rib. the pathophysiology of ctrv joint problems has not been completely elucidated, but in a recent histological study, immunoreactivity was confirmed for specific neuropeptides within the ctrv joint tissue samples, which suggests that the ctrv joint is generating pain. on the other hand, a partial resection model of the ctrv joint in canines demonstrated that the ctrv joint participates as a stabilizer of the thoracic spine along with the thoracic intervertebral disk and costovertebral joint. this finding suggests that ctrv joint dysfunction may affect or be affected by thoracic spine destabilization. the small number of patients and lack of a control group impaired this study, and we did not conduct long - term follow - up. we can not suggest specific diagnostic methods that identify ctrv joint problems among various overlapping anatomical structures, other than typical tenderness and related clinical manifestations. therefore, a more controlled study is needed to determine the exact source of pain, such as a staged diagnostic block in the surrounding structures of the thoracic spine. in conclusion, we demonstrated clinical improvements in thoracic back pain patients after a ctrv joint injection. ultrasound - guided ctrv joint injections reduced patients ' pain scores and led to a high level of satisfaction at short - term follow - up in patients with suspected ctrv joint - generated pain. the results of this study suggest that ctrv injection should be considered as a therapeutic option to solve complex problems of thoracic back pain in selected patients. | because of its anatomical location and function, the costotransverse (ctrv) joint can be a source of thoracic back pain. in this retrospective observational study, we evaluated the clinical effectiveness of the ctrv joint injection in thoracic back pain patients with suspected ctrv joint problems. we enrolled 20 thoracic back pain patients with localized tenderness that was provoked by the application of pressure on the affected ctrv joints. we injected it with 0.5 ml of a ropivacaine and triamcinolone mixture at each level. the mean pre - injection pain score decreased by 37.9% (7.2 1.5 to 4.5 1.7, p < 0.001) two weeks after ctrv joint injection. in addition, 70% of patients reported an excellent or good level of satisfaction. we demonstrated that an ultrasound - guided injection of the ctrv joint reduced patients ' pain scores and led to a high level of satisfaction at short - term follow - ups in patients with suspected ctrv joint problems. |
cerebrotendinous xanthomatosis (ctx) is an autosomal recessive inborn error of lipid metabolism manifesting characteristically with neurological dysfunction, juvenile cataract and tendinous xanthoma. it is a treatable disorder which often remains unrecognized till late in its course due to its protean manifestations and lack of suspicion on part of treating specialists. an estimated prevalence of 1 in 50,000 to 70,000 population has been mentioned in some of the published studies. if similar prevalence is presumed for indian population, one would expect to have 12000 to 20000 such patients in india. in practice this could be due to lower prevalence of the ctx gene in indian population or under - recognition of it. the disorder has variable manifestations leading to misdiagnosis particularly early in the course when there are no xanthomas and the clinical presentation appears similar to other childhood and young adult onset neurological diseases. we report here two cases of ctx who had different presentations and remained undiagnosed till they had significant permanent physical impairment. one of the patients, in whom genetic analysis was done, had a novel mutation of ctx gene. a 50-year - old commerce graduate with degree in law presented to us with insidious onset gradually progressive spastic diplegia for 20 years followed later by gradually progressive bladder dysfunction, poor scholastic performance, and a single seizure. pedigree analysis revealed absence of any family history in previous two generations. on examination, he had firm, non - tender swellings over ankles, patella, and elbows bilaterally [figure 1 ]. detailed higher mental function assessment revealed impairment of attention, new learning and visual memory with normal language functions. the patient had severe spasticity in both lower limbs with brisk deep tendon reflexes in both the upper and the lower limbs, extensor plantar responses, and normal sensory examination. photograph showing xanthomatous swelling over achilles and elbows (marked with arrows) blood investigations revealed a normal complete hemogram (including hemoglobin, total leukocyte count, differential leukocyte count), liver and kidney function tests, serum electrolytes and, triglyceride and cholesterol levels. x - rays of ankle, knee, and elbow showed homogenous soft tissue swelling. mri brain showed t2-weighted hyperintensities in dentate nucleus regions bilaterally and generalized cerebral and cerebellar atrophy [figure 2 ]. plasma cholestanol levels were found to be raised [16.8g / ml ; normal laboratory reference range : 4.2 1.2 (s.d) ] suggesting cerebrotendinous xanthomatosis. the genetic analysis of cyp27a1 gene of the patient was carried out by the method of polymerase chain reaction and sequencing of both the dna strands was done. the analysis revealed heterozygous mutation in exon 6 with substitution of cytosine by thymine at 1151 position (c. 1151c > tp.p384l) and a novel mutation in exon 1 of cyp 27 gene with substitution of thymine by cytosine at nucleotide position 2 of the gene (c. 2 t > cp.m1 t). the mutation in exon 6 is already described as disease causing while the mutation in exon 1 is previously unreported as per the current human gene mutation database. the mutation causes loss of a highly conserved start codon and likely results in loss of functional protein. the patient is receiving atorvastatin besides physical therapy and chenodeoxycholic acid (cdca) is being arranged as it is not marketed in india. mri brain showing t1w hypointensities and t2w hyperintensities in bilateral cerebellar hemispheres in dentate nuclei region the second case was of a 28-year - old male who was born of a non - consanguineous marriage, had delayed psychomotor milestones with mental retardation, congenital cataract, and recurrent episodes of diarrhea since early childhood. he had history of progressive spastic diplegia, tremulousness in both upper limbs and scanning speech. pan - sensory impairment was noted below knee with impaired limb coordination in upper limbs. patient 's skin biopsy of swelling over achilles confirmed xanthoma [figure 3 ]. mri brain showed altered signals in bilateral cerebral peduncles, ventral pons, both the middle cerebellar peduncles and medullary pyramids. based on the clinical, radiological, and biopsy findings of the achilles tendon swelling, diagnosis of ctx was confirmed. h & e, stain of xanthoma over achilles tendon region showing foam cells (arrow head) and multinucleate giant cell (arrow) cerebrotendinous xanthomatosis is an autosomal recessive disorder of lipid metabolism resulting from homozygous or compound heterozygous mutations in cyp27a1 gene on chromosome 2q. the mutation results in deficient sterol 27 hydroxylase activity with resultant impaired formation of cdca and accumulation of metabolic intermediary, cholestanol [figure 4 ]. the cholestanol starts depositing in lipophilic tissues including brain, spinal cord, lens of eyes, and tendons. the patients also develop premature atherosclerosis secondary to cholesterol and cholestanol deposits in the vessel wall. the first description of the clinical features of the disease is attributed to van bogaert. certain ethnic groups such as sephardims of moroccon origin have been reported to have high mutant gene carrier frequency of 1:108. mathew. noted one carrier of cyp27 gene mutation r362c among 115 control white population. they estimated that prevalence of ctx gene mutation r362c among the white ethnic population to be 1.9 per 10. it is estimated that the prevalence of ctx is 3 - 5 per 10 population in whites. the cases presented here remained undiagnosed for more than 20 years after the clinical manifestations despite having been examined by various specialists. if the prevalence of gene mutation in indian population is similar to the whites, assuming 1.2 billion indian populations, the estimated number of ctx cases in india should be around twenty thousands. our case reports reinforce the possibility of misdiagnosis or underdiagnosis of the condition in indian population. schematic representation of chenodeoxycholic acid synthesis from cholesterol cerebrotendinous xanthomatosis can present to different specialists because of its multisystem involvement. it may manifest in neonatal and early childhood period with chronic diarrhea, congenital or juvenile cataract, delayed developmental milestones, mental retardation and occasionally, neonatal cholestatic jaundice. tendon adult patients may manifest with spastic quadriparesis, diplegia, paraparesis, bulbar paresis, cerebellar ataxia, extrapyramidal symptoms, progressive cognitive deterioration, and peripheral neuropathy. high index of suspicion alone can help detect the condition in early stages and prevent subsequent neurological complications and permanent disability. cerebrotendinous xanthomatosis shares presence of xanthoma and premature atherosclerosis with other lipid storage disorders including familial hypercholesterolemia and sitosterolemia. progressive neurological dysfunction and early cataract are seen in cerebrotendinous xanthomatosis only. both our patients had t2 hyperintensities in cerebellar hemispheres with prominent involvement of dentate nuclei on mri brain. in addition to cerebellar hyperintensities, ctx patients may have multiple white matter t2 hyperintensities in cerebral hemispheres and, cerebral and cerebellar atrophy. spinal cord mri may also show evidence of white matter hyperintensities due to xanthomatous deposition. mri spine was done in the first patient and did not show any abnormality. despite significant changes in cerebellum on mri, significant ataxia was not noted either historically or on examination in our first patient. biopsy of tendon xanthoma or subcutaneous swellings often reveals foam cells and touton giant cells. spindle - shaped clefts are seen in the xanthomas on hematoxylin and eosin staining and rod shaped collections are seen on electron microscope. studies have shown that early institution of cdca therapy can prevent development of ctx phenotype. it has been observed that the combination of cdca and hmg co a inhibitor simvastatin is more effective in reducing cholestanol levels and normalizing ldl cholesterol. the diagnosis of ctx should be suspected and ruled out in all cases of congenital or juvenile cataract, spastic diplegia, paraparesis, quadriparesis or ataxia or combinations of these features particularly on a background of chronic diarrhea even in the absence of tendinous xanthomas, which often do not appear till 2 decade of life. raised plasma cholestanol levels are diagnostic of the disease early in course. genetic testing in presymptomatic family members of affected patients should be done and cdca therapy instituted to prevent the disease manifestations. a 50-year - old commerce graduate with degree in law presented to us with insidious onset gradually progressive spastic diplegia for 20 years followed later by gradually progressive bladder dysfunction, poor scholastic performance, and a single seizure. pedigree analysis revealed absence of any family history in previous two generations. on examination, he had firm, non - tender swellings over ankles, patella, and elbows bilaterally [figure 1 ]. detailed higher mental function assessment revealed impairment of attention, new learning and visual memory with normal language functions. the patient had severe spasticity in both lower limbs with brisk deep tendon reflexes in both the upper and the lower limbs, extensor plantar responses, and normal sensory examination. photograph showing xanthomatous swelling over achilles and elbows (marked with arrows) blood investigations revealed a normal complete hemogram (including hemoglobin, total leukocyte count, differential leukocyte count), liver and kidney function tests, serum electrolytes and, triglyceride and cholesterol levels. x - rays of ankle, knee, and elbow showed homogenous soft tissue swelling. mri brain showed t2-weighted hyperintensities in dentate nucleus regions bilaterally and generalized cerebral and cerebellar atrophy [figure 2 ]. plasma cholestanol levels were found to be raised [16.8g / ml ; normal laboratory reference range : 4.2 1.2 (s.d) ] suggesting cerebrotendinous xanthomatosis. the genetic analysis of cyp27a1 gene of the patient was carried out by the method of polymerase chain reaction and sequencing of both the dna strands was done. the analysis revealed heterozygous mutation in exon 6 with substitution of cytosine by thymine at 1151 position (c. 1151c > tp.p384l) and a novel mutation in exon 1 of cyp 27 gene with substitution of thymine by cytosine at nucleotide position 2 of the gene (c. 2 t > cp.m1 t). the mutation in exon 6 is already described as disease causing while the mutation in exon 1 is previously unreported as per the current human gene mutation database. the mutation causes loss of a highly conserved start codon and likely results in loss of functional protein. the patient is receiving atorvastatin besides physical therapy and chenodeoxycholic acid (cdca) is being arranged as it is not marketed in india. mri brain showing t1w hypointensities and t2w hyperintensities in bilateral cerebellar hemispheres in dentate nuclei region the second case was of a 28-year - old male who was born of a non - consanguineous marriage, had delayed psychomotor milestones with mental retardation, congenital cataract, and recurrent episodes of diarrhea since early childhood. he had history of progressive spastic diplegia, tremulousness in both upper limbs and scanning speech. pan - sensory impairment was noted below knee with impaired limb coordination in upper limbs. mri brain showed altered signals in bilateral cerebral peduncles, ventral pons, both the middle cerebellar peduncles and medullary pyramids. based on the clinical, radiological, and biopsy findings of the achilles tendon swelling, diagnosis of ctx h & e, stain of xanthoma over achilles tendon region showing foam cells (arrow head) and multinucleate giant cell (arrow) cerebrotendinous xanthomatosis is an autosomal recessive disorder of lipid metabolism resulting from homozygous or compound heterozygous mutations in cyp27a1 gene on chromosome 2q. the mutation results in deficient sterol 27 hydroxylase activity with resultant impaired formation of cdca and accumulation of metabolic intermediary, cholestanol [figure 4 ]. the cholestanol starts depositing in lipophilic tissues including brain, spinal cord, lens of eyes, and tendons. the patients also develop premature atherosclerosis secondary to cholesterol and cholestanol deposits in the vessel wall. the first description of the clinical features of the disease is attributed to van bogaert. certain ethnic groups such as sephardims of moroccon origin have been reported to have high mutant gene carrier frequency of 1:108. mathew. noted one carrier of cyp27 gene mutation r362c among 115 control white population. they estimated that prevalence of ctx gene mutation r362c among the white ethnic population to be 1.9 per 10. it is estimated that the prevalence of ctx is 3 - 5 per 10 population in whites. the cases presented here remained undiagnosed for more than 20 years after the clinical manifestations despite having been examined by various specialists. if the prevalence of gene mutation in indian population is similar to the whites, assuming 1.2 billion indian populations, the estimated number of ctx cases in india should be around twenty thousands. our case reports reinforce the possibility of misdiagnosis or underdiagnosis of the condition in indian population. schematic representation of chenodeoxycholic acid synthesis from cholesterol cerebrotendinous xanthomatosis can present to different specialists because of its multisystem involvement. it may manifest in neonatal and early childhood period with chronic diarrhea, congenital or juvenile cataract, delayed developmental milestones, mental retardation and occasionally, neonatal cholestatic jaundice. tendon adult patients may manifest with spastic quadriparesis, diplegia, paraparesis, bulbar paresis, cerebellar ataxia, extrapyramidal symptoms, progressive cognitive deterioration, and peripheral neuropathy. high index of suspicion alone can help detect the condition in early stages and prevent subsequent neurological complications and permanent disability. cerebrotendinous xanthomatosis shares presence of xanthoma and premature atherosclerosis with other lipid storage disorders including familial hypercholesterolemia and sitosterolemia. progressive neurological dysfunction and early cataract are seen in cerebrotendinous xanthomatosis only. both our patients had t2 hyperintensities in cerebellar hemispheres with prominent involvement of dentate nuclei on mri brain. in addition to cerebellar hyperintensities, ctx patients may have multiple white matter t2 hyperintensities in cerebral hemispheres and, cerebral and cerebellar atrophy. spinal cord mri may also show evidence of white matter hyperintensities due to xanthomatous deposition. despite significant changes in cerebellum on mri, significant ataxia was not noted either historically or on examination in our first patient. biopsy of tendon xanthoma or subcutaneous swellings often reveals foam cells and touton giant cells. spindle - shaped clefts are seen in the xanthomas on hematoxylin and eosin staining and rod shaped collections are seen on electron microscope. studies have shown that early institution of cdca therapy can prevent development of ctx phenotype. it has been observed that the combination of cdca and hmg co a inhibitor simvastatin is more effective in reducing cholestanol levels and normalizing ldl cholesterol. the diagnosis of ctx should be suspected and ruled out in all cases of congenital or juvenile cataract, spastic diplegia, paraparesis, quadriparesis or ataxia or combinations of these features particularly on a background of chronic diarrhea even in the absence of tendinous xanthomas, which often do not appear till 2 decade of life. raised plasma cholestanol levels are diagnostic of the disease early in course. genetic testing in presymptomatic family members of affected patients should be done and cdca therapy instituted to prevent the disease manifestations. | cerebrotendinous xanthomatosis (ctx) is an autosomal recessive disorder of bile acid metabolism manifesting typically with the triad of neurological dysfunction, tendon xanthoma, and early onset cataract. the diagnosis is often missed and delayed as the patients do not manifest all the classical features. early recognition and initiation of chenodeoxycholic acid therapy with hydoxymethylglutaryl coenzyme - a (hmg - co - a) inhibitors is critical to prevent irreversible neurological damage and permanently disabled existence. we report about two patients, both of whom remained undiagnosed for more than 20 years. genetic analysis in one of the patients revealed a novel genetic mutation in one of the homologous genes. the patient was found to have heterozygous mutation of ctx gene with a novel mutation in exon 1 of cyp27a1 gene. |
lentinula edodes was cultured in 500 ml of sterile potato dextrose broth at 25 for 28 days with standing. as shown in fig. 1, the culture filtrate exhibited antibacterial activity, forming a clear zone of 22 mm on the paper disk method against ralstonia solanacearum, a causal agent of tomato bacterial wilt disease. to purify the antibacterial compound, the culture filtrate was subjected to octadecyl - silica column chromatography and eluted with different concentrations from 5% to 100% methanol. none of the 50-l aliquots of methanol eluate showed an antimicrobial effect on the bacteria, but the water fraction that passed through the column maintained antibacterial activity. the ph of the passed fraction was 3.9, suggesting the presence of an organic acid. therefore, qualitative analyses of organic acids from the culture filtrate of l. edodes were carried out by high - performance liquid chromatography (hplc). hplc was performed on a hitachi chromaster apparatus (tokyo, japan), which consisted of a pump, autosampler, column oven, uv - vis detector, and hpx-87 column (i.d. 4.6 300 mm, particle size of 5 m, aminex therapeutics, kenmore, wa, usa) ; temperature was maintained at 25. the flow rate was 0.6 ml / min. the mobile phase was 4 mm h2so4 in water for a total running time of 25 min. qualitative analysis of organic acids in the culture filtrate of l. edodes was performed by hplc and the results were compared with those of authentic organic acids. oxalic acid was a dominant component in the culture filtrate of l. edodes, accounting for 50% of the hplc analysis data. in addition to oxalic acid, phytic acid, malonic acid, and fumaric acid were major components. quantitative analysis of oxalic acid in the culture filtrate of l. edodes was carried out under the same hplc conditions as were used in qualitative analyses. for quantitative analysis, a standard curve of oxalic acid was prepared in the concentration range of 200~2,000 g / ml using four different concentrations. the coefficient of the correlation value for the standard curve of oxalic acid was 0.9982. thus, the content of oxalic acid in the culture filtrate of l. edodes was 0.07%. louis, mo, usa) was tested for its antibacterial activity against r. solanacearum using the paper disk method. 5,000mg / l) were added to the filter paper disks (5 mm diameter), placed on nutrient agar, and overlaid with soft agar (0.8%) mixed with bacterial cells (5 10). clear zones that had formed around the paper disks were evaluated as growth inhibition of the bacterial cells. the antibacterial activities of nine different organic acids present in the culture filtrate of l. edodes shown in fig. 2 were examined against r. solanacearum. each clear zone of 34, 27, and 12 mm was formed on concentrations of 5,000, 100, and 500 mg / l, but not on other concentrations of oxalic acid. antibacterial activity on 500mg / l oxalic acid was compared to the positive control, tetracycline (30mg / l). furthermore, the antibacterial effect of oxalic acid was evaluated against eight different phytopathogenic bacteria : xanthomonas campestris pv. vesicatoria, escherichia coli, and a gram - positive bacteria strain, bacillus subtilis. different concentrations of oxalic acid from 300 to 100mg / l were added to nutrient broth containing different bacterial cells and cultured at 28 for 24 hr. as shown in table 1, oxalic acid exhibited antibacterial activities against the bacterial strains tested at the minimum inhibitory concentration (mic) of 250mg / l, except x. campestris pv. vesicatoria were the most sensitive, and cell growth was inhibited at an mic of 200mg / l. interestingly, oxalic acid, like antibiotics such as tetracycline, had a bactericidal effect and the growth of bacterial cells could not be recovered on nutrient media. in preliminary experiments, the ph of media containing oxalic acid was ph 3.5~4.5 ; we further evaluated whether the antibacterial activity of oxalic acid was a result of low ph. the ph 3.5~4.5 media were adjusted with hcl and the r. solanacearum cells were cultured in the media. the bacterium was grown in the media, although the growth rate was slightly different corresponding to ph (data not shown), indicating that low ph is not main factor affecting antibacterial activation. taken together, the results suggest that oxalic acid is critical factor responsible for antibacterial activity. oxalic acid, a saturated short chain dicarboxylic acid, occurs naturally in many plants and fungi and forms insoluble calcium or magnesium oxalate crystals, soluble sodium, or potassium oxalate. in previous study, different organic acids were identified in japanese apricot fruit by hplc analysis and their antibacterial activities were determined against e. coli, b. subtilis, staphylococcus aureus, and streptococcus suis. however, oxalic acid showed relatively low antibacterial activities towards these bacterial species with an mic of 1,300mg / l compared to the mic of 500 mg / l found in this study. oxalic acid produced by plant pathogenic fungi provide a low ph, which results in maximal activities of fungal enzymes that degrade the cell wall of the host plant. in artificial cultivation of l. edodes mushroom, l. edodes may degrade wood sawdust by producing lignocellulytic enzymes (laccase, cellulase, and xylanase) and the resulting product, glucose, is used in energy metabolism. the sms after 4 cycles of harvest was obtained from a mushroom farm, mixed with water (1 : 3 ratio w / v), and incubated with shaking for 2 hr. the mixture was then filtered through two layers of miracloth (calbiochem, la jolla, ca, usa), centrifuged for 10 min at 5,000 g and the supernatant was used as the water extract of sms. water extract from the natural mushroom substrate that had not been inoculated with l. edodes was used as a control. to quantitatively analyze oxalic acid, the one peak at 6.63 min indicating oxalic acid was detected at 1000 mau in the water extract of a sms sample, but not in the water extract from natural mushroom substrate. thus, the extract of sms of l. edodes may be an eco - friendly control agent for bacterial plant diseases. | the culture filtrate of lentinula edodes shows potent antimicrobial activity against the plant pathogenic bacteria ralstonia solanacearum. bioassay - guided fractionation was conducted using diaion hp-20 column chromatography, and the insoluble active compound was not adsorbed on the resin. further fractionation by high - performance liquid chromatography (hplc) suggested that the active compounds were organic acids. nine organic acids were detected in the culture filtrate of l. edodes ; oxalic acid was the major component and exhibited antibacterial activity against nine different phytopathogenic bacteria. quantitative analysis by hplc revealed that the content of oxalic acid was higher in the water extract from spent mushroom substrate than in liquid culture. this suggests that the water extract of spent l. edodes substrate is an eco - friendly control agent for plant diseases. |
evidence for the putative association between exposure to phthalates and risk of type 2 diabetes (t2d) is now increasing. for example, association of urinary phthalate metabolites with t2d has been shown in swedish, mexican, and united states populations [36 ]. it has been reported that rats exposed to the phthalate di-(2-ethylhexyl) phthalate (dehp) develop reversible hyperglycemia, hypoinsulinemia, and symptoms of diabetes. similarly, developmental exposure to phthalates is known to be associated with -cell dysfunction and glucose abnormality in rats. there also exists additional evidence implicating phthalate exposure in the pathogenesis of insulin resistance [4, 6, 912 ]. despite this body of evidence, it is currently unknown whether severity of t2d and likelihood of its complications may also be altered depending on exposure to phthalates. conceivably, sustained and long - term exposure to phthalates can hasten the diabetic process and thereby precipitate the occurrence of diabetic complications. thus, it can be conjectured that even after the onset of t2d is triggered, the severity of diabetes, as manifested by its complications, may also be modulated by environmental challenges. alternatively or concomitantly, it is also plausible that acceleration of progress to retinopathy subsequent to phthalate exposure may be a result of a direct effect on the retina. it is instructive in this regard that dehp is known to partake in retinal vessel remodeling. also, dimethyl phthalate (dmp), diethyl phthalate (dep), and dehp have been shown to influence the activity of retinal aldolase. however, the relative contribution of phthalate exposure to these two possible mechanisms is currently unknown. in this study, we hypothesized that phthalate exposure is associated with eye - related complications in subjects with t2d. by choosing participants who already have t2d, we attempted to focus on the direct association of phthalates with ocular complications after t2d sets in. to test this hypothesis, we examined the association of twelve phthalate metabolites in urine with the risk of self - reported eye affliction / retinopathy in the publicly available and nationally representative sample of individuals with diabetes recruited in the national health and nutrition examination survey (nhanes) 20012010. nhanes is an annual survey conducted by the national center of health statistics of the centers for disease control and prevention (cdc). detailed description of the nhanes 20012010 survey and sampling strategies can be found online at http://www.cdc.gov / nchs / nhanes.htm/. the selection of participants for the present study is schematically shown in figure 1. in this dataset of 52,195 responders, urinary phthalates were measured in 13,288 (25.5%) individuals. (n = 955) as a response of yes to one or more of the following questions : other than during pregnancy, have you ever been told by a doctor or health professional that you have diabetes or sugar diabetes ? or are you now taking insulin ? or are you now taking diabetic pills to lower blood sugar ? we excluded individuals who refused to answer the question, did not know the answer, or had a missing value. to maximize the likelihood of capturing participants with diabetes, we also included undiagnosed diabetes cases which were defined as glycated hemoglobin value 6.5%. using this criterion we then proceeded to increase the likelihood that the reported or undiagnosed cases of diabetes are indeed t2d from the 1,077 participants with diabetes. we defined t2d as presence of diabetes in participants whose age at screening as well as age at diagnosis of diabetes was at least 20 years. using these criteria these cases were recruited in the nhanes over the 10-year period spanning 20012010 and had data on self - reported eye affliction / retinopathy. self - reported eye affliction / retinopathy was defined as an affirmative answer to the following question : has a doctor ever told you that diabetes has affected your eyes or that you had retinopathy ? an estimated 13.87% of the 1,004 study participants reported this outcome. we examined the validity of this outcome by comparing it with the results of ophthalmologist 's reports of detailed eye and retinal examination. the ophthalmological assessment was available only for the nhanes 2005 - 2006 and nhanes 2007 - 2008 cycles. these data are based on images of two fundus examinations captured using a nonmydriatic digital camera and the grading was based on the early treatment diabetic retinopathy study severity scale [1618 ]. the images were graded as no retinopathy, mild nonproliferative retinopathy (npr), moderate / severe npr, and proliferative retinopathy (pr). pr was defined as presence of neovascularization on the retinal surface or abnormal growth of new retinal blood vessels into the vitreous. urinary phthalate metabolites have been measured in a random subsample of nhanes participants (http://www.cdc.gov/exposurereport/ and [19, 20 ]). briefly, frozen urine samples (20c) were assayed using a combination of solid - phase extraction, high performance liquid chromatography, and tandem mass spectrometry. concentrations below the corresponding limit of detection (lod) were replaced by lod/2. although the nhanes 20012010 dataset contained information on 15 phthalate metabolites (table 1), complete measurements on all the included individuals were available for the following 12 phthalate metabolites : mono - n - butyl phthalate (mbp), mono - cyclohexyl phthalate (mcp), mono - ethyl phthalate (mep), mono-(2-ethyl)-hexyl phthalate (mehp), mono - isononyl phthalate (mnp), mono - n - octyl phthalate (mop), mono - benzyl phthalate (mbzp), mono - n - methyl phthalate (mnm), mono-(3-carboxypropyl) phthalate (mcpp), mono-(2-ethyl-5-hydroxyhexyl) phthalate (mehhp), mono-(2-ethyl-5-oxohexyl) phthalate (meohp), and mono - isobutyl phthalate (mibp). distribution of the urinary phthalate concentrations in the study participants is shown in table 1. in addition to urinary concentrations of 12 phthalate metabolites, we considered the following potential confounders : age, sex, race, marital status, educational attainment, poverty income ratio, physical activity (measured as metabolic equivalent hours and categorized as recommended by), glycated hemoglobin levels, total serum cholesterol, serum high - density lipoprotein cholesterol, serum triglycerides, blood pressure, duration of diabetes, total calorie intake (estimated from dietary questionnaires), and obesity (defined on the basis of body mass index, bmi). distribution of these variables in the study participants and the coding schemes used in analyses are shown in table 1. to ensure that all the selected study participants are represented in all the multivariable models, we coded the missing values of confounders as zero. all the analyses were conducted using the stata 12.0 (statacorp, college station, tx) software package. to ensure a normal distribution of urinary phthalate metabolites and the use of a common metric for all the urinary phthalates, we performed a two - step transformation of the urinary concentrations of phthalate metabolites. in the first step, we corrected for urinary dilution by calculating log ratio of the phthalate metabolite concentration and urinary creatinine concentration. in the second step, we inverse - normalized this log ratio by (i) ranking the values, (ii) creating a cumulative density function based on the ranks, and (iii) using the invnorm () function in stata to create a variable distributed as n(0,1). the histograms of raw values, values corrected for dilution, and the inverse - normalized values for each urinary phthalate metabolite are shown in figures s1s15 in supplementary material available online at http://dx.doi.org/10.1155/2016/7269896. to examine the validity of self - reported retinopathy, we estimated cohen 's kappa. to assess the robustness of the observed associations with self - reported eye affliction / retinopathy, we ran 5000 replicates using monte carlo simulations. in these simulations, we applied the misclassification rates observed in the validity subsample to the whole sample and estimated the association using the full logistic regression model that included all the covariates mentioned earlier. we used the svy set of commands to adjust for the sampling weights and design variables as recommended by the centers for disease control (http://www.cdc.gov/nchs/tutorials/). descriptive statistics such as means and proportions were also adjusted for the survey design variables. the association analyses employed univariate and multivariable logistic regression and were conducted using the logistic subcommand of the svy command. for the univariate analyses, we corrected the significance values for multiple comparisons using the bonferroni method. hypothesis of for dose - response relationship was tested using the armitage test for linear trend. when each phthalate metabolite was separately regressed on the study outcome in a univariate fashion (table 2), only mop was significantly associated with eye affliction / retinopathy such that one standard deviation increase in urinary mop was associated with a 1.39 times higher likelihood (bonferroni - corrected p = 0.048) of eye affliction / retinopathy in participants with diabetes. to test the robustness of this observation, we first conducted a series of multivariable analyses since there was a complex pattern of correlations among the 12 phthalate metabolites (table s1). for the multivariable analyses, we conducted a forward stepwise logistic regression retaining all the included covariates in each step. firstly we included all the 12 urinary phthalates as covariates and found that mop was significantly associated with the study outcome. stepwise addition of all the other demographic and clinical variables did not influence the independent association of mop. the results from the final model including all 26 covariates in addition to mop are shown in table s2. the final model identified three significant predictors of self - reported eye affliction / retinopathy : mop (or 2.02, 95% ci 1.223.35), low hdl cholesterol (or 0.54, 95% ci 0.330.89), and duration of diabetes (or 1.95, 95% ci 1.622.33). data on self - reported eye affliction / retinopathy and ophthalmological examination was available on 285 participants with t2d (table 4). in these participants, the prevalence of mild nonproliferative retinopathy, moderate / severe nonproliferative retinopathy, and proliferative retinopathy was 23.8, 7.7, and 1.6%, respectively. the proportion of self - reported eye affliction / retinopathy was 11%, 15%, 46%, and 48% in participants who had no retinopathy, mild npr, moderate / severe npr, and pr, respectively. there was an 82.01% agreement between these two outcomes which translated into a cohen 's kappa of 0.31 (p = 1.2 10). as evidenced from the results shown in table 4, 19 participants (design - corrected proportion 4.92%) who had moderate / severe npr or pr were missed by self - reported retinopathy / eye affliction indicating possible underreporting. on the other hand, there were 33 (design - corrected proportion 11.2%) participants who reported eye affliction / retinopathy but did not have corroborative imaging data. since ophthalmologically determined retinopathy was available in only 285 participants, the full set of association analyses could not be run on this smaller subsample. instead, we generated 5000 replicates using a monte carlo procedure in which the cases and controls of self - reported eye affliction / retinopathy were randomly reshuffled based on the misclassification rates (11.2% and 4.92%) observed in the validation subsample. for each of these replicates, we ran the full logistic regression model shown in table 3 (model 16) and determined distribution of the regression coefficient for the association of inverse - normalized mop with self - reported eye affliction / retinopathy. we observed (figure 2) that the estimated regression coefficient followed a normal distribution with a mean of 0.4947 which translates to an or of 1.64 (95% ci 1.082.50). these results imply that even if one accounts for possible errors of misclassifying of cases and controls of retinopathy due to self - reporting, the association of mop would still be significant. we next examined if there was evidence for a dose - response relationship of the inverse - normalized urinary mop concentration and prevalence of eye affliction / retinopathy. we found that indeed there was a clearly significant linear increase in the prevalence based on the dose of mop (figure 3). the prevalence of self - reported eye affliction / retinopathy steadily increased from 0.07 (in participants with > 1 sd below mean mop) to 0.21 (in participants with > 1 sd above mean mop). armitage test for linear trend showed a significantly linear association (p = 0.004) between urinary mop and self - reported retinopathy / eye affliction. since mop is a rarer metabolite of dnop, we investigated whether the observed concentrations were confounded by the ability of the assay to detect this metabolite in urine. we found that in 73% of the participants included in this study the urinary concentration of mop was above the lower limit of detection (0.84 ng / ml). secondly, we created a variable to capture the molar sum of dnop derivatives (mop and mcpp) and tested the association of the molar sum of dnop with self - reported eye affliction / retinopathy. we found that this association was not statistically significant (or 0.92, 95% ci 0.761.12, p = 0.393). our results indicate a specific, robust, independent, and statistically significant association of urinary mop concentration with the likelihood of self - reported eye affliction / retinopathy in a nationally representative sample of participants with diabetes from the united states. interestingly, of the three metabolites of dehp, mehp, mehhp, and meohp, none showed a statistically trending association (in the multivariable adjusted model). since mop is a metabolite of dnop, our study has identified exposure to dnop as a potentially important contributor to ocular complications of diabetes. it is intriguing, however, that the other metabolite of dnop (mcpp) was not significantly associated with self - reported eye affliction / retinopathy. in the metabolism of dnop, mop is the result of the first step of hydrolysis while mcpp is the result of the next step of oxidation. it is conceivable, for example, that the enzymatic progression from the stage of hydrolysis to oxidation may be dysfunctional or subfunctional in dr resulting in an increase in mop without significantly affecting the concentration of the downstream metabolite (mcpp). direct in vitro and ex vivo studies need to be undertaken to demonstrate these postulated perturbations in the metabolic pathway of phthalates but our results point towards this interesting and putative hypothesis. our results also indicate that phthalate exposure may be associated with not only an increased likelihood of t2d but also its complications. notably, our sample included non - dr diabetic patients as controls and, therefore, the observed associations are indicative of an epidemiological link between dr and mop rather than between underlying diabetes and mop. while making these and other conclusions from this study, it must be considered that these results only provide associative evidence and therefore causal conclusions need to be refrained from. it is also noteworthy that we observed an association of low hdl cholesterol with self - reported retinopathy. it is generally believed that low hdl is not associated with diabetic retinopathy [2325 ]. for example, in a study by sasongko., it was found that of all the routinely used lipid measures, low hdl provided the most significant and independent contribution to prediction of dr. other studies from united states, china, and india also provide supportive evidence to the association of low hdl and dr. it is instructive, in this regard, that the global multicentric case - control study of dr and the italian riace study posit that the observed association between low hdl and dr can be partly explained on the basis of coexisting chronic kidney complications of diabetes. we did not have data on coexisting diabetic nephropathy and therefore can not comment on the possibility of a nephropathy driven association between low hdl and dr. together, published literature does not offer a definitive answer on the association of low hdl with dr. future studies need to carefully address this issue using a combination of meta - analytical and mechanistic approaches. if indeed mop is a player in the complex web of diabetic retinopathy then a biological rationale for such a finding deserves consideration. diabetic retinopathy typically results in neovascularization of retinal vessels with a consequent bleeding into the vitreous and/or macular edema. this process has a strong genetic basis. in an elegant recent review, murea. enlisted eight genes that have been previously implicated in the pathogenesis of diabetic retinopathy. phthalates have been shown to alter expression of or signaling through several of these genes [3340 ] and one of these genes partakes in the pathway orchestrated by the peroxisome proliferator - activated receptor gamma gene (ppar), a known target of all phthalates including mop. thus, there may be a genetic basis to the possible precipitation of diabetic retinopathy by exposure to mop. also, using an in silico approach, jananie. recently demonstrated that phthalates can bind angiotensin ii type 1 (at1) receptor which plays a central role in diabetic retinopathy. action of at1 in the retinal microvessels and pericytes can contribute to diabetic retinopathy [4244 ]. whether this mechanism is operational in exposure however, the plethora of animal and human studies that point towards a consistent association of some phthalates with retinopathy provide an indirect (and, admittedly, insufficient) evidence for a biological basis to the association of mop with diabetic retinopathy observed in this study. although recall bias can be expected to be low for this outcome, such a bias can not be eliminated. validation of this outcome based on retinal imaging studies also indicated that some misclassification was evident. however, our analyses using monte carlo simulation indicate that the potential misclassification due to an alloyed outcome used in this study is unlikely to negate the association of mop with diabetic retinopathy. second, urinary concentrations of phthalate metabolites only indirectly imply environmental exposure. the pattern, dose, and modes of actual exposure to phthalates can not be discerned from this study. thus, whether the associations reflect a cumulative risk due to sustained exposure can not be reliably answered from these data. third, since the temporal sequence of phthalate exposure and complications of diabetes is unknown, the evidence presented here can at best be considered associative and not causal. urinary phthalate concentrations are considered to be a reasonable biomarker of chronic exposure to phthalates. since the biological effects of phthalate exposure are likely reversible, our results beckon a critical and intensive look into the potential role of mop in diabetic retinopathy. the ubiquity of dnop in products like flooring tiles and cosmetics entails that future studies need to carefully dissect out the putative epidemiological link observed in this study. | background. an epidemiological association between exposure to phthalates and type 2 diabetes (t2d) is known. however, the potential role of environmental phthalates in the complications of t2d is unknown. methods. using data from the national health and nutrition examination survey (nhanes) 20012010, we studied the association of 12 urinary phthalate metabolites with self - reported eye affliction / retinopathy in 1,004 participants with diabetes. data from retinal imaging was used to validate this outcome. independence of the phthalatest2d association was studied by adjusting for age, sex, race, marital status, educational attainment, poverty income ratio, physical activity, glycated hemoglobin levels, total serum cholesterol, serum high - density lipoprotein cholesterol, serum triglycerides, blood pressure, duration of diabetes, total calorie intake, and obesity. results. self - reported eye affliction / retinopathy had 82% accuracy with cohen 's kappa of 0.31 (p < 0.001). urinary mono - n - octyl phthalate (mop) was independently associated with the likelihood of self - reported eye affliction / retinopathy in subjects with t2d after accounting for all the confounders. this significance of this association was robust to the potential misclassification in cases and controls of retinopathy. further, a significant dose - response relationship between mop and self - reported eye affliction / retinopathy was demonstrable. conclusions. we show a novel epidemiological link between the environment and diabetic complications in nhanes 20012010 participants. |
to understand the precise mechanism of transcriptional regulation of a gene, it is essential to identify and analyze its transcriptional start sites (tsss), which are located in the vicinity of its potential promoter regions. several genome - wide studies, including ours, have identified multiple tsss or their corresponding alternative promoters of each gene (13). it is even unknown whether they represent no more than intrinsic biological errors or cloning artifacts. to understand biological relevance of those divergent tsss, we have developed dbtss, datebase of transcriptional start sites, and continued its updates since 2001 (4). dbtss provides tss information, which was determined using our cap - site detection method, oligo - capping (5). to obtain the genome - wide data of tss information, rna sequences immediately downstream of the tsss are sequenced with an illumina massively parallel sequencing platform as tss tags. on the other hand, a large number of systematic epigenomic studies are underway, aiming at comprehensive understanding of chromatin conditions in the genome. recently, a group of the encode project published a chromatic map of nine representative cell types from the chip - seq analyses of nine chromatin markers for various types of histone modifications (8). integration of such kind of data with the tss data would be useful for both studies. in fact, we too have generated similar types of chip - seq data for the studies of individual cells as listed in our web site. similarly, rna - seq data from several subcellular components, such as nucleus, cytoplasm, and polysome fractions, are useful to further characterize the tss data. meanwhile, human genome re - sequencing projects including exome sequencing projects have been also massively accumulating single - nucleotide variation (snv) data (9,10) although understanding their biological consequences is still difficult. since we believe that many of genetic disorders should be attributed to malfunctions in transcriptional regulations, the link between snps and the transcriptional information based on tsss, histone modification status, and transcripts must be established. in this report, we report three main progresses in dbtss. first, we expanded our tss - seq data by 3-fold, so that a major part of human adult and embryonic tissues are covered. second, we added various types of transcriptomics data that can be useful to further interpret tss information. for example, we integrated our original rna - seq data of subcellular - fractionated rnas (11) as well as the chip - seq data of histone modifications, rna polymerase ii and several transcriptional regulatory factors (1214) in cultured cell lines to collectively understand the relationship between tss, transcript dynamics and epigenetic factors. some of the external epigenomic data, such as those by the encode project (8), are also added. third, we associated our tss data with snv data to identify snv candidates that may be responsible for disorders of transcriptional regulation. we believe that such integration provides in - depth biological insight of divergent tsss in vivo. in this update, we added new tss data that were derived from our tss - seq experiment. now dbtss contains 418 146 632 tss tags, collected from 28 tissues or cell types, including 16 kinds of human adult tissues, 5 kinds of human fetal tissues and 7 kinds of cultured cells (table 1). these tss tags were clustered into subgroups with 500-bp bins to define tss clusters (tscs) as putative promoter unit (see ref. 15 for more detail). as a default viewer setting, we adopted tscs with the expression levels higher than 5 ppm (particles or tags per million ; > 5 ppm tscs), which exists from 11 300 to 36 718 in varying cell types (table 2). in several cell lines, we collected tss - seq information under different experimental conditions such as before or after the stimulation by cytokines or hypoxic shocks. also, a total of 70 386 438 tss - seq tag data from several developmental stages and cultured cells are included in mice. users can search tsss where different expressions are observed between different tissue types or culture conditions (figure 1b). (a) users can use a refseq i d for the simplest search (red box in the figure). search condition window. in this case, users can search tsss that are overexpressed after il4 stimulation by 2-folds, with their expression level higher than 5 ppm, showing h3k4me3 signals, and having nearby dbsnp data. (c) users can search tsss around a given snp or any genomic position (upper window). snps that are neighboring with known genes can be sought, too (bottom window). table 1.statistics of tss - seq datacategoryno. of cell types or tissuesno. of total conditionno. of tss - seqhuman adult tissues1620138 864 978human fetal tisssues5541 744 136human cell lines723237 537 518human total2848418 146 632mouse embrio1438 897 846mouse cell lines3331 488 592mouse total4770 386 438total3255488 533 070 table 2.tscs corresponding to ncbi refseq genes and snp informationtss - seq tagstotal tscstscs in refseqtsc > 5 ppmoverlap refseq (> 5 ppm)db_snp (> 5 ppm)jpt / yri / chs / ceu (> 5 ppm)hek29320 686 169193 140137 51811 33810 23411 2853930/5471/4391/3476ramos31 022 974371 759239 30811 455922711 4184036/5433/4340/3513beas2b98 761 770708 912440 30227 62820 38672202993/3979/3194/2167dld148 580 850462 724272 17119 94116 96519 8787084/9735/7731/6211mcf715 785 949172 834120 69511 79010 38311 7434094/5645/4432/3584tig318 780 087198 129144 62211 89310 51211 8474186/5857/4674/3711hela3 919 71999 24174 71911 300971011 2624187/5787/4705/3734adult tissue138 864 9781 496 409911 87236 71826 02350 67416 874/22 996/18 163/14 927fetal tissue41 744 136822 577572 94132 53326 77332 38610 400/14 192/11 088/9260samples : category of samples, tss - seq tags : tag number in each category, total tscs : observed tsc number, tscs in refseq : tscs overlapping with the refseq transcribed region (including their 50 k bp upstream region), tsc > 5 ppm : number of tscs whose expression level is higher than 5 ppm, overlap refseq (5 ppm) : > 5ppm tscs which overlap with the refseq transcribed region, db_snp (> 5 ppm) : number of tscs which contain snps in dbsnp, jpt / yri / chs / ceu (> 5 ppm) : number of tscs which contain ethnic snps (jpt : japanese in tokyo, ceu : utah residents with northern and western european ancestry from the ceph collection, chs : chinese in singapore, and yri : yoruba in ibadan) (a) users can use a refseq i d for the simplest search (red box in the figure). in this case, users can search tsss that are overexpressed after il4 stimulation by 2-folds, with their expression level higher than 5 ppm, showing h3k4me3 signals, and having nearby dbsnp data. (c) users can search tsss around a given snp or any genomic position (upper window). snps that are neighboring with known genes can be sought, too (bottom window). statistics of tss - seq data tscs corresponding to ncbi refseq genes and snp information samples : category of samples, tss - seq tags : tag number in each category, total tscs : observed tsc number, tscs in refseq : tscs overlapping with the refseq transcribed region (including their 50 k bp upstream region), tsc > 5 ppm : number of tscs whose expression level is higher than 5 ppm, overlap refseq (5 ppm) : > 5ppm tscs which overlap with the refseq transcribed region, db_snp (> 5 ppm) : number of tscs which contain snps in dbsnp, jpt / yri / chs / ceu (> 5 ppm) : number of tscs which contain ethnic snps (jpt : japanese in tokyo, ceu : utah residents with northern and western european ancestry from the ceph collection, chs : chinese in singapore, and yri : yoruba in ibadan). in addition to the tss - seq data, we also included rna - seq tags in the data of cultured cells. for example, in dld-1 cell data, rna - seq data from total rna, nuclear, cytoplasm, polysome, and the argonaute complex - associated fractions are stored (15). users can search and browse the transcriptional levels of each gene in each fraction to understand the dynamics of its transcripts. to further understand the relationship between tsss and chromatin states of the surrounding regions, we integrated chip - seq data of histone modifications : h3k4me3 and h3ac for active chromatin markers and h3k27me3 for a silent chromatin marker. data of the binding sites of rna polymerase ii and several transcription factors, such as stat6 in ramos and beads2b cells and hif1a in dld-1 cells, are also included. particularly, we added the above - mentioned data of chromatin states in nine human cell lines (8). in addition to the dbsnp data, we obtained exome data for four ethnic groups from the ncbi site (ftp://ftp.ncbi.nlm.nih.gov/1000genomes/ftp) and generated snvs uniquely called for these ethnic groups. we called 687 881 jpt (japanese in tokyo), 646 522 ceu (utah residents with northern and western european ancestry from the ceph collection) 578 299 chs (chinese in singapore) and 1 120 302 yri (yoruba in ibadan) snvs from 73, 64, 90 and 82 individual 's exome data, respectively. also, we added genome - wide association studies (gwas) data which connect 5800 snvs with clinical information (16). we integrated the internal and external tss - seq, rna - seq, chip - seq and snv data with each other. table 2 (and the statistics page of our web site) represents the statistics of each data and the overlap between them. it should be noted that all of the active markers do not always overlap. in some cases, active chromatin markers these cases may be interesting targets for future analysis of potentially stepwise transcriptional regulations. from a population genetic point of view, it may be also intriguing to further characterize tsss which are observed in an ethnic group - specific manner with solid supports from chip - seq and rna - seq data in cultured cells. for example, users can use a refseq i d for the simplest search (figure 1a). users can narrow down the targets by considering expression levels within a particular cell types or fold expression changes between different cell types (figure 2a). in the main viewer, users can recalculate tag counts by varying genomic regions (figure 2b and c). users can also consider overlap of the chip - seq data and rna - seq data with the tss - seq data (figure 2a). particularly, using the snp search function, users can input an snv in a particular category and search tsss in its surrounding region. conversely, users can search whether there are any tsss that have snvs of various categories in its surrounding regions (figures 1b and 2d as search results). (a) overview of tss - seq and chip - seq for nm_013293, transformer 2 alpha homolog. there are three major putative alternative promoters (ap4, ap10 and ap15) in dld1 cells. the expression of ap10 under the normoxia condition (21%) is relatively low compared with that under the hypoxia condition (1%). using check boxes, users can also check the tss - seq and chip - seq results in other tissues. (b) function of recalculating tag counts by specifying desired genomic regions. in this case, 1.5 ppm tsc specific to normoxia and 10.6 ppm tsc to hypoxia are observed. an ethnic snp (chs) and two dbsnps (rs11523571 and rs41273990) are also found in this region. searching chr1:159680868 based on the input window (figure 1c) also leads to a similar result. (a) overview of tss - seq and chip - seq for nm_013293, transformer 2 alpha homolog. there are three major putative alternative promoters (ap4, ap10 and ap15) in dld1 cells. the expression of ap10 under the normoxia condition (21%) is relatively low compared with that under the hypoxia condition (1%). using check boxes, users can also check the tss - seq and chip - seq results in other tissues. (b) function of recalculating tag counts by specifying desired genomic regions. in this case, 1.5 ppm tsc specific to normoxia and 10.6 ppm tsc to hypoxia are observed. an ethnic snp (chs) and two dbsnps (rs11523571 and rs41273990) are also found in this region. searching chr1:159680868 based on the input window (figure 1c) also leads to a similar result. all of the raw data are freely and anonymously available from our download site at ftp://ftp.hgc.jp / pub / hgc / db / dbtss / dbtss_ver8. we expect more genome - wide data to become available not only for tss - seq data but also for rna - seq and chip - seq data from humans, mice and other model organisms in the near future. also, tens of thousands of snv data for different ethnic groups are being generated without further biological information except their potential disease associations. we believe that, by the integration of the data, our dbtss will continue to serve as a unique database for a wide variety of researchers : both for researchers analyzing transcriptional regulations of individual genes as well as for those analyzing comprehensive transcriptional regulatory networks from a systems biological point of view. in this update, we added new tss data that were derived from our tss - seq experiment. now dbtss contains 418 146 632 tss tags, collected from 28 tissues or cell types, including 16 kinds of human adult tissues, 5 kinds of human fetal tissues and 7 kinds of cultured cells (table 1). these tss tags were clustered into subgroups with 500-bp bins to define tss clusters (tscs) as putative promoter unit (see ref. 15 for more detail). as a default viewer setting, we adopted tscs with the expression levels higher than 5 ppm (particles or tags per million ; > 5 ppm tscs), which exists from 11 300 to 36 718 in varying cell types (table 2). in several cell lines, we collected tss - seq information under different experimental conditions such as before or after the stimulation by cytokines or hypoxic shocks. also, a total of 70 386 438 tss - seq tag data from several developmental stages and cultured cells are included in mice. users can search tsss where different expressions are observed between different tissue types or culture conditions (figure 1b). (a) users can use a refseq i d for the simplest search (red box in the figure). tss - seq detailed search, users will obtain the search condition window. in this case, users can search tsss that are overexpressed after il4 stimulation by 2-folds, with their expression level higher than 5 ppm, showing h3k4me3 signals, and having nearby dbsnp data. (c) users can search tsss around a given snp or any genomic position (upper window). snps that are neighboring with known genes can be sought, too (bottom window). table 1.statistics of tss - seq datacategoryno. of cell types or tissuesno. of total conditionno. of tss - seqhuman adult tissues1620138 864 978human fetal tisssues5541 744 136human cell lines723237 537 518human total2848418 146 632mouse embrio1438 897 846mouse cell lines3331 488 592mouse total4770 386 438total3255488 533 070 table 2.tscs corresponding to ncbi refseq genes and snp informationtss - seq tagstotal tscstscs in refseqtsc > 5 ppmoverlap refseq (> 5 ppm)db_snp (> 5 ppm)jpt / yri / chs / ceu (> 5 ppm)hek29320 686 169193 140137 51811 33810 23411 2853930/5471/4391/3476ramos31 022 974371 759239 30811 455922711 4184036/5433/4340/3513beas2b98 761 770708 912440 30227 62820 38672202993/3979/3194/2167dld148 580 850462 724272 17119 94116 96519 8787084/9735/7731/6211mcf715 785 949172 834120 69511 79010 38311 7434094/5645/4432/3584tig318 780 087198 129144 62211 89310 51211 8474186/5857/4674/3711hela3 919 71999 24174 71911 300971011 2624187/5787/4705/3734adult tissue138 864 9781 496 409911 87236 71826 02350 67416 874/22 996/18 163/14 927fetal tissue41 744 136822 577572 94132 53326 77332 38610 400/14 192/11 088/9260samples : category of samples, tss - seq tags : tag number in each category, total tscs : observed tsc number, tscs in refseq : tscs overlapping with the refseq transcribed region (including their 50 k bp upstream region), tsc > 5 ppm : number of tscs whose expression level is higher than 5 ppm, overlap refseq (5 ppm) : > 5ppm tscs which overlap with the refseq transcribed region, db_snp (> 5 ppm) : number of tscs which contain snps in dbsnp, jpt / yri / chs / ceu (> 5 ppm) : number of tscs which contain ethnic snps (jpt : japanese in tokyo, ceu : utah residents with northern and western european ancestry from the ceph collection, chs : chinese in singapore, and yri : yoruba in ibadan). a) users can use a refseq i d for the simplest search (red box in the figure). tss - seq detailed search, users will obtain the search condition window. in this case, users can search tsss that are overexpressed after il4 stimulation by 2-folds, with their expression level higher than 5 ppm, showing h3k4me3 signals, and having nearby dbsnp data. (c) users can search tsss around a given snp or any genomic position (upper window). snps that are neighboring with known genes can be sought, too (bottom window). statistics of tss - seq data tscs corresponding to ncbi refseq genes and snp information samples : category of samples, tss - seq tags : tag number in each category, total tscs : observed tsc number, tscs in refseq : tscs overlapping with the refseq transcribed region (including their 50 k bp upstream region), tsc > 5 ppm : number of tscs whose expression level is higher than 5 ppm, overlap refseq (5 ppm) : > 5ppm tscs which overlap with the refseq transcribed region, db_snp (> 5 ppm) : number of tscs which contain snps in dbsnp, jpt / yri / chs / ceu (> 5 ppm) : number of tscs which contain ethnic snps (jpt : japanese in tokyo, ceu : utah residents with northern and western european ancestry from the ceph collection, chs : chinese in singapore, and yri : yoruba in ibadan). in addition to the tss - seq data, we also included rna - seq tags in the data of cultured cells. for example, in dld-1 cell data, rna - seq data from total rna, nuclear, cytoplasm, polysome, and the argonaute complex - associated fractions are stored (15). users can search and browse the transcriptional levels of each gene in each fraction to understand the dynamics of its transcripts. to further understand the relationship between tsss and chromatin states of the surrounding regions, we integrated chip - seq data of histone modifications : h3k4me3 and h3ac for active chromatin markers and h3k27me3 for a silent chromatin marker. data of the binding sites of rna polymerase ii and several transcription factors, such as stat6 in ramos and beads2b cells and hif1a in dld-1 cells, are also included. particularly, we added the above - mentioned data of chromatin states in nine human cell lines (8). in addition to the dbsnp data, we obtained exome data for four ethnic groups from the ncbi site (ftp://ftp.ncbi.nlm.nih.gov/1000genomes/ftp) and generated snvs uniquely called for these ethnic groups. we called 687 881 jpt (japanese in tokyo), 646 522 ceu (utah residents with northern and western european ancestry from the ceph collection) 578 299 chs (chinese in singapore) and 1 120 302 yri (yoruba in ibadan) snvs from 73, 64, 90 and 82 individual 's exome data, respectively. also, we added genome - wide association studies (gwas) data which connect 5800 snvs with clinical information (16). we integrated the internal and external tss - seq, rna - seq, chip - seq and snv data with each other. table 2 (and the statistics page of our web site) represents the statistics of each data and the overlap between them. it should be noted that all of the active markers do not always overlap. in some cases, active chromatin markers these cases may be interesting targets for future analysis of potentially stepwise transcriptional regulations. from a population genetic point of view, it may be also intriguing to further characterize tsss which are observed in an ethnic group - specific manner with solid supports from chip - seq and rna - seq data in cultured cells. for example, users can use a refseq i d for the simplest search (figure 1a). users can narrow down the targets by considering expression levels within a particular cell types or fold expression changes between different cell types (figure 2a). in the main viewer, users can recalculate tag counts by varying genomic regions (figure 2b and c). users can also consider overlap of the chip - seq data and rna - seq data with the tss - seq data (figure 2a). snp search function, users can input an snv in a particular category and search tsss in its surrounding region. conversely, users can search whether there are any tsss that have snvs of various categories in its surrounding regions (figures 1b and 2d as search results). (a) overview of tss - seq and chip - seq for nm_013293, transformer 2 alpha homolog. there are three major putative alternative promoters (ap4, ap10 and ap15) in dld1 cells. the expression of ap10 under the normoxia condition (21%) is relatively low compared with that under the hypoxia condition (1%). using check boxes, users can also check the tss - seq and chip - seq results in other tissues. (b) function of recalculating tag counts by specifying desired genomic regions. in this case, 1.5 ppm tsc specific to normoxia and 10.6 ppm tsc to hypoxia are observed. an ethnic snp (chs) and two dbsnps (rs11523571 and rs41273990) are also found in this region. searching chr1:159680868 based on the input window (figure 1c) also leads to a similar result. (a) overview of tss - seq and chip - seq for nm_013293, transformer 2 alpha homolog. there are three major putative alternative promoters (ap4, ap10 and ap15) in dld1 cells. the expression of ap10 under the normoxia condition (21%) is relatively low compared with that under the hypoxia condition (1%). using check boxes, users can also check the tss - seq and chip - seq results in other tissues. (b) function of recalculating tag counts by specifying desired genomic regions. in this case, 1.5 ppm tsc specific to normoxia and 10.6 ppm tsc to hypoxia are observed. an ethnic snp (chs) and two dbsnps (rs11523571 and rs41273990) are also found in this region. searching chr1:159680868 based on the input window (figure 1c) also leads to a similar result. all of the raw data are freely and anonymously available from our download site at ftp://ftp.hgc.jp / pub / hgc / db / dbtss / dbtss_ver8. we expect more genome - wide data to become available not only for tss - seq data but also for rna - seq and chip - seq data from humans, mice and other model organisms in the near future. also, tens of thousands of snv data for different ethnic groups are being generated without further biological information except their potential disease associations. we believe that, by the integration of the data, our dbtss will continue to serve as a unique database for a wide variety of researchers : both for researchers analyzing transcriptional regulations of individual genes as well as for those analyzing comprehensive transcriptional regulatory networks from a systems biological point of view. funding for open access charge : grant - in - aid for publication of scientific research results ; program for improvement of research environment for young researchers from the ministry of education, culture, sports, science and technology (mext), japan. | to support transcriptional regulation studies, we have constructed dbtss (database of transcriptional start sites), which contains exact positions of transcriptional start sites (tsss), determined with our own technique named tss - seq, in the genomes of various species. in its latest version, dbtss covers the data of the majority of human adult and embryonic tissues : it now contains 418 million tss tag sequences from 28 tissues / cell cultures. moreover, we integrated a series of our own transcriptomic data, such as the rna - seq data of subcellular - fractionated rnas as well as the chip - seq data of histone modifications and the binding of rna polymerase ii / several transcription factors in cultured cell lines into our original tss information. we also included several external epigenomic data, such as the chromatin map of the encode project. we further associated our tss information with public or original single - nucleotide variation (snv) data, in order to identify snvs in the regulatory regions. these data can be browsed in our new viewer, which supports versatile search conditions of users. we believe that our new dbtss will be an invaluable resource for interpreting the differential uses of tsss and for identifying human genetic variations that are associated with disordered transcriptional regulation. dbtss can be accessed at http://dbtss.hgc.jp. |
macular hole (mh) is a full - thickness defect of the retinal tissue involving the anatomical fovea. the advent of vitrectomy surgery and subsequent refinements of surgical techniques have led to improved visual outcomes.1,2 currently, mh treatment includes small - gauge pars plana vitrectomy with internal limiting membrane (ilm) peeling and placement of intraocular air or gas tamponade, followed by facedown posturing by the patient. the single surgery anatomical closure rate of mh is > 90%.3 despite a high success rate, significant controversy exists over the duration of facedown posturing.4,5 apart from the inconvenience, prolonged facedown positioning imposes physical, mental, and medical risks that can be a major burden for elderly patients. diagnosis of mh closure in gas - filled eyes is important in cases selected for long periods of posturing. recently, various studies have indicated that monitoring of mh closure through the air bubble in patients with a gas - filled eye could be achieved by postoperative optical coherence tomography (oct).3 this method could be useful in cases selected for long periods of facedown posturing.6 however, good quality oct images in gas - filled eyes are not always obtainable. fundus autofluorescence (faf), derived from the lipofuscin - laden retinal pigment epithelium, has been introduced as a new tool for the diagnosis and evaluation of mh surgical outcomes.7,8 in normal eyes, autofluorescence of the retinal pigment epithelium underlying the fovea is decreased due to the absorption of excitation light by the yellow macular pigment in the overlying retina. an increased intensity of an autofluorescence spot in the macula is consistent with a loss of foveal tissue, especially in a complete full - thickness mh.7 faf disappearance from the mh could occur after successful surgical repair.8 an ultrawide - field scanning laser ophthalmoscope with a green (532 nm) and a red (633 nm) laser (optos 200tx ; optos plc, dunfermline, scotland, uk) has been developed and is widely used in clinical ophthalmology. the device scans up to 200 and has an image capture time of 0.25 second in nonmydriatic conditions. the efficacy of optos in diabetic retinopathy has been reported,9 and a recent report showed that faf changes detected with optos was also useful in evaluating the characterization of rhegmatogenous retinal detachment.10 in this study, we examined the diagnostic utility of optos in evaluating mh closure in gas - filled eyes. a total of 18 consecutive eyes of 18 patients with unilateral mhs, who underwent vitrectomy between july 2013 and october 2015, were included in the study. full preoperative ophthalmologic examinations including best - corrected visual acuity, slit lamp examination, fundus examination, axial length measurement with the iol master (carl zeiss meditec ag ; jena, germany), spectral domain - oct (sd - oct) (hra ; heidelberg engineering gmbh, dossenheim, germany) or oct rs-3000 (nidek, gamagori, japan), and optos 200tx faf (optos plc) were performed for every patient. this study was approved by the oshima hospital of ophthalmology ethics committee and was performed in accordance with the ethical standards laid down by the declaration of helsinki. all patients underwent surgery with vitrectomy and posterior hyaloid dissection, epiretinal membrane excision when present, and ilm peeling (ilm dissection with a diameter not less than 2,500 m), and was assisted in all cases with brilliant blue g staining and a final fluid air exchange. the brilliant blue g solution (coomassie bbg 250 ; sigma - aldrich co., st louis, mo, usa) was prepared at a concentration of 0.25 mg / ml in bss plus (alcon, fort worth, tx, usa). gas exchange was performed with 20% sulfur hexafluoride (sf6) to tamponade the retina immediately after the vitrectomy. on preoperative day 1 and postoperative days 1, 2, and 7, oct and optos 200tx faf were imaged in all patients. these images were compressed into high - quality jpeg files and evaluated independently by two independent masked retina specialists (sn and ra) for the status of the mh at the indicated postoperative days. when the estimations of the two observers were different, a third observer (he) who was also masked to the clinical status would estimate the status. in this study, when mh closure was confirmed by oct, the facedown position was discontinued, although the patients were instructed to avoid a supine position. when oct showed the mh to be open or when it was inconclusive, the facedown position was maintained, even if decreased autofluorescence was observed in optos. faf images from optos were used for the quantitative analysis in the fovea by evaluating an area that is two - disc diameters in size. each faf image was saved as a jpeg file and cropped to two - disc diameter size with standardized illumination and contrast using adobe photoshop cs3 (adobe systems, san diego, ca, usa). a horizontal line was drawn through the center of the mh in imagej 1.42k (national institutes of health, usa) using the oct image or the color fundus photo preoperatively. in all cases, this line was carefully determined in postoperative images using location of retinal vessels and reference to the preoperative image. the intensity of each faf image on the drawn line in the fovea was calculated with imagej 1.42k. the maximum and minimum values were configured as 100 and 0, respectively. from the values, the intensity of the mh autofluorescence was calculated as a percentage of the preoperative oct image or the color fundus photo. the intensity graphs were created using excel 2008 (microsoft corporation, redmond, wa, usa). all patients underwent surgery with vitrectomy and posterior hyaloid dissection, epiretinal membrane excision when present, and ilm peeling (ilm dissection with a diameter not less than 2,500 m), and was assisted in all cases with brilliant blue g staining and a final fluid air exchange. the brilliant blue g solution (coomassie bbg 250 ; sigma - aldrich co., st louis, mo, usa) was prepared at a concentration of 0.25 mg / ml in bss plus (alcon, fort worth, tx, usa). gas exchange was performed with 20% sulfur hexafluoride (sf6) to tamponade the retina immediately after the vitrectomy. on preoperative day 1 and postoperative days 1, 2, and 7, oct and optos 200tx faf were imaged in all patients. these images were compressed into high - quality jpeg files and evaluated independently by two independent masked retina specialists (sn and ra) for the status of the mh at the indicated postoperative days. when the estimations of the two observers were different, a third observer (he) who was also masked to the clinical status would estimate the status. in this study, facedown posturing continued until mh closure including bridge formation was confirmed by oct. when mh closure was confirmed by oct, the facedown position was discontinued, although the patients were instructed to avoid a supine position. when oct showed the mh to be open or when it was inconclusive, the facedown position was maintained, even if decreased autofluorescence was observed in optos. faf images from optos were used for the quantitative analysis in the fovea by evaluating an area that is two - disc diameters in size. each faf image was saved as a jpeg file and cropped to two - disc diameter size with standardized illumination and contrast using adobe photoshop cs3 (adobe systems, san diego, ca, usa). a horizontal line was drawn through the center of the mh in imagej 1.42k (national institutes of health, usa) using the oct image or the color fundus photo preoperatively. in all cases, this line was carefully determined in postoperative images using location of retinal vessels and reference to the preoperative image. the intensity of each faf image on the drawn line in the fovea was calculated with imagej 1.42k. the maximum and minimum values were configured as 100 and 0, respectively. from the values, the intensity of the mh autofluorescence was calculated as a percentage of the preoperative oct image or the color fundus photo. the intensity graphs were created using excel 2008 (microsoft corporation, redmond, wa, usa). to evaluate the utility of faf imaging by optos in estimating mh status in the gas - filled eye, the study included 18 consecutive eyes of 18 patients (nine eyes of nine females and nine eyes of nine males) with unilateral mhs. one eye had a stage 1b mh (5.6%), three eyes had a stage 2 mh (16.7%), five eyes had a stage 3 mh (27.8%), and nine eyes had a stage 4 mh (50.0%). the size of the mhs ranged from 82 to 706 m (average standard deviation [sd ] ; 390172 m). preoperatively, one patient with stage 1b mh showed small but definitive increased autofluorescence in the fovea compared with the surrounding foveal area (case 6). all three patients with stage 2 mh showed intense autofluorescence at the site of the hole at preoperative optos imaging. clear increased autofluorescence signal at the mh area could not be shown with preoperative optos imaging in two cases of the stage 3 and two cases of the stage 4 mh, respectively (40.0% and 22.2%) (table 2). on postoperative day 1, 11 of 18 eyes (61.1%) had optos images that were of a quality that could be used for quantitative analysis of mh closure (table 3). among the seven postoperative optos - undetectable cases, two cases (cases 3 and 4) the remaining five eyes (cases 8, 10, 12, 14, and 15) did not show interpretable images in spite of the gas fill being over 60% of the vitreous cavity. quantitative analysis in cases with clear faf images showed that the faf intensity before surgery was 75.4%16.7% (mean sd), whereas the intensity was 45.5%17.2% (mean sd) 1 day after surgery (n=14 and 11, respectively) (table 4). the postoperative status of the mh could be determined by oct in eleven of 18 eyes (61.1%) on day 1 after the surgery (table 2). the finding between the faf and oct was consistent in eleven of 18 eyes (61.1%) on postoperative day 1 (table 3). on postoperative day 2, nine of 18 eyes (50.0%) could be imaged by optos. in six eyes (33.3%), a clear faf image could not be obtained due to the gas water interface being in the mh area. the reason for optos imaging failure in the other three eyes was unknown (16.7%). oct assessable images could be obtained in eight cases on postoperative day 2 (table 2). on postoperative day 7, 17 of 18 eyes (94.4%) could be imaged by optos, and decreased faf signals were obtained in the macula of most cases (table 2). in one eye (case 1), faf demonstrated persistent small hyperfluorescence in the fovea. quantitative analysis showed that the faf intensity was 17.1%13.6% (mean sd) 7 days after surgery (table 4). in contrast, clear images of the macula were obtained in all eyes by oct. a 65-year - old woman had a stage 3, 316 m mh in the right eye (figure 1a). the study of preoperative faf images obtained with optos revealed an autofluorescence signal corresponding to the mh (figure 1b and c). on day 1 after vitrectomy in accordance with the observation, a foveal autofluorescence signal could not be detected by optos in the sf6 gas - filled eye (figure 1e and f). on postoperative day 7 after the gas disappeared, oct confirmed that the hole was closed with small subfoveal space (figure 1 g). optos also could not detect any significant foveal autofluorescence signal (figure 1h and i). quantitative analysis showed a gradual decrease in faf intensity during mh closure after the surgery (figure 1j l, table 4). a 64-year - old woman had a stage 4 mh in the left eye (figure 2a). study of faf images obtained with optos showed an increased autofluorescence signal corresponding to the mh (figure 2b and c). this patient underwent surgery with pars plana vitrectomy and posterior hyaloid dissection and ilm peeling assisted with brilliant blue g staining, and a final air gas exchange with 20% sf6 gas. on postoperative day 1, oct showed bridge formation (foveal detachment) (figure 2d). study of faf images obtained with optos showed reduced but persistent autofluorescence in the macula in the sf6 gas - filled eye (figure 2e and f). after reabsorption of the gas, the oct examination confirmed foveal detachment (figure 2 g). faf imaging by optos showed a ring of hypoautofluorescence surrounding the central area of relative hyperautofluorescence (figure 2h and i). the quantitative analysis confirmed high faf intensity (91.5%) before the operation and decreased faf intensity (55.3% and 43.4%) 1 and 7 days after the surgery, respectively (figure 2j l, table 4). a 65-year - old woman had a stage 3, 316 m mh in the right eye (figure 1a). the study of preoperative faf images obtained with optos revealed an autofluorescence signal corresponding to the mh (figure 1b and c). on day 1 after vitrectomy, oct was not identifiable clearly (figure 1d). in accordance with the observation, a foveal autofluorescence signal could not be detected by optos in the sf6 gas - filled eye (figure 1e and f). on postoperative day 7 after the gas disappeared, oct confirmed that the hole was closed with small subfoveal space (figure 1 g). optos also could not detect any significant foveal autofluorescence signal (figure 1h and i). quantitative analysis showed a gradual decrease in faf intensity during mh closure after the surgery (figure 1j l, table 4). a 64-year - old woman had a stage 4 mh in the left eye (figure 2a). study of faf images obtained with optos showed an increased autofluorescence signal corresponding to the mh (figure 2b and c). this patient underwent surgery with pars plana vitrectomy and posterior hyaloid dissection and ilm peeling assisted with brilliant blue g staining, and a final air gas exchange with 20% sf6 gas. on postoperative day 1, oct showed bridge formation (foveal detachment) (figure 2d). study of faf images obtained with optos showed reduced but persistent autofluorescence in the macula in the sf6 gas - filled eye (figure 2e and f). after reabsorption of the gas, the oct examination confirmed foveal detachment (figure 2 g). faf imaging by optos showed a ring of hypoautofluorescence surrounding the central area of relative hyperautofluorescence (figure 2h and i). the quantitative analysis confirmed high faf intensity (91.5%) before the operation and decreased faf intensity (55.3% and 43.4%) 1 and 7 days after the surgery, respectively (figure 2j l, table 4). a 65-year - old woman had a stage 3, 316 m mh in the right eye (figure 1a). the study of preoperative faf images obtained with optos revealed an autofluorescence signal corresponding to the mh (figure 1b and c). on day 1 after vitrectomy, oct was not identifiable clearly (figure 1d). in accordance with the observation, a foveal autofluorescence signal could not be detected by optos in the sf6 gas - filled eye (figure 1e and f). on postoperative day 7 after the gas disappeared, oct confirmed that the hole was closed with small subfoveal space (figure 1 g). optos also could not detect any significant foveal autofluorescence signal (figure 1h and i). quantitative analysis showed a gradual decrease in faf intensity during mh closure after the surgery (figure 1j l, table 4). a 64-year - old woman had a stage 4 mh in the left eye (figure 2a). study of faf images obtained with optos showed an increased autofluorescence signal corresponding to the mh (figure 2b and c). this patient underwent surgery with pars plana vitrectomy and posterior hyaloid dissection and ilm peeling assisted with brilliant blue g staining, and a final air gas exchange with 20% sf6 gas. on postoperative day 1, oct showed bridge formation (foveal detachment) (figure 2d). study of faf images obtained with optos showed reduced but persistent autofluorescence in the macula in the sf6 gas - filled eye (figure 2e and f). after reabsorption of the gas, the oct examination confirmed foveal detachment (figure 2 g). faf imaging by optos showed a ring of hypoautofluorescence surrounding the central area of relative hyperautofluorescence (figure 2h and i). the quantitative analysis confirmed high faf intensity (91.5%) before the operation and decreased faf intensity (55.3% and 43.4%) 1 and 7 days after the surgery, respectively (figure 2j l, table 4). the diagnosis of idiopathic full - thickness mh is usually made by biomicroscopic examination and oct. the surgical repair of mh involves pars plana vitrectomy with removal of the ilm and filling of the vitreous cavity with gas followed by facedown posturing by the patient. in most facilities, actual anatomical closure of the mh after surgery is confirmed mainly by oct. recent studies have demonstrated the ability of oct to successfully image the macula through gas and to guide the duration of facedown posturing.3,6,8,11 however, a few studies have reported mh reopening after oct - guided releasing of facedown posturing.12 arima reported a case of an anatomically closed mh reopening, 2 days after the release of facedown positioning where oct showed anatomical closure of the mh, but bright hyperfluorescence in the fovea was detected by faf imaging.13 thus, oct and faf imaging may exhibit discrepant findings in some cases. furthermore, in this study, either faf or oct in six of 18 eyes (33.3%) was not identifiable on postoperative day 1 (table 3). therefore, this current study suggests that faf imaging by optos, and oct could be complementary tools in estimating postoperative mh status. in one case, optos detected reduced but persistent auto - fluorescence in the macula in an sf6 gas - filled eye in spite of oct showing bridge formation (case 1). although this case did not result in reopening of the mh after release of the facedown positioning, reopening of a closed mh with bridge formation might occur in some cases. in another case (case 2), reduced autofluorescence was detected on optos faf imaging on day 7 after surgery in spite of bridge formation on oct. the difference in faf intensity between case 1 and case 2 might be due to the thickness of the bridging retinal tissue. the bridging tissue in case 1 may have been too thin to block the excitation wave light. the faf image early after mh surgery could be an important indicator of the degree of mh closure. furthermore, our finding suggests that maintenance of the facedown position may be required depending on faf intensity. however, further investigations with larger sample sizes would be necessary to determine the threshold of reduced autofluorescence that would predict a possible reopening of a closed mh after surgery. as gas - filled eyes shift toward high myopia,14 oct images have to be taken with a 20 diopter adjustment.11 oct imaging also requires a high level of technical skill, and there is a steep learning curve involved in obtaining good quality oct images of gas - filled eyes. in this study, on postoperative day 1, oct images and faf images could be obtained from 11/18 eyes and 11/18 eyes, respectively. in three cases, optos - imaged faf could thus be a helpful imaging modality in evaluating mh closure and complementing oct analysis. however, the image quality might be affected in patients with postsurgical corneal conditions, increased intraocular pressure, or inflammation of anterior segment. in this study, four of the 18 mhs it is possible that the area of decreased foveal autofluorescence in these cases represents the floating of the operculum or the cuff of neurosensory retinal detachment surrounding the mh. non - optos faf devices are unable to obtain good quality images in gas - filled eyes. the myopic shift in gas - filled eyes may be a reason why clear faf images can not be obtained with non - optos faf devices. optos is widely used in clinical ophthalmology because of the wide - angle view of the retina that it captures.9,10 the system also includes an auto - focusing technology, which enables images to be taken regardless of the axial length.15 therefore, the use of optos to obtain high - quality images of a gas - filled eye circumvents the technical challenges posed by other fundus cameras or the oct. changing the orientation of the subject s face or the eye fixation may improve the faf image quality. in this study, lid retractors were not used in optos imaging, which may have been useful based on a previous report utilizing an eyelid speculum in optos imaging,16 and this technique may be used in future studies to obtain better quality faf images. in conclusion, we report that optos can provide high - quality faf images from gas - filled eyes after mh surgery in some cases. faf imaging using optos and subsequent quantitative analysis of these images might be a useful tool for the surgeon in deciding the release of the patient from facedown posturing. | background and objectiveexisting ophthalmoscopy methods are unable to obtain clear fundus autofluorescence (faf) images in gas - filled eyes. the purpose of this study was to evaluate the capability of wide - field laser ophthalmoscopy (optos) in obtaining faf images in gas - filled eyes for the assessment of macular hole (mh) closure after surgery.methodsthis was an interventional case series. eighteen consecutive patients with unilateral mh underwent vitrectomy with internal limiting membrane peeling and 20% sulfur hexafluoride gas tamponade. faf images using optos were recorded preoperatively and postoperatively (days 1, 2, and 7).resultson postoperative days 1, 2, and 7, faf images were obtained from 11/18 (61.1%), 9/18 (50.0%), and 17/18 eyes (94.4%), respectively, using optos. the quality of faf images using optos was sufficient to determine mh closure in 9/18 (50.0%) of gas - filled eyes postoperatively. quantitative analysis of faf images was helpful in determining complete or partial closure of the mh.conclusionfaf imaging using optos might be a useful adjunct to optical coherence tomography as a supportive method to guide the release from facedown posturing in some cases of mh. |
diabetes, as the most common metabolic disease, causes complications such as eye, renal, neurological, and cardiovascular disorders. costs associated with the treatment of diabetes is approximately 132 billion dollars per year, and its prevalence is increasing by about 6% in the world each year. now, according to official statistics of the world health organization, 190 million people suffer from this disease worldwide. by 2025, it is estimated that this number would be more than 330 million people and that by 2030, the share of developing countries would be 77.6% of the total number of diabetic patients in the world. recent evidence from the international diabetes federation indicates that diabetes currently affects 246 million people worldwide. diabetes incidence in iran is 5 - 16/3%, and for type 2 in 2010, it was approximately 8%. studies have indicated that direct and indirect costs of diabetes and its complications are very high, compared to other diseases. diabetes is responsible for the deaths of hundreds of thousands of people in the world annually ; its prevalence in tehran is equal to 7.2% in the population aged 30 and more, and it is 6.5% in isfahan city. considering high and growing prevalence of type 2 diabetes in isfahan as the most common metabolic disease, with its inestimable direct and indirect costs, its microvascular complications such as retinopathy, nephropathy, and neuropathy, and its macrovascular complications such as cardiovascular disease. it is necessary to pay more attention to this disease and identify easy and cheap ways to prevent it. an important point is that education of people and timely treatment can reduce the incidence of this disease and, to some extent, can prevent its debilitating complications. most deaths of diabetic patients are due to cardiovascular diseases such as myocardial infarction, which are preventable by glycemic control, blood pressure control, and lipid profile regulation. because education of such patients in iran does not receive enough attention, patient - centered and community - based interventions with focus on education and its continuance and support of patients can impact significantly on blood glucose control, enhance the quality of life, increase patient satisfaction with care and treatment, and create patient knowledge and awareness. because patient education plays an important role in the control of diabetes, identification and use of simple and inexpensive ways to educate these patients amount to necessities of a community. usually for chronic diseases such as cardiovascular disease, diabetes, and the like, the method of information therapy is a useful option. information therapy is a new method that provides health - related information for patients and enables patients to make informed decisions about their health and treatment and be a partner in the healing process. this method helps reduce the usage rate of health services and consequently reduces the cost of treatment. information therapy is a new tool with potential for physicians that can improve knowledge and decision making of patient and the relationship between patient and physician. the most important advantage of this method for someone who receives information therapy is the ability to provide self - care as it allows people to take care of themselves and reduce the use of health - care services and medical care costs. by providing information therapy one of the methods of information therapy is nonattendance and distance training. because of the elimination of the limitation of time and space and the facility of establishing a relationship with clients, the possibility of reduction in diabetes complications has been proven with proper glycated hemoglobin (hba1c) control, and there are promising prospects for cardiovascular complications of this disease. in the recent years, the treatment techniques of diabetes and its complications are more accurate, and public access is more extensive. relying on the importance of patient education, can speak more confidently about the physical aspects of disease ideal control. but we should not forget that all of these are only part of a comprehensive treatment plan for any type of chronic and serious disease. one of the most valuable services that a medical librarian can provide is helping people understand their information needs. visiting a good therapist, who by asking skillful questions enables patients to speak about things that are bothering them, can be very similar to an interaction with a physician. in information therapy, medical librarians work with treatment teams and based on the specific information needs of each patient, they share information with patients or their family. this new service and patient participation in their treatment is not possible unless the patient informed, and by providing reliable and evidence - based medical information become powerful and autonomous in the care moments. for more than two decades information services of medical librarians in the health are classified into two categories : direct and indirect services. direct role of medical librarians involves providing health - care information about the control and prevention of diseases, public health, complementary medicine, effects and side effects of drugs, fitness, and nutrition directly to patients and the public without any recommendation for decision making, without any prescribed information by the health - care provider such as a doctor. when information has been prescribed by a physician, librarians can play an indirect role in their support ; it means providing the right, new, evidence - based, and referred information to patients which has been confirmed by a physician. providing information to health - care providers can also be included in the indirect role of librarians in the information therapy process. on the other hand, many studies have indicated educational programs for diabetic patients improves blood sugar and are effective in its control. afshar and izadi showed in their research that education has been successful in controlling blood sugar. masoudi alavi. concluded that after the educational intervention, a statistically significant reduction in hba1c was observed in both the intervention and control groups, and patient satisfaction increased in both groups ; the increase in patient satisfaction in the intervention group was significantly higher than the control.. showed in their study that telephone follow - up by nurses led to improved diabetic diet adherence and reduced hba1c in patients with type 2 diabetes. have shown in their research that education and remote follow - up via telephone and sms services by nurses have significant effects on blood glucose control in patients with diabetes. zer. showed that the health scores of diabetic patients who had participated in the training program were twice that of other patients. adolfsson. concluded in their research that empowerment through group education can improve knowledge and awareness in diabetic patients in relation to glucose control despite the recurrent nature of diabetes. showed that local educational programs of diabetes self - management in general prevents from increased hba1c and can have positive effects on patients with high hba1c levels. al - khawaldeh. in their study concluded that enhanced self - efficacy and self - management are essential and important components of diabetes education programs. moreover, behavioral counseling and interventions skills are vital for patients so that they are able to manage their diabetes. according to what has been discussed, it seems that information therapy for diabetic patients in isfahan city by medical librarians along with the treatment team can be effective in reducing blood glucose levels in these patients. so, this study is trying to investigate the influence of non - attendance information therapy on the control of hba1c in type 2 diabetic patients in isfahan city. the present study is an interventional semiexperimental one, which was conducted with pretest and post - test and two - group design. statistical population was 6,000 diabetics patients of isfahan endocrine and metabolism research center, of whom 64 people with type 2 diabetes according to this formula n = (z + z) (2s)/d were randomly selected and with respect to age, sex, education, and hba1c levels were divided into intervention and control groups (32 persons per group). the diabetes center members were required to participate in six sessions on diabetes education and its care. after this period, these patients, according to their wishes, although not required, sometimes refer to the isfahan endocrine and metabolism research center. in this study, we wanted to continue education of diabetes self - management and content of the six sessions of education of the diabetes center as nonattendance information therapy and investigate its effect. criteria for entry into the groups were having a medical record in the mentioned center, willingness to participate in the information therapy plan, and also previous participation in the six education sessions at the center. exclusion criteria were unwillingness to participate in the information therapy plan and no previous participation in the education sessions at the isfahan endocrine and metabolism research center. at the end of the sixth session in the center, personal data, addresses, and phone numbers from both groups were taken and a consent form was completed. then, their hba1c levels were checked and recorded. after that, the intervention group received training under the information therapy plan for two months to enable their self - care by mailing the training package and sending messages to their mobile phones, the text content of which was confirmed by medical experts. the training packages included 8 brochures and 24 messages whose content matched with the content of the six sessions at the isfahan endocrine and metabolism research center and had been approved by the second supervisor (doctor). every patient received one training package and three messages in a week. after passing the nonattendance information therapy period (two months) and after the incubation period (for its effect) for statistical analysis, t - test, paired t - test and chi - square and mann whitney u tests were used. mean ages of the control and intervention groups were 53 7.3 and 52.6 8.2 years, respectively. the results of the t - test showed that there was no significant difference in mean age between the two groups (p = 0.79) [table 1 ]. distribution of age in the control and intervention groups in terms of gender, 41 were men and 23 were women. the chi - square test showed that there was no significant difference between the groups in sex distribution (p = 0.79). the mann whitney u test showed that the level of education of the two groups did not differ significantly (p = 0.86) ; so, there was no significant difference between the intervention and control groups before the intervention in terms of age, sex, and also the main variables of education and training and hba1c level [table 2 ]. demographic characteristics of the control and intervention groups table 3 shows the comparison of hba1c levels in both intervention and control groups before and after information therapy intervention. independent t - tests showed that before intervention, the hba1c mean reported no significant differences between the two groups (p = 0.97). but after intervention, the hba1c mean was significantly lower in the intervention group than the control group (p = 0.048). the paired t - test showed that in the control group there was no significant difference in hba1c mean (p = 0.37) before and after information therapy intervention. however, in the intervention group after the intervention, the hba1c mean was significantly reduced (p < 0.001) [table 3 ]. comparison of mean glycosylated hemoglobin (hba1c) between intervention and control groups before and after information therapy figure 1 shows the change more clearly. hba1c : glycosylated hemoglobin table 4 shows the mean change in hba1c scores in two groups. independent t - tests showed that the mean change in hba1c was significantly higher in the intervention group than the control group (p = 0.01). the results of this study showed that there were no significant difference statistically between the intervention and control groups in terms of confounding variables that may have an influence on the study results ; in other words, the two groups were similar in terms of sex, age, education, previous training, as well as initial hba1c levels. so, significant differences in the independent variables in the intervention group before and after intervention showed a positive effect of the educational follow - up and information therapy in patients with type 2 diabetes. the findings showed that after nonattendance information therapy by training packages and sms service, the hba1c mean was significantly decreased in the intervention group. these findings are in agreement with the findings of afshar and izadi, which reported that training had been successful in controlling blood sugar. they were also in agreement with the research of masoudi - alavi., which showed that after the intervention in both intervention and control groups there was a statistically significant reduction in hba1c levels. also concluded that training in diet is effective in lowering blood sugar in type 2 diabetic patients. zakerimoghadam and colleagues showed that follow - up through telephone by nurses results in improved adherence to the diabetic diet, reducing the hba1c in patients with type 2 diabetes. the findings of the present study is in agreement with various foreign studies, including adolfsson., who reported that empowerment through group training improves knowledge and awareness of diabetic patients for control of blood sugar despite the recurrent nature of the disease ; rygg. also showed that local educational diabetes self - management programs in general prevents an increase in hba1c and can have an impact on patients with high hba1c levels. however, the results of the study by wong., investigating the effect of telephone follow - up by nurses on diabetes status, were inconsistent with the result of our study as there was no difference between the hba1c levels in the intervention and control groups. it seems that long interval between calls and short duration of the follow - up calls in the study of wong. also, findings of the present study is unlike the research of maljanian., which reported that telephone follow - up and the training during three months had no effect on the hba1c level and the quality of life of diabetic patients and it only promoted instruction adherence of the diabetes association and medical care. the present study showed that a simple and inexpensive way such as nonattendance information therapy is effective in reducing hba1c in diabetic patients. in a diabetic patient, treatment will not be effective unless the patient clearly understands the nature of his disease and take steps to deal with it. nonattendance education and follow - up and reminder instructions for the care and treatment of diabetic patients will lead to improved results in the treatment process, and consequently, the number of visits of these patients to medical centers and the related direct and indirect costs will be reduced. therefore, it is suggested to conduct nonattendance information therapy for diabetic patients in all diabetes research centers by competent authorities with the co - operation of medical librarians and also use e - mail and sms systems in diabetes research centers to facilitate nonattendance information therapy process. | introduction : patient education plays an important role in the control of diabetes. nonattendance education, enabling elimination of limitations caused by time and space and facilitating the relationship between patient and care liaison is an effective, simple, and cheap method. the aim of this study is determination of the effects of nonattendance information therapy on the control of glycosylated hemoglobin (hba1c) in type 2 diabetic patients in isfahan.materials and methods : the present study was an interventional semi experimental study with pretest and post - test and control groups. statistical population were type 2 diabetics patients of the isfahan endocrine and metabolism research center, of whom 64 people were randomly selected and divided into intervention and control groups. first, the preliminary data were collected using the hba1c test in patients. then, the intervention group received training package and short message service (sms) for eight weeks. after one - month incubation period, hba1c was again determined in both groups. data were analyzed using t - test, paired t - test and mann whitney u and chi - square tests.results:results showed that diabetes patients hba1c in the intervention group was significantly lower after the intervention through training packages and sms service compared to before the intervention (p < 0.001). comparison of the two groups showed that there was a significant difference in the hba1c between the intervention and control groups (p = 0.048).conclusion : follow - up of education of patients with type 2 diabetes through training packages and sms services had significant effects on the control of the patients hba1c. also due to the low cost and high effectiveness of this method, it is recommended to health - care providers and treatment groups. this study also showed that having medical librarians along with treatment group can have a positive effect on the type 2 diabetic patients health. |
severe combined immunodeficiency (scid), characterized by extremely low or absent t cell production, defective t cell function and absent antibody responses, can be caused by defects in any of several genes and if untreated leads to early death due to infections [1, 2 ]. population based newborn screening for scid has been recommended to identify affected infants before the onset of devastating infections so that effective treatment can be provided [36 ]. a newborn screening test for scid, now implemented in several states, ascertains t - cell receptor excision circles (trecs), dna byproducts of t cell antigen receptor gene rearrangement, as a biomarker of normal t cell development [5, 710 ]. trecs are measured by quantitative pcr (qpcr) of dna isolated from infant dried blood spot (dbs) samples universally collected in nurseries. for infants with undetectable or low trecs, or with unsatisfactory dna amplification, a differential white blood count and analysis of lymphocyte subsets by flow cytometry are obtained to establish the absolute number of nave t cells, after which further clinical and laboratory evaluations are performed to arrive at a definitive diagnosis. beyond typical scid cases, trec screening has detected a spectrum of infants with inadequate numbers of diverse, autologous t cells. as predicted before the start of screening, leaky scid and omenn syndrome, both due to hypomorphic mutations in scid genes, have been found in infants with low trecs, as have cases of digeorge syndrome / chromosome 22q11 deletion in which a substantial degree of thymic insufficiency exists. in addition, secondary causes of t lymphocytopenia have included abnormal loss of t cells from the peripheral circulation, such as with chylothorax or hydrops. a challenging and less anticipated category of cases with abnormal trec results has been the infants with persistent t lymphocytopenia of 3001,500 t cells/l, no maternal t cell engraftment, and absence of identified deleterious mutations in common scid genes. these infants have been designated as combined immunodeficiency (cid) or scid variants by trec newborn screening programs. cid or scid variant cases have been of particular interest, providing an opportunity to discover previously unappreciated causes of newborn t lymphocytopenia. in the absence of clues to narrow the number of potential candidate genes to account for variant scid in asymptomatic infants who appear healthy, high throughput deep sequencing may be useful ; this approach has led to gene identification in other primary immunodeficiencies [1113 ]. using whole exome sequencing (wes), we found two infants with variant scid who had deleterious mutations in the ataxia telangiectasia mutated (atm) gene. prompted by the prospective discovery of these patients diagnoses and a recent report of low trecs in archived dbs from cases of ataxia telangiectasia (at), we reviewed 13 cases of at in our clinical cohorts. by retrieving their residual dbs samples taken in the newborn nursery and measuring their trec numbers, we showed that over half of at patients could be identified as abnormal, making at a secondary target of scid screening. infants v003 and v004 were identified as positive by routine california scid screening by trec test and confirmed to have t lymphocytopenia. informed consent for research, including cellular immune studies and wes, was obtained for the infants and their parents under approved protocols at children s hospital los angeles (chla) and the university of california san francisco (ucsf). additional patients from the pediatric immunology services at chla and ucsf were enrolled with institutional review board approval. genomic dna from edta anticoagulated whole blood was prepared using a gentra puregene blood kit (qiagen usa : germantown, md). libraries were prepared by ligating a pair of truseq adaptors (illumina : san diego, ca) to genomic dna sheared to a mean fragment size of 200300 bp (s2 sonicator, covaris : woburn, ma). libraries with these adaptors and barcode sequences were enriched with 10 cycles of pcr. for infant v003 and parents, exon capture was performed by pooling 500 ng of each of 6 libraries incubated with illumina truseq version 2 biotinylated exon - encoded dna oligonucleotides for 20 h. for v004 and parents, exon capture was performed by incubation with a roche nimblegen version 3 capture array. exon - enriched dna was captured with streptavidin - labeled magnetic beads, washed and eluted. after 10 cycles of dna amplification, the exome libraries were sequenced (hiseq2000, llumina). paired 100 bp end reads were generated (> 50 m reads / subject), to yield an average of > 65 reads covering the targeted regions with > 90 % covered by at least 10 reads. the resulting files were converted to compressed binary format (bam), sorted by coordinate, indexed, and marked for pcr duplicate reads using the picard toolkit (http://picard.sourceforge.net). bam files were processed to reduce artifacts and improve call accuracy using gatk software (v 1.4.15) [16, 17 ]. specifically, local realignment was performed around known insertion or deletion (indel) locations, and base quality scores were re - calibrated using co - variates such as position in read and sequencing chemistry effect. variants were called using the gatk unifiedgenotyper. the called single nucleotide polymorphisms (snps) had their scores re - calibrated by variant quality score recalibration (vqsr) using the exomes in this report plus 24 others sequenced at our site. hapmap v3.3 and the omni chip array sets from the 1,000 genomes project (october, 2011 release) were training data, and hapmap 3.3 provided truth sites [18, 19 ]. a truth sensitivity cutoff of 99 % was used. for indel recalibration and quality selection, we used qd 200.0. additional annotations including region, effect, dbsnp 135 and 1,000 genomes membership and omim phenotype were added using custom scripts. atm exons 7, 10, 39, and 46 were amplified from genomic dna using published primers (labeled as exons 9, 12, 41, and 48 in thorstenson.) with an m13 extension 5 to each reverse primer as follows : atme7_forward 5 - gta aaa cga cgg cca gtc agc ata cca ctt cat aac tgatme7_reverse 5 - tca tat cct cct aaa gaa cacatme10_forward 5 -tgt gat gga ata gtt ttc aaatme10_reverse 5- gta aaa cga cgg cca gtt gtg atg gaa tag ttt tca aatme39_forward 5- tgt ggt ttt tgg gaa ttt gtaatme39_reverse 5- gta aaa cgacgg cca gtt gtg gtt ttt ggg aat ttg taatme46_forward 5 - gta aaa cga cgg cca gtt ctt gtc act aca aaa gtt cct ttatme46_reverse 5 - tct ttt tcc ctc agg ctt tc. atme7_forward 5 - gta aaa cga cgg cca gtc agc ata cca ctt cat aac tg atme7_reverse 5 - tca tat cct cct aaa gaa cac atme10_forward 5 -tgt gat gga ata gtt ttc aa atme10_reverse 5- gta aaa cga cgg cca gtt gtg atg gaa tag ttt tca a atme39_forward 5- tgt ggt ttt tgg gaa ttt gta atme39_reverse 5- gta aaa cgacgg cca gtt gtg gtt ttt ggg aat ttg ta atme46_forward 5 - gta aaa cga cgg cca gtt ctt gtc act aca aaa gtt cct tt atme46_reverse 5 - tct ttt tcc ctc agg ctt tc. sequencing was performed with the m13 forward primer, and results were compared with reference atm ng_009830.1, using sequencher 4.10.1 software (gene codes cooperation : ann arbor, mi). residual dbs originally collected for routine nbs and stored at 20 by the genetic disease laboratory (gdl) of the california department of public health (cdph) were retrieved, and trec and -actin gene copy number determined by the gdl newborn screening laboratory, using the protocol of chan and puck modified for high throughput and implemented by perkinelmer, inc (lab within a lab at california department of public health, richmond, ca ; parent company based in hershey, pa) with cutoff values as reported. infants v003 and v004 were identified as positive by routine california scid screening by trec test and confirmed to have t lymphocytopenia. informed consent for research, including cellular immune studies and wes, was obtained for the infants and their parents under approved protocols at children s hospital los angeles (chla) and the university of california san francisco (ucsf). additional patients from the pediatric immunology services at chla and ucsf were enrolled with institutional review board approval. genomic dna from edta anticoagulated whole blood was prepared using a gentra puregene blood kit (qiagen usa : germantown, md). libraries were prepared by ligating a pair of truseq adaptors (illumina : san diego, ca) to genomic dna sheared to a mean fragment size of 200300 bp (s2 sonicator, covaris : woburn, ma). libraries with these adaptors and barcode sequences were enriched with 10 cycles of pcr. for infant v003 and parents, exon capture was performed by pooling 500 ng of each of 6 libraries incubated with illumina truseq version 2 biotinylated exon - encoded dna oligonucleotides for 20 h. for v004 and parents, exon capture was performed by incubation with a roche nimblegen version 3 capture array. exon - enriched dna was captured with streptavidin - labeled magnetic beads, washed and eluted. after 10 cycles of dna amplification, the exome libraries were sequenced (hiseq2000, llumina). paired 100 bp end reads were generated (> 50 m reads / subject), to yield an average of > 65 reads covering the targeted regions with > 90 % covered by at least 10 reads. the resulting files were converted to compressed binary format (bam), sorted by coordinate, indexed, and marked for pcr duplicate reads using the picard toolkit (http://picard.sourceforge.net). bam files were processed to reduce artifacts and improve call accuracy using gatk software (v 1.4.15) [16, 17 ]. specifically, local realignment was performed around known insertion or deletion (indel) locations, and base quality scores were re - calibrated using co - variates such as position in read and sequencing chemistry effect. variants were called using the gatk unifiedgenotyper. the called single nucleotide polymorphisms (snps) had their scores re - calibrated by variant quality score recalibration (vqsr) using the exomes in this report plus 24 others sequenced at our site. hapmap v3.3 and the omni chip array sets from the 1,000 genomes project (october, 2011 release) were training data, and hapmap 3.3 provided truth sites [18, 19 ]. a truth sensitivity cutoff of 99 % was used. for indel recalibration and quality selection, we used qd additional annotations including region, effect, dbsnp 135 and 1,000 genomes membership and omim phenotype were added using custom scripts. atm exons 7, 10, 39, and 46 were amplified from genomic dna using published primers (labeled as exons 9, 12, 41, and 48 in thorstenson.) with an m13 extension 5 to each reverse primer as follows : atme7_forward 5 - gta aaa cga cgg cca gtc agc ata cca ctt cat aac tgatme7_reverse 5 - tca tat cct cct aaa gaa cacatme10_forward 5 -tgt gat gga ata gtt ttc aaatme10_reverse 5- gta aaa cga cgg cca gtt gtg atg gaa tag ttt tca aatme39_forward 5- tgt ggt ttt tgg gaa ttt gtaatme39_reverse 5- gta aaa cgacgg cca gtt gtg gtt ttt ggg aat ttg taatme46_forward 5 - gta aaa cga cgg cca gtt ctt gtc act aca aaa gtt cct ttatme46_reverse 5 - tct ttt tcc ctc agg ctt tc. atme7_forward 5 - gta aaa cga cgg cca gtc agc ata cca ctt cat aac tg atme7_reverse 5 - tca tat cct cct aaa gaa cac atme10_forward 5 -tgt gat gga ata gtt ttc aa atme10_reverse 5- gta aaa cga cgg cca gtt gtg atg gaa tag ttt tca a atme39_forward 5- tgt ggt ttt tgg gaa ttt gta atme39_reverse 5- gta aaa cgacgg cca gtt gtg gtt ttt ggg aat ttg ta atme46_forward 5 - gta aaa cga cgg cca gtt ctt gtc act aca aaa gtt cct tt atme46_reverse 5 - tct ttt tcc ctc agg ctt tc. sequencing was performed with the m13 forward primer, and results were compared with reference atm ng_009830.1, using sequencher 4.10.1 software (gene codes cooperation : ann arbor, mi). residual dbs originally collected for routine nbs and stored at 20 by the genetic disease laboratory (gdl) of the california department of public health (cdph) were retrieved, and trec and -actin gene copy number determined by the gdl newborn screening laboratory, using the protocol of chan and puck modified for high throughput and implemented by perkinelmer, inc (lab within a lab at california department of public health, richmond, ca ; parent company based in hershey, pa) with cutoff values as reported. newborns in california are screened for scid and classified as positive if trec copy number is 5 with -actin > 5,000 copies, or trec copy number is between 6 and 25 with -actin > 10,000 copies. the tests are classified as incomplete and are repeated if there are low trecs, but also low copies of the -actin gene segment amplified as a control. t cells are measured by flow cytometry in cases that are positive or that have two incomplete dbs samples. infants v003 and v004 were unrelated, healthy females born at term following normal pregnancies. family history for both was negative for immune disease or consanguinity. lymphocyte flow cytometry was ordered for infant v003 after two dbs yielding incomplete results, while v004 had an initial positive result with 21 trecs and 13,300 -actin copies (table i).table iimmunologic phenotype of infants identified by scid newborn screeninginfantv003v004age3 m5.5 m21 days7 mtrec (normal > 25)421beta - actin (> 10,000)10,00013,300wbc (5,00019,500 cells/l)7,1003,5304,5005,320alc (2,50016,500)1,6002,1892,0003,032cd3 t cells (2,5505,500)9966151,0601,397cd4 t - helper cells (1,6004,000)749502600777cd8 t - cytotoxic cells (5601,700)185172340400cd3/cd4/cd45ra (1,2003,700)456360cd3/cd4/cd45ro (60900)299180cd19 b cells (3002,000)52844401,232cd16/56 nk cell (1701,100)3471,605300381igg (165781 mg / dl)156414iga (25154) 0.1 iu / ml)0.073.73anti - h. influenzae type b (> 1 mcg / ml) 2,000 and > 300, respectively), and the number of cd45ra nave cd4 t cells was low. low t and b cell numbers persisted, and low igg levels with failure to produce antibodies after vaccination led to institution of immunoglobulin replacement and trimethoprim - sulfamethoxazole antibiotic prophylaxis. lymphocyte proliferation to phytohemagglutinin and tcr v diversity assessed by spectratyping were normal, but an epstein barr virus transduced b cell line from the patient had only half normal phosphorylation of stat5 in response to il-2, suggesting an intrinsic lymphocyte impairment. later, at age 1416 months, v003 was reported by her mother to have unsteady gait ; physical examination first showed mild truncal ataxia at 20 months. infant v004 had flow cytometry at 21 days of age, showing only 1,060 t cells, with low cd45ra nave helper cd4 t cells (table i). b and nk cell numbers and lymphocyte proliferation were normal, but v spectratyping showed decreased t cell diversity (not shown). as with infant v003, in vitro phosphorylation of stat5 after il-2 activation was diminished, but not absent, as would be the case in scid due to defects in the il-2 receptor common chain or janus kinase 3 [9, 24 ]. physical examination of infant v004 has been normal to date, but she did not mount robust antibody to t - cell dependent protein - conjugated h. influenzae vaccination. to investigate the genetic etiology underlying their observed immunodeficient status, dna samples from v003, v004 and their parents were subjected to wes to generate a list of small nucleotide polymorphism (snp) and small insertion or deletion (indel) variants. these lists were filtered to retain successively fewer candidate variants as shown for snps and indels separately for each infant in fig. after initial quality filtering and removal of variants common enough to be found in dbsnp 135 and the 1,000 genomes database, further filters were applied to keep only the non - synonymous variants lying in captured exonic and splice - site regions of genes, and only those variants with a high (> 30) genotype quality score (fig. 1wes variants filtering paths. a trapezoids represent filters with resulting numbers of variants retained after each step indicated by a circled digit. starting with initial total variant lists (1), filters were applied for quality (2) and then to keep rare alleles (3) that alter splice sites or produce non - synonymous codon changes and are absent in local exomes (4). subsequent strategies were : focusing on variants from a list of genes associated with t cell phenotypes (yellow shading, 5) ; or demanding a recessive inheritance pattern (red shading, 6). b numbers of variants retained for the exome of each proband, v003 and v004, after each filtering step in a, showing individual numbers of snps / indels, left, and genes harboring variants, right. for steps (7) and (8), the final lists of genes at step (8) are as follows : for v003 atm, pcdh15, phf2 ; for v004 atm, eys, pcdp1, prune2, sh3d21, tshz3, ttn., genes with rare variants conforming to a recessive disease model in the family trio wes variants filtering paths. a trapezoids represent filters with resulting numbers of variants retained after each step indicated by a circled digit. starting with initial total variant lists (1), filters were applied for quality (2) and then to keep rare alleles (3) that alter splice sites or produce non - synonymous codon changes and are absent in local exomes (4). subsequent strategies were : focusing on variants from a list of genes associated with t cell phenotypes (yellow shading, 5) ; or demanding a recessive inheritance pattern (red shading, 6). b numbers of variants retained for the exome of each proband, v003 and v004, after each filtering step in a, showing individual numbers of snps / indels, left, and genes harboring variants, right. for steps (7) and (8), number of genes containing candidate variants in each proband are shown. the final lists of genes at step (8) are as follows : for v003 atm, pcdh15, phf2 ; for v004 atm, eys, pcdp1, prune2, sh3d21, tshz3, ttn., 2 variants, both in the atm gene., genes with rare variants conforming to a recessive disease model in the family trio we then further limited the disease gene candidates either by function of gene products (fig. 1a, yellow) or by genetic segregation (red). for functional selection, we used a list of 49 candidate genes involved in t cell development or reported to be defective in human primary t cell deficiencies by the international union of immunological sciences committee on primary immunodeficiency. in infant v003, this filtering left three heterozygous variants, of which two were frameshift deletions within the atm gene. aligned sequence reads supporting one of these, variant c1787delaa (k468fs), a two base deletion in atm exon 10, are illustrated in fig. evidence for the second atm variant of v003, c6238dela (f1952fs), a single base deletion in exon 39, was equally robust (not shown).fig. a aligned paired - end reads from whole exome sequence, chr11:108121571108121612, viewed with savant genome browser. center black bars indicate deletion of c1787delaa, k468fs, found in 27 of the 54 reads that include this sequence. colored rectangles, mismatch base calls compared to reference genome (judged to be artifacts because of singular occurrence). b sanger genomic reverse sequence confirming the heterozygous deletion. c reference and mutated amino acid codons, showing the frameshift, which led to 17 missense codons followed by a termination sequence evidence for a heterozygous exon 10 2-bp deletion of atm in infant v003. a aligned paired - end reads from whole exome sequence, chr11:108121571108121612, viewed with savant genome browser. center black bars indicate deletion of c1787delaa, k468fs, found in 27 of the 54 reads that include this sequence. colored rectangles, mismatch base calls compared to reference genome (judged to be artifacts because of singular occurrence). b sanger genomic reverse sequence confirming the heterozygous deletion. c reference and mutated amino acid codons, showing the frameshift, which led to 17 missense codons followed by a termination in infant v004, inclusion in the t cell gene list yielded only two heterozygous snps, both in atm, c.1260c > t (p292l), and c.7064c > t (r2227c). these results suggested disease - causing compound heterozygosity in both v003 and v004. by the segregation filtering method, we retained gene altering variants fitting a homozygous or compound heterozygous model of recessive inheritance - that is, those genes for which the patient inherited one rare allele from each parent - using exome data from each infant / parent trio. by this method, 5 candidate genes remained in the family of v003 and 9 in the family of v004. by filtering out variants also found in the unrelated local exomes used for vqsr infant v003 shared the atm mutation k468fs with her mother and f1952fs with her father, while v004 shared p292l with her mother and r2227c with her father. the other genes harboring parentally shared variants were not associated with any recognized immunologic phenotype. sanger sequencing confirmed the atm mutations seen by exome sequencing for both v003 (fig. 2b) and v004, as well as in both sets of parents (results not shown). subsequent to the sequence findings, both infants underwent measurement of serum alpha fetoprotein (afp) levels ; elevated afp compared to age - adjusted normal ranges is a reliable marker for at in children. v003 at age 16 months had afp 307 g / l, while v004 at 7 months had 112 g / l (normal range for these ages, 880 g / l). western blot showed absent atm protein in both patients (data not shown). to test whether t lymphocytopenia detected by low trecs is common in infants with atm mutations, medical records of california - born patients with at followed at chla and ucsf over the past 25 years were reviewed, and their residual neonatal dbs were retrieved by the cdph for trec testing. as summarized in table ii, 13 patients with at, 9 females and 4 males, none of whom had been suspected to have at at birth, had their newborn dbs samples retrieved. upon testing, 7 samples had trecs 25 ; thus newborn screening would have flagged these at patients in infancy to receive follow - up lymphocyte immunophenotyping. the at patients ethnic distribution was not different from the overall distribution of california births.table iicharacteristics of at patients whose newborn dried blood spots were tested for trecspatientethnicitysexnewborn dbsage at first symptomsage at diagnosis of atfirst immune panel, showing absolute lymphocyte number/l at indicated ageearliest afp, g / l (age) normal 2 yimmune and hematologic manifestationscurrent age or age at deathtrecactinresultagetcd4 tbnk1whitef37,690positive2y2y 9m2y 9m1,162995n / an / a131 (2y)lymphoma (6 y), granulomatous skin lesions (3 y)d. 6y 3m2multiplef765,500positive1y2y 8m2y 8m681373n / an / a60 (2y 8m)bronchiectasis16y3whitem1416,400positive5y5y 8m5y 8m4067042175310 (12y 3m)splenic granulomas (24 y), osteomyelitis (19 y), abdominal burkitt lymphoma (14 y)25y4hispanicf1933,500positive8y11y 6m11y 6m740475142n / a62.8 (11y 7m)none14y 11m5hispanicm2220,500positive1y3y m3y 4m443227173421108.1 (3y 4m)granulomatous skin lesions6y 2m6hispanicm2436,800positive1y 5m1y 7m19m29512510021016.8 (1y 7m)none8y 0m7asianf2531,900positive1y11yn / an / an / an / an / a164 (11y 11m)large b - cell lymphoma (13 y) ; fungal pneumonia (14 y)17y8hispanicf2814,900normal1y 4m3y 5m3y 5m33818012825586.8 (3y 7m)none6y 2m9hispanicf3061,300normal3y11y 10m12y46327747n / a174 (11y 10m)bronchiectasis12y 2m10hispanicm3934,700normal2y7y 3m7y 9m1,069534196n / a127 (7y 6m)recurrent hsv ; relapsed aml (10 y)d. 10y 3m11asianf4126,100normal10m1y 6m23m176132522n / a48.7 (1y 9m)none2y 6m12hispanicf10188,100normal1y 2m2y 11m6y 11m861369284n / a210 (6y 8m)none8y 4m13hispanicf21637,300normal2y 6m4y 6m4y 6m1,571428238n / a37.6 (4y 6m)chronic sinusitis and otitis7y 6 m characteristics of at patients whose newborn dried blood spots were tested for trecs all 13 at patients initially presented with symptoms of ataxia and abnormal gait between ages 12 months and 8 years (median 17 months). patients experienced a delay from 2 months to 10 years between onset of symptoms and at diagnosis, which occurred between ages 1.5 and 12 years (median age 3 years 5 months). at diagnosis all 13 at patients had high serum afp concentrations, from 16.8 g / l at 1 year 7 months (patient 6) to 310 g / l at 12 years 3 months (patient 3). afp levels in utero and at birth are high, but fall to 25 copies), there were no significant differences in age at presentation with neurological symptoms, afp levels, total cd3 t cell counts, or time between symptom onset and diagnosis, though our t cell information from retrospective chart review did not provide lymphocyte subset data in infancy ; the median age for the first recorded immune panel was 4 years. interestingly, there was a correlation between trec copy numbers in the patients archived newborn dbs and their subsequently measured cd4 t cell counts (r = 064). further analysis with a larger sample of at patients might reveal additional relationships between newborn trec numbers and phenotypic clinical and laboratory features of at. newborns in california are screened for scid and classified as positive if trec copy number is 5 with -actin > 5,000 copies, or trec copy number is between 6 and 25 with -actin > 10,000 copies. the tests are classified as incomplete and are repeated if there are low trecs, but also low copies of the -actin gene segment amplified as a control. t cells are measured by flow cytometry in cases that are positive or that have two incomplete dbs samples. infants v003 and v004 were unrelated, healthy females born at term following normal pregnancies. family history for both was negative for immune disease or consanguinity. lymphocyte flow cytometry was ordered for infant v003 after two dbs yielding incomplete results, while v004 had an initial positive result with 21 trecs and 13,300 -actin copies (table i).table iimmunologic phenotype of infants identified by scid newborn screeninginfantv003v004age3 m5.5 m21 days7 mtrec (normal > 25)421beta - actin (> 10,000)10,00013,300wbc (5,00019,500 cells/l)7,1003,5304,5005,320alc (2,50016,500)1,6002,1892,0003,032cd3 t cells (2,5505,500)9966151,0601,397cd4 t - helper cells (1,6004,000)749502600777cd8 t - cytotoxic cells (5601,700)185172340400cd3/cd4/cd45ra (1,2003,700)456360cd3/cd4/cd45ro (60900)299180cd19 b cells (3002,000)52844401,232cd16/56 nk cell (1701,100)3471,605300381igg (165781 mg / dl)156414iga (25154) 0.1 iu / ml)0.073.73anti - h. influenzae type b (> 1 mcg / ml) 2,000 and > 300, respectively), and the number of cd45ra nave cd4 t cells was low. low t and b cell numbers persisted, and low igg levels with failure to produce antibodies after vaccination led to institution of immunoglobulin replacement and trimethoprim - sulfamethoxazole antibiotic prophylaxis. lymphocyte proliferation to phytohemagglutinin and tcr v diversity assessed by spectratyping were normal, but an epstein barr virus transduced b cell line from the patient had only half normal phosphorylation of stat5 in response to il-2, suggesting an intrinsic lymphocyte impairment. later, at age 1416 months, v003 was reported by her mother to have unsteady gait ; physical examination first showed mild truncal ataxia at 20 months. infant v004 had flow cytometry at 21 days of age, showing only 1,060 t cells, with low cd45ra nave helper cd4 t cells (table i). b and nk cell numbers and lymphocyte proliferation were normal, but v spectratyping showed decreased t cell diversity (not shown). as with infant v003, in vitro phosphorylation of stat5 after il-2 activation was diminished, but not absent, as would be the case in scid due to defects in the il-2 receptor common chain or janus kinase 3 [9, 24 ]. physical examination of infant v004 has been normal to date, but she did not mount robust antibody to t - cell dependent protein - conjugated h. influenzae vaccination. to investigate the genetic etiology underlying their observed immunodeficient status, dna samples from v003, v004 and their parents were subjected to wes to generate a list of small nucleotide polymorphism (snp) and small insertion or deletion (indel) variants. these lists were filtered to retain successively fewer candidate variants as shown for snps and indels separately for each infant in fig. after initial quality filtering and removal of variants common enough to be found in dbsnp 135 and the 1,000 genomes database, further filters were applied to keep only the non - synonymous variants lying in captured exonic and splice - site regions of genes, and only those variants with a high (> 30) genotype quality score (fig. 1a, step 4).fig. a trapezoids represent filters with resulting numbers of variants retained after each step indicated by a circled digit. starting with initial total variant lists (1), filters were applied for quality (2) and then to keep rare alleles (3) that alter splice sites or produce non - synonymous codon changes and are absent in local exomes (4). subsequent strategies were : focusing on variants from a list of genes associated with t cell phenotypes (yellow shading, 5) ; or demanding a recessive inheritance pattern (red shading, 6). b numbers of variants retained for the exome of each proband, v003 and v004, after each filtering step in a, showing individual numbers of snps / indels, left, and genes harboring variants, right. for steps (7) and (8), number of genes containing candidate variants in each proband are shown. the final lists of genes at step (8) are as follows : for v003 atm, pcdh15, phf2 ; for v004 atm, eys, pcdp1, prune2, sh3d21, tshz3, ttn., genes with rare variants conforming to a recessive disease model in the family trio wes variants filtering paths. a trapezoids represent filters with resulting numbers of variants retained after each step indicated by a circled digit. starting with initial total variant lists (1), filters were applied for quality (2) and then to keep rare alleles (3) that alter splice sites or produce non - synonymous codon changes and are absent in local exomes (4). subsequent strategies were : focusing on variants from a list of genes associated with t cell phenotypes (yellow shading, 5) ; or demanding a recessive inheritance pattern (red shading, 6). b numbers of variants retained for the exome of each proband, v003 and v004, after each filtering step in a, showing individual numbers of snps / indels, left, and genes harboring variants, right. for steps (7) and (8), the final lists of genes at step (8) are as follows : for v003 atm, pcdh15, phf2 ; for v004 atm, eys, pcdp1, prune2, sh3d21, tshz3, ttn., 2 variants, both in the atm gene., genes with rare variants conforming to a recessive disease model in the family trio we then further limited the disease gene candidates either by function of gene products (fig., we used a list of 49 candidate genes involved in t cell development or reported to be defective in human primary t cell deficiencies by the international union of immunological sciences committee on primary immunodeficiency. in infant v003, this filtering left three heterozygous variants, of which two were frameshift deletions within the atm gene. aligned sequence reads supporting one of these, variant c1787delaa (k468fs), a two base deletion in atm exon 10, are illustrated in fig. evidence for the second atm variant of v003, c6238dela (f1952fs), a single base deletion in exon 39, was equally robust (not shown).fig. a aligned paired - end reads from whole exome sequence, chr11:108121571108121612, viewed with savant genome browser. center black bars indicate deletion of c1787delaa, k468fs, found in 27 of the 54 reads that include this sequence. colored rectangles, mismatch base calls compared to reference genome (judged to be artifacts because of singular occurrence). c reference and mutated amino acid codons, showing the frameshift, which led to 17 missense codons followed by a termination sequence evidence for a heterozygous exon 10 2-bp deletion of atm in infant v003. a aligned paired - end reads from whole exome sequence, chr11:108121571108121612, viewed with savant genome browser. center black bars indicate deletion of c1787delaa, k468fs, found in 27 of the 54 reads that include this sequence. colored rectangles, mismatch base calls compared to reference genome (judged to be artifacts because of singular occurrence). b sanger genomic reverse sequence confirming the heterozygous deletion. c reference and mutated amino acid codons, showing the frameshift, which led to 17 missense codons followed by a termination in infant v004, inclusion in the t cell gene list yielded only two heterozygous snps, both in atm, c.1260c > t (p292l), and c.7064c > t (r2227c). these results suggested disease - causing compound heterozygosity in both v003 and v004. by the segregation filtering method, we retained gene altering variants fitting a homozygous or compound heterozygous model of recessive inheritance - that is, those genes for which the patient inherited one rare allele from each parent - using exome data from each infant / parent trio. by this method, 5 candidate genes remained in the family of v003 and 9 in the family of v004. by filtering out variants also found in the unrelated local exomes used for vqsr, these numbers were reduced to 3 and 7 genes, respectively. infant v003 shared the atm mutation k468fs with her mother and f1952fs with her father, while v004 shared p292l with her mother and r2227c with her father. the other genes harboring parentally shared variants were not associated with any recognized immunologic phenotype. sanger sequencing confirmed the atm mutations seen by exome sequencing for both v003 (fig. 2b) and v004, as well as in both sets of parents (results not shown). subsequent to the sequence findings, both infants underwent measurement of serum alpha fetoprotein (afp) levels ; elevated afp compared to age - adjusted normal ranges is a reliable marker for at in children. l, while v004 at 7 months had 112 g / l (normal range for these ages, 880 g / l). to test whether t lymphocytopenia detected by low trecs is common in infants with atm mutations, medical records of california - born patients with at followed at chla and ucsf over the past 25 years were reviewed, and their residual neonatal dbs were retrieved by the cdph for trec testing. as summarized in table ii, 13 patients with at, 9 females and 4 males, none of whom had been suspected to have at at birth, had their newborn dbs samples retrieved. upon testing, 7 samples had trecs 25 ; thus newborn screening would have flagged these at patients in infancy to receive follow - up lymphocyte immunophenotyping. the at patients ethnic distribution was not different from the overall distribution of california births.table iicharacteristics of at patients whose newborn dried blood spots were tested for trecspatientethnicitysexnewborn dbsage at first symptomsage at diagnosis of atfirst immune panel, showing absolute lymphocyte number/l at indicated ageearliest afp, g / l (age) normal 2 yimmune and hematologic manifestationscurrent age or age at deathtrecactinresultagetcd4 tbnk1whitef37,690positive2y2y 9m2y 9m1,162995n / an / a131 (2y)lymphoma (6 y), granulomatous skin lesions (3 y)d. 6y 3m2multiplef765,500positive1y2y 8m2y 8m681373n / an / a60 (2y 8m)bronchiectasis16y3whitem1416,400positive5y5y 8m5y 8m4067042175310 (12y 3m)splenic granulomas (24 y), osteomyelitis (19 y), abdominal burkitt lymphoma (14 y)25y4hispanicf1933,500positive8y11y 6m11y 6m740475142n / a62.8 (11y 7m)none14y 11m5hispanicm2220,500positive1y3y m3y 4m443227173421108.1 (3y 4m)granulomatous skin lesions6y 2m6hispanicm2436,800positive1y 5m1y 7m19m29512510021016.8 (1y 7m)none8y 0m7asianf2531,900positive1y11yn / an / an / an / an / a164 (11y 11m)large b - cell lymphoma (13 y) ; fungal pneumonia (14 y)17y8hispanicf2814,900normal1y 4m3y 5m3y 5m33818012825586.8 (3y 7m)none6y 2m9hispanicf3061,300normal3y11y 10m12y46327747n / a174 (11y 10m)bronchiectasis12y 2m10hispanicm3934,700normal2y7y 3m7y 9m1,069534196n / a127 (7y 6m)recurrent hsv ; relapsed aml (10 y)d. 10y 3m11asianf4126,100normal10m1y 6m23m176132522n / a48.7 (1y 9m)none2y 6m12hispanicf10188,100normal1y 2m2y 11m6y 11m861369284n / a210 (6y 8m)none8y 4m13hispanicf21637,300normal2y 6m4y 6m4y 6m1,571428238n / a37.6 (4y 6m)chronic sinusitis and otitis7y 6 m characteristics of at patients whose newborn dried blood spots were tested for trecs all 13 at patients initially presented with symptoms of ataxia and abnormal gait between ages 12 months and 8 years (median 17 months). patients experienced a delay from 2 months to 10 years between onset of symptoms and at diagnosis, which occurred between ages 1.5 and 12 years (median age 3 years 5 months). at diagnosis all 13 at patients had high serum afp concentrations, from 16.8 g / l at 1 year 7 months (patient 6) to 310 g / l at 12 years 3 months (patient 3). afp levels in utero and at birth are high, but fall to 25 copies), there were no significant differences in age at presentation with neurological symptoms, afp levels, total cd3 t cell counts, or time between symptom onset and diagnosis, though our t cell information from retrospective chart review did not provide lymphocyte subset data in infancy ; the median age for the first recorded immune panel was 4 years. interestingly, there was a correlation between trec copy numbers in the patients archived newborn dbs and their subsequently measured cd4 t cell counts (r = 064). further analysis with a larger sample of at patients might reveal additional relationships between newborn trec numbers and phenotypic clinical and laboratory features of at. newborn screening by trecs was developed with scid as its primary target, but a spectrum of conditions are also identified that feature clinically significant t lymphocytopenia, defined by the california newborn screening program as < 1,500 t cells/l or a lack of cd45ra nave t cells. through screening with the trec assay, two apparently healthy california newborns with unexplained lymphocytopenia were identified to have at, with deleterious mutations in the atm gene detected by wes and confirmed by sanger sequencing, afp elevation and undetectable atm protein expression. the abnormal trec screening results in infants v003 and v004 allowed physicians to avoid exposure to live attenuated rotavirus vaccine, contraindicated in infants with t cell immunodeficiency. although t and b cell immunodeficiency is a well recognized, but variable feature of at patients of older ages, the degree of immune compromise in early infancy has not been documented. our early diagnosis of at has provided an opportunity to observe prospectively the evolution of immunological, neurological and malignant features of at. clinical features of at include the loss of motor milestones between 1 and 2 years of age, with falls, slurred speech, oculomotor apraxia, and truncal instability. laboratory features often include absent iga and always include elevated serum afp, though the interpretation of afp requires age - adjustment because this fetal serum protein remains abundant in infancy [26, 27 ]. magnetic resonance imaging of the brain is unhelpful in early disease, as cerebellar abnormalities become apparent only after 2 years. increased cellular radiosensitivity is found in at, though assays may not differentiate at from other defects with defective dna repair. thus, low trecs and t lymphocytopenia detected by newborn screening can be the earliest and simplest warning that at may be present, and validates the hypothesis of borte., who suggested that at might be detectable by prospective trec screening, based on their survey measuring trec and kappa chain b cell excision circles in archival dbs from patients with known primary immunodeficiencies. the large size of the atm gene, with 63 exons encoding an mrna of 13,147 nucleotides, makes genomic sequencing costly and laborious ; thus, wes may become a cost effective first line approach to examine this gene. our study demonstrates the value of a short - list of gene candidates of interest when searching deep sequencing data for rare, disease - causing variants. by focusing on a list of genes known to be associated with t - cell deficiencies, we were able to single out clinically relevant mutations with a minimum of other filters. the variants in our families occurred in regions with excellent coverage, as illustrated by the abundant bidirectional reads in fig. however, high - quality coverage of all at exons by wes or other deep sequencing is not guaranteed. in the exome datasets of our infants and their parents, between one and four atm exons had < 15x coverage, arguably inadequate to make accurate variant calls. as quality, coverage and affordability of deep sequencing improve, the chance of missing a variant will decline, but should be considered when using wes clinically. another consideration regarding deep sequencing analysis is the utility of filtering against local exome data other than dbsnp and 1,000 genomes. thus we observed large numbers of indels compared to snps persisting through the first 3 steps of our analysis (fig. also, indel calling is less reliable than snp calling ; filtering indel variants against unrelated local exomes reduces the rate of local errors and artifacts. infant v003 s gene mutations have not been previously reported, but, like most atm mutations, produce early stop codons, predicted to lead to nonsense mediated decay of mrna. one of infant v004 s mutations has been reported ; the other is predicted to be damaging by snap and polyphen-2 algorithms [32, 33 ]. we have demonstrated the ability to diagnose at within the context of a newborn screening program and have provided documentation for the first time of sufficiently low t cell counts in two infants with at in the first months of life that live vaccines should be avoided. the incidence of at has been reported to be between 1:40,000 and 1:100,000 births with high rates in some populations due to founder mutations. the two infants reported here are the only newborns found to have at in the first 18 months of california s scid newborn screening program, in which over 740,000 infants were screened. with 7 of 13 at cases (54 %) from archived samples having abnormal trec screening, california has the largest number of annual births of any state in the u.s., and population - based newborn screening with trecs will provide a prospective, unbiased method to establish the incidence of at. the detection of at by newborn screening illuminates challenges surrounding the inclusion of new tests into a public health screening program. currently there is no cure for at ; the neurological deterioration is progressive and irreversible such that patients usually become wheelchair dependent by their teenage years, and lifespan is decreased. in addition, patients with at have increased risk for malignancies, attributed to compromise of dna repair mechanisms. consistent with the reported 3040 % lifetime risk and 1015 % childhood or early adulthood risk of lymphoid malignancy, 3 of our 13 retrospective patients had lymphomas and one had leukemia [37, 38 ]. atm heterozygous mutation carriers also have an increased risk of breast cancer as well as other epithelial malignancies [39, 40 ]. thus, although screening programs are designed to identify newborns with diseases for which there are treatments, the early diagnosis of at as a secondary target of trec screening for scid can provide important information for family and genetic counseling. patients should avoid undue irradiation and should be monitored for malignancy as well as protected from infection, while carriers of atm mutations should be made aware of their own increased risk of cancer, and any anti - cancer therapy they require should be tailored in light of their increased sensitivity to radiation. a diagnosis of at by newborn screening can also help parents plan for the care of the affected child and obtain genetic counseling when considering future pregnancies. furthermore, while there is currently no effective treatment for at, multiple lines of research are aiming towards that goal. antioxidant therapies [4244 ], and more recently hdac4 inhibition, have shown promise in mouse models ; and in vitro experiments with human cells suggest that for some patients atm function could be restored using mutation - targeted therapy. for any of the potential therapies under development, early intervention made possible by identification through newborn screening would be most likely to show benefit by allowing therapy to take effect before extensive degeneration of relevant tissues has occurred. this study demonstrates that t lymphocytopenia revealed by newborns trec screening can be an identifying feature of at. we also show the utility of exome sequencing to arrive at a gene diagnosis for infants with variant scid or cid. although there is no current cure for the progressive neurological impairment of at, early detection provides information to improve patient management and offer family genetic counseling. whole exome sequencing coupled with newborn screening in an ethnically diverse, large population will reveal unbiased data about rare diseases associated with t lymphocytopenia. | purposesevere combined immunodeficiency (scid) is characterized by failure of t lymphocyte development and absent or very low t cell receptor excision circles (trecs), dna byproducts of t cell maturation. newborn screening for trecs to identify scid is now performed in several states using pcr of dna from universally collected dried blood spots (dbs). in addition to infants with typical scid, trec screening identifies infants with t lymphocytopenia who appear healthy and in whom a scid diagnosis can not be confirmed. deep sequencing was employed to find causes of t lymphocytopenia in such infants.methodswhole exome sequencing and analysis were performed in infants and their parents. upon finding deleterious mutations in the ataxia telangiectasia mutated (atm) gene, we confirmed the diagnosis of ataxia telangiectasia (at) in two infants and then tested archival newborn dbs of additional at patients for trec copy number.resultsexome sequencing and analysis led to 2 unsuspected gene diagnoses of at. of 13 older at patients for whom newborn dbs had been stored, 7 samples tested positive for scid under the criteria of california s newborn screening program. at children with low neonatal trecs had low cd4 t cell counts subsequently detected (r = 0.64).conclusionst lymphocytopenia in newborns can be a feature of at, as revealed by trec screening and exome sequencing. although there is no current cure for the progressive neurological impairment of at, early detection permits avoidance of infectious complications, while providing information for families regarding reproductive recurrence risks and increased cancer risks in patients and carriers. |
at the linkage level, organisations may develop protocols to facilitate referral or collaboration to deal with patients ' needs. however, the organisations continue to function within their respective jurisdictions, responsibility and operational rules. in canada, since the health care system is universal and mainly publicly funded, there are already many initiatives and programmes in the health care system that integrate services at the linkage level. at the other end of the spectrum, the full integration level, the integrated organisation is responsible for all services, either under one structure or by contracting some services with other organisations. many examples of this level of isd programmes have been developed. in the united states, the california on lok project gave rise to the pace (program of all inclusive care for the elderly) projects. in canada, the choice (comprehensive home option of integrated care for the elderly) project in edmonton is an adaptation of the pace projects. these programmes are built around day centres where the members of the multidisciplinary team who evaluate and treat the clients are based. clients are selected according to relatively strict inclusion (degree of disability compatible with admission to a nursing home) and exclusion (e.g. behavioural problems) criteria. these systems usually function in parallel with the socio - health structures in place. services are delivered by structures operated by the system or by external structures linked through contracts (hospitals, specialised medical care, long - term care institutions). an evaluation of these programmes in the usa showed that they have an impact on the number and duration of short - term hospitalisations, the number of admissions to long - term institutions, drug use, mortality and the cost of services. however, this study did not include any specific control groups and the data from the pace projects were only compared to national statistics for groups whose comparability is questionable. in northern italy, barnabei. showed with a randomised controlled trial that a programme of integrated social and medical care and case management is effective in reducing admission to institutions and functional decline in older people living in the community. the social hmo in the united states and the sipa (systme de services intgrs pour personnes ges en perte d'autonomie) project in montreal are also integrated services but do not include a day centre. however, home care services are provided by personnel hired by or under contract with the organisation. all these fully integrated models are nested in the usual health and social services in a particular area but are run in parallel to them. they do not involve significant changes to the structure or processes of existing services, except for the negotiation of protocols for referring clients to isd and the provision of some services not covered by isd. the other level of integrated care, co - ordination, involves the development and implementation of defined structures and mechanisms to manage the complex and evolving needs of patients in a co - ordinated fashion. system and to adapt its operations and resources to the agreed requirements and processes. at this level, the isd system is not only nested in the health care and social service system but is embedded within it. figure 1 and table 1 compare the co - ordination model with the full integration model. (continuous - lined boxes means that the organisations are fully independent in their structure and management ; dotted - lined boxes means that part of the autonomy of the organisations is transferred to the integrated structure.) the prisma (program of research to integrate the services for the maintenance of autonomy) project in the province of quebec is an example of this type of integrated care. this article will describe in more detail the integrated care mechanisms and tools developed and implemented by prisma. the mechanisms refer to (1) co - ordination between decision - makers and managers at the regional and local level, and the use of (2) a single entry point, (3) a case management process and (4) individualised service plans. the tools refer to (5) a single assessment instrument coupled with a management system based on the clients ' functional autonomy, and (6) a computerised clinical chart for communicating between institutions and clinicians for client monitoring purposes. these tools not only facilitate the delivery of services adapted to the clients ' needs but can also continuously monitor the resources and manage the supply of services effectively and efficiently. since this model of co - ordinated system was developed to fit in a publicly funded health care system, capitation budgeting is not an essential component and funding of the system can be included as part of the agreement between organisations. (continuous - lined boxes means that the organisations are fully independent in their structure and management ; dotted - lined boxes means that part of the autonomy of the organisations is transferred to the integrated structure.) the prisma (program of research to integrate the services for the maintenance of autonomy) project in the province of quebec is an example of this type of integrated care. this article will describe in more detail the integrated care mechanisms and tools developed and implemented by prisma. the mechanisms refer to (1) co - ordination between decision - makers and managers at the regional and local level, and the use of (2) a single entry point, (3) a case management process and (4) individualised service plans. the tools refer to (5) a single assessment instrument coupled with a management system based on the clients ' functional autonomy, and (6) a computerised clinical chart for communicating between institutions and clinicians for client monitoring purposes. these tools not only facilitate the delivery of services adapted to the clients ' needs but can also continuously monitor the resources and manage the supply of services effectively and efficiently. since this model of co - ordinated system was developed to fit in a publicly funded health care system, capitation budgeting is not an essential component and funding of the system can be included as part of the agreement between organisations. first, at the strategic level (governance), by creating a joint governing board (table de concertation) of all health care and social services organisations and community agencies where the decision - makers agree on the policies and orientations and what resources to allocate to the integrated system. second, at the tactical level (management), a service co - ordination committee, mandated by the board and comprising public and community service representatives together with older people, monitors the service co - ordination mechanism and facilitates adaptation of the service continuum. finally, at the operational level (clinical), a multidisciplinary team of practitioners surrounding the case manager evaluates clients ' needs and delivers the required care. the single entry point is the mechanism for accessing the services of all the health care institutions and community organisations in the area for the frail senior with complex needs. it is a unique gate which older people, family caregivers and professionals can access by telephone or written referral. a link is established with the health info line available to the general population in quebec seven days a week, 24 hours a day. clients are referred to the isd system after a brief needs assessment [triage ] to ensure they meet the eligibility criteria for the integrated system (table 2). otherwise, they are referred to the relevant service. we have developed a quick 7-question screening instrument (prisma-7) to be used by professionals and nonprofessionals to identify clients who should present moderate to severe functional decline and be eligible for isd. this screening tool is used for triage either at the single - entry point or in volunteer agencies, and health and social services (emergency rooms, physicians ' offices, home care services, etc.). the case manager is responsible for doing a thorough evaluation of the client 's needs, planning the required services, arranging to admit the client to these services, organising and co - ordinating support, directing the multidisciplinary team of practitioners involved in the case, and monitoring and re - evaluating the client. in a randomised trial, eggert. showed that case management is more effective, if the case manager is not just a service broker but is also actively and directly involved in delivering the services to the client in his / her area of expertise. family physicians should be one of the case manager 's primary collaborators because, in addition to being the main medical practitioner, they are pivotal in regard to access to and co - ordination of specialised medical services. on the other hand, the case manager relieves family physicians of some of their burden by facilitating access to and co - ordinating the rest of the social and health interventions. the individualised service plan results from the overall assessment of the client and summarises the prescribed services and target objectives. it must be led by the case manager and established at a meeting of the multidisciplinary team including all the main practitioners involved in caring for the older person. in services or programmes where multidisciplinary meeting processes are already in place, the case manager joins this process without duplication. the individualised service plan includes the intervention plans of each of the practitioners and must be reviewed periodically. it must allow for evaluating the needs of clients either at home or in institutions. the smaf (systme de mesure de l'autonomie fonctionnelle functional autonomy measurement system) is a 29-item scale developed according to the who classification of disabilities. it measures functional ability in five areas : activities of daily living (adl) (7 items), mobility (6 items), communication (3 items), mental functions (5 items) and instrumental activities of daily living (iadl) (8 items). for each item, the disability is scored on a 5-point scale : 0 (independent), 0.5 (with difficulty), 1 (needs supervision), 2 (needs help), 3 (dependent). the resources available to compensate for the disability are also evaluated and a handicap score is deducted. the stability of the resources is also assessed. a disability score (out of 87) the smaf must be administered by a health professional who scores the subject after obtaining the information either by questioning the subject and proxies, or by observing and even testing the subject. this instrument has been submitted to a number of validity and reliability studies [20, 21 ]. correspondence of the smaf score with the required nursing - care time and the cost of long - term care, either at home or in different institutional settings, has also been established. a case - mix classification system based on the smaf has also been developed [23, 24 ]. fourteen iso - smaf profiles were generated using cluster analysis techniques in order to define groups that are homogeneous in regard to their profiles, but heterogeneous in other respects. these analyses were carried out with the data from a provincial study done by our team on nearly 2000 subjects living at home or in different types of residential facilities. by linking the evaluation of the iso - smaf autonomy profile of an older person to the amount and cost of the resources that person requires, based on his / her living situation (community - living, institutionalised, etc.), it is a quick and easy task to monitor the clinical, administrative and research data. these profiles are used to establish the admission criteria to the different institutions and to calculate the required budget of the institutions, given the autonomy of their clientele. in a health care system with multiple sources of funding, in addition, implementation of an integrated system like this requires the deployment of a continuous information system and the use of computerised tools to facilitate communications and ensure the continuity of services. through a computerised clinical chart (ccc), all the practitioners have quick access to complete, continuously updated information and can inform the other clinicians of the client 's progress and changes in the intervention plan. the ccc is part of the management system and thus provides an interface between the clinical information and the management information. a ccc called the sigg (systme d'information gronto - griatrique) has been developed and implemented in a pilot project in victoriaville (quebec, canada). this shareable, clinical chart is common to all the professionals in the service continuum for the older person. it uses the quebec ministry of health and social services internet network and lotus notes. the prisma group has implemented this model in two clsc (centre local de services communautaires) territories in the victoriaville region (the bois - francs project). the purpose of this pilot project was to evaluate, using a quasi - experimental design, the implementation and impact of this model for community - living clienteles. two cohorts of subjects in the study (n=272) and control (n=210) areas were followed and evaluated annually over a three - year period (1997 to 2000). one of the main outcomes of the study, functional decline, was defined as either death, institutionalisation and significant increase in disabilities (difference of 5 points or more on the smaf scale). in the study, there were fewer people who experienced a functional decline in the study group for those with moderate to severe disability at entry but not for the ones with mild disability. this effect was significant at 12 months (49.1% vs. 31.3 p=0.002) and tended to remain at 24 months (35.9 vs. 25.9 p=0.066). desire to be institutionalised showed a significant decrease in the experimental group at 12 and 24 months. caregivers ' burden was significantly lower in the study group than in the control group at 12 and 24 months. although the utilisation pattern of acute care hospitals was similar, the risk of returning to the emergency room within 10 days after a first visit or after discharge from an acute care hospital was significantly greater in the control group. the risk of being institutionalised tended to be greater in the control group (rr=1.44 ; p=0.06). based on these preliminary results, the group is now extending this model to clsc territories in other regions in the eastern townships that present different environments : presence of multiple and university institutions, urban versus rural, presence or absence of an acute care hospital. the evaluation of the implementation focuses on the process of implementing the mechanisms and tools and how they function. the objective is to explain the variations observed between the different implementation settings using a case study approach developed by yin. the questions that are documented try to define the extent to which the clientele using the services corresponds to the clientele initially targeted ; if the services delivered correspond to those planned ; and if the delivery procedure corresponds to the one initially defined. other questions focus on evaluating the process itself and identifying its strengths and weaknesses in order to reinforce or correct some of the elements comprising the new mechanisms and tools. the unit of analysis [case ] is each of the selected clsc territories involved in the study. the main variables and dimensions studied are : involvement of the decision - makers in the implementation ; whether the main users have the same understanding of the mechanisms or tools ; the population reached, productivity achieved, delays encountered, sources of references, time breakdown in relation to the mechanism functions, clinician - client interactions, problems identified, facilitating factors, etc. data are collected from the policy - makers, managers, clinicians, as well as clients and informal care - givers using different methods (interviews, focus groups, surveys). effectiveness is being evaluated using a quasi - experimental design (pre - test, multiple post - tests with control group). as opposed to the bois - francs pilot project where efficacy was measured using service users as subjects, this study measures the effectiveness by selecting a sample of older individuals at risk of using the services. it employs a different sampling strategy from that used to recruit the users in the system. although it requires a larger sample size, this strategy will enable us to measure the real populational effectiveness and to estimate the system penetration rate (accessibility). using a list from the quebec health insurance board, a sample of people over 75 were selected and sent a postal questionnaire already developed and validated by our team. the responses to this questionnaire or the fact of not returning it establish a risk of presenting a significant functional decline over the next year. since the annual incidence of functional decline in this group is estimated to be 48%, it is probable that the great majority of subjects selected in this way will contact the socio - health network during the two years of the study. after being informed of the study and agreeing to participate, the subjects were evaluated at pretest (t0) and will be reassessed annually over a two - year period (t1, t2). the variables measured are : functional autonomy (smaf), satisfaction in regard to the services received, client empowerment, caregivers ' burden, utilisation of health services and social services, and drug use. an economic analysis is also being performed. since it is embedded within the usual health care and social services system, this model could be more appropriate to the canadian universal and publicly funded health care system than the fully integrated models tested so far. however, it requires a shift from the traditional institution - based approach to a client - centred approach and tremendous efforts in co - ordination at all levels of the organisations. the on - going studies will show if the model could be generalised to other areas with different characteristics and show data on its impact on clienteles and cost. other studies are planned to focus on specific aspects of the model : an evaluation of the ccc to describe its perceived usefulness by the older people and the clinicians and its real use ; a socio - political analysis of the different roles played by the provincial, regional and local levels to facilitate or constrain the implementation of integrated care ; and the development of a conceptual framework to assess quality of health care and social services in an integrated care model. the next step will be to test the model elsewhere in canada and in other countries. in other health system contexts, the mechanisms and tools will probably have to be adapted. for example, in multi - payer systems, the management tool (iso - smaf profiles) could be used for funding or in the capitation payment calculation. finally, the prisma group is also a unique partnership experience between researchers, managers and policy - makers. representatives of the managers and decision - makers form an integral part of the research team and are participating at every stage in the studies, i.e. developing the protocol, finding funding, conducting the studies and analysing and presenting the results. this exceptional partnership is possible because of the close links developed over the last few years between the researchers, managers and decision - makers. these productive experiences are proof of the value of the links between these different groups. in addition to periodical general meetings where all the projects are discussed, the researchers and managers are divided into mixed project teams to develop and conduct the various studies. representatives of the managers and decision - makers and members of the research team also participate in discussions with the regional health and social services boards involved in implementing the mechanisms and tools used in the research programme. seminars and colloquia are organized to present and discuss the results of the studies or to review methodological questions. this partnership ensures the relevancy of the research projects and fosters the quick translation of research findings into better interventions, services and policies. prisma [program of research to integrate the services for the maintenance of autonomy ] is a partnership between two research teams [research centre on aging in sherbrooke and laval university geriatric research team in quebec city ] and several health organisations in the province of quebec : ministry of health and social services, five regional health and social services boards [estrie, mauricie - centre - du - qubec, laval, montrgie, quebec city ], and the sherbrooke geriatric university institute. prisma is funded by the canadian health services research foundation, the fonds de la recherche en sant du qubec [frsq ], and the partnering organisations. many projects run by the prisma group are also funded by the canadian institutes of health research.the prisma group includes the following members : rjean hbert, md mphil ; nicole dubuc, phd ; dani;agele blanchette, phd ; gina bravo, phd ; martin buteau, dsc ; johanne desrosiers, ot phd ; marie - france dubois, phd ; maxime gagnon, msc ; linda millette, md msc[c ] ; pascale morin, rd phd ; michel rache, msc ; michel tousignant, pht phd ; anne veil, msw ; alain villeneuve, dba [research centre on aging, sherbrooke ] ; pierre j. durand, md msc ; andr tourigny, md mba ; any bussi;agere, mps ; diane morin, rn, phd ; daniel pelletier, mps ; line robichaud ot phd ; louis rochette, msc ; mich;agele st - pierre, phd ; aline vzina, phd [geriatric research team, quebec city ] ; louis demers, phd ; mich;agele houpert, msc[c ] ; judith lavoie, msc [tl - universit, quebec city ] ; pierre bergeron, md phd ; philippe de wals, md phd [national public health institute, quebec ] ; anne lemay, phd [chum / montreal university health centre, montreal ] ; lysette trahan, phd [quebec ministry of health and social services ] ; mich;agele paradis, msc [quebec regional board ] ; linda dieleman [estrie regional board ] ; irma clapperton ; jocelyn lavalle ; liane lafleur, mba [laval regional board ] ; line beauchesne, msc ; lucie bonin, md msc ; danile laurin, phd ; hlaine - annie roy, msw ; [mauricie - centre - du - qubec regional board ] ; hung nguyen [montrgie regional board ]. | abstractpurposeprisma is an innovative co - ordination - type integrated service delivery system developed to improve continuity and increase the efficacy and efficiency of services, especially for older and disabled populations.descriptionthe mechanisms and tools developed and implemented by prisma include : (1) co - ordination between decision - makers and managers, (2) a single entry point, (3) a case management process, (4) individualised service plans, (5) a single assessment instrument based on the clients ' functional autonomy, and (6) a computerised clinical chart for communicating between institutions for client monitoring purposes.preliminary resultsthe efficacy of this model has been tested in a pilot project that showed a decreased incidence of functional decline, a decreased burden for caregivers and a smaller proportion of older people wishing to be institutionalised.conclusionthe on - going implementation and effectiveness study will show evidence of its real value and its impact on clienteles and cost. |
hypoxic - ischemic encephalopathy due to birth asphyxia is the major factor that induces neuronal cell injury before, during, and after birth. although there has been marked development in the fields of obstetrics and neonatology, the incidence of and mortality caused by hypoxic - ischemic encephalopathy remain elevated (1, 2). low birth weight infants and infants with intrauterine growth retardation, who have already experienced intrauterine repetitive hypoxic distress, seem to have milder hypoxic - ischemic brain damage than full - term newborns who are exposed to a lethal ischemic insult in the perinatal period. therefore, some have suggested the possibility that sublethal hypoxic episodes afford a protective effect against further hypoxic exposure (3, 4). hypoxic preconditioning has been described in the brain, heart, retina, and other tissues. (5) first showed that exposure of neonatal rat pups to hypoxia alone (8% oxygen for 3 hr) protected these animals 1 day later from a stroke induced by combined hypoxia / ischemia. although the mechanisms of preconditioning are still undetermined, it appears to require synthesis of new rna and proteins during the reoxygenation period before permanent ligation (6, 7). (8) explained that the difference in the degree of hypoxia and the delay between preconditioning and ischemia determines the efficacy of preconditioning. this study was conducted to evaluate the protective effect of hypoxic preconditioning on hypoxic - ischemic injury in the neonatal rat and the persistence of a protective window after hypoxic preconditioning. furthermore, this study investigated the relationship between the downregulation of apoptosis and its possible role in the protective effect of hypoxic preconditioning. the survival rates, as well as ratios of lipid to n - acetyl aspartate (naa) and lipid to creatine (cr), were investigated in positive cells, and morphologic changes were investigated with proton (h) magnetic resonance spectroscopy (mrs) and terminal deoxynucleotidyl transferase - mediated dutp - biotin nick end - labeling (tunel) staining. a total of 231 seven - day - old sprague - dawley newborn rats weighing 12 - 18 grams were divided into six groups : preconditioned 6 hr before hypoxic - ischemic injury (pre-6 hr, n = 30), 12 hr before (pre-12 hr, n = 32), 1 day before (pre-1 day, n = 34), 3 days before (pre-3 day, n = 41), 6 days before (pre-6 day, n = 43), and control without preconditioning (n = 51). all experimental animal protocols were approved by the university of ulsan college of medicine iacuc committee (2008 - 12 - 011). the rats were preconditioned by hypoxic exposure (8% oxygen/92% nitrogen) for three hours at 37. exhaled carbon dioxide was removed through a one - way valve in the lower part of a hypoxic chamber. rats were returned to their home cages at room temperature immediately after the hypoxic preconditioning. control group rats were isolated during preconditioning, and then they were also returned to their home cages. using ligation of the right common carotid artery, 6 hr, 12 hr, 1, 3, and 6 days after receiving three hours of hypoxic preconditioning, all rats, including the control group, 5) under isoflurane anesthesia, and the rats were submitted to hypoxia (8% oxygen, 92% nitrogen) for 3 hr under the same conditions as in the preconditioning procedure. after the hypoxic - ischemic insult, the specimens were returned to their home cages at room temperature. h mrs was performed 24 hr after hypoxic - ischemic insult, and the results were examined using bruker biospec 4.7 tesla mri / mrs system (bruker, fallanden, switzerland). the voxel for mrs was located in the parietotemporal area, and the volume of a single voxel was 3 3 4 l. the spin echo signal was detected by tr = 3,000 msec, te = 30 msec, and ns = 128, and the h2o signal was inhibited by a chemical shift selective (chess) sequence. spectra were analyzed semi - quantitively using an aspect 3000 computer and tomikon software (bruker, fallanden, switzerland). in order to measure the ratios of lipid to naa and lipid to cr as early predictors of apoptosis, the h spectra of naa, creatine, choline, and lipids were assessed at 2.02, 3.03, 3.24, and 1.3 ppm, respectively. to differentiate between lipids and lactate at 1.3 ppm, the presence of the peak at te = 135 msec was analyzed. one day after the hypoxic - ischemic injury, the rats were anesthetized with an intra - abdominal ketamine (2 mg / kg) injection following h mrs examination. the rats were then perfused through the left ventricle of the heart with heparinized saline (2 unit heparin/1 ml saline, 2.5 ml / g) followed by buffered paraformaldehyde (4% in phosphate buffer, ph 7.4). two rats in each group were sacrificed one day after hypoxic - ischemic injury, and these brains were examined for histologic study using tunel staining. tunel staining for the detection of dna fragmentation produced during cell apoptosis began with the digestion of deparaffinized sections using proteinase k, blocking solution, and permeabilization solution. these sections were then stained with the tunel reaction mixture (50 l) of an in situ cell death detection kit (boehringer - mannheim, germany). the remaining rats were morphologically evaluated on the fourteenth day after hypoxic - ischemic injury and scored according to the following scale of modified palmer 's classification (9) : 0, no difference between the two hemispheres in shape and size ; 1, mild reduction of the right hemisphere volume only ; 2, volume reduction and atrophic changes with small cysts in the right hemisphere ; 3, moderate atrophic changes of the right hemisphere ; and 4, severe atrophic changes of the right hemisphere. the results were expressed as mean standard deviation, and the statistics were calculated using an unpaired t - test, one way anova, tukey 's test, dunn 's method, mann - whitney u test, and pearson 's correlation with the threshold of significance at 0.05. a total of 231 seven - day - old sprague - dawley newborn rats weighing 12 - 18 grams were divided into six groups : preconditioned 6 hr before hypoxic - ischemic injury (pre-6 hr, n = 30), 12 hr before (pre-12 hr, n = 32), 1 day before (pre-1 day, n = 34), 3 days before (pre-3 day, n = 41), 6 days before (pre-6 day, n = 43), and control without preconditioning (n = 51). all experimental animal protocols were approved by the university of ulsan college of medicine iacuc committee (2008 - 12 - 011). the rats were preconditioned by hypoxic exposure (8% oxygen/92% nitrogen) for three hours at 37. exhaled carbon dioxide was removed through a one - way valve in the lower part of a hypoxic chamber. rats were returned to their home cages at room temperature immediately after the hypoxic preconditioning. control group rats were isolated during preconditioning, and then they were also returned to their home cages. using ligation of the right common carotid artery, 6 hr, 12 hr, 1, 3, and 6 days after receiving three hours of hypoxic preconditioning, all rats, including the control group, 5) under isoflurane anesthesia, and the rats were submitted to hypoxia (8% oxygen, 92% nitrogen) for 3 hr under the same conditions as in the preconditioning procedure. after the hypoxic - ischemic insult, the specimens were returned to their home cages at room temperature. h mrs was performed 24 hr after hypoxic - ischemic insult, and the results were examined using bruker biospec 4.7 tesla mri / mrs system (bruker, fallanden, switzerland). the voxel for mrs was located in the parietotemporal area, and the volume of a single voxel was 3 3 4 l. the spin echo signal was detected by tr = 3,000 msec, te = 30 msec, and ns = 128, and the h2o signal was inhibited by a chemical shift selective (chess) sequence. spectra were analyzed semi - quantitively using an aspect 3000 computer and tomikon software (bruker, fallanden, switzerland). in order to measure the ratios of lipid to naa and lipid to cr as early predictors of apoptosis, the h spectra of naa, creatine, choline, and lipids were assessed at 2.02, 3.03, 3.24, and 1.3 ppm, respectively. to differentiate between lipids and lactate at 1.3 ppm, the presence of the peak at te = 135 msec was analyzed. one day after the hypoxic - ischemic injury, the rats were anesthetized with an intra - abdominal ketamine (2 mg / kg) injection following h mrs examination. the rats were then perfused through the left ventricle of the heart with heparinized saline (2 unit heparin/1 ml saline, 2.5 ml / g) followed by buffered paraformaldehyde (4% in phosphate buffer, ph 7.4). two rats in each group were sacrificed one day after hypoxic - ischemic injury, and these brains were examined for histologic study using tunel staining. tunel staining for the detection of dna fragmentation produced during cell apoptosis began with the digestion of deparaffinized sections using proteinase k, blocking solution, and permeabilization solution. these sections were then stained with the tunel reaction mixture (50 l) of an in situ cell death detection kit (boehringer - mannheim, germany). the remaining rats were morphologically evaluated on the fourteenth day after hypoxic - ischemic injury and scored according to the following scale of modified palmer 's classification (9) : 0, no difference between the two hemispheres in shape and size ; 1, mild reduction of the right hemisphere volume only ; 2, volume reduction and atrophic changes with small cysts in the right hemisphere ; 3, moderate atrophic changes of the right hemisphere ; and 4, severe atrophic changes of the right hemisphere. the results were expressed as mean standard deviation, and the statistics were calculated using an unpaired t - test, one way anova, tukey 's test, dunn 's method, mann - whitney u test, and pearson 's correlation with the threshold of significance at 0.05. here we confirmed the preconditioning groups, followed by 8% oxygen hypoxia, reduced brain injury in newborn rats. the pre-6 hr, pre-12 hr, and pre-1 day hypoxic preconditioning groups had significantly higher survival rates when evaluating 152 rats among 231 : control (25 rats, 47%) ; pre-6 hr (26 rats, 86%) ; pre-12 hr (32 rats, 100%) ; pre-1 day (32 rats, 94%) ; pre-3 day (19 rats, 45%) ; and pre-6 day (18 rats, 42%) (fig. we performed h mrs study after hypoxic - ischemic insult ; and then lipid / naa (n - acetyl aspartate) and lipid / cr (creatine) ratios were measured as early predictors of apoptosis. the pre-6 hr, pre-12 hr, and pre-1 day hypoxic preconditioning groups also had lower lipid / naa and lipid / cr ratios on h mrs in comparison with the pre-3 day and pre-6 day (p < 0.05, fig. 3). the lipid / naa ratios in control, pre-6 hr, pre-12 hr, pre-1 day, pre-3 day and pre-6 day ratios were 8.05 2.95, 4.64 2.78, 5.25 2.58, 5.05 1.86, 7.09 2.87, and 6.19 2.62, respectively. results of the histologic evaluation demonstrated that the hypoxic preconditioning attenuated hypoxic - ischemic induced apoptosis in newborn brain. on day 1, the groups that received hypoxic preconditioning 6 hr, 12 hr, and 1 day prior to hypoxic - ischemic injury had fewer tunel - positive apoptotic cells present than in the control group, which received no preconditioning. in addition, the h mrs characteristics are shown and lower lipid peaks are observed in the preconditioning groups in fig. we monitored the structural damage in rats of different groups on the fourteenth day after hypoxic - ischemic injury. the morphologic scores of the preconditioning groups were also significantly lower than those of the control group (p < 0.05, fig. 5). the morphologic scores in the control, pre-6 hr, pre-12 hr, pre-1 day, pre-3 day, and pre-6 day groups were 3.28 0.84, 1.12 1.21, 1.59 1.46, 1.69 1.15, 2.95 1.08, and 2.61 1.54, respectively, at day 14. the high lipid / naa and lipid / cr ratios on h mrs were obtained on the first day after hypoxic - ischemic insult and were predictive of morphologic changes (fig. however, the h mrs and morphologic scores of the pre-3 day and pre-6 day preconditioning groups did not show a protective effect on survival rates or on lipid / naa and lipid / cr ratios. both fetal and neonatal asphyxia can cause cerebral hypoxic - ischemic injury, resulting in severe neurologic sequelae and death. survivors of perinatal asphyxia frequently have moderate to severe brain injury for which there is currently no promising therapy. reports published in the last decade have made increasing use of the term hypoxic or ischemic preconditioning to describe an adaptive response to subsequent lethal events (3). focusing on protective strategies to prevent and reduce hypoxic - ischemic injury, sublethal stimuli, such as hypoxia (4), hyperthermia (10), hypothermia (11), glutamate and seizure (12), oxygen - glucose deprivation (13), receptor blockade / activation (14), and cytokine (15), have been reported. the mechanism of the brain tolerance induced by hypoxic preconditioning has not been fully elucidated, but three major mechanisms have been proposed. first, the formation of various types of prolonged neuronal adaptive responses is associated with the activation of the cell genome and protein synthesis. these include stress proteins (heat shock proteins [hsp ]) (10) ; immediate early genes (c - fos, c - jun, krox-24 [ngfi - a ]) (16) ; transcription factors (hypoxia - inducible factor-1 [hif-1 ]) (17) ; nfb (nuclear factor b) (18) ; camp response element - binding protein (creb) (19) ; neuromodulatory peptides for survival, differentiation, and plasticity ; growth factors (igf-1, brain - derived neurotrophic factor [bdnf ], ngf) (20) ; and anti - apoptotic genes (bcl-2, bcl - xl, bax) (3). in particular, an important role is played by immediate early gene superfamilies whose protein products are transcription factors that regulate the expression of phenotype - specific late genes. a rather long time (24 hr or more) between mild preconditioning episodes and severe hypoxia is needed for the maximal protective effect of preconditioning to appear, which suggests the involvement of neuronal gene expression and de novo protein synthesis in the mechanisms of brain tolerance (7). for example, kitagawa. (21) found that sublethal ischemia at 1 and 2 days prior to an ischemic insult that is normally lethal to neurons was neuroprotective in vivo. our data, however, showed significant neuroprotective effects in the pre-6 hr hypoxic preconditioning group. this suggests that the factors mentioned above could be induced to influence the tolerance of the brain in as little as 6 hr, and the persistence of the protective effect lasted 3 days after preconditioning. the second possibility involves the roles of signaling proteins (no, erk1/2, akt) (22). no, an important proximal component of the transduction pathway of adaptive genomic changes, was shown to induce diverse paracrine actions in the central nervous system. third, the roles of molecules, including adenosine a1 receptors (23) and manganese superoxide dismutase (mnsod) (24) have suggested a possible mechanism. finally, activation of n - methyl - d - aspartate (nmda) receptors has been shown to be involved in the development of both adaptive and pathological brain reactions. paradoxically, subtoxic concentrations of nmda protect neurons against glutamate - mediated excitotoxicity (1, 14). the major mechanism of nmda receptor - medicated tolerance after preconditioning involves hsp induction ; bcl-2 gene expression ; activation of adenosine a1 receptors ; release and synthesis of bdnf by activation of nfb ; trkb receptor, akt activation through phosphoinositide 3-kinase, and calmodulin - dependent protein kinase ; and downregulation of the mixed - lineage kinase 3 (mlk3)-c - jun n - terminal kinase (jnk) signaling pathway via akt activation (22). according to data from the present study, the pre-6 hr, pre-12 hr, and pre-1 day hypoxic preconditioning groups had significantly higher survival rates than those of the control group. these results suggest that preconditioning was effective in myocardial cells as well as cells in the brain. mild, short ischemic events in myocytes contributed to the resistance to subsequent, lethal ischemic exposure, which reduced cardiac infarction and improved cardiac function during reperfusion after ischemic insult. it is well known that apoptosis plays an important role in hypoxic - ischemic brain injury and that the apoptotic cell counts are closely related to the degree of brain injury (25). evidence of fragmented and condensed dna is apparent using the tunel method of in situ dna end labeling. cantagrel. (3) found a decrease in the number of apoptotic cells 24 and 48 hr after the insult using a newborn rat model of hypoxic preconditioning (8% oxygen/92% nitrogen, 3 hr, 37) on day 6 followed by carotid ligation and hypoxic insult on day 7, suggesting that regulation of apoptotic cell death is one of the mechanisms involved in the tolerance to hypoxia - ischemia that is induced by hypoxic preconditioning. using the same experimental model, we investigated the preconditioning effects in the present study. mrs allows for the study of brain biochemistry and metabolism and for indirect characterization of the composition of brain tissue in vivo. mrs has become an important adjunct to diagnostic structural imaging that permits more accurate and earlier determination of prognosis after hypoxia - ischemia in the newborn (26 - 28). furthermore, non - invasive in vivo h mrs of a hypoxic - ischemic brain might reveal apoptosis (29). in h mrs of hypoxic - ischemic newborn rat brains, the prominent lipid peaks at 1.3 ppm appear early, and the apoptotic cell counts might be correlated with the intensity of the lipid peak and the ratios of lipid / naa and lipid / cr. therefore, the ratios of lipid / naa and lipid / cr could be used as an early indicator to diagnose hypoxic - ischemic injury by apoptosis and also to assess the effect of preconditioning on decreasing apoptosis. we have used h mrs in vivo for the purpose of early detection in the hypoxic - ischemic brain injury model (30). additionally, the results of the current study demonstrate that the increase of the lipid peak at 24 hr following hypoxic - ischemic injury is correlated with morphologic scores. we suggest a promising future for early in vivo detection of apoptosis using h mrs, which can be used to develop new therapeutic methods to attenuate apoptosis in the hypoxic - ischemic injured brain. in conclusion, using h mrs, tunel staining, and morphologic scores, this study showes that hypoxic preconditioning 6 hr to 1 day before hypoxic - ischemic injury produces higher survival rates and the presence of neuroprotective effects against subsequent hypoxic - ischemic injury. however, hypoxic preconditioning in the pre-3 day & pre-6 day groups does not show these protective effects. the downregulation of apoptotic cell death may be a mechanism underlying the protective effects of hypoxic preconditioning in the newborn rat brain. protective effect of hypoxic preconditioning on hypoxic - ischemic injured newborn rats hyun - kyung park, in - joon seol and ki - soo kim brief episodes of hypoxia before subsequent lethal ischemic events could be helpful. the protective effect of hypoxic preconditioning on hypoxic - ischemic brain injury was investigated in the neonatal rat. our study showed that hypoxic preconditioning 6-hr to 1-day before hypoxic - ischemic injury increased survival rates and had neuroprotective effects against subsequent hypoxic - ischemic injury. | brief episodes of cerebral hypoxia - ischemia cause transient ischemic tolerance to subsequent ischemic events that are otherwise lethal. this study was conducted to evaluate the protective effect of hypoxic preconditioning on hypoxic - ischemic injury in the neonatal rat and the persistence of a protective window after hypoxic preconditioning. the rats were preconditioned with hypoxia (8% oxygen, 92% nitrogen) for three hours, subjected to ischemia using ligation of the right common carotid artery, and then exposed to another three hours of hypoxia. using proton magnetic resonance spectroscopy, terminal deoxynucleotidyl transferase - mediated dutp - biotin nick end - labeling (tunel) staining, and morphologic scores, this study shows that hypoxic preconditioning 6-hr to 1-day before hypoxic - ischemic injury increases survival rates and has neuroprotective effects against subsequent hypoxic - ischemic injury. the mechanism of the protective effects of hypoxic preconditioning in the newborn rat brain may involve downregulation of apoptotic cell death. |
she had experienced dizziness and dyspnea on exertion (nyha class iii) for 1 year prior to presentation at our institute, occurring once to twice per month. her medical history included hypertension, which had been treated with aspirin 100 mg / day, isosorbide dinitrate 40 mg / day, and furosemide 20 mg / day. transthoracic echocardiography (tte) showed severe calcification and thickening of the aortic valve, with an aortic valve area of approximately 0.6 cm, a maximum transaortic valvular velocity of 6.2 m / sec, a peak / mean pressure gradient of the aortic valve of 152/92 mmhg, and a left ventricular ejection fraction of 52%. computed tomography (ct) revealed mild atherosclerosis in the aorta and in both common iliac arteries. a coronary angiogram showed a normal coronary artery, and her euroscore ii was 3.58%. evaluation of her airway showed adequate head extension, mouth opening, and jaw protrusion, and a mallampatti score of 1 to 2. (ph : 7.36, paco2 : 49 mmhg, pao2 : 144 mmhg, hco3 : 28.0 mmhg, saturation : 99%) anesthetic monitoring consisted of a 5-lead electrocardiogram (ecg), bispectral index (bis), pulse oximetry, cerebral oximetry, and capnography. a co2 analyser probe was placed close to a nostril to increase accuracy of end - tidal carbon dioxide tension (etco2) measurement. sedation was induced with midazolam 1 mg, fentanyl 50 g and dexmedetomidine (precedex, hospira, lake forest, il, usa) loading of 50 g (1 g / kg). arterial cannulation was inserted into the left radial artery for continuous pressure monitoring and a central venous catheter was inserted into the right internal jugular vein. two external defibrillator pads were attached to the patient for the early treatment of atrial fibrillation or ventricular fibrillation. bis was monitored from 60 to 90, and the patient 's consciousness status was carefully assessed by the anesthesiologist. following dexmedetomiine infusion, her blood pressure (bp) and heart rate (hr) showed minimal changes within the normal ranges. respiratory function showed consistent saturation of 98 - 100% and etco2 of 20 - 40 mmhg with a respiratory rate (rr) of > 10. a tee probe was not inserted to a patient, and only fluoroscopy and aortography guidance was used for the procedure. because the cardiologists of our institution inserted the femoral sheath without a local anesthetic block, the patient received fentanyl 50 g prior to femoral vessel manipulation. a pacing wire was inserted into the right ventricular apex via the left femoral vein, and a pigtail catheter was inserted into the aortic root via the left femoral artery. right femoral artery cannulation was performed to insert an introducer catheter. before balloon valvuloplasty, the patient received intravenous heparin 1 mg / kg to achieve an activated coagulation time of > 250 sec. irvine, ca, usa) was placed at the aortic annulus under rvp, and the optimal prosthetic valve position was confirmed by fluoroscopic angiogram. when rvp was performed, the patient 's systolic bp dropped below 45 - 55 mmhg, and ventricular fibrillation occurred with the patient 's hr reaching 200 bpm. bp and hr restored to the previous levels of 110/55 mmhg and 50 - 60 bpm, respectively, after termination of pacing. the tavi procedure was terminated 5 - 10 minutes later, immediately after withdrawal of the catheter from femoral vessels. the total length of the intervention time was 80 min and the total anesthesia time was 110 min. after the procedures, patient 's bis value was 80 - 90, and she was transferred to the intensive care unit (icu) for close hemodynamic monitoring. on arrival at the icu, the patient 's level of consciousness was assessed according to the glasgow coma scale and richmond agitation sedation scale ; she showed scores of 15 and -2, respectively, which suggests a " light sedation " (patients awakens with eye opening and eye contact, but not sustained). in addition, she showed spontaneous respiration, and saturation was maintained 98 - 100%. two hours after arrival in the icu, the patient showed an alert mental status. the patient was transferred to the general ward on postoperative day 3, after her vital signs were stable and she showed normal sinus rhythm without atrioventricular (av) blockage. tte showed that the prosthetic aortic valve was functioning well, with no paravalvular and transvalvular leakage. she was found to be positive for scrub typhus antibody on postoperative day 16 and was administered empirical therapy with doxycycline 100 mg. on postoperative day 21, she was discharged without complications. she had developed dyspnea 1 year earlier and complained of recently developed resting dyspnea with orthopnea. she had hypertension, diabetes mellitus, and osteoporosis and was being treated with an angiotensin - converting enzyme inhibitor 4 mg / day, furosemide 40 mg / day, statin 10 mg / day, and glimepiride 2 mg / day. the patient received aspirin 100 mg / day and clopidogrel 300 mg / day the day before the tavi. the aortic valve area was 0.48 cm, a peak / mean pressure gradient was 111/67 mmhg, and the maximum transaortic valvular velocity was 5.3 m / sec. her left ventricular ejection fraction was 62%, and her aortic root annulus diameter was 19 mm. ct showed mild atherosclerosis in the descending thoracic aorta and in both common and internal iliac arteries. fentanyl (50 g) was administered via injection, and dexmedetomidine was loaded at a dose of 60 g (1 g / kg). her left radial artery was cannulated, and a central venous catheter was inserted into her right internal jugular vein. she was given 1 mg midazolam and a 25 g bolus of fentanyl and was continuously infused with dexmedetomidine 0.5 her bp decreased to 120/55 mmhg from a baseline bp of 160/80 mmhg, and her hr reduced from 80 bpm to 60 bpm after injection of dexmedetomidine, midazolam and fentanyl. the patient had a bis level of 60 - 90, saturation of 98 - 100%, and etco2 of 25 - 40 mmhg. the tavi procedure was aided by fluoroscopy and aortograpy. the patient was given heparin 1 mg / kg to achieve an activated coagulation time of > 250 seconds. without using rvp, a 29 mm core valve (medtronic, minneapolis, minnesota, usa) was advanced over a guide - wire and deployed within the annulus under fluoroscopic guidance and serial aortography to validate the position of the valve. once valve competence was assessed, dexmedetomidine infusion was discontinued. the patient became alert immediately after completion of the procedure and was transferred to the icu with a bp of 112/54 mmhg, saturation of 99 - 100%, and no complaints of dizziness of dyspnea. on postoperative day 3, the patient underwent tee and a coronary valve ct, which revealed non - specific findings. an ecg, however, showed that a left bundle branch block (lbbb) rhythm alternated with a first degree av block rhythm and was accompanied by an increase in hr to 115 - 220 bpm. the patient had no symptoms corresponding to these arrhythmias, and she was transferred to the general ward on the next day postoperative day 4. continuous ecg monitoring was maintained and av block rhythm was constantly checked using a 12-lead ecg. ecg continuously showed lbbb and the first degree av block. on the 7th day after tavi her prosthetic valve motion was well preserved with mild perivalvular aortic regurgitation (ar). she had experienced dizziness and dyspnea on exertion (nyha class iii) for 1 year prior to presentation at our institute, occurring once to twice per month. her medical history included hypertension, which had been treated with aspirin 100 mg / day, isosorbide dinitrate 40 mg / day, and furosemide 20 mg / day. transthoracic echocardiography (tte) showed severe calcification and thickening of the aortic valve, with an aortic valve area of approximately 0.6 cm, a maximum transaortic valvular velocity of 6.2 m / sec, a peak / mean pressure gradient of the aortic valve of 152/92 mmhg, and a left ventricular ejection fraction of 52%. computed tomography (ct) revealed mild atherosclerosis in the aorta and in both common iliac arteries. a coronary angiogram showed a normal coronary artery, and her euroscore ii was 3.58%. evaluation of her airway showed adequate head extension, mouth opening, and jaw protrusion, and a mallampatti score of 1 to 2. (ph : 7.36, paco2 : 49 mmhg, pao2 : 144 mmhg, hco3 : 28.0 mmhg, saturation : 99%) anesthetic monitoring consisted of a 5-lead electrocardiogram (ecg), bispectral index (bis), pulse oximetry, cerebral oximetry, and capnography. a co2 analyser probe was placed close to a nostril to increase accuracy of end - tidal carbon dioxide tension (etco2) measurement. sedation was induced with midazolam 1 mg, fentanyl 50 g and dexmedetomidine (precedex, hospira, lake forest, il, usa) loading of 50 g (1 g / kg). arterial cannulation was inserted into the left radial artery for continuous pressure monitoring and a central venous catheter was inserted into the right internal jugular vein. two external defibrillator pads were attached to the patient for the early treatment of atrial fibrillation or ventricular fibrillation. bis was monitored from 60 to 90, and the patient 's consciousness status was carefully assessed by the anesthesiologist. following dexmedetomiine infusion, her blood pressure (bp) and heart rate (hr) showed minimal changes within the normal ranges. respiratory function showed consistent saturation of 98 - 100% and etco2 of 20 - 40 mmhg with a respiratory rate (rr) of > 10. a tee probe was not inserted to a patient, and only fluoroscopy and aortography guidance was used for the procedure. because the cardiologists of our institution inserted the femoral sheath without a local anesthetic block, the patient received fentanyl 50 g prior to femoral vessel manipulation. a pacing wire was inserted into the right ventricular apex via the left femoral vein, and a pigtail catheter was inserted into the aortic root via the left femoral artery. right femoral artery cannulation was performed to insert an introducer catheter. before balloon valvuloplasty, the patient received intravenous heparin 1 mg / kg to achieve an activated coagulation time of > 250 sec. irvine, ca, usa) was placed at the aortic annulus under rvp, and the optimal prosthetic valve position was confirmed by fluoroscopic angiogram. when rvp was performed, the patient 's systolic bp dropped below 45 - 55 mmhg, and ventricular fibrillation occurred with the patient 's hr reaching 200 bpm. bp and hr restored to the previous levels of 110/55 mmhg and 50 - 60 bpm, respectively, after termination of pacing. the tavi procedure was terminated 5 - 10 minutes later, immediately after withdrawal of the catheter from femoral vessels. the total length of the intervention time was 80 min and the total anesthesia time was 110 min. after the procedures, patient 's bis value was 80 - 90, and she was transferred to the intensive care unit (icu) for close hemodynamic monitoring. on arrival at the icu, the patient 's level of consciousness was assessed according to the glasgow coma scale and richmond agitation sedation scale ; she showed scores of 15 and -2, respectively, which suggests a " light sedation " (patients awakens with eye opening and eye contact, but not sustained). in addition, she showed spontaneous respiration, and saturation was maintained 98 - 100%. two hours after arrival in the icu, the patient showed an alert mental status. the patient was transferred to the general ward on postoperative day 3, after her vital signs were stable and she showed normal sinus rhythm without atrioventricular (av) blockage. tte showed that the prosthetic aortic valve was functioning well, with no paravalvular and transvalvular leakage. she was found to be positive for scrub typhus antibody on postoperative day 16 and was administered empirical therapy with doxycycline 100 mg. on postoperative day 21, she was discharged without complications. she had developed dyspnea 1 year earlier and complained of recently developed resting dyspnea with orthopnea. she had hypertension, diabetes mellitus, and osteoporosis and was being treated with an angiotensin - converting enzyme inhibitor 4 mg / day, furosemide 40 mg / day, statin 10 mg / day, and glimepiride 2 mg / day. the patient received aspirin 100 mg / day and clopidogrel 300 mg / day the day before the tavi. the aortic valve area was 0.48 cm, a peak / mean pressure gradient was 111/67 mmhg, and the maximum transaortic valvular velocity was 5.3 m / sec. her left ventricular ejection fraction was 62%, and her aortic root annulus diameter was 19 mm. ct showed mild atherosclerosis in the descending thoracic aorta and in both common and internal iliac arteries. fentanyl (50 g) was administered via injection, and dexmedetomidine was loaded at a dose of 60 g (1 g / kg). her left radial artery was cannulated, and a central venous catheter was inserted into her right internal jugular vein. she was given 1 mg midazolam and a 25 g bolus of fentanyl and was continuously infused with dexmedetomidine 0.5 g / kg / hr. her bp decreased to 120/55 mmhg from a baseline bp of 160/80 mmhg, and her hr reduced from 80 bpm to 60 bpm after injection of dexmedetomidine, midazolam and fentanyl. the patient had a bis level of 60 - 90, saturation of 98 - 100%, and etco2 of 25 - 40 mmhg. the patient was given heparin 1 mg / kg to achieve an activated coagulation time of > 250 seconds. without using rvp, a 29 mm core valve (medtronic, minneapolis, minnesota, usa) was advanced over a guide - wire and deployed within the annulus under fluoroscopic guidance and serial aortography to validate the position of the valve. once valve competence was assessed, dexmedetomidine infusion was discontinued. the patient became alert immediately after completion of the procedure and was transferred to the icu with a bp of 112/54 mmhg, saturation of 99 - 100%, and no complaints of dizziness of dyspnea. on postoperative day 3, the patient underwent tee and a coronary valve ct, which revealed non - specific findings. an ecg, however, showed that a left bundle branch block (lbbb) rhythm alternated with a first degree av block rhythm and was accompanied by an increase in hr to 115 - 220 bpm. the patient had no symptoms corresponding to these arrhythmias, and she was transferred to the general ward on the next day postoperative day 4. continuous ecg monitoring was maintained and av block rhythm was constantly checked using a 12-lead ecg. ecg continuously showed lbbb and the first degree av block. on the 7th day after tavi her prosthetic valve motion was well preserved with mild perivalvular aortic regurgitation (ar). several previous studies have compared mac and ga use during tavi and described the advantages and limitations of mac. in those studies, anesthesiologists used the sedative agents such as propofol, remifentanil, fentanyl, ketamine, and midazolam. recently a report described the use of dexmedetomidine as an alternative to sedative agents during tavi under mac. dexmedetomidine is a highly selective alpha-2 agonist with sedative, analgesic properties, making it an anesthetic adjuvant. it is associated with minimal respiratory depression, reduced opioid requirements, and decreased hemodynamic changes. furthermore, it can be used for cooperative sedation in which well - sedated patients can be awakened by stimulation for clinical assessment or cooperation. for these reasons, however, rapid injections of dexmedetomidine have been found to induce transient hypertension, bradycardia, and tachycardia. to avoid these effects, in the present cases, we slowly infused a loading dose of 1 g / kg over 10 minutes, followed by a continuous dose 0.5 g / kg / hr, and this regimen showed no hemodynamic instability. in the present cases, therefore, we gave an additional bolus of midazolam 1 mg and/or fentanyl 25 - 50 g before sheath dilatation and rvp to prevent unexpected movements and to ensure patient comfort. patient immobility and an adequate level of sedation are crucial in achieving successful valve implantations with or without rvp. pre - emptive therapy with vasopressor such as norepinephrine, epinephrine, and phenylephrine, is important to treat hypotension and facilitate recovery after rvp. in our patients, bp, hr and cardiac rhythm were restored to baseline levels within 10 - 15 seconds after termination of rvp. airway obstruction or respiratory depression may develop under mac, which cause adverse effects, especially in older as patients who have significant comorbidities, such as cardiac dysfunction and pulmonary disease. in the two present cases, we assessed the patients ' airways before the procedures to determine whether they were suitable for mac. the patients ' respiratory functions were maintained at rates of > 10, and spo2 was 98 - 100%. etco2 was 20 - 40 mmhg, and the difference between etco2 and paco2 was 15 - 20 mmhg. in addition, the procedures were performed by experienced cardiac anesthesiologists in a hybrid room, and all appropriate equipment was prepared in case of emergency procedures. tee can be used during tavi procedures to assess cardiac function and prosthetic valve size and position. however, intra - procedural tee may be limited under mac. in the present patients, tee was used as a pre - procedural evaluation, while only fluoroscopy, and not intra - procedural tee, was performed during the procedure. the patient in the second case developed an av block and lbbb on post - procedural ecg, which seem to be complications associated with corevalve implantation rather than with dexmedetomidine administration on the basis of following consideration. firstly, the conduction abnormality appeared on post - procedure day 3, secondly, there were no ecg abnormalities during the tavi. tavi with the corevalve prosthesis results in a high incidence of total av block requiring permanent pacemaker insertion and new - onset lbbb. hence, cardiologists should monitor post - procedural ecg and, if necessary, insert a temporary or permanent pacemaker. in conclusion, ga and mac can both be used in transfemoral tavi, depending on the individual patient and procedural details. successful tavi performed under mac with dexmedetomidine is feasible with close monitoring, appropriate drug titration, and fluoroscopic guidance without intra - procedural tee monitoring. | percutaneous trans - catheter aortic valve implantation (tavi) is recommended for inoperable patients with severe aortic stenosis at high risk for conventional aortic valve replacement. originally, tavi was mostly performed under general anesthesia. here we describe two cases of transfemoral tavi performed under monitored anesthesia care (mac) with dexmedetomidine. dexmedetomidine provides sedation, analgesia with minimal respiratory depression. although mac during transfemoral tavi has limitations, such as unexpected patient movement and difficulty in intra - procedural use of transesophageal echocardiography, mac with dexmedetomidine is feasible with close monitoring, fluoroscopic guidance and the participation of experienced anesthesiologists. |
gastrointestinal duplication cysts (gidc) are rare congenital malformations that predominantly present before the age of 2 years. they are infrequently found within the colon with only a small number having been described in adults. colonic duplication cysts most commonly cause obstruction or perforation. we present the very rare case of a colonic duplication cyst causing bowel ischaemia in an elderly female. a 74-year - old female, with a background history of hypertension and hypothyroidism, presented to the emergency department with a 6-h history of sudden onset generalized abdominal pain. blood tests were normal except for a mild neutrophilia (7.5 10/l) and lactate of 2.5 mmol / l. a computed tomography (ct) scan of her abdomen and pelvis was performed which showed abnormal loops of small bowel along the right side of the abdomen with mesenteric fat stranding. a partially calcified structure was noted lying medial to the loops of bowel (figs 1 and 2). intraoperative findings revealed an ischaemic loop of small bowel wrapped around a mass in the mesentery adjacent to the sigmoid colon (fig. the patient underwent a partial small bowel resection with primary anastomosis and en - bloc resection of the mass with the formation of an end colostomy. the final histological revealed a 6 4 4 cm cystic colonic duplication cyst separate but adjacent to the sigmoid colon. the wall of the cyst was sclerotic with occasional lymphoid aggregates, smooth muscle and neurovascular bundles (fig. figure 1:axial ct scan showing a partially calcified structure adjacent to loops of small bowel. figure 2:coronal ct scan showing a partially calcified structure adjacent to loops of small bowel. figure 3:intraoperative image black arrow pointing to intra - abdominal calcified structure. figure 4:haemolysin and eosin stain showing occasional lymphoid aggregates, smooth muscle and neurovascular bundles within the duplication cyst. haemolysin and eosin stain showing occasional lymphoid aggregates, smooth muscle and neurovascular bundles within the duplication cyst. gastrointestinal duplication cysts are rare congenital malformations the aetiology of which is not fully understood. they are predominantly found in childhood with 6780% presenting before the age of 2 years [1, 2 ]. to the best of our knowledge, they can occur at any point along the gastrointestinal tract from oral cavity to anus with the majority found within the abdominal cavity (75%). they most commonly involve in the ileum (60%) but can also occur in the stomach, oesophagus and colon. duplication cysts are found adjacent to or within the gi tract and and consist of two types, cystic duplications (80%) and tubular duplications (20%). the cysts vary in size and have been reported as large as 20 15 cm. multiple theories have proposed the reason for the development of gidc, recanalization, split notochord, environmental factors or presence of embryologic diverticula [47 ]. colonic duplication cysts are rare and account for a very small percentage of duplication cysts. the exact percentage is not fully known but one of the largest studies of 73 patients over a 22-year period found that gi duplication cysts accounted for 6.8%. colonic duplication cysts are often asymptomatic but can present with abdominal pain, vomiting or bleeding. however, our case demonstrates small bowel ischaemia as a rare complication of duplication cyst. in this case, ischaemia resulted from a segment of small bowel becoming adherent to the cyst with resulting vascular compromise. colonic duplication cysts can be identified using endoscopic ultrasound, ct or rarely contrast enema. other case reports describe a cystic gas filled structure that may be identified on plain abdominal x - ray. this is due the fact that intraoperatively, it is often difficult to differentiate duplication cysts from other causes of mesenteric masses. in our case, surgical resection is often only considered in those that are symptomatic, however, some would advocate removal due to the potential to avoid complications including the possibility of neoplastic transformation [9, 10 ]. in conclusion, | colonic duplication cysts are rare congenital malformations that predominantly present before the age of 2 years. we report the case of a 74-year - old lady who presented with sudden onset abdominal pain. a computed tomography scan noted a calcified structure adjacent to abnormal loops of bowel. intraoperative findings revealed an ischaemic loop of small bowel wrapped around a mass in the mesentery adjacent to the sigmoid colon. final histology revealed a colonic duplication cyst. colonic duplication cysts are rare entities that most commonly cause obstruction or perforation. we present the very rare case of a colonic duplication cyst causing bowel ischaemia in an elderly female. |
three plasmodial species were present : plasmodium falciparum, p. vivax, and p. malariae. plasmodium falciparum was the dominant species until the beginning of the 1950s, when p. vivax became more important. the most affected areas were those, which were most inundated, mainly the plains comprising swamps, which are the main breeding sites of the major vector in morocco : anopheles labranchiae. drainage and irrigation projects, undertaken at the beginning of the twentieth century have contributed to the reduction of malaria transmission, but disease was still common in the early 1960s. in 1965, a national malaria control program was launched. it was based on vector control using ddt indoor residual spraying and parasite reservoir control by treatment and chemoprophylaxis. the effect on p. falciparum transmission was rapid, and the last autochthonous case was notified in 1973., morocco undertook the process for certification of malaria - free status according to who criteria. however, morocco still reports, every year, about one hundred of imported malaria cases. these are mainly detected in large urban areas, mainly casablanca and rabat, but also fes and agadir. most of imported cases are p. falciparum originating from sub - saharan africa [2, 3 ]. thus, malaria resurgence in morocco remains a risk, because vectors are present in formerly malarious areas and because parasites are regularly imported. this risk may change with climatic and environmental modifications as well as increasing numbers of malaria parasite carriers coming from sub - saharan africa. the prevention of the reappearance of malaria transmission in morocco relies on surveillance which should be based on a spatial and temporal stratification with respect to malariogenic risk factors, that is, vulnerability, receptivity, and infectivity. the receptivity takes into account all parameters of the vectorial capacity of anopheline population (density, trophic behaviour, longevity, and duration of sporogonic cycle of plasmodium). the infectivity (closely related to vectorial competence) is the ability of a particular parasite species to replicate in a given vector. national malaria control program has a considerable inventory of entomological and parasitological information, and several areas of high risk are regularly studied and monitored [5, 6 ]. analysis of these data in association with environmental factors could reduce the need for very detailed field studies and allow generating maps of vector distribution and transmission risk. these would be useful for decision makers, by rapidly elucidating spatial patterns and permitting a better orientation and targeting of control measures. in this study, we analyze the receptivity of the loukkos area, one of the largest wetlands in northern morocco, to quantify and map malaria transmission risk in this region by integrating biological and environmental factors derived from high spatial resolution imagery. the study was carried out in the loukkos area located in northwestern morocco on the atlantic coast. it is situated between northwestern coordinate 350947n, 060916e and southeastern coordinate 345655n, 055958e (figure 1). this region is characterized by a typically mediterranean climate, with marked oceanic influences (low annual thermic amplitude and frequent dew). the total annual rainfall ranges from 600 and 800 mm concentrated mainly between october and april. the monthly mean low and high temperatures are ranging from 6 to 32c, respectively, during winter and summer. the landscape is composed of wetlands (coastal lagoons, swamps and rice fields), forests, and agricultural zones. two study sites, in two different biotopes, were chosen to carry out this study. (1) boucharen site located in an artificial wetland with controlled hydrology and characterized by the presence of large rice cultivation surfaces in continuous extension. (2) beggara site located in a shallow swampy zone fed by river water and subject to the atlantic ocean tidal influence. fifteen potential breeding sites, in each study area, located in the main biotopes potentially suitable to an. mosquito larvae, when present, were collected using plankton nets, counted and preserved in ethanol before being identified morphologically. indoor resting mosquitoes were collected monthly from february to november in 4 stations (animal shelters) for two consecutive nights in each site. landing catches were made, in each station, on two volunteers, one indoor and one outdoor from 08:00 pm to 05:00 am during two consecutive nights. these catches were made monthly from april to november in boucharen and only in june and july in beggara. human biting rate was estimated as the average number of bites per person per night. five cdc light traps were deposited in each site during two consecutive nights in five different stations (animal shelters). all larvae and adults collection stations were georeferenced using a global positioning system receiver (gps). vectorial capacity (vc) was calculated using the formula of garrett - jones derived from the works of macdonald : (1)vc = ma2pnlnp, where m is vector - host ratio, that is, the anopheline density in relation to man, a is vector biting rate, the average number of people bitten by one mosquito in one day, p is daily survival rate, calculated as p = pu (where p is the parous rate and u is the trophogonic cycle duration in days), and n is length in days of the sporogonic development. parity rate : was calculated during every session of capture according to the method of detinova. trophogonic cycle : the duration of this cycle was calculated using the formula : u = f1/(t g1) where f1 and g1 are parameters depending on humidity and t is the temperature. f1 and g1 correspond respectively to 36.5c - days and 9.9c, for a relative humidity of 7080% which characterizes the study region. n = f2/(t g2) where f2 and g2 are parameters depending on plasmodium species and are experimentally evaluated, respectively, at 111c - days and 16c for p. falciparum and at the 105c - days and 14.5c for p. vivax. temperature above which the sporogony is impossible is estimated at 18c and 16c for p. falciparum and p. vivax, respectively. labranchiae infectivity to p. vivax and p. falciparum was estimated based on literature, respectively, to 0.5 [1, 13, 14 ] and 0.05 [15, 16 ]. to identify and map the landscape units within the study area, a spot satellite image taken on july 22, this image allows the study area (14 24 km) to be covered in one scene with a spatial resolution of 10 10 meters. the classification of this image resulted in a land use map depicting the ecological units likely to impact anopheles abundance (figure 1). meteorological data recorded by the meteorological station of loukkos were used. mean monthly temperatures were used for the estimation of sporogonic and trophogonic cycle durations. relative humidity average is considered in the calculation of the duration of the trophogonic cycle. to spatially characterize the risk, we used the approach developed by tran. based on a mosquito abundance regression logistic model which consists in using the results of the followup of larvae in different ecological units to identify specific larvae breeding sites (rice fields, different wetlands) and quantify the relationship between land cover, larvae presence, and adult abundance. analysis of larval data led to the definition of a larval index that was defined as the probability of observing larvae in a point at least once during the mosquito season and was estimated by applying the logistic transformation to each pixel, within the image of the study area, according to its biotope. an adult abundance index was generated from the larval index and was also calculated for each pixel in the study area. this adult index was defined for each trap location as the mean value of the larval index within a buffer radius of 300 m around the trap location. hence, the model developed by tran. was applied to the land cover map of the loukkos region. it resulted in a map of a larval index, corresponding to the probability of observing an. comparison of the adult abundance index and the number of indoor resting anophelines really captured in the same pixel showed a highly significant linear regression (r = 0.71, p <.01), allowing us to infer adult abundance index for each pixel in the loukkos. from this, the human biting rate (ma) was inferred from a comparison between mean of indoor resting anopheline catches and mean of human landing catches. captures conducted the same night, in the same area and in pixels of the same adult abundance index class, were compared. comparison showed a significant linear relationship between indoor resting anopheline and human landing catches (r = 0.61, p <.01). subsequently, monthly predictive maps of human biting rate (ma) were produced. integration of the other vectorial capacity parameters (see section 2.2.3), which are supposed to vary in time but not in space, as well as an. labranchiae susceptibility to plasmodium species, p. falciparum, and p. vivax has allowed the spatiotemporal mapping of the entomological risk for each plasmodium species. during this study, a total of 10168 adult anophelines among which 1239 in beggara and 8928 in boucharen were caught by the three collection methods. catches by cdc light traps were very irregular with low production : only 405 mosquitoes were collected by this method while 7574 were cached on man and 2188 by indoor resting catches. molecular identification of 325 mosquitoes (3% of the total), collected in the two study sites by the three collection methods, showed that they all belong to an. total density is higher in boucharen, even development seems to begin earlier in beggara. peak densities are observed in may and june, respectively, in beggara and boucharen sites. labranchiae species and 861 culicinae larvae belonging to culex and aedes genera. anopheles. labranchiae larvae were collected in the two study sites (boucharen and beggara) mostly in rice fields and swamps with, respectively, 80% and 56% of positive sites. the human blood index was measured on 354 fed females : only eleven mosquitoes were engorged on man. parity rate : a total of 621 unfed females collected throughout the study period were dissected to estimate this parameter. table 3 recapitulates the results of different parameters used in the calculation of the vectorial capacity during the study period in the two study sites. entomological risk for p. vivax estimated during july and august are represented in figures 2 and 3. this risk is unimportant during the whole year for p. falciparum, presenting a maximum in august (figure 4) while it is significant for p. vivax from may to october peaking in august. labranchiae, the only representative of maculipennis group in morocco [5, 6, 18, 19 ], was the only anopheline species collected. labranchiae finds its preference area in the atlantic plains in the northwestern of the country where it constitutes more than 95% of the anopheline populations. the study site, a large wetland situated in the plain of loukkos, contains various types of larval habitats favourable to the development of mosquitoes particularly the swamps and rice fields, considered as preferred breeding sites of an. the proximity of human and animal (particularly bovine) agglomerations to the breeding sites makes this zone a very favourable biotope for the development of this species. in fact, the most dramatic malaria epidemics in the history of morocco were registered in this area when an. labranchiae was involved in the transmission of p. vivax, as well as of p. falciparum and p. malariae. these results corroborate those of sicault. as well as those of guy and holstein who demonstrated that an. labranchiae has no spontaneous ovarian diapauses in morocco ; it may lay eggs even in winter in moderate temperature. the increase of temperatures in resting shelters increases the activity of females after the winter [22, 24 ]. in this study, the rice fields (boucharen area) became favourable to the development of larvae from their inundation in may, and larvae densities reached their maximum in june / july during the rice growing phase. in this period, wide areas of the swampy zones dried out or became unsuitable to the development of larvae because of their exploitation by man or standing about by cattle. so, the complex swamp / rice field assures an. labranchiae development in the region of loukkos all over the year. rice fields offer, by their biotic and abiotic characteristics, a much more favourable biotope to an. the results of this study confirm these earlier observations ; in june, the receptivity of boucharen village located near rice fields is 6 times higher than receptivity of beggara situated near swamps. malaria transmission risk assessment or entomological risk is based on the evaluation of the vectorial capacity and vectorial competence of local vectors as defined by garrett - jones and shidrawi. the vectorial capacity is the best measure to quantify receptivity to malaria in a given region. thus, the entomological risk corresponds to the product of the vectorial capacity and the infectivity (vectorial competence for a particular plasmodial species or strain). it is in the same area time, space and plasmodium species dependant. to study its variations in loukkos, we characterized it spatially according to environmental markers generated from satellite images. examination of the risk maps so produced shows that transmission risk varies strongly by environmental determinants which control the presence and extension of breeding sites and consequently the presence of vector and its density. indeed, rice fields are known for their role in maintaining a high entomological risk in the mediterranean basin as this was demonstrated by several authors [5, 2931 ]. the risk variations in time are mainly influenced by climatic determinants, particularly the temperature which modulates the vectorial capacity of the vector populations by increasing their densities (by shortening their development cycle) and by decreasing the duration of trophogonic and sporogonic cycles. results obtained in this study clearly indicate that transmission risk is strongly higher for p. vivax than for p. falciparum in the various biotopes and during all the transmission period, which is extending from may to october. results of the current study suggest a possible transmission of p. falciparum, although low in loukkos. the findings are at odds, with earlier reports of the apparent refractoriness of european an. such a different vectorial capacity between allopatric populations of the same species has been found in a number of other anopheline species. fluviatilis t are known to act as vectors of malaria in iran and pakistan but seem not to be vectors in india [3335 ]. even though entomological risk is very high in loukkos region, the malaria vulnerability (risk of presence of infective plasmodium carriers) is very low. although morocco notifies every year about one hundred imported malaria cases, these are notified particularly in big cities where transmission conditions are absent. the probability that these carriers contact the vector in the potential transmission zones during the transmission season is very low. since 1996, only 5 imported malaria cases were notified in larache city, about 10 km from the study area (ministry of health, unpublished data). the most worrying situation would result from the exposure of local vectors to imported p. vivax. indeed the number of cases of p. vivax imported malaria is in net growth ; it increased from 1.5% (1/69) in 2003 to 9% (13/142) in 2008. it is therefore important to maintain strict surveillance, particularly for importation of p. vivax to areas with environmental characteristics similar to loukkos. in synthesis the increase of african populations arriving clandestinely from malaria endemic zones and the perpetual change of land cover factors can lead to increased malaria risk. continuous contact with exotic strains of plasmodium could in the long term lead to the selection and/or to the adaptation of strains able to develop better in local vectors. this indicates the need for focused, but flexible, epidemiological surveillance to prevent any possibility of encounters between potential vectors and gametocyte carriers. the present work can be considered as a public health tool designated to help manage malaria risk and to better use available resources by focusing actions to areas most at risk. | malaria resurgence risk in morocco depends, among other factors, on environmental changes as well as the introduction of parasite carriers. the aim of this paper is to analyze the receptivity of the loukkos area, large wetlands in northern morocco, to quantify and to map malaria transmission risk in this region using biological and environmental data. this risk was assessed on entomological risk basis and was mapped using environmental markers derived from satellite imagery. maps showing spatial and temporal variations of entomological risk for plasmodium vivax and p. falciparum were produced. results showed this risk to be highly seasonal and much higher in rice fields than in swamps. this risk is lower for afrotropical p. falciparum strains because of the low infectivity of anopheles labranchiae, principal malaria vector in morocco. however, it is very high for p. vivax mainly during summer corresponding to the rice cultivation period. although the entomological risk is high in loukkos region, malaria resurgence risk remains very low, because of the low vulnerability of the area. |
transient mid- and basal ventricular ballooning is a new variant of transient left ventricular (lv) ballooning similar to transient lv apical ballooning syndrome, mainly due to emotional and physical stress (1 - 4). these cases showed similar clinical courses to transient lv apical ballooning syndrome and catecholamine - mediated myocardial dysfunction, which might be a potential mechanism of this syndrome. a 32-yr - old man presented to our emergency department with palpitation and squeezing chest pain. the initial electrocardiogram showed slight st segment elevation in the inferior leads (lead ii, iii, and avf). echocardiography demonstrated a dilated mid portion of the lv and lv ejection fraction was 35% (fig. 1a, b). however, the motion of the lv apex was normal. during the echocardiographic examination, serum level of creatinine kinase (ck) was 177 iu / l (normal range : 55 - 170 iu / l), ck - mb was 7.8 ng / ml (normal range : 0 - 4 ng / ml), and troponin i was 1.74 ng / ml (normal range : 0 - 0.05 ng / ml). the serum level of n - terminal - pro - b type natriuretic peptide (nt - pro - bnp) was increased up to 733 pg / ml (normal range : < 200 pg / ml). the coronary arteries were free of any organic stenosis and lv showed severe hypokinesia in the mid to basal portion. 2). to rule out pheochromocytoma, we performed abdominal computerized tomography (ct). an abdominal ct showed an approximately 7.88.310.0 cm large septated cystic mass with irregular wall enhancement of the right adrenal gland (fig. his serum catecholamine levels were reported as follows : epinephrine, 436.1 pg / ml (normal range : 0 - 140 pg / ml) and norepinephrine, 509.7 pg / ml (normal range : 0 - 450 pg / ml). the urinary catecholamine levels were as follows : epinephrine, 1,530.6 g / day (normal range : 10 - 20 g / day), norepinephrine, 1,048.8 g / day (normal range : 15 - 80 g / day) and metanephrine, 22,107.4 g / day (normal range : 52 - 341 g / day). from these findings, the patient was treated with oral furosemide, an oral alpha blocker (prazosin) and intravenous nitrate infusion. the follow up echocardiography after three days of treatment showed marked improvement of lv systolic function and decreased lv dimension. the lv ejection fraction was 59%, with no regional wall motion abnormalities (fig. he underwent successful surgical resection and the pathologic findings of the excised adrenal gland were compatible with pheochromocytoma (fig. 3b). a 47-yr - old man with no cardiac history presented with hypotension after resection of an inverted papilloma of the left nasal cavity. he was treated with subcutaneous epinephrine (epinephrine hcl 1 mg / ample) injection, about 0.2 - 0.3 ml, to the nasal mucosa to prevent excessive bleeding. after the injection, his systolic blood pressure and heart rate transiently increased to 170 mmhg and 140/min, respectively, then decreased to 50 mmhg and 70/min, respectively. echocardiography demonstrated a dilated lv, akinesia of the basal to mid portion of the lv with sparing of apex and lv ejection fraction was 28% (fig. 4a, b). serum level of ck was 248 iu / l (normal range : 55 - 170 iu / l), ck - mb was 9.1 ng / ml (normal range : 0 - 4 ng / ml), and troponin i was 5.4 ng / ml (normal range : 0 - 0.05 ng / ml). the serum level of nt - pro - bnp was increased up to 4,277 pg / ml (normal range : < 200 pg / ml). no coronary angiography was performed because of the low probability of coronary arterial disease and a normal preoperative treadmill exercise test. a follow - up echocardiogram obtained after two days of conservative treatment showed normalized lv size and function (fig. treatment with -blocker and angiotensin converting enzyme inhibitor was initiated and continued for about one month after normalization of lv function. a 32-yr - old man presented to our emergency department with palpitation and squeezing chest pain. the initial electrocardiogram showed slight st segment elevation in the inferior leads (lead ii, iii, and avf). echocardiography demonstrated a dilated mid portion of the lv and lv ejection fraction was 35% (fig. 1a, b). however, the motion of the lv apex was normal. during the echocardiographic examination, serum level of creatinine kinase (ck) was 177 iu / l (normal range : 55 - 170 iu / l), ck - mb was 7.8 ng / ml (normal range : 0 - 4 ng / ml), and troponin i was 1.74 ng / ml (normal range : 0 - 0.05 ng / ml). the serum level of n - terminal - pro - b type natriuretic peptide (nt - pro - bnp) was increased up to 733 pg / ml (normal range : < 200 pg / ml). the coronary arteries were free of any organic stenosis and lv showed severe hypokinesia in the mid to basal portion. 2). to rule out pheochromocytoma, we performed abdominal computerized tomography (ct). an abdominal ct showed an approximately 7.88.310.0 cm large septated cystic mass with irregular wall enhancement of the right adrenal gland (fig. his serum catecholamine levels were reported as follows : epinephrine, 436.1 pg / ml (normal range : 0 - 140 pg / ml) and norepinephrine, 509.7 pg / ml (normal range : 0 - 450 pg / ml). the urinary catecholamine levels were as follows : epinephrine, 1,530.6 g / day (normal range : 10 - 20 g / day), norepinephrine, 1,048.8 g / day (normal range : 15 - 80 g / day) and metanephrine, 22,107.4 g / day (normal range : 52 - 341 g / day). from these findings, the patient was treated with oral furosemide, an oral alpha blocker (prazosin) and intravenous nitrate infusion. the follow up echocardiography after three days of treatment showed marked improvement of lv systolic function and decreased lv dimension. the lv ejection fraction was 59%, with no regional wall motion abnormalities (fig. he underwent successful surgical resection and the pathologic findings of the excised adrenal gland were compatible with pheochromocytoma (fig. a 47-yr - old man with no cardiac history presented with hypotension after resection of an inverted papilloma of the left nasal cavity. he was treated with subcutaneous epinephrine (epinephrine hcl 1 mg / ample) injection, about 0.2 - 0.3 ml, to the nasal mucosa to prevent excessive bleeding. after the injection, his systolic blood pressure and heart rate transiently increased to 170 mmhg and 140/min, respectively, then decreased to 50 mmhg and 70/min, respectively. echocardiography demonstrated a dilated lv, akinesia of the basal to mid portion of the lv with sparing of apex and lv ejection fraction was 28% (fig. 4a, b). serum level of ck was 248 iu / l (normal range : 55 - 170 iu / l), ck - mb was 9.1 ng / ml (normal range : 0 - 4 ng / ml), and troponin i was 5.4 ng / ml (normal range : 0 - 0.05 ng / ml). the serum level of nt - pro - bnp was increased up to 4,277 pg / ml (normal range : < 200 pg / ml). no coronary angiography was performed because of the low probability of coronary arterial disease and a normal preoperative treadmill exercise test. a follow - up echocardiogram obtained after two days of conservative treatment showed normalized lv size and function (fig. treatment with -blocker and angiotensin converting enzyme inhibitor was initiated and continued for about one month after normalization of lv function. we report two cases of transient mid- and basal ventricular ballooning syndrome associated with catecholamines. transient mid- and basal ventricular ballooning is a new variant of the transient lv apical ballooning syndrome, which is also known as " stress cardiomyopathy ", and includes transient cardiac contractile abnormalities and heart failure precipitated by acute emotional or physical stress. however, the involvement of the lv 's mid- and basal ventricle with sparing of the apical segment is the unique finding of this variant (1, 2). proposed potential mechanisms of transient lv apical ballooning are multivessel epicardial spasm, microvascular dysfunction of the coronary arteries, impaired fatty acid metabolism, myocarditis and catecholamine - mediated myocardial dysfunction (1, 3, 5). although the mechanism underlying the association between sympathetic stimulation and myocardial stunning is unknown, several proposed mechanisms have been proposed including coronary arterial spasm (6) and direct injury to myocytes (7). moreover, transient lv apical ballooning can be observed in patients with excessive circulating catecholamines with pheochromocytoma (8). although there was a case report of apical ballooning associated with pheochromocytoma, our report seems to be the first to demonstrate that mid- and basal ventricular ballooning is related with high blood catecholamines (8). one possible explanation is different distribution of sympathetic nerves (9) and dissimilar density of sympathetic nerves in the heart (10), which make the apex more vulnerable to a sudden increase in circulating catecholamine levels. in our cases, the patients showed severe mid- and basal ventricular dysfunction from transient elevation of blood catecholamine level probably due to hemorrhagic necrosis of the adrenal pheochromocytoma and injection of epinephrine, respectively. the variations in segmental involvement regardless of coronary anatomy in patients with excessive catecholamine levels may suggest different susceptibility to sympathetic stimulation from individual to individual. | the exact pathogenesis of transient mid- and basal ventricular ballooning, a new variant of transient left ventricular (lv) ballooning, remains unknown. we report two cases of transient mid- and basal ventricular ballooning associated with catecholamines. these cases suggest that catecholamine - mediated myocardial dysfunction might be a potential mechanism of this syndrome. |
blood cells are subject to constant depletion through cellular senescence, bleeding, and other causes, and must be replenished throughout life. most blood cells are produced in the bone marrow, which is also the site where self - renewing stem cells reside. in contrast, t lymphocytes are produced not in the bone marrow, but in the thymus, although t lymphopoiesis still relies on the bone marrow because the thymus contains no self - renewing cells (1, 2). postnatally, the blood represents the immediate source of thymic progenitors, ostensibly from a pool of circulating stem or progenitor cells released by the bone marrow. recruitment of these circulating progenitors into the thymus is not a constant process (3), and it has even been proposed that release of thymus progenitors into the blood is coordinated with the requirements of the thymus (4). thus, there appears to be an unexplained communication between bone marrow and thymus, allowing the bone marrow to respond in a purposeful fashion to maintain the integrity of t cell reconstitution. since the time it became evident that the thymus had no self - renewing capacity, the nature of cells that seed the thymus has been questioned. the thymus is seeded by cells with unrestricted lineage capacity, or at least the capacity to generate multiple lineages, which are then relegated to the t cell lineage by virtue of their exposure to the thymic stromal microenvironment. in the other scenario, commitment to the t cell lineage occurs before thymic entry, and the thymus specifically solicits those cells with t cell potential from a larger pool of progenitors in the blood (fig., there is little debate that the thymic microenvironment is necessary for other processes, including proliferation, differentiation into various t cell sublineages, survival, and antigen receptor selection. however, these two scenarios imply substantially different roles for the thymus in the process of t lineage commitment ; if t lineage commitment occurs before entry, there is little point in studying lineage commitment signals in the thymus (or in thymocytes). in this respect, it is worth noting that although signaling through notch, an archetypal mediator of asymmetric lineage decisions, is clearly required for t cell production by the thymus (5, 6), this requirement does not necessarily invoke a lineage commitment signal. for instance, cells at later stages of intrathymic differentiation continue to require notch signals, even though they are already restricted to the t cell lineage (79). thus, it is possible that less mature thymocytes also utilize notch to support functions other than lineage commitment. canonical t cell progenitors (those that satisfy a variety of well - established criteria ; see diversity among lymphoid section) make up the consensus pathway. nonetheless, it remains unclear whether the blood - borne cells that initiate this sequence are already restricted to the t cell lineage or remain multipotent (especially with respect to b and t cells), and it remains possible that thymic - homing cells include a mixture of both. in addition, other progenitors that do not behave conventionally also enter the thymus. the role of these cells is not well understood, but the cells may give rise to specialized subtypes of t cells or serve to condition the thymic microenvironment. thus, although many progenitor cell types home to the thymus and may contribute to the intrathymic t cell pool, each may differ qualitatively and quantitatively in their contribution to peripheral t cells. one of the best ways to resolve the nature versus nurture question is to determine the lineage potential of thymus - seeding cells before thymic entry, i.e., while still in the bone marrow and/or blood. some time ago, kondo and colleagues described a bone marrow progenitor that could give rise to t or b cells, but not myeloid cells (10), thus identifying a common lymphoid progenitor (clp) that was consistent with the long speculated bifurcation between lymphoid and myeloid lineages. this important discovery underscores a common dilemma in such endeavors, namely, distinguishing between what cells can do when placed under ideal (artificial) conditions from what the same cells normally do under biological conditions. although clp gives rise to both t and b lymphocytes when properly enticed (1012), there is little evidence to suggest that such cells home to the thymus or efficiently make t cells under normal circumstances. a recent article also failed to identify clp - like cells circulating in the blood (13), although both our laboratories not only find clp - like cells in blood, but also find that they are highly efficient at generating t cells under appropriate conditions (unpublished data). nonetheless, evidence that these blood - borne clps enter the thymus is lacking, and their contribution to the mature t cell pool remains undefined. the question of whether the thymus is seeded by a progenitor possessing both b and t lymphoid potential has also been called into question by other studies. for a number of years, it has been known that cloned fetal thymocytes, fetal blood, and/or fetal liver cells exhibit the capacity to make t cells, natural killer (nk) cells, and/or dendritic cells (dc) in vitro, but generally lack a precursor cell that has both t and b cell capacity (1417). more recently, one of our laboratories has found that a similar situation exists during postnatal differentiation ; thymic progenitors exhibiting characteristics typical of archetypal t cell progenitors (defined below) were able to generate t cells or nk lineage cells (11) as well as cells expressing macrophage / monocytic markers, but could not make any b cells either in vivo or in vitro. as discussed in that paper, the inability of these cells to generate b cells implies that the thymus is either colonized by progenitors that have already lost b cell potential or that b cell potential is lost instantaneously upon entry into the thymus. the latter is difficult to accept, given that various studies indicate that lineage commitment is more of a rheostat than a switch (for review see references 1820). the former is called into question by recent studies of blood progenitors, which suggest that t cell progenitor activity comes from within a pool of cells (lineage cd117 sca-1) that have multilineage potential (13). however, clonal responses were not analyzed in that study, and it remains possible that t cell progenitor activity derives from a subset of unipotent cells that share a common phenotype with multipotent cells. it is also important to note that nk cell potential (at least) persists among t - not - b cell progenitors in the postnatal thymus (11), although again clonal analyses have yet to be performed and it remains possible that nk cell and t cell lineages arise from discrete precursors within this population. an evolving issue for attempts to identify thymus - seeding progenitors is that there appears to be several, and perhaps numerous, populations that can give rise to t cells. in the postnatal thymus, there are atypical progenitor populations that have the capacity to make both b and t lineage cells (11) but lack many of the other hallmarks that define a stereotypical t cell progenitor. when cultured under conditions that support robust proliferation of t cell progenitors (21), most of these cells fail to proliferate, exhibit normal kinetics of differentiation, and/or to undergo a normal sequence of developmental events. nonetheless, these populations are clearly present in the normal thymus and, in fact, home to the thymus rapidly in response to administration of lineage - depleted bone marrow cells into nonirradiated, normal recipients (11). their purpose in the thymus is not known : it is possible that some of them give rise to alternate thymic lineages (for example, nkt cells or regulatory t cells), or that they have functions such as conditioning the thymic microenvironment (22). in any case, the presence of distinct populations in the bone marrow and the thymus that have qualitatively different t cell progenitor capacities dictates that in order to be defined, without qualification, as in addition to the ability to generate immature and mature t lineage cells, true t cell progenitor populations must home to the thymus under competitive (steady - state) conditions, undergo extensive proliferation (in vivo or in vitro), demonstrate orderly progression through all the known stages of thymic lymphoid differentiation, and display normal kinetics of transit during this differentiation process. identifying progenitors that possess t lineage potential and also satisfy these additional criteria is an essential first step in defining which cells to focus on for analysis of non t lineage potential, and therefore is fundamental in defining the role of the thymus in the divergence of b and t cell lineages and the specification of t cell fate. progenitors that do not satisfy all of these criteria may still have the capacity to make t cells, but defining such cells as t cell progenitors without further qualification can only serve to further confuse this complex issue. a further difficulty in identification of true t cell progenitors, whether from the bone marrow, blood, or thymus, is that definitions generated by various research groups are not uniform. this is natural, since each group approaches the problem differently and in some cases seminal observations arise by chance and therefore can not be devised to fit existing criteria. nonoverlapping definitions are particularly an issue for the genetic reporter strains that have been essential in dissecting the lymphoid progenitor question, including mice expressing the thy-1.1 allele (23), human cd25 under the control of the pre - t promoter (24), or egfp controlled by the rag1 or ccr9 promoters (reference 12 and see benz and bleul on page 21 of this issue). integration of these new progenitor definitions into existing ones from other laboratories is difficult, involving the acquisition and expansion of reporter strains and the duplication of tedious and reagent - dense experiments. however, it does seems prudent for authors of all future studies to compare their definitions to a set of consensus markers including cd44, cd117, cd135, and sca-1, and desirable, where possible, to evaluate other promising markers, such as cd24 (11), cd27 (12), cd45r (24), and cd62l (26). one of the most important concepts emerging from this field is that there is more than one distinct progenitor subtype that can give rise to t cells, although the relative efficiency at which t cells are made from each can fluctuate in response to a variety of factors. different progenitor cells certainly have inherent qualitative differences and may also behave differently depending on the experimental circumstances used. however, as described earlier, it is important to remember that lineage commitment is a gradual process, not an instantaneous one. coupled with the fact that hematopoietic cells are migratory, it is likely that even within a single progenitor cell type, individual cells may differ as a consequence of which environments they have been exposed to and for how long. thus, everyone is probably at least partially correct in their conclusions ; the bad news is that no one is likely to be completely correct. | the thymus manufactures new t cells throughout life but contains no self - renewing potential. instead, replenishment depends on recruitment of bone marrow derived progenitors that circulate in the blood. attempts to identify thymic - homing progenitors, and to assess the degree to which they are precommitted to the t cell lineage, have led to complex and sometimes conflicting results. as described here, this probably reflects the existence of multiple distinct types of t cell lineage progenitors as well as differences in individual experimental approaches. |
we investigate li, na and k cryolite melts by raman spectroscopy and dft. a slight red shift of main raman peaks is observed in the row li, na, k. a decrease of the half - widths of peaks is observed in the same row. the effect of alkali cation nature on the properties of fluoroaluminate melts alf3 + mf (m = li, na, or k) is an important problem related directly to industrial aluminum electrolysis. this problem was repeatedly addressed at thermodynamical and spectroscopic levels (see ref. for review), mostly to understand the effects of lif additives on cryolite properties. lif is a typical minor component of sodium - based melts used in order to increase their conductivity. some data on the influence of lithium ions on sodium intercalation and (feebly) on overvoltage values for both cathodic alumina reduction, and anodic carbon oxidation processes were reported as well. later an obvious interest in kf additives, as well as in potassium - based fluoroaluminate melts arose due to the problem of low - temperature aluminum production. inert (low - consumable) anode material, because the melt temperature plays a major role in anode degradation, and acidic potassium fluoroaluminates provide the lowest possible temperature. a deeper insight into melt ionic composition is expected to be helpful to balance useful and harmful effects of additives by combining some of them. a number of experimental studies were performed by raman spectroscopy for molten mixtures (mostly fluoroaluminates with flinak, csf kf and naf kf eutectics) [36 ]. the results of other studies (where the effect of different cations was addressed separately) have been reported in refs. most of the systematic data on cation effects are still available only for rather high cryolite ratios (cr)1 of ca. 23, close to the values typical for industrial scale electrolysis. a thorough investigation of the interplay between cr and cation nature effects is, therefore, a topical issue. experimental data obtained for fluoroaluminate melts by raman spectroscopy are usually discussed using language adopted from coordination chemistry, i.e. the formation of different al(iii) fluorocomplexes (including polymeric forms) is assumed. that is why a quantum chemical modelling of fluoroaluminates is very useful to facilitate the interpretation of experimental spectra. computational approaches at molecular level provide detailed information on the geometry and electronic structure of certain species existing in the melt ; ionic equilibria in wide cr intervals and complexation kinetics can be investigated as well. only a few attempts were made so far to model alkali cation effect on molecular structure of cryolite melts considering small neutral associates (see, e.g., refs. the level of these studies was restricted to ab initio scf and density functional theory (dft). raman spectroscopy forms a natural link between molecular modelling and the prediction of microscopic properties of melts, as calculated spectra can be employed to verify model results. in our previous communication we have reported some results on a combined experimental and computational study of sodium cryolite in 13 cr intervals, in order to demonstrate the possibilities of such an approach. in what follows we present experimental raman spectra for lithium and potassium cryolites melts in the same range and discuss the reasons of cation - dependent spectral behaviour on the basis of molecular - level treatment of assumed equilibria. the experimental raman spectra and results of quantum chemical modelling are reported in section 3. spectroscopic measurements were performed using a fiber - optics raman spectrometer ramanrxn1 with a long - focal - length optics (work distance is 430 mm). the reagents (x3alf6 and alf3 of pure grade, and xf (x = li, k) of extra pure grade, chimmed, russia) were used to prepare melts at different temperatures (see table 1). for xf containing crystallized water, the preliminary heat treatment in platinum crucible at 500 this procedure, as well as any additional purification of all reagents, demonstrated no effect on the spectra. we can assume that all volatile impurities could be deleted in the course of melting, and the traces of other (less volatile) impurities could not affect spectral behavior of predominating fluoroaluminate components. the water ir filter prevented the spectrometer optics from ir radiation and destructive vapor of the melt. test measurements in one and the same melt at different temperatures shown that overheat of 100 grades reckoned from the melting temperature does change slightly the form and ratios of peaks. for a quantitative analysis of spectra we employed a deconvolution of the experimental bands (gaussian - lorenzian fit, the accuracy of band maxima determination is strongly dependent on the degree of bands overlap. namely, for a separate band in the vicinity of 620 cm this accuracy is surely below 1 cm. for two other bands, the shift of bands resulting from overheat never exceeded 12 cm, i.e. it is comparable with the accuracy of determination of maxima positions. for ca. 4 cm area of melt / air interface, changes of melt composition with time (induced by evaporation) were not drastic. this can be concluded from comparison of spectra registered subsequently with 10 min long intervals (at least three spectra were collected for each sample). typical time interval between temperature stabilization (after melting) and registration of spectra never exceeded 20 min. the changes of spectra with time (if any) were negligible as compared to observed cr effects. to say more quantitatively, the intensity of the most characteristic band at 620 cm decreased for na > k. according to theory developed in ref. vt, which may explain additionally the cation effect on experimental spectra at acidic fluoroaluminate melts (see table 6). of course, this explanation is purely qualitative, as temperature effect predicts only 6% decreasing the v values when going from li to k. we have reported experimental raman spectra obtained for alf3 + mf melts (m = li and k) and compared these data with our previous results (spectra for m = na). clear trends in the row li na k can be observed from a comparative analysis of the experimental data. the cation effect at molecular level on the structure of fluoroaluminates in the melt was addressed using dft calculations of model clusters and constructing the potential energy surfaces ; raman spectra were calculated and compared with experimental data. local minima on the energy surfaces are sensitive to the cluster size which is an obvious shortcoming of our approach ; this can be overcome in molecular dynamics simulations of melts. we believe that such an approach is robust enough and can be employed to make useful predictions. it is significantly more difficult to estimate possible error bars of calculated model quantities as compared with the experimental data. errors might result from several sources : computational level, size of clusters, continuum approach to describe medium effects. that is why the main accent of our modeling is put on qualitatively interesting aspects of the problem. as follows from the results of quantum chemical modeling, lithium, sodium and potassium cations entail a number of important differences in the geometry of ionic associates, equilibrium and kinetics of al(iii) complexation. this enables, in turn, to explain qualitatively the main trends in the experimental raman spectra (band frequency shift and change of band half - width) in the sequence li the molecular nature of stabilization of less - coordinated al(iii) complex forms in k containing melts (observed in experiment) remains, however, a challenge for future studies. a tempting next step would be to devise a set of pair potentials from dft calculations in order to use them in classical molecular dynamics (md) simulations of the melts. some results on md simulations of cryolite melts have been reported recently [2730 ]. raman spectra were modeled as well ; the authors addressed structure of bridged al the molten cryolite was simulated, however, at a very limited number of cr values ; no attempts were made so far to compute its ionic conductivity and to investigate the cation effect on the structure and dynamics of the melts with the help of md simulations. as for additional experimental support, it can follow from nmr data (see, e.g., ref.). | graphical abstracthighlights we investigate li, na and k cryolite melts by raman spectroscopy and dft. a slight red shift of main raman peaks is observed in the row li+, na+, k+. a decrease of the half - widths of peaks is observed in the same row. fluoroaluminates and their complexation kinetics play an important role. |
waldenstrom 's macroglobulinemia / lymphoplasmacytic lymphoma (wm / lpl) is a lymphoproliferative disease characterized by an igm gammopathy and accounts for approximately 2% of all hematological malignant neoplasms. it has an indolent course and the main causes of death are bone marrow hematopoietic failure, infection, embolism and heart failure. wm / lpl patients have a median age of 65 years and a male predominate with a ratio of male : female of 2:1. transformation to higher - grade non - hodgkin lymphoma (nhl) and therapy - related myelodysplasia / acute leukemia (t - mds / aml) have been described in case reports and small series. however, only two reports of patients with wm transformed to hodgkin disease. in this study, we reported the first case in china with a patient who was initially diagnosed as wm / lpl and then presented with wm / lpl and classical hodgkin 's lymphoma (hdhl) simultaneously 3 years later. a 74-year - old man was admitted to our department in june 2011 with the diagnosis of wm. he had fatigue, dizziness, night sweat and enlargement of left cervical lymph nodes for approximately 2 months. g / l (reference range, 0.4 g / l-2.3 g / l) and serum immunofixation electrophoresis showed an igm - kappa monoclonal peak. bone marrow aspiration and biopsy confirmed the diagnosis of wm with the appearance of lymphoid plasma cells. the patient was diagnosed as wm and treated with 3 courses of fludarabine and cyclophosphamide (fc) from october 2008 to december 2008. he achieved complete remission with the disappearance of malignant lymphoid cells in the bone marrow and normal igm immunoglobulin level. he developed giddiness and fatigue in april 2011, and subsequently headache, pain on the left neck, blurred vision, night sweat and fever in the following weeks, and was admitted to our department. physical examination revealed enlarged lymph nodes in the supraclavicular, anterior cervical and submandibular areas, but no hepatosplenomegaly. laboratory examinations were shown as follows : complete blood count revealed white blood cells (wbc) at 5.110/l (reference range, 3.9 - 10.010/l), hemoglobin at 90 g / l, and platelets at 31610/l (reference range 100 - 30010/l), and igm at 31.8 other tests included : elevated serum -2 microglobulin (4.07 mg / l ; reference range, 0.40 - 0.49 mg / l), increased erythrocyte sedimentation rate (esr) (125 mm / h ; reference range, 0.00 - 40.00 mm / h),and high serum content of -light chain (6.45 karyotype analysis showed 46, xy, and the test for igh rearrangement was positive. however, lymph node biopsy revealed that the lacunar cells were positive for cd30, cd15, cd20 and pax-5 and negative for lca, consistent with the diagnosis of classical hodgkin 's lymphoma (mixed cell type) (fig. moreover, pet / ct confirmed the enlargement of several lymph nodes in the cervical and submandibular region and a tumoral mass in posterior wall of nasopharynx of the size of 1.61.3 cm, and the highest standardized uptake value (suv) was 11.2 in all involved sites. the patient was then diagnosed as classical hodgkin 's lymphoma, mixed cell type with wm. he received the abvd regime (vinblastine, dacarbazine, bleomycin and doxorubicin) since june, 2011. fever, blurred vision and night sweat were relieved, and cervical lymph nodes were reduced 30% in volume after one cycle of treatment. the dose of doxorubicin was reduced by 75% due to atrial premature beats on a 24-hour holter electrocardiogram. unfortunately, his symptom progressed 1 month after the second cycle of the abvd regimen. the patient was treated with the r - gemox (decitabine and oxaliplatin) regimen as salvage chemotherapy. however, he did not respond to chemotherapy and was complicated with sepsis, and finally succumbed to respiratory failure on november 11, 2011. b (cd15 positive) and c (cd30 positive) show the lacunar cells of left supraclavicular lymph node biopsy specimen. wm is a distinct b - cell disorder, which is characterized by the accumulation of clonally lymphoplasmacytic cells mainly infiltrating the bone marrow and secreting a monoclonal igm protein. wm is considered lymphoplasmacytic lymphoma (lpl) by the world health organization (who) classification system. the majority of lpl / wm patients have an indolent course. previous studies have reported that patients may develop higher - grade nhl, such as diffuse large b cell lymphoma (dlbcl), therapy - related myelodysplasia / acute leukemia (t - mds / aml) and immunoblastic sarcoma, especially those patients who have been previously treated with nucleoside analogs. however, only two cases of lpl / wm who developed to hl have been reported. here, we report a patient initially diagnosed as lpl / wm and presented with lpl / wm and hl simultaneously 3 years later. our patient was diagnosed as wm in 2008 confirmed by the presence of monoclonal igm detected on serum protein electrophoresis and the appearance of lymphoid plasma cells in bone marrow aspirate and biopsy. lymph node biopsy revealed large cells with the immunochemistry of hl (mixed cell type). interestingly, lymphoid plasma cells but not malignant large cells were found in his bone marrow and monoclonal igm can also be detected on serum protein electrophoresis. therefore, we defined that the patient had lpl / wm and hl simultaneously, maybe in the process of transformation from lpl / wm to hl. patients with chronic lymphocytic leukemia / small lymphocytic lymphoma who transform to a high - grade lymphoma, such as hl, hairy cell leukemia and nhl, especially dlbcl are described as richter 's syndrome. although the exact pathogenesis of richter 's syndrome is still unclear, many mechanisms have been raised, including transformation triggered by viral infections, such as epstein - barr virus (ebv), the expression of genes that inhibit cancer development and chromosomal abnormalities. these abnormalities mainly include trisomy 12, structural abnormalities at 11q and mutations in tumor suppressor genes, especially in p53 and p16ink4a. ebv can be detected in reed - sternberg cells of about 35% of hd patients, but whether ebv can cause hd was not confirmed. in our case, the patient was negative for ebv transcription and had a normal karyotype, which may suggest that ebv infections and the expression of genes implicated in cancer development and chromosomal abnormalities were probably not involved in the pathogenesis of lpl / wm transforming to hl. reported an increased incidence of transformation to high - grade nhl and the development of t - mds / aml among patients with wm treated with nucleoside analogs. ling. also reported two cases of wm with unusually aggressive transformation with treatment of cladribine. however, lin. reported an incidence of 13% among 92 patients who developed dlbcl, and these patients were associated with poor prognosis and ebv was not involved in the pathogenesis of transformation. although risk factors for developing the transformation in patients with lpl / wm are poorly understood at present, the transformation of our patient may be due to the use of nucleoside analogs - based chemotherapy (the fc regimen) at the initial treatment. in summary, we report a rare case who presented with hl and lpl / wm simultaneously 3 years after the initial diagnosis of lpl / wm. evolution to hl may be attributed to exposure to nucleoside analogs. | abstractwaldenstrom 's macroglobulinemia / lymphoplasmacytic lymphoma (wm / lpl) is a low - grade b - cell non - hodgkin 's lymphoma with an indolent clinical course. higher - grade non - hodgkin lymphoma (nhl) and therapy - related myelodysplasia / acute leukemia (t - mds / aml) have been reported in patients with wm / lpl in previous studies. however, only two cases with wm / lpl were reported to develop to hodgkin lymphoma (hl). here, we report the first case of wm / lpl who developed classical hl simultaneously 3 years after initial nucleoside analog - based chemotherapy. |
gastrointestinal stromal tumours (gists) are mesenchymal neoplasms of the gastrointestinal tract (gi) that can arise anywhere from the oesophagus to the rectum. gists accounts for less than 1% of all gi tumours ; however they are the most common mesenchymal neoplasms of the gi tract. population - based studies from different european countries report annual incidence rates ranging from 6.5 to 20 cases per million [26 ]. these studies also report an increase in the incidence of gists over the last two decades due to the improvement in their diagnosis and registration. few gists manifest as abdominal emergencies, such as gi haemorrhage, intestinal obstruction, or tumour perforation. although acute abdomen due to gist perforation into the peritoneal cavity is rare, a few cases of peritonitis caused by perforation of small - intestinal gists have been reported in the literature [829 ]. together with a review of the published cases, here we report a case of an elderly patient with acute abdomen due to spontaneous perforation of a gist located in the jejunum. an 82-year - old man was admitted to the emergency unit of our hospital with fever, vomiting, diarrhoea and diffuse abdominal pain that had been experienced for the previous 24 h. the patient had a history of arterial hypertension. vital signs were within normal limits and the rectal temperature was 38.6 c. on examination, laboratory tests showed elevated white cell count (13.430/mm3, 92.7% neutrophils) and hyper - glycaemia (221 mg dl). abdominal plain radiography showed gas - fluid levels in the small bowel without free intraperitoneal air. an abdominal enhanced computed tomography (ct) scan was urgently performed and revealed the presence of a 6 5.5 5.5 cm solid mass in the left upper quadrant towards the umbilicus, adherent to a jejunal loop, and surrounded by free fluid. a small layer of free extraluminal air was also seen close to the margin of the mass (fig. the diagnosis of a perforation of a gist located in the jejunum wall was suspected. intraoperative findings showed free purulent exudate in the abdominal cavity and a diffuse inflammatory reaction of the peritoneum. the mass was located 10 cm away from the treitz ligament, on the antimesenteric side of the first jejunal loop (fig. a segmental resection of the jejunum containing the mass was performed followed by a mechanical end - to - side anastomosis, peritoneal irrigation and toilet, and finally the placement of multiple drainages. the post - operative course was uneventful and the patient was discharged 10 days after surgery. on histological examination, the specimen was described as a solid smooth grey and white mass (maximum diameter of 7 cm) arising from the wall of the resected segment of small bowel. the mitotic index was 16/50 high - power field (hpf) and the ki-67 value was 15%. 3b), focal positive for h - caldesmon, negative for smooth muscle actin, desmin, cd34, s-100, ema and cytocheratine. all these findings led to the diagnosis of gist of the jejunum, which was classified as high - risk using the prognostic classification by fletcher.. as soon as the diagnosis was confirmed by histology, the patient was started on imatinib mesylate therapy at the daily oral dose of 400 mg and he is currently under follow - up after six months of therapy. nearly a third of patients with gists are asymptomatic and the diagnosis is made incidentally during surgical, endoscopic, radiologic procedures or at autopsy. most of the symptomatic patients present with vague, nonspecific abdominal pain or discomfort, sometimes associated with nausea and vomiting. more frequently, gists larger than 4 cm may produce symptoms secondary to obstruction or gi bleeding. the bleeding can be either chronic, often leading to anaemia, or acute with episodes of haematemesis or melena. very few cases manifest as other abdominal emergencies, such as haemoperitoneum secondary to intra - abdominal tumour rupture, or peritonitis secondary to tumour perforation. however gist perforation seems to occur more frequently in the small bowel compared to other anatomic sites. our literature search revealed 21 cases of acute abdomen with diffuse or localized peritonitis caused by spontaneous perforation of small intestine gists (table 1) [929 ]. they are not included in table 1 because the details of the cases are not available. in another two cases not listed in table 1, spontaneous ruptures of the small intestinal gists were associated with an intraperitoneal abscess without perforation to the intestinal lumen. of the 22 cases reported in table 1, including this case, 16 patients were male and five were female. gist perforation can occur irrespective of the age, since the patients ages ranged from 22 up to 82 years. the jejunum was the more common location of perforation compared to the ileum (15 vs 4). in three other cases it was not specified at what level of the small intestine the perforated gist was located. the small intestinal gist was associated with hepatic metastases, in the other two cases small bowel gists were multifocal. diffuse peritonitis was described in 19 patients, while in the remaining three the intraoperative presentation was a localized abscess adjacent to the anatomical site of the perforated gist. despite perforation and concomitant peritonitis, all authors but one reported good post - operative outcome. the only death occurred on postoperative day four due to septic shock in a complicated case of perforated gist of the ileum with concomitant small bowel necrosis secondary to internal herniation. a possible explanation of the very low morbidity and mortality in the reported cases is the relatively low peritoneal contamination due to the predominant proximal anatomic site of perforation, and the expeditious resuscitation followed by emergency surgical intervention in all patients with diffuse peritonitis. the only exception was a terminal ileostomy performed in the very compromised patient reported by zben.. complete resection can be achieved in approximately 85% of patients and the estimated incidence of recurrence or metastasis after radical surgery is 50%. using the widely accepted prognostic classification proposed by fletcher., based on the size of the tumour and the mitotic count, gists are classified as very low-, low-, intermediate- and high - risk for potential malignancy. joensuu and coll have proposed a classification that includes tumour rupture and tumour site (table 2) [3739 ]. based on this risk classification, all the patients with tumour rupture should be considered at high risk for recurrence. imatinib mesylate, a tyrosine kinase inhibitor, has been found to be beneficial after radical resective surgery of high - risk gists. more recently, adjuvant therapy with imitinab has been used with wider indications, such as intermediate - risk tumours with size > 3 cm and primary tumours with rupture or perforation.. of the 21 surviving patients reported in table 1, 13 were treated with imatinib, including our case. in the other eight cases, there is no mention of imatinib therapy. follow - up data are reported for 12 patients, of which 10 were on imatinib adjuvant therapy. all but one were alive without recurrence, with a follow - up ranging from six to 48 months. this patient had multiple hepatic metastases at the time of surgery and later developed peritoneal dissemination. at that time (2001) imatinib was still an investigational chemiotherapic agent and the patient was treated with this experimental therapy with dramatic clinical improvement. emergency presentation with a gist is not uncommon and one of its manifestations is acute abdomen secondary to intraperitoneal rupture or perforation of a primary gist of the small intestine. since there is an increased risk of recurrence after spontaneous intraperitoneal rupture / perforation of gists, patients should be evaluated by a multidisciplinary team in order to assess the indications for imatinib adjuvant therapy and for close monitoring and follow - up. ma is associate professor of the department of clinical, surgical, diagnostic and paediatric sciences, university of pavia, and director of the department of general surgery, varzi hospital ; mg, ps, em and dz are staff assistants of the department of general surgery, varzi hospital ; sr is staff assistant of the radiology service, voghera hospital ; vp is staff assistant of the department of oncology, irccs san matteo hospital foundation, pavia ; lc is staff assistant of the unit of general surgery 1, irccs san matteo hospital foundation, pavia and assistant professor of the department of clinical, surgical, diagnostic and paediatric sciences, university of pavia. ma, mg, ps, em, dz collected and analyzed the data and were involved in the care of the patient. sr and vp were involved in the interpretation of the diagnostic and oncologic data, in the diagnosis and in the follow - up of the patient. all the authors contributed equally to drafting the article or revising it critically for important intellectual content and for the final approval of the version to be published. written informed consent was obtained from the patient for publication of this case report and any accompanying images. a copy of the written consent is available for review by the editor - in - chief of this journal. | highlightsemergency presentation of a gist is not uncommon and one of its manifestations is acute abdome.the cases described in the literature are the tip of the iceberg and spontaneous rupture or perforation of gists are a far more frequent first presentation of this rare tumour.the jejunum was the more common location of perforation compared to the ileum.emergency surgery is mandatory and should achieve radical resection. |
endosymbionts are normally asexual and transmitted by uniparental vertical inheritance (sachs., 2011). multicellular organisms thus have a single mitochondrial genotype and those that have photosynthesis rely on a single clone of plastids. the evolution of such obligate symbiotic mutualisms has strong elements of partner commitment driven by kin selection, because exclusive association of hosts with a single symbiont genotype ensures that its services to growth and survival of the host will benefit clone mates that are vertically transmitted when hosts reproduce (frank, 1994, doebeli and knowlton, 1998, sachs., 2004, the same logic implies that hosts and symbionts are potentially in conflict over the mode of symbiont transmission (frank, 1996, douglas, 2008), as symbionts would always benefit from additional horizontal transmission. however, hosts might suffer fitness losses from this form of commitment - disloyalty and therefore suppress symbiont investments in sexual reproduction (frank, 1996, leigh, 2010). when, despite such host efforts, symbiont lineages manage to co - infect hosts and compete for resources, hosts will be under selection to monitor symbiont genetic diversity and eliminate additional symbiont lineages when such competition implies a net loss of cumulative symbiont service to the host (frank, 1996). the classical mitochondrial and plastid endosymbioses are so integrated with their host cells that their reduced genomes preclude any form of non - symbiotic life, and the same is true for many obligate and facultative endosymbionts with less reduced genomes (mccutcheon and moran, 2012). many of these interactions likely represent adaptive endpoints of host - symbiont coevolution, where host - symbiont conflicts were resolved in favor of the hosts (mccutcheon and moran, 2012, wernegreen, 2012) or symbiont (werren., 2008), but their advanced stage of symbiosis normally precludes direct tests of evolutionary conflict theory over symbiont mixing because co - evolved symbionts can often not be reared in vitro. the fungus - growing ants offer a feasible model system to do such tests, because they have multiple obligate mutualisms, including fungus gardens (schultz and brady, 2008, mikheyev., 2010) and cuticular actinobacteria (cafaro., 2011, andersen., 2013) that are ectosymbionts for individual ants, but endosymbionts for the ant colonies. this implies that partners can be reared in vitro without each other 's interference for sufficient periods of time to quantify antagonism between symbiont clones, monitor host reactions to alternative symbionts, and relate observed differences to the genetic characteristics of the interactants (armitage., 2011, bot. attine ant colonies have never been found to rear a multi - clone fungus garden (apterostigma, dentinger., 2009 ; cyphomyrmex, green., 2002 and mehdiabadi., 2012 ; atta, mueller., there was substantial genetic variation among fungus - garden clones across sympatric colonies, consistent with vertical transmission and normal patterns of variation of mitochondrial and plastid organelles across individual animals and plants (e.g. embley and martin, 2006). however, in contrast to these cellular endosymbionts, there may be considerable horizontal transfer of symbionts when territories of founding attine colonies overlap, consistent with species belonging to the same genus often sharing clades of symbionts (green., 2002, poulsen and boomsma, 2005, de fine licht and boomsma, 2011, de fine licht and boomsma, 2014, mehdiabadi., 2012), while sympatric attine ant genera normally rear distinct fungal symbiont clades (mueller and gerardo, 2002, dentinger., 2009, vo., 2009,. horizontal swaps of fungus - garden symbionts between colonies of the same or closely related attine ant species may reduce the efficiency of co - evolutionary adaptation at the lowest taxonomic level, but allows ant lineages to replace an asexual crop symbiont that is compromised by genetic load or another form of maladaptation to prevailing ecological conditions (mueller, 2002). however, as long as fungus - garden clones are thriving, they will also be under selection to actively defend their ant - care monopoly (bot. such defenses are expected to evolve when the threat to be replaced is real, i.e., any hostility of this kind should target non - self symbiont genotypes belonging to the clade of symbionts that can in fact partake in a viable symbiosis with a focal attine ant species. the acromyrmex echinatior and acromyrmex octospinosus populations in gamboa, panama appear to co - exploit the same clade of fungus - garden symbionts in sympatry (bot. resident fungus gardens of these ant species have been shown to maintain their clonal integrity by a combination of behavioral adaptations in the ants to remove and kill alternative fungus clones (bot., 2001,, 2009) and by the expression of somatic incompatibility reactions between clones from different acromyrmex colonies reared together on the same agar plates (poulsen and boomsma, 2005). these incompatibilities correlated with amplified fragment length polymorphism (aflp) genetic distances between pairs of fungal symbionts, a pattern that also applied to the fecal fluid of acromyrmex large workers fed with fungus from their own and other colonies (poulsen and boomsma, 2005). however, founding a. octospinosus queens readily accept fungal clones from other colonies, suggesting that this stage offers a special window for horizontal transfers even though signs of reduced performance with a novel symbiont taken from a mature colony were also found (poulsen., 2009). this study suggested that incompatibility mechanisms might be different for sympatric atta leaf - cutting ants, which tend to rear different fungal symbionts (mikheyev., 2007, kooij., 2015) and whose queens never forage during colony founding and therefore have negligible likelihood of encountering alternative symbionts. somatic (in)compatibilities between plated fungi of panamanian acromyrmex species are expressed in a gradual manner that correlates with aflp genetic distances between pairs of clones (poulsen and boomsma, 2005). in basidiomycetes, somatic incompatibilities are generally induced by allorecognition so that strains are increasingly likely to be incompatible when they are more genetically different (may, 1988, worrall, 1997). rayner, 1991, worrall, 1997) and usually involve dark pigmentation in the interaction zone (rayner., 1984), changes in septal maintenance, or blockage of septa precluding the movement of cytoplasm, and can lead to programmed cell death (rayner, 1991). the underlying genetic mechanisms remain largely unknown, but multiple loci appear to be involved (worrall, 1997) and their expression may be linked to sexual incompatibility genes (van der nest., 2009, if sex occurs at all, it is extremely rare in the fungal symbionts of higher attine ants (fisher., 1994a, fisher., 1994b, pagnocca., 2001, mueller, 2002, mikheyev., 2006) recent work further indicated that the symbionts of panamanian leaf - cutting ants are multi - genomic chimeras (kooij., 2015), which likely explains why incompatibility patterns between panamanian acromyrmex symbionts appear to be gradual (poulsen and boomsma, 2005), we aimed to further our understanding of the biological factors governing somatic incompatibility among strains of attine ant fungal symbionts. we focused on comparing sympatric mature colonies of panamanian atta colombica and acromyrmex echinatior leaf - cutting ants to address the following questions : (1) do plated fungal symbionts of a. echinatior and a. colombica express similar somatic incompatibility reactions when confronted with symbionts from other colonies ? (2) to what extent does the intensity of these reactions differ within and between the two genera ? (3) to what extent is the intensity of these reactions correlated with genetic distance between the fungal symbionts ? (4) are sympatric colonies of a. echinatior and a. colombica rearing the same or overlapping set(s) of fungal symbionts or are they associated with distinct lineages of the leucoagaricus gongylophorus symbiont ? the same set of symbionts would be expected if horizontal symbiont transmission between the two ant genera were more frequent than natural divergence of lineages via mutation and genetic drift. in contrast, segregated lineages would be expected when horizontal transmission between genera is absent because co - adaptations in l. gongylophorus strains reared by a. echinatior and a. colombica would preclude that horizontal symbiont swaps between genera are viable. finally, we also compared two different sets of genetic markers (aflps and microsatellites) and different time spans between plate inoculation and scoring of incompatibilities to evaluate the robustness of our conclusions. fungal cultivars were isolated from nine acromyrmex echinatior colonies (ae150a, ae160, ae168, ae263, ae266, ae322, ae356, ae394, ae488) and nine atta colombica colonies (ac-2006 - 27, ac-2009 - 42, ac-2009 - 46, ac-2011 - 2, ac-2011 - 3, ac-2012 - 1, ac-2012 - 2, ac-2012 - 8, ac-2012 - 31) living sympatrically in gamboa, panama, and grown on 39 g l potato dextrose agar (sigma aldrich, st louis, mo, usa) with the addition of 5 g l yeast extract, 15 mg l tetracycline and 12 mg l streptomycin. for each of the colonies a single isolate was obtained as it has been shown before that both acromyrmex (poulsen and boomsma, 2005) and atta (mueller., 2010) maintain their fungal symbiont in a monoculture. dna of each fungal strain was extracted using the qiagen (venlo, the netherlands) dneasy plant tissue extraction kit and stored at 20 c until further analysis. the gamboa sampling site was the same as where most previous colonies of the copenhagen fungus - growing ant research program have been collected, including the acromyrmex colonies studied by bot., 2001, poulsen and boomsma, 2005, mikheyev., 2007, richard. (2009). to calculate the genetic distance between each of the fungal strains we used two different methods : aflp and ten microsatellite markers (a128, a1030, a1132, a1151, b12, b447, c101, c126, c647 and d115 developed by scott. (1995) with two selective primer combinations (eco - acc + mse - cat and eco - acc + mse - cac). microsatellite markers were analyzed using pcr with 5 l vwr red taq dna polymerase master mix (vwr international, haasrode, belgium), 0.25 l forward and reverse primer each, 1.5 l ddh2o and 1 l dna, and a program of 5 min denaturing at 95 c, followed by 14 cycles of 30 s denaturing at 95 c, 30 s annealing at 6858 c with a touchdown of 0.5 c per cycle, and 30 s extension at 72 c followed by 20 cycles of 30 s denaturing at 95 c, 30 s annealing at 58 c and 30 s extension at 72 c, and finally a 15 min extension at 72 c. both aflp and microsatellite amplification products were analyzed on an abi 3130xl (applied biosystems, nrum, denmark) sequencer. specific allele scorings (aflp : table s1 ; microsatellites : table s2) were obtained by analyzing chromatograms in genemapper 4.0 (applied biosystems, nrum, denmark). the program populations 1.2.32 (langella, 2001) was used to calculate fst values for the microsatellite data and nei 's standard genetic distance (ds) for the aflp data, followed by neighbor joining phylogenetic analyses with 500 bootstrap replicates for each of the two types of markers. was used to analyze population structure for the aflp data with the following settings : an optimized k = 3 tested with the online program structure harvester (earl and vonholdt, 2011), a burn - in period of 1,000,000 iterations followed by 10,000,000 mcmc iterations, an admixture model, and independent allele frequencies among populations with = 0.78. the 20 runs that we obtained were merged with the greedy analysis with 1,000,000 repeats in the clumpp software (jakobsson and rosenberg, 2007), and visualized with distruct (rosenberg, 2004). microsatellite data were further analyzed using the package poppr (kamvar., 2014) in r (r core team, 2013), to evaluate the discriminatory power of our markers and verify clonality of the symbionts. to test whether plated fungi showed (in)compatibility, cultures of all 18 fungi were paired in all possible (171) combinations with four replicate pairings for each combination. for each pair, small tufts of mycelium (ca. 2 mm) were placed at a distance of 1.5 cm from each other on a 5 cm petri dish with 39 g l potato dextrose agar with the addition of 5 g l yeast extract and 35 g l agar. the growth medium was selected in a pilot study testing somatic incompatibility reactions for control (self) encounters on this and three alternative media, which showed that the used pdya medium most consistently avoided discolorations in controls (fig s1). incompatibility reactions were assessed after 6, 8, and 10 weeks and scored using the semi - quantitative scale described by poulsen and boomsma (2005) : 0 = demarcation zone absent, 1 = demarcation zone weak but present, 2 = demarcation zone broad and distinct, and 3 = strong demarcation zone with consistent brown or black coloration of mycelium. these four scores are consistent with the variation in somatic (in)compatibility reactions that are typically found in free - living basidiomycetes, where they usually occur in a more stepwise manner as explained above (worrall, 1997). all scorings were done blindly by randomly assigning numbers to each plate, after which two of the authors did the initial assessment and a third author blindly checked combinations for which the first two authors did not agree on the score. degrees of (in)compatibility were subsequently compared with genetic distances between pairs of fungal clones using mantel and partial mantel tests for dissimilarity matrices (mantel) (r core team, 2013) with 99,999 permutations in the community ecology package : ordination, diversity and dissimilarities vegan correlations were forced through the origin based on the fact that the controls were (0,0). figures were created using plot with repeated symbols by size (sizeplot) in the plotrix package (lemon, 2006). for acromyrmex symbionts, consistent scorings of somatic incompatibility were obtained 8 weeks after agar plates were inoculated. coloration contrasts were not fully developed after 6 weeks, so mantel correlation coefficients between incompatibility and genetic distance after 6 weeks remained low (fig s2). beyond 8 weeks, mantel correlation coefficients continued to improve, but contaminations and medium desiccation problems affected scoring accuracy 10 weeks after inoculation (fig s2) so that almost 5% of the replicates were lost. as scoring results at 8 weeks were approximately the same as the 2-months between inoculation and scoring in poulsen and boomsma (2005), our main results for the 8 weeks observation period are presented, to remain as comparable as possible with that previous study on somatic incompatibilities between a. echinatior and a. octospinosus fungal symbionts collected at the same sampling site more than 10 yr earlier. however, for atta symbionts a comparable result was only obtained when using microsatellite markers, as aflp markers produced mantel correlation coefficients close to zero for all observation periods (fig s2). using the 8 weeks data, somatic incompatibilities increased with increasing aflp genetic distances between fungi (mantel r = 0.463, p = 0.003, fig 1a) in acromyrmex symbionts, but not in atta symbionts (mantel r = 0.164, p = 0.792, fig 1c). when using genetic distances based on microsatellite markers, both acromyrmex (mantel r = 0.469, p = 0.003, fig 1b) and atta (mantel r = 0.312, p = 0.032, fig 1d) symbionts had incompatibilities that increased with genetic distance, but less of the incompatibility variance was explained in atta than in acromyrmex. the a. echinatior results were consistent with the results obtained by poulsen and boomsma (2005), but in that study the variance explained by the mantel coefficient was larger (r = 0.855 ; p < 0.0001). however, when the 10-weeks scorings for acromyrmex symbiont pairings was used there was a correlation closer to the one obtained after 2 months by poulsen and boomsma (2005) (microsatellites : mantel r = 0.596, p < 0.001 ; aflp : mantel r = 0.654, p < 0.001). overall, the atta fungi had smaller genetic distances when calculated from the microsatellite marker data (0.09 0.01 se), but larger genetic distances when using aflp markers (0.26 0.03 se) compared to acromyrmex (0.13 0.01 se and 0.17 0.02 se, respectively), which is reflected in the average number of aflp bands observed (atta : 37.3 1.8 se ; acromyrmex 30.9 1.5 se ; this implied that aflp and microsatellite genetic distances (fst) were only comparable for acromyrmex symbionts (r = 0.932 ; fig s3), whereas correlations decreased in comparisons between acromyrmex and atta symbionts and became very low in comparisons involving only colonies of atta colombica (fig s3). independent of the type of genetic marker used, mean genetic distances were higher in comparisons between acromyrmex and atta (microsatellites : 0.20 0.01 se ; aflp : 0.36 0.02 se) than in comparisons within the ant genera (means ses given above ; microsatellite markers : f2,168 = 45.127, p < 0.0001 ; aflp : f2,168 = 22, also the average incompatibility scores were different for comparisons within and across ant species (genera) (= 12.236 ; df = 2 ; p < 0.01). this difference was mostly due to less strongly expressed somatic incompatibilities between atta symbionts, because the stronger mean reactions among acromyrmex symbionts alone and between acromyrmex and atta symbionts were not significantly different from each other (w = 1635, p = 0.338). after pooling data across the entire range of genetic distances, the increase in somatic incompatibility with genetic distance was completely absent (aflp : mantel r = 0.045, p = 0.595, fig 2a) or no longer significant (microsatellites : mantel r = 0.153, p = 0.213, fig 2b). specific evaluation of the microsatellite genetic differences between the fungal symbionts of atta and acromyrmex colonies showed that they were completely separated (fig 3, fig s4 and supplementary information), consistent with earlier findings by mikheyev. calculations for the standardized indexes of association, a linkage test (rd, with 999 permutations), accounting for the number of loci sampled, were significant for the acromyrmex symbionts (ia = 1.451, p = 0.001 ; rd = 0.184, p = 0.001) and for the atta symbionts (ia = 1.923, p = 0.001 ; rd = 0.319, p = 0.001). however, because only seven out of nine atta symbionts had independent multi - locus genotypes, with three being identical (see supplementary information for details), a clone - correction was applied. this showed that the standardized index of association for the atta symbionts remained highly significant (ia = 0.744, p = 0.037 ; rd = 0.119, p = 0.009), consistent with all multi - locus genotypes being clonal. rooting the symbiont tree with a sympatric fungal symbiont of trachymyrmex zeteki (fig s5) and by constructing a minimum spanning network (fig 3) and a upgma tree (supplementary information), both based on bruvo genetic distances, confirmed that the symbionts belonged to separate clades, although bootstrap values for the rooted tree were low. mirror imaging of trees obtained by microsatellite and aflp markers showed almost complete congruence for the acromyrmex fungi, but more noisy correspondence for the atta fungal symbionts (fig s6), confirming that these markers were less reliable predictors of somatic incompatibilities for atta symbionts. furthermore, structure analyses of the aflp data showed that four of the nine acromyrmex symbionts were similar to the atta symbionts (fig 4), and these were the same four symbionts that were most closely related to atta symbionts in the rooted tree based on microsatellites (fig s5). the results of our study show that sympatric panamanian colonies of a. echinatior and a. colombica rear genetically different lineages of the leaf - cutting ant garden symbiont l. gongylophorus and that microsatellite markers appear to predict genetic (in)compatibility better than aflp markers. however, even when using microsatellite markers, the correlation between somatic incompatibility and neutral - marker - based genetic distance in atta is noisier than in acromyrmex. this shows that incompatibility reactions correlate only with genetic distances among fungal strains that have a realistic probability of being horizontally transferred, and not between more distant clades that are apparently unsuitable as symbionts for the sister genus of leaf - cutting ants. the results of our study confirm the earlier findings by poulsen and boomsma (2005) showing that somatic (in)compatibility of panamanian acromyrmex symbionts is predictable from pairwise genetic distances for aflp markers (figs 1a, c) and that the same result can be obtained with more specific microsatellite markers (figs 1b, d). they also indicate that incompatibility reactions between separate clades of fungal symbionts, maintained by the two different genera of leaf - cutting ants, can no longer be predicted by neutral genetic markers. this lack of genetic signal may be due to incompatibility being ultimately caused by allelic variation at unknown loci (worrall, 1997) that only correlate with neutral markers when there is recent common ancestry. this is only likely for local fungal symbiont lineages that are exploited by a single metapopulation of attine ants that share a joint pool of symbionts because each ant colony can in principle establish a viable symbiosis with each of these fungal genotypes. we expect somatic incompatibility to be actively maintained by selection only in populations where the ants have the possibility to acquire multiple genetically different symbionts that are viable alternatives. such selection would then be driven by resident fungus - garden symbionts being under selection to defend their monopoly against alternative strains that might be secondarily introduced, and with the active support of the farming ants that would lose fitness when maintaining multiple lineages of the same symbiont that compete for their attention rather than serving their hosts unconditionally (frank, 1996, bot., 2001). when symbionts belong to different clades that no longer mix or exchange genes, each symbiont lineage is only a viable symbiont for one lineage of ant farmers or the other, but not for both. our finding that a. colombica and a. echinatior maintain separated clades of fungal symbionts (figs 3 and s5) suggests that l. gongylophorus has, in fact, been split into an atta and acromyrmex clade after its monophyletic origin 23 mya (mikheyev., 2010). the two species of leaf - cutting ants that we investigated rear representatives of these symbiont lineages that appear adapted to being, respectively, an atta and acromyrmex symbiont. the fact that the fungal symbionts of panamanian leaf - cutting ants appear to have split in two monophyletic clades, is consistent with the results of a recent study that experimentally swapped fungus - garden symbionts between sympatric trachymyrmex septentrionalis and atta texana from texas, usa (seal., 2012). although these ants are at the northern edge of the attine ant distribution (mueller., 2011a), and likely to have lower genetic variation among their symbionts, they share even less common ancestry than the panamanian fungal symbionts of our present study (mueller., 2011b). the swapped fungal symbionts thus could only serve as viable mutualists for either trachymyrmex or atta, but not both, which was confirmed in the published experiments showing that : (1) alternative fungus gardens were not always rejected by the ants but were never adopted as a viable alternative symbiont, because the ants were able to grow their original symbiont back from minuscule remnants that the authors had been unable to remove. (2) none of the t. septentrionalis colonies ever produced virgin queens when they maintained an a. texana fungal symbiont, consistent with the new combination being non - viable for transmitting ant or fungal genes to future generations. a follow - up study transplanting a. texana fungus to colonies of both t. septentrionalis and trachymyrmex turrifex confirmed that these trachymyrmex species can not enter into viable symbiosis with l. gongylophorus symbionts and that the virgin queens produced on swapped gardens had poor fat reserves making it unlikely that they could successfully found colonies (seal and mueller, 2013). to make further progress, it would be desirable to identify the genes that are directly responsible for somatic incompatibility, as this would allow direct studies on the signatures of selection and specialization across the clades of higher attine ant symbionts. in another, non - eusocial model system of fungus - growing insects, the sirex wood wasp, a range of genes are involved in somatic incompatibility reactions among lineages of the associated fungus, including fusion and recognition genes and genes that mediate cellular damage, stress response, and programmed cell death (van der nest., 2011). whether these genes have homologs or analogs in attine ant fungal symbionts remains to be explored, as the sirex symbiont amylostereum areolatum belongs to a distantly related clade of basidiomycetes (binder and hibbett, 2002), and their respective domestication histories may have implied that recognition systems were lost and gained over evolutionary time. incompatibility reactions among atta symbionts were significantly weaker and less predictable from neutral fst marker values than similar reactions between acromyrmex - associated fungi. one possible explanation for this difference could be that there is a fundamental difference in colony founding in the sense that acromyrmex queens forage during colony founding similar to all more basal attine ants, whereas atta queens have secondarily evolved claustral colony founding. this implies that newly - mated atta queens close off their nest cavity to raise the first worker cohort purely on their body reserves, so that new colonies will only be opened by these workers 80100 d after they were founded (weber, 1969, fernandez - marin and wcislo, 2005). however, acromyrmex queens not only forage for leaf fragments to manure their incipient fungus garden but, particularly when they have lost their garden, also for a replacement garden of another incipient colony whose queen is out foraging (poulsen., 2009), an option that is unavailable for founding atta queens. although swapping of incipient fungus gardens with a fungus garden fragment from a mature acromyrmex colony was relatively unconstrained during early colony founding, it is likely that stronger mutual commitment between a founding queen and her resident fungus garden builds up in a matter of weeks, including stronger incompatibility reactions in case one of the first workers brings in an unrelated fungus garden fragment from a neighboring nest (poulsen., 2009). this can never happen in the 80100 d during which atta colonies remain closed, removing selection for expressing incompatibility mechanisms during colony founding. why neutral markers should be weaker predictors of somatic incompatibility in atta colonies after workers start foraging remains unclear. imprinting of workers on the odor of a resident fungus garden is a possibility (seal., 2012), but it seems unclear why such mechanisms should differ between atta and acromyrmex symbionts and why that should reduce selection for more direct defenses by fungal symbionts against being replaced. fungus gardens of panamanian acromyrmex colonies differ in chemical profiles (richard. these differences do not correlate with genetic distances and comparable data for sympatric atta colonies are lacking. another hypothesis may be that mature atta colonies have hundreds of fungus gardens, whereas sympatric mature acromyrmex colonies have one or a few at best. this may make a difference in the likelihood of a resident fungus garden symbiont being replaced by an accidentally imported small fragment of fungus garden from a neighboring colony, so that less accurate recognition systems suffice in mature colonies of atta but not in acromyrmex. finally, there could also be a technical explanation for the incompatibility differences between the fungal symbionts of atta and acromyrmex. we found similar variation for the symbiont - specific microsatellite markers obtained from panamanian symbiont samples (scott., 2009) and the general aflp markers for acromyrmex symbionts, but enhanced variation in aflp peaks for atta symbionts, relative to acromyrmex symbionts (fig s3). this suggests that dna from other organisms may have been amplified with the aflps and that such other organisms were only present in atta symbiont cultures. in principle, this could be viral (pearson., 2009), bacterial (suen., 2010) or prion (wickner. | obligate mutualistic symbioses rely on mechanisms that secure host - symbiont commitments to maximize host benefits and prevent symbiont cheating. previous studies showed that somatic incompatibilities correlate with neutral - marker - based genetic distances between fungal symbionts of panamanian acromyrmex leaf - cutting ants, but the extent to which this relationship applies more generally remained unclear. here we showed that genetic distances accurately predicted somatic incompatibility for acromyrmex echinatior symbionts irrespective of whether neutral microsatellites or aflp markers were used, but that such correlations were weaker or absent in sympatric atta colombica colonies. further analysis showed that the symbiont clades maintained by a. echinatior and a. colombica were likely to represent separate gene pools, so that neutral markers were unlikely to be similarly correlated with incompatibility loci that have experienced different selection regimes. we suggest that evolutionarily derived claustral colony founding by atta queens may have removed selection for strong incompatibility in atta fungi, as this condition makes the likelihood of symbiont swaps much lower than in acromyrmex, where incipient nests stay open because queens have to forage until the first workers emerge. |
sugar - sweetened beverages (ssb) are drinks sweetened with various forms of sugars that add calories and include, but are not limited to, soda, fruit ades and fruit drinks, and sports (sd) and energy drinks (ed). on average, ssb intake contributes approximately 300 kilocalories to the daily intake of adolescents 1219 years old in the united states. regular consumption of these caloric drinks can increase the risk for obesity and dental caries. in particular, sports and energy drinks (seds) are relatively new products that are increasingly marketed to adolescents. furthermore, purchase and consumption of these drinks by adolescents appear to be common [1, 5, 6 ]. in 2010, the proportion of high school students who consumed sds and eds at least once per day was 16% and 5%, respectively. sds contain carbohydrates, minerals, and electrolytes and are often marketed for the purpose of rehydration. however, drinking water alone provides adequate replenishment in most instances other than prolonged vigorous exercise. both sds and eds have been associated with increased dental erosion, due to their acidity and the presence of citric acid. eds carry additional consequences due to their stimulatory and performance enhancing effects, derived from ingredients such as caffeine, taurine, guarana, and carbohydrate sweeteners [11, 12 ]. unlike caffeinated soda drinks, eds do not have restrictions on their caffeine concentration nor are they required to have undergone safety testing or placement of appropriate warning labels [6, 13 ]. as a result, the caffeine content found in eds can range from 50 mg to 505 mg per can, which can be as much as that found in 14 12-ounce cans of a typical soda. consumption of eds may put adolescents at risk of adverse effects of caffeine (elevated heart rate, anxiety, and sleep disturbances) and in one study their consumption was associated with coconsumption of alcohol. a significant proportion of us adolescents regularly consume eds, ranging from 28% of 12- to 14-year - olds to 34% of 18- to 24-year - olds. the problem extends to other countries, as evidenced by one study in germany that found 23% of adolescents regularly consuming eds at 45 years), sex, race and ethnicity (non - hispanic white versus all others), weight status determined by body mass index (bmi) calculated from self - reported height and weight (normal / underweight bmi 10 years), number of total patients per week (100 versus > 100), and perceived financial status of their patients (very poor to poor, poor to low middle, low middle to middle, middle to upper middle, and upper middle to affluent). the overall prevalence among health care providers giving regular counseling to adolescent patients regarding sds, eds, or both was assessed. chi - square analysis was used to examine differences in the prevalence of regular sed counseling among participants with different personal and medical practice - related characteristics. multivariate logistic regression was conducted to determine characteristics independently associated with the three outcomes of interest : regular sd counseling, regular ed counseling, and regular comprehensive sed counseling. the significance level was p < 0.05 and the selection criterion for bivariate inclusion in the multivariate model was p < 0.20. all analyses were conducted using sas 9.2 statistical software (sas institute inc.). approximately three - fourths of providers were non - hispanic whites, with a relatively even distribution in sex and age (table 1). thirty - seven percent of providers were overweight and 15% were obese, 55% reported high fruit and vegetable intake (at least 5 cups per day, 4 days per week), and 29% engaged in 30 minutes or more of physical activity 5 days per week. nearly 60% had been practicing for greater than ten years and approximately half (46%) had teaching privileges. the majority (66%) of the participants worked in a group practice, 57% had five or less physicians in their practice, and 58% saw 100 patients per week. overall, 41% of participants provided regular (always or often) sd counseling compared to 55% for ed counseling (table 1). female providers exhibited a higher prevalence of regular counseling, at 47% and 61% for sds and eds, respectively, compared to 35% and 49% by male providers. pediatricians had the highest prevalence of regular sd counseling at 55% compared to internists who had the lowest prevalence at 30%. for regular ed counseling, nurse practitioners had the highest prevalence at 65% compared to internists at 43%. multivariate modeling found that regular sd counseling was independently associated with being female (adjusted odds ratio (aor) : 1.41 [95% confidence interval (95% ci) : 1.071.88 ]), high fruit and vegetable intake (aor : 1.63 [95% ci : 1.242.13 ]), type of provider, and group practices compared to individual practices (aor : 0.66 [95% ci : 0.470.93 ]) (table 2). in the model, compared to pediatricians as the reference group, fgps (aor : 0.46 [95% ci : 0.330.64 ]), internists (aor : 0.33 [95% ci : 0.180.59 ]), and nurse practitioners (aor : 0.55 [95% ci : 0.360.83 ]) all had significantly lower odds of providing regular sd counseling. similar to sd counseling, multivariate modeling found that regular ed counseling was independently associated with being female (aor : 1.40 [95% ci : 1.061.84 ]), high fruit and vegetable intake (aor : 1.68 [95% ci : 1.302.16 ]), internists as compared to pediatricians (aor : 0.56 [95% ci : 0.330.96 ]), and group practices compared to individual practices (aor : 0.68 [95% ci : 0.480.95 ]) (table 2). other provider types did not significantly differ from pediatricians in the adjusted model. with regard to either regular sd counseling or ed counseling, no statistically significant differences were found with regard to race / ethnicity, bmi, years of practice, teaching privileges, number of physicians per practice, number of patients seen per week, or the provider 's perception of their patient population 's socioeconomic level (table 2). the third outcome of this study is regular comprehensive sed counseling, which had an overall prevalence of 34% (table 1). the prevalence of counseling was higher in female providers at 41% compared to male providers at 28%. among the types of health care providers, pediatricians and nurse practitioners had the highest prevalence of comprehensive sed counseling at 43% and 42%, respectively, compared to fgps at 29% and internists at 23%. multivariate modeling found regular comprehensive sed counseling to be independently associated with being female (aor : 1.44 [95% ci : 1.071.93 ]), high fruit and vegetable intake (aor : 2.05 [95% ci : 1.542.73 ]), fgps (aor : 0.58 [95% ci : 0.410.82 ]) and internists (aor : 0.37 [95% ci : 0.200.70 ]) compared to pediatricians, and group practices (aor : 0.59 [95% ci : 0.420.84 ]) compared to individuals practices (table 2). we found that one - third of health care providers in this study reported comprehensive sed counseling, indicating that the majority of health care providers who see adolescent patients were not providing both sd and ed counseling regularly. stratification by provider type found that pediatricians had the highest prevalence of regular comprehensive sed counseling (43%). this is lower than the ssb counseling rate of 65% reported by pediatricians in the 2006 aap periodic survey of fellows. however, differences may be attributed to the topic counseled upon (sed versus ssb) and how frequent counseling in addition to pediatricians, nurse practitioners also exhibited high counseling rates ; these two provider types comprised the two highest prevalences of sd only, ed only, and comprehensive sed counseling. the finding that pediatricians typically exhibited the highest rates of sed counseling is congruent with previous studies, which have also have found that pediatricians more frequently assessed weight status and provided behavioral counseling than family / general practitioners [19, 20 ]. nurse practitioners were more likely to provide sed counseling than fgps and internists in this study. one possible reason for this difference may be that, in this study, fgps and internists tend to see fewer pediatric patients per week (fgps / internists : 23.3 pediatric patient versus nurse practitioners : 29.4 versus pediatricians : 105.0) and may have less experience counseling adolescents. another reason, though not investigated in this study, is the amount of time nurse practitioners have to spend with each patient and the complexity of cases they manage. regardless of the cause of the disparities, fgps and internists likely play significant roles in the health maintenance of adolescent patients. gaps in counseling practices can be addressed through changes in the training and continuing education of providers. currently, the differences in provider access and familiarity of aap counseling recommendations are unknown. this study also found that the prevalence of regular sd counseling and of regular ed counseling significantly differed overall, at 41% and 55%, respectively. specifically, this difference varied by provider type, with internists exhibiting the greatest discrepancy (21% higher for ed counseling) in comparison to pediatricians (1% higher for ed counseling). these findings suggest that there are provider - specific differences in how the health risks of these two drink types are perceived and managed. we found that being female, having high fruit and vegetable intake, being a pediatrician, and operating in an individual practice compared to a group practice were associated with greater odds of providing regular comprehensive sed counseling. some variables identified in previous studies [21, 22 ] to be related to physician counseling behaviors such as race / ethnicity and bmi were not found to be associated in this study. compared to bleich., this study contained a similar proportion of overweight / obese providers at 52%, with the difference being a focus on adolescent patients with whom providers may be more motivated to counsel on obesity - related topics. given the relatively low rates of regular sed counseling, increasing provider awareness of the health effects of sed is the first step in addressing this issue. secondly, studies have found that various factors affect how inclined and confident a clinician is in terms of providing health behavior counseling [23, 24 ]. one study found that physicians with normal bmis were more likely to discuss weight loss and were more confident in offering diet and exercise suggestions. another study found that clinicians who personally struggle with making healthy choices, such as avoiding calorically dense foods and beverages, may find it more difficult to counsel patients on that topic. our data supports that hypothesis, with providers of high fruit and vegetable intake having higher prevalence of sed counseling. encouraging health care providers to lead healthy lives may contribute to higher prevalences of sed counseling. ideally, providers can be role models for healthy behavior and create a supportive environment in their clinic or hospital setting and in the community - at - large for patients and families. other studies have found that another barrier to offering effective health counseling is a lack of training or confidence in behavioral counseling. techniques such as motivational counseling [26, 27 ], the 5 a 's heuristic, and multistage models provide frameworks from which effective changes can be encouraged. investigators have developed and examined curricula for medical students and residents [3032 ], comprised of lectures and opportunities to practice counseling techniques that resulted in subjective increases in confidence in providing health behavior counseling. lastly, physicians can also learn from effective community - based interventions and adapt them for their patients. for example, physicians can consider explaining caloric information to adolescent patients in terms of physical activity equivalents, a promising intervention used for youth in baltimore. first, the sample was drawn from an opt - in database, which is subject to selection bias and may not be representative of health care providers nationally. second, the responses regarding counseling behaviors are subjective and susceptible to recall bias ; thus, reported prevalence may not reflect actual practices. third, there is limited evidence for or against the assignment of the sometimes response to the not regular counseling group ; however, the data was also analyzed with sometimes assigned to the regular counseling group and the results were not significantly different. finally, the survey did not assess barriers that prevented sd and/or ed counseling from being offered. with the prevalence of regular comprehensive sed counseling at 34%, increased efforts must be applied to educate adolescent patients and not overlook the associated health risks of high consumption of these beverages. additional research is required to assess physician opinions and barriers that stand in the way of providing regular counseling. research and training can help teach health care providers of various disciplines pertinent information and effective counseling techniques for patients and parents. | objective. to examine the proportion of health care providers who counsel adolescent patients on sports and energy drink (sed) consumption and the association with provider characteristics. methods. this is a cross - sectional analysis of a survey of providers who see patients 17 years old. the proportion providing regular counseling on sports drinks (sds), energy drinks (eds), or both was assessed. chi - square analyses examined differences in counseling based on provider characteristics. multivariate logistic regression calculated adjusted odds ratios (aor) for characteristics independently associated with sed counseling. results. overall, 34% of health care providers regularly counseled on both seds, with 41% regularly counseling on sds and 55% regularly counseling on eds. on adjusted modeling regular sed counseling was associated with the female sex (aor : 1.44 [95% ci : 1.071.93 ]), high fruit / vegetable intake (aor : 2.05 [95% ci : 1.542.73 ]), family / general practitioners (aor : 0.58 [95% ci : 0.410.82 ]) and internists (aor : 0.37 [95% ci : 0.200.70 ]) versus pediatricians, and group versus individual practices (aor : 0.59 [95% ci : 0.420.84 ]). modeling for sd- and ed - specific counseling found similar associations with provider characteristics. conclusion. the prevalence of regular sed counseling is low overall and varies. provider education on the significance of sed counseling and consumption is important. |
we examined electronic patient records from the health improvement network (thin) database, a source of detailed clinical information about patient primary care consultations (3). in the united kingdom, 98% of the population is registered with a general practitioner (a primary care physician who provides advice, treatment, and prescriptions and acts as a gatekeeper to specialist services) (4). participating practices enter demographic and clinical data into the practice database by using vision software (www.inps4.co.uk) every time a consultation takes place, generating a longitudinal medical record. since 1990, symptoms, diagnoses, treatments, and referrals have been recorded by use of a hierarchical system of > 103,000 read codes (5). prescriptions are recorded by using multilex (www.fdbhealth.co.uk/solutions/multilex) drug codes, which link each drug formulation to the british national formulary, a compendium of drugs arranged by system into 15 chapters. thin contains the medical records of > 3.7 million current patients (3) and is broadly representative of the uk population (6). prescription and consultation rates in the dataset are comparable to those recorded by national statistics and external data sources (7,8). adequacy of death data recording is assessed by identifying the date at which mortality rates recorded by the practice correspond to the national age and sex standardized mortality rate, defined as the acceptable mortality recording date. we included data from patients from practices that met acceptable mortality recording criteria and were fully computerized (9). persons were eligible for study inclusion if they were registered with a participating practice from january 1, 1995, through december 31, 2010. read code lists were developed to identify patients seeking care for impetigo. a drug code list was developed to identify patients for whom topical fusidic acid (with or without a topical steroid) was prescribed, based on the relevant chapter of the british national formulary. to assess time trends, we included the first consultation for impetigo for patients 014 years of age or the first prescription of fusidic acid ; that is, patients were counted once per practice. patients left the study on the earliest of the following dates : date of consultation or prescription, date of leaving the practice, date of death, or date the study ended (december 31, 2010). we used poisson regression to calculate the incidence of first consultations or prescriptions per year. the denominator was the total number of person - years contributed by patients in the sample population for each corresponding calendar year. the thin program of anonymized data provision for researchers was approved by the national health service south east multi - centre research ethics committee in 2002. this study was approved by the thin scientific review committee, reference 11504. during 19952010, a total of 130,095 children 014 years of age were seen by a general practitioner for impetigo. the annual incidence of infection increased from 1,646 (95% ci 1,5611,733) consultations per 100,000 person - years in 1995 to 3,106 (95% ci 3,0483,165) per 100,000 person - years in 2001 (figure 1) and declined thereafter to 1,447 (95% ci 1,4131,481) per 100,000 person - years in 2010. the incidence of fusidic acid prescription for the total population increased from 1,287 (95% ci 1,2561,318) prescriptions per 100,000 person - years in 1995 to 2,308 (95% ci 2,2892,326) per 100,000 person - years in 2003 and stabilized thereafter (figure 2). prescriptions were most frequently issued for children 014 years of age ; incidence peaked in 2003 at 4,911 (95% ci 4,8434,980) prescriptions per 100,000 person - years. at the peak of the epidemic, there were 130,000 more general practitioner consultations for impetigo ; this estimate is based on an estimated population of 9,375,100 children < 15 years of age in england in 1995 and 9,282,700 in 2001 (10). rates of general practitioner consultation for impetigo among children 014 years of age, united kingdom, 19952010. a major increase in impetigo in children in the united kingdom was not detected by routine surveillance data. the increased number of infections placed a substantial burden on primary care, adding 130,000 consultations in england at the peak of transmission. in england and wales, the royal college of general practitioners research and surveillance centre network of 100 sentinel practices is responsible for alerting general practitioners to major trends in the incidence of common conditions, such as influenza and impetigo, by providing weekly reports of disease incidence (11). although this system detected a comparable increase in the rate of consultations for impetigo, the data were not used to alert general practitioners about a major increase in impetigo in the community because the system is designed to detect rapid changes in incidence, such as occur during an influenza epidemic. over the past decade, several european countries have reported a rise in general practice consultations for impetigo (12,13). many european countries have established primary care databases, such as the information system for the development of research in primary care in catalonia (14) or the health search database in italy (15). although these databases are smaller than the thin database (3), they are sufficiently large and well - established to be used to examine national consultation and prescription trends in infectious diseases, offering the potential for a powerful international infectious disease surveillance network. this study s strengths lie in its scale and the fact that the database is nationally representative, containing the medical records of 6% of the uk population (4). the study s limitations lie in the fact that the database was designed for patient management, not research. we acknowledge that comparing population rates for consultation and prescriptions might not accurately represent the association between prescriptions and disease at an individual level. patients seeking consultation for impetigo were identified by diagnostic read codes, and some general practitioners might prescribe fusidic acid without recording a diagnostic code. identifying patients by read code alone might underestimate incidence, whereas identifying patients by prescription data might lead to an overestimation because drugs are not always prescribed for a single condition. the actual incidence of impetigo in the community might have been higher because not all persons with impetigo would have consulted a general practitioner. although changes in health - seeking behavior or data recording could underlie the increased consultation rate, the fact that similar increases were reported from other primary and secondary care datasets suggests that our findings are not artifacts. the read codes used to identify patients were unchanged throughout the study period, and we are unaware of any changes in clinical practice that would lead to an increased tendency to diagnose impetigo. impetigo is frequently dismissed as a mild infection that spontaneously resolves with a good outcome (1). by contrast, this study suggests that an undetected increase in impetigo in the community drove a major increase in hospital admissions of children in england from 19891990 through 20032004. awareness of this epidemic by general practitioners could have triggered development of specific guidelines on the management of this condition, potentially improving treatment outcomes and reducing hospital admissions. routinely collected primary care data are an underused and potentially rich source of information about infectious diseases in the community. we should do more to find novel ways of incorporating this information into international surveillance networks and using it to guide evidence - based treatment and prescribing decisions in primary care. | using a primary care database, we identified a major increase in impetigo in the united kingdom during 19952010. despite a doubled rate of primary care consultations, this increase was not identified by routine surveillance. primary care databases are a valuable and underused source of surveillance data on infectious diseases. |
vein of galen aneurismal malformation (vogam) is a rare congenital anomaly with a reported incidence of 30% of pediatric malformations. jaeger., first published a reported case of vogam in literature. in the choroidal stage of development of the cerebral vascular system, the venous drainage is mainly by the median venous structure, the mprosv of markowski. vein of galen aneurismal malformation is a cluster of arteriovenous fistulas (avfs) draining into the dilated median vein of the prosencephalon. the arterial feeders are usually from the anterior and posterior choroidal arteries, the pericallosal artery, and the transmesencephalic branches of the basilar tip. the vogam is mainly divided into mural and choroidal types. in the mural type (simple type), there is a direct high flow shunt located within the wall. in the choroidal type (complex type) clinically, the mural types of vogam present later in infants with macrocephaly or failure to thrive and may be associated with mild cardiac failure or asymptomatic cardiomegaly. the choroidal types of vogam usually cause heart failure in newborns. on account of a sustained increased flow, enlarged arteries can be associated with aneurysms or occlusions. on the venous side, impairment of the outflow causes increased venous hypertension and increases the risk of intracranial hemorrhage. communicating hydrocephalus occurs due to impaired absorption of the cerebrospinal fluid by the arachnoid villi. on account of a sustained increased flow, enlarged arteries can be associated with aneurysms or occlusions. on the venous side, impairment of the outflow causes increased venous hypertension and increases the risk of intracranial hemorrhage. communicating hydrocephalus occurs due to impaired absorption of the cerebrospinal fluid by the arachnoid villi. eventually the child was followed up with serial imaging. computed tomography (ct) and magnetic resonance (mr) imaging [figure 1 ] done at the time of presentation showed the vogam in the posterior interhemispheric fissure, causing a compression of the third ventricle and the cerebral aqueduct, leading to hydrocephalus. subsequently, an angiogram was done, which revealed a mural type of vogam [figure 2a, b ]. contrast - enhanced mr images showing the vein of galen aneurysm in the midline causing compression of the third ventricle, leading to hydrocephalus pre - embolization vertebral artery angiogram of the anteroposterior and lateral views (a, b), demonstrating the mural type of fistula (arrow), post embolization vertebral artery angiogram (c, d) shows complete obliteration of the fistula with good visualization of the posterior cerebral artery (arrow) procedure : under general anesthesia, using the retrograde seldinger technique, right femoral access was taken. using a 5f envoy guiding catheter, the right posterior cerebral artery (pca) was selectively catheterized with a micro catheter and micro wire combination (excel14 / transcend wire) and the fistula was occluded, with 80% n - butyl cyanoacrylate (nbca). a post - procedure angiogram revealed complete occlusion of the fistula and better visualization of the pca branches [figure 2c, d ]. the child had an uneventful course in the ward. on the follow - up of three months, six months, and on the two - year follow - up there was significant decrease in the macrocrania, with normal developmental milestones for age. a 15-month - old female child presented with abnormally dilated neck veins and delayed milestones. numerous arterial feeders were seen from the bilateral posterior choroidal arteries [figure 3a, b, c ]. in order to reduce the risk of contrast and fluid overload, and to prevent the occurrence of hyperperfusion breakthrough, endovascular treatment in multiple sessions was planned. in the first session, the right posterior choroidal feeders were embolized with 33 and 24% nbca causing 25% flow reduction in the fistulous connection. the child was asked to come for follow - up after three months. at the age of 18 months, a second session of embolization of the left posterior choroidal feeders was done using 33 and 25% nbca resulting in 50% reduction in the flow [figure 4a, b ]. the child tolerated the procedure well and was followed up after a three - month interval. digital substraction angiogram showing multiple feeders from the posterior choroidal arteries (arrows) in the choroidal type of vogam (a, b), with venous drainage into a large ectatic vein (c) vertebral angiogram demonstrating reduction in the flow to the malformation after the third session of embolization by nbca (arrow) (a, b). further reduction in the flow following the fifth session of embolization (c, d) at the age of 21 months a third session of embolization was done. the right posterior choroidal feeder was accessed and embolized with 25% nbca. on a post - procedure angiogram there was 60% reduction in the abnormal flow through the vogam. on account of the contrast dose limitation, the child was taken up for the fourth session after an 11-day interval and embolization of the residual thalamoperforators and posterior choroidal feeders was done with 25% nbca with 70% decrease in the flow to the vogam. the child tolerated the session well and was followed up after a two - month interval. the child was taken up for the fifth session, and embolization of the residual feeders was done [figure 4c, d ]. the child was doing well, with clinical improvement in the symptoms and normal developmental milestones for the age, on a follow - up of two years. eventually the child was followed up with serial imaging. computed tomography (ct) and magnetic resonance (mr) imaging [figure 1 ] done at the time of presentation showed the vogam in the posterior interhemispheric fissure, causing a compression of the third ventricle and the cerebral aqueduct, leading to hydrocephalus. subsequently, an angiogram was done, which revealed a mural type of vogam [figure 2a, b ]. contrast - enhanced mr images showing the vein of galen aneurysm in the midline causing compression of the third ventricle, leading to hydrocephalus pre - embolization vertebral artery angiogram of the anteroposterior and lateral views (a, b), demonstrating the mural type of fistula (arrow), post embolization vertebral artery angiogram (c, d) shows complete obliteration of the fistula with good visualization of the posterior cerebral artery (arrow) procedure : under general anesthesia, using the retrograde seldinger technique, right femoral access was taken. using a 5f envoy guiding catheter, the right posterior cerebral artery (pca) was selectively catheterized with a micro catheter and micro wire combination (excel14 / transcend wire) and the fistula was occluded, with 80% n - butyl cyanoacrylate (nbca). a post - procedure angiogram revealed complete occlusion of the fistula and better visualization of the pca branches [figure 2c, d ]. the child had an uneventful course in the ward. on the follow - up of three months, six months, and on the two - year follow - up there was significant decrease in the macrocrania, with normal developmental milestones for age. a 15-month - old female child presented with abnormally dilated neck veins and delayed milestones. numerous arterial feeders were seen from the bilateral posterior choroidal arteries [figure 3a, b, c ]. in order to reduce the risk of contrast and fluid overload, and to prevent the occurrence of hyperperfusion breakthrough, endovascular treatment in multiple sessions was planned. in the first session, the right posterior choroidal feeders were embolized with 33 and 24% nbca causing 25% flow reduction in the fistulous connection. the child was asked to come for follow - up after three months. at the age of 18 months, a second session of embolization of the left posterior choroidal feeders was done using 33 and 25% nbca resulting in 50% reduction in the flow [figure 4a, b ]. the child tolerated the procedure well and was followed up after a three - month interval. digital substraction angiogram showing multiple feeders from the posterior choroidal arteries (arrows) in the choroidal type of vogam (a, b), with venous drainage into a large ectatic vein (c) vertebral angiogram demonstrating reduction in the flow to the malformation after the third session of embolization by nbca (arrow) (a, b). further reduction in the flow following the fifth session of embolization (c, d) at the age of 21 months a third session of embolization was done. the right posterior choroidal feeder was accessed and embolized with 25% nbca. on a post - procedure angiogram there was 60% reduction in the abnormal flow through the vogam. on account of the contrast dose limitation the child was taken up for the fourth session after an 11-day interval and embolization of the residual thalamoperforators and posterior choroidal feeders was done with 25% nbca with 70% decrease in the flow to the vogam. the child tolerated the session well and was followed up after a two - month interval. the child was taken up for the fifth session, and embolization of the residual feeders was done [figure 4c, d ]. the child was doing well, with clinical improvement in the symptoms and normal developmental milestones for the age, on a follow - up of two years. recent advances in the field of intervention neuroradiology has changed the treatment and prognosis of children with vogam.[58 ] therapeutic options available for a vogam include no treatment, open surgery, endovascular treatment, and stereotactic radiosurgery. the therapeutic options should be individualized with consideration of age, clinical manifestation of the lesion and the angioarchitecture. in the past nbca is the embolic agent used which is premixed with lipiodol (to increase the radio opacity) in our first case, which was a mural type of fistula, we used high concentrated glue, which was fast setting and highly effective in the high flow variety. in our second case, staged embolization is done, to reduce the risk of fluid overload and prevent the perfusion breakthrough. in this child 33 and 25% nbca allowed precise targeting of the av shunt and was preferred over other embolic agents like microcoils. the presence of pre - existing brain damage characterized by severe atrophy and parenchymal calcifications indicated a poor prognosis, with bad outcomes the transvenous approach was described in literature, but it was associated with increased risks of bleeding complications., described the largest cohort study of these lesions treated using the endovascular method, at their center, in a period of 11 years, with variable outcomes. introduction of endovascular techniques modified the prognosis in patients with vogam. with the recent advances of technology of endovascular management, these children can be well - managed with a fairly good outcome. the treatment plan was individualized with consideration to the clinical manifestations and angioarchitecture of the lesion. the goal of the treatment was clinical improvement of the patients presenting with the symptoms and not an angiographic cure. | vein of galen aneurysm malformation is one of the most difficult intracranial vascular lesions to manage. the difficulty in management of its shunts is aggravated by the hemodynamic effect and shunting reflected in the growing brain of infants and children. at present, the therapeutic options are widened by the opening of a new horizon in intervention neuroradiology. we present a series of two case reports of these malformations treated using the endovascular method, at our institution. |
frequent exacerbations of chronic obstructive pulmonary disease (copd) can lead to considerable declines in functioning and increased health care costs (nih 2003). there is no generally accepted definition for acute exacerbations of copd (aecopd) though most studies use the criteria of worsening symptoms accompanied by a change in the medical regimen (pauwels 2004). while the follow - up of patients after aecopd has primarily focused on the resolution of symptoms, improvement in lung function and gas exchange, and changes in inflammatory biomarkers (seemungal 2001 ; aaron 2002), investigations into the precise patterns of physical activity recovery following aecopd have been minimal or notably absent. findings from studies that rely on caregiver report or retrospective recall suggest that patients have considerable functional impairment after aecopd, especially those episodes that require hospitalizations (connors 1996 ; garcia - aymerich 2003). connors and colleagues (1996) found that more than 50% of patients hospitalized for an aecopd continued to require assistance with their activities of daily living (adl) six months after discharge. a lower level of self - reported physical activity was strongly associated with increased risk of hospital readmissions for an aecopd in one recent prospective study (garcia - aymerich 2003). qualitative interviews in another study showed that aecopd have a negative impact on adl in 91% of the sample with more than half of these patients stopping activities altogether during their aecopd (vogelmeier 2004). electronic devices such as accelerometers are suitable for long term monitoring and are capable of detecting and recording daily activities that are likely difficult for patients to recall and quantify with typical self - reports (matthews 2002). accelerometers have been used on a short - term basis, anywhere from one (coronado 2003 ; behnke 2005) to five days (steele 2003 ; pitta 2005), often to validate self - reports of physical activity or prescribed exercise. it is important to note that patients who experienced aecopd have typically been excluded from physical activity monitoring studies. therefore, the purpose of this exploratory study was to determine the feasibility and acceptability of using an accelerometer device to characterize physical activity patterns surrounding outpatient - treated acute exacerbations of copd (aecopd) over a 4-month period. patients who met the following criteria were enrolled : 1) moderate to severe stable copd (forced expiratory volume in one second [fev1]%<50% and fev1/forced vital capacity [fvc]<70%) confirmed by spirometry ; 2) experienced at least two aecopd that were treated on an outpatient basis in the previous 12 months ; 3) absence of illnesses such as bronchiectasis, active malignancies or other end stage diseases ; 4) ability to speak, read and write english ; and 5) no plans to engage in any new physical activities over the next 4 months. subjects were recruited directly from an academic medical center and veteran s administration chest clinics and referrals from a copd support group. we anticipated that if moderate to severely ill patients with copd who had at least 2 outpatient - treated aecopd in the previous 12 months were recruited, they would potentially experience at least one aecopd within the 4-month monitoring time frame (seemungal 2000 ; garcia - aymerich 2003). the study protocol was approved by the institutional review board and each subject gave informed written consent. the first author made visits to subjects homes to obtain written consent, perform spirometry, administer baseline paper - pencil questionnaires, demonstrate appropriate use of the activity monitor, and instruct on completion of a daily symptom diary. subjects wore the rt3 tri - axial accelerometer (stayhealthy, monrovia, ca, usa) during waking hours by clipping the device to the waist area, on the nondominant side, upon awakening and removing it when they retired to bed for approximately 112 consecutive days. two telephone calls were made within the first week to ensure that subjects did not have any difficulty wearing the device. biweekly visits to subjects homes or to other mutually agreed places were made to obtain symptom diaries, download the physical activity data to a laptop computer, record health resource use, install fresh batteries, and provide a small honorarium. the pager - size rt3 measured physical activity in three dimensions : anteroposterior, vertical, and mediolateral. the device sampled movements at a rate of 10hz and analog - to - digital converted data were recorded every second and then summed to produce 1-minute movement epochs of vector magnitude units (vmu). an earlier model of this device (tritrac, stayhealthy, monrovia, ca, usa) was tested in patients with copd who participated in pulmonary rehabilitation and concurrent validity was established (steele 2003). we operationally defined copd exacerbations as a sustained worsening of at least two major symptoms (dyspnea, sputum volume, and sputum purulence) or increase in one major and one minor symptom (wheezing, cough, sore throat, and nasal discharge / congestion) for at least two consecutive days and accompanied by changes in the medical regimen (seemungal 2000 ; pauwels 2004). a prescription for or self - directed treatment with a course of oral prednisone or antibiotics were considered a change in the usual medical regimen. improvements in symptoms for at least 3 consecutive days (better rating on the symptom diary) indicated the start of recovery. the medical research council (mrc) dyspnea scale (fletcher 1959) and the seattle obstructive lung disease questionnaire (soldq) were administered at baseline (tu 1997). exit interviews were conducted during the final home visit to assess acceptability of the protocol and other difficulties not previously identified. physical activity data were converted into a microsoft excel (microsoft, redmond, wa, usa) file for data reduction and analysis. the mean vmu per minute was calculated by dividing the total vmu for the day by the number of minutes the device was worn. we did not remove time spent riding in a motor vehicle since unpublished data showed inconsequential differences in mean vmu calculations (steele pers comm). adherence data were calculated as a percentage of the number of days the device was worn out of 112 days. plots were made of the physical activity data for each subject with aecopd noted on the plots based on the symptom diaries and medications used. nine subjects were screened with eight (5 women and 3 men) who enrolled and completed the study. subjects had a mean age of 71 4, fev1% predicted of 40% 16%, fev1/fvc ratio of 45 7, and mrc dyspnea score of 2.3 0.9. a majority of the subjects were caucasian (88%), attended at least some college (63%), and were retired (88%). baseline soldq scores were 38.8 14.9, 69.6 20.3, 75.0 21.7, and 64.1 10.4 for the physical, emotional functioning, coping skills, and treatment satisfaction scales, respectively. overall, mean adherence to the 112-day monitoring protocol was 97.6% (range 92%100%) while adherence to wearing the device for at least 10 hours per day was slightly less at 91.5% (range 75%99%). there were two incidents of battery failure with two different devices ; a total of 23 days of data were lost. the adherence rate was impressive especially in light of the consistent report by subjects that remembering to wear the device was most challenging. i would see the pager on my kitchen table and would pass by it repeatedly during the day but somehow it does nt cross my mind to clip it on until later when i all of sudden realize that i should be doing that. the daily average vmu per minute for the sample was 117.8 47 (range 61.4184.1). we primarily relied on visual analyses of graphical plots since no meaningful statistical analyses could reliably be performed on the physical activity data from a relatively few number of aecopd events and with the degree of intra- and inter - individual, day - to - day variability that we observed. data for one relatively less disabled 72-year old female subject who did not have any aecopd during the 4 month is shown in figure 1. figure 2 shows data for a 75-year old male subject who had three episodes of greater symptoms than usual and one meeting our aecopd criteria. similar to the other four subjects who experienced at least one aecopd, there was a tendency for decreased activity when increased symptoms were logged. however, there were no consistent downward trends in activity before, during, or after aecopd that were remarkably different from the day - to - day variability. this exploratory study showed that patients with copd were willing and able to undergo 4 months of continuous physical activity monitoring, the longest recording duration that we are aware of in the copd literature. we found that the rt3 device captured substantial intra - individual fluctuations in daily physical activity. finally, the preliminary data suggest that patients who experienced aecopd and were treated at home did not have noticeable changes in their physical activity patterns before, during, or after the aecopd episode that was distinguishable from their usual day - to - day variability. our main goal was to assess if patients with moderate to severe copd would wear an unobtrusive monitoring device that could potentially yield useful information about their health over an extended period. the overall adherence rate was quite remarkable considering the challenges that other investigators typically face when accelerometer devices are used for brief periods to ascertain exercise participation (dubbert 2002). the high adherence rate could be attributed to our weekly contact, but also that a majority of the subjects were retired and very conscientious in implementing routines to help them remember to wear the device. this finding should provide some measure of support to researchers to further explore the possible use of like devices to passively monitor and detect dynamic changes in patients physical functioning over time. aside from the extraneous artifact that typically accompany accelerometry recordings, the variability in day - to - day physical activity was, to some degree expected, since previous reports described patients experiences of good and bad days. the findings from two studies that used an accelerometer (pitta 2005) and pedometer (schonhofer 1997) for shorter monitoring periods confirm our observations. the mean coefficient of variation for walking time in 18 patients with copd over five days was 28% 14% and was as high as 89% 35% with lying time. the degree of variability was not associated with disease severity or age (pitta 2005). the report by sandland and colleagues (2005) of no significant differences in mean activity counts within subjects over a seven day window were probably reflective of intra - individual variations. one group of investigators actually reported strikingly similar patterns of variability in daily self - reported health status over the course of a year for patients who were discharged from a hospital for aecopd (verbrugge 1989). the tremendous within - subjects variation in accelerometry - derived activity data may perhaps have significant drawbacks for use, both as a reliable summary outcome measure from therapies and as a potential though very preliminary, our findings suggest that patients whose aecopd are milder and are managed outside of a hospital did not have visible changes in their physical activity pattern before, during, or after their aecopd episodes. only one study presented in the form of an abstract actually measured physical activity in patients with copd during aecopd but regrettably, those aecopd episodes, presumably more severe, were treated in a hospital (pitta 2004). patients were monitored on days 2 and 7 of their hospitalization and 1 month after discharge for 12 hours using a tri - axial activity monitor worn on a belt with a second sensor attached to the leg. unlike the rt3, the leg sensor provided a second positioning coordinate, allowing the device to differentiate between different positions. these patients walked significantly less and spent more time in the lying position one month after discharge compared with stable, but unmatched copd patients. although matching can account for a limited number of factors that influence physical activity, comparisons of activity patterns should ideally be made using patients previous stable states which we have attempted to do. they found that in a cohort of patients with copd who were followed over an 8-year period had rapid recoveries from outpatient - treated aecopd as measured by self - reports of time spent outdoors. it is important to note that our sample may not be characteristic of the general copd population since three of the four unique aecopd occurred in three subjects who were on a stable self - directed exercise regimen for more than 6 months prior to enrolling in the study. also, the rt3, designed to be worn at the waist, does not have adequate specificity in capturing decrements in certain other potentially important adl that may be negatively impacted by aecopd. our findings suggest that patients with copd will wear a pager - sized accelerometer that continuously tracks their physical activity over time, both during the stable state and with aecopd episodes. the results of this study extend current knowledge regarding the comparable within - subjects variability in physical activity associated with outpatient - treated aecopd as in the stable state. a major caveat to our observations is that the sample was rather small with few events and we were not able to subject the data to rigorous statistical tests. however, we recommend additional work in this area, first, identifying a simple device, unhampered by sophisticated data reduction and analysis algorithms that yield reliable ambulatory physical activity data and secondly, conduct of a larger study to document the natural pattern of physical activity across the illness continuum including periods of stable illness, during aecopd, and recovery from these acute events. an understanding of these specific activity patterns might aid in the development of more timely and responsive interventions aimed at minimizing functional declines in this patient population. | purposeto determine the feasibility of using an accelerometer to characterize physical activity patterns (pa) surrounding chronic obstructive pulmonary disease (copd) exacerbations (aecopd) in patients with copd for 16 weeks.methodspatients with copd (n = 8) wore the rt3, a triaxial accelerometer (stayhealthy, monrovia, ca) during waking hours and kept daily symptom diaries. the mean vector magnitude unit (vmu) per minute was calculated by dividing the total vmu for the day by the number of minutes the device was worn. descriptive statistics were used and plots were made showing pa for each subject with aecopd markers based on symptom diaries and health resource utilization.resultssample characteristics were : age 71 4 ; 5 females ; forced expiratory volume in one second (fev1)% predicted : 40% 16% ; fev1/forced vital capacity : 45 7 ; and medical research council dyspnea scale : 2.3 0.9. overall adherence to the monitoring protocol was 97.6% (range 92%100%) while adherence to wearing the device for at least 10 hours per day was 91.5% (range 75%99%). mean vector magnitude units per minute was 117.8 47 (range 61.4184.1). seven exacerbations were captured over a total of 896 person - days of monitoring. there were substantial intra - individual fluctuations in daily pa during both the stable state and with outpatient treated exacerbations.conclusionspatients with copd were able to adhere to a 16-week activity monitoring protocol and reported a willingness to wear such a device for an extended period of time if the data yield important and useful information for themselves and their health provider. future work will need to focus first, on validating other promising devices that produce higher quality pa data and second, replicate this monitoring protocol with a larger sample of copd patients over a longer period. |
logistic regression has been widely used by caribbean and/or latin american scholars to examine parameters and weights of determinants of self - reported health status[17 ] or life satisfaction. embedded in the use of logistic regression in the study of self - reported (rated) health is the dichotomisation of health status. self - rated health status is a likert scale variable ranging from very poor to very good health status. this denotes that the dichotomisation of self - reported health must address where moderate health status should be placed. the dichotomisation of self - reported health status brings into focus the issue of a cut - off and the validity of one 's choice. by categorising an ordinal measure (i.e., self - reported health) into a dichotomous one, another important issue which is unresolved in the choice of a cut - off is the subjective with which caribbean scholars have continued to make their decision. their decision as to what constitutes bad or good (including excellent) health is not purely subjective, as this practice is global one. the decision of a cut - off can not be subject to international norm if there is no rationale for this approach. caribbean scholars can not merely follow tradition in their choice of conceptualisation and operationalisation of a measure, as this is not a scientific enough rationale for the use of a particular measure. some scholars have opined that self - reported health status should remain a likert scale measure or in its continuous form as against the dichotomisation of the measure[1517 ]. the work of finnas. showed that the five - point likert scale variable of self - reported health status can be dichotomised. however, there are some methodological issues that must be considered. finnas and colleagues study revealed that the cut - off point of bad versus good self - reported health and the decision as to where moderate self - reported health status be placed does not depend on age. however, when the categorisation of poor self - reported health excludes moderate self - reported health, the covariate of marital status and educational level were found to be highly age - dependent. within the context of the aforementioned findings, caribbean scholars need to examine these issues within the available health data in order to be able to empirically make a choice of 1) dichotomisation or 2) non - dichotomisation of self - reported health status. the discourse on whether or not to dichotomise self - reported health status is unresolved., therefore, dichotomising the measure simply because it has been done so by non - caribbean scholars in developed nations is not a sufficient rationale for following suit in latin america and the caribbean. latin america and the caribbean are developing nations whose socio - economic situations are different from those in first world countries, emphasising the justification of why latin america and caribbean scholars should examine self - reported health data in order to concretise their choice of dichotomisation or not. jamaica, which is a part of latin america and the caribbean, has been collecting self - reported health data since 1988, and these data have been used repeatedly by scholars to aid public health programmes. an extensive review of the literature did not find a single study that has examined the validity of dichotomisation of self - reported health status. the same was also found for the wider caribbean, suggesting that scholars have been keeping with the tradition and the practice of using the scholarly information from the developed nations when it comes to dichotomised self - reported health status. the current study fills this gap in the literature, and will be used to guide public health practitioners and other users of self - reported health data on jamaicans. the objectives of the study are : 1) evaluate which cut - off point should be used for self - reported health status ; 2) assess whether dichotomisation of self - reported data should be practiced ; 3) ascertain any disparity in dichotomisation by some covariates (i.e., marital status, age cohort, social class) ; and 4) examine the odds of reporting poor or moderate - to - very poor self - reported health status if one has an illness. this study used secondary cross - sectional survey data, which was collected between may and august, 2007. the jamaica survey of living conditions (jslc), which is used for this study, is a joint research conducted by the planning institute of jamaica (pioj) and the statistical institute of jamaica (statin). it is a standard exercise ; the jslc 's sample is a proportion of the labour force survey (lfs). in 2007, it was one - third of the lfs. for 2007, the current study extracted 1,583 respondents from the larger sample as the focus was on participants aged 46 + years. this design was a two - stage stratified random sampling design where there was a primary sampling unit (psu) and a selection of dwellings from the primary units. the psu is an enumeration district (ed), which constitutes a minimum of 100 residences in rural areas and 150 in urban areas. this means that the country was grouped into strata of equal size based on dwellings (eds). based on the psus, a listing of all the dwellings was made, and this became the sampling frame from which a master sample of dwellings was compiled, which in turn provided the sampling frame for the labour force. a total of 620 households were interviewed from urban areas, 439 from semi - urban areas and 935 from rural areas, which constituted 6,783 respondents. the jslc is a modification of the world bank 's living standards measurement study household survey. face - to - face interviews over the aforementioned period were used to collect the data. a structured questionnaire was used and already trained interviewers were then trained again specifically for this task. the questions covered demographic characteristics, household consumption, health status, health care - seeking behaviour, illnesses, education, housing, social welfare and related programmes, and inventory of durable goods. data were stored, retrieved and analyzed using spss - pc for windows version 16.0. cross tabulations were done to examine non - metric dependent and independent variables, which provided the percentages. percentages were computed for dichotomous health statuses (i.e., very poor or poor health status, and the other very poor to moderate health status) ; these were employed for calculating the odds ratio in each dichotomisation of self - reported health status. among men aged 46 - 54 years, 37.7% of those who reported an illness rated their health status as very poor or poor, as compared to 7.3% of those who did not indicate an illness. hence, the odds ratio of very poor - to - poor health status was 7.7 [(37.7/62.3)/(7.3/92.7) ] indicating that men who reported an illness also have 8 times as high odds of reporting very poor or poor health status than those who did not report a dysfunction. in age cohort 46 - 54 years, the percentage of men who reported very poor, poor or moderate health status was 81.4% compared to 39.9% of those who did not report an illness. hence, the odds ratio of very poor, poor or moderate health status versus non - very poor to moderate health status was 9.6 [(81.4/18.6)/ (31.2/68.8) ]. the current study expanded on the work of finnas., which examined some of the methodological challenges in self - reported data in finland. 's study in a number of respects, such as : 1) their work used age cohort 35 - 64 years while this study used 45 - 85 + years ; 2) self - reported illness was included among the covariates in the examination of self - reported (rated) health status ; and 3) social class and access (or lack of access) to material resources play a critical role in directly and indirectly influencing health, and so this was added to this paper. although higher education plays a vital role in health status, 2% of the sample had tertiary level education and of this, 0.2% was older than 45 years. most of the sample had at most primary level education (87.3%), which means that the role of tertiary education would contribute marginally to this sample. hence, the researcher excluded it from the covariate analysis of self - reported health status. self - reported illness status is a dummy variable, where 1 = reporting an ailment or dysfunction or illness in the last 4 weeks, which was the survey period, 0 = no self - reported ailments, injuries or illnesses. while self - reported ill - health is not an ideal indicator of actual health conditions, because people may underreport, it is still an accurate approximation of ill - health and mortality. self - reported health status (or health status) was measured by the question : generally, how would you describe your health currently ? the options were : very good, good, moderate (or fair), poor, and very poor. age group was classified as children (aged less than 15 years), youth (aged 15 through 25 years), and other age cohorts ranging in 5 year intervals from 26 - 30 years, et cetera. medical care - seeking behaviour was taken from the question : has a health care practitioner, healer, or pharmacist been visited in the last 4 weeks ? medical care - seeking behaviour, therefore, was coded as a binary measure where 1=yes and 0= otherwise. social class is measured using income quintile where it ranges from poorest 20% to wealthiest 20%. the distribution of the different age cohorts for each sex based on self - reported health status is given in figures 1a and 1b. figures 1a and 1b will be used to argue the case for a cut - off point for the dichotomisation of self - reported health status in jamaica. health status (in %) by age cohort controlled for sex (i.e. male) health status (in %) by age cohort controlled for sex (i.e. female) it is well established in biomedical literature that there is a strong negative correlation between health and age ; the current study using self - reported health status by different age cohort controlled for sexes revealed that good health decreases as the individual ages and that more women beyond 80 years old reported very good health status compared to men in the same age cohorts. health status, therefore, can be simply explained by age cohorts, and the aforementioned findings show that sex must be taken into consideration among the covariates in order to comprehend the effects of particular demographic variables on the statistical interpretations of health data. the other covariates must include education level, marital status, area of residence, and social class. the issue of dichotomising self - reported health status continues to be debated in jamaica as researchers continue to grapple with whether to use very poor - to - poor health status versus moderate - to - very poor health status. the issue of using moderate health in poor or good health status is critical as this will aid researchers in understanding whether there should be a cut - off point and where it should be, as this is the crux of the interpretation of the logistic regression model. based on figure 1, the very poor - to - poor health status is marginal at ages below 46 years, and so for the purpose of dichotomisation, ages 46 years and older will be used. this study used secondary cross - sectional survey data, which was collected between may and august, 2007. the jamaica survey of living conditions (jslc), which is used for this study, is a joint research conducted by the planning institute of jamaica (pioj) and the statistical institute of jamaica (statin). it is a standard exercise ; the jslc 's sample is a proportion of the labour force survey (lfs). in 2007, it was one - third of the lfs. for 2007, the current study extracted 1,583 respondents from the larger sample as the focus was on participants aged 46 + years. this design was a two - stage stratified random sampling design where there was a primary sampling unit (psu) and a selection of dwellings from the primary units. the psu is an enumeration district (ed), which constitutes a minimum of 100 residences in rural areas and 150 in urban areas. this means that the country was grouped into strata of equal size based on dwellings (eds). based on the psus, a listing of all the dwellings was made, and this became the sampling frame from which a master sample of dwellings was compiled, which in turn provided the sampling frame for the labour force. a total of 620 households were interviewed from urban areas, 439 from semi - urban areas and 935 from rural areas, which constituted 6,783 respondents. the jslc is a modification of the world bank 's living standards measurement study household survey. face - to - face interviews over the aforementioned period were used to collect the data. a structured questionnaire was used and already trained interviewers were then trained again specifically for this task. the questions covered demographic characteristics, household consumption, health status, health care - seeking behaviour, illnesses, education, housing, social welfare and related programmes, and inventory of durable goods. data were stored, retrieved and analyzed using spss - pc for windows version 16.0. cross tabulations were done to examine non - metric dependent and independent variables, which provided the percentages. percentages were computed for dichotomous health statuses (i.e., very poor or poor health status, and the other very poor to moderate health status) ; these were employed for calculating the odds ratio in each dichotomisation of self - reported health status. among men aged 46 - 54 years, 37.7% of those who reported an illness rated their health status as very poor or poor, as compared to 7.3% of those who did not indicate an illness. hence, the odds ratio of very poor - to - poor health status was 7.7 [(37.7/62.3)/(7.3/92.7) ] indicating that men who reported an illness also have 8 times as high odds of reporting very poor or poor health status than those who did not report a dysfunction. in age cohort 46 - 54 years, the percentage of men who reported very poor, poor or moderate health status was 81.4% compared to 39.9% of those who did not report an illness. hence, the odds ratio of very poor, poor or moderate health status versus non - very poor to moderate health status was 9.6 [(81.4/18.6)/ (31.2/68.8) ]. the current study expanded on the work of finnas., which examined some of the methodological challenges in self - reported data in finland. 's study in a number of respects, such as : 1) their work used age cohort 35 - 64 years while this study used 45 - 85 + years ; 2) self - reported illness was included among the covariates in the examination of self - reported (rated) health status ; and 3) social class and access (or lack of access) to material resources play a critical role in directly and indirectly influencing health, and so this was added to this paper. although higher education plays a vital role in health status, 2% of the sample had tertiary level education and of this, 0.2% was older than 45 years. most of the sample had at most primary level education (87.3%), which means that the role of tertiary education would contribute marginally to this sample. hence, the researcher excluded it from the covariate analysis of self - reported health status. self - reported illness status is a dummy variable, where 1 = reporting an ailment or dysfunction or illness in the last 4 weeks, which was the survey period, 0 = no self - reported ailments, injuries or illnesses. while self - reported ill - health is not an ideal indicator of actual health conditions, because people may underreport, it is still an accurate approximation of ill - health and mortality. self - reported health status (or health status) was measured by the question : generally, how would you describe your health currently ? the options were : very good, good, moderate (or fair), poor, and very poor. age group was classified as children (aged less than 15 years), youth (aged 15 through 25 years), and other age cohorts ranging in 5 year intervals from 26 - 30 years, et cetera. medical care - seeking behaviour was taken from the question : has a health care practitioner, healer, or pharmacist been visited in the last 4 weeks ? medical care - seeking behaviour, therefore, was coded as a binary measure where 1=yes and 0= otherwise. social class is measured using income quintile where it ranges from poorest 20% to wealthiest 20%. the distribution of the different age cohorts for each sex based on self - reported health status is given in figures 1a and 1b. figures 1a and 1b will be used to argue the case for a cut - off point for the dichotomisation of self - reported health status in jamaica. health status (in %) by age cohort controlled for sex (i.e. male) health status (in %) by age cohort controlled for sex (i.e. female) it is well established in biomedical literature that there is a strong negative correlation between health and age ; the current study using self - reported health status by different age cohort controlled for sexes revealed that good health decreases as the individual ages and that more women beyond 80 years old reported very good health status compared to men in the same age cohorts. health status, therefore, can be simply explained by age cohorts, and the aforementioned findings show that sex must be taken into consideration among the covariates in order to comprehend the effects of particular demographic variables on the statistical interpretations of health data. the other covariates must include education level, marital status, area of residence, and social class. the issue of dichotomising self - reported health status continues to be debated in jamaica as researchers continue to grapple with whether to use very poor - to - poor health status versus moderate - to - very poor health status. the issue of using moderate health in poor or good health status is critical as this will aid researchers in understanding whether there should be a cut - off point and where it should be, as this is the crux of the interpretation of the logistic regression model. based on figure 1, the very poor - to - poor health status is marginal at ages below 46 years, and so for the purpose of dichotomisation, ages 46 years and older will be used. this study used secondary cross - sectional survey data, which was collected between may and august, 2007. the jamaica survey of living conditions (jslc), which is used for this study, is a joint research conducted by the planning institute of jamaica (pioj) and the statistical institute of jamaica (statin). it is a standard exercise ; the jslc 's sample is a proportion of the labour force survey (lfs). in 2007, it was one - third of the lfs. for 2007, the current study extracted 1,583 respondents from the larger sample as the focus was on participants aged 46 + years. this design was a two - stage stratified random sampling design where there was a primary sampling unit (psu) and a selection of dwellings from the primary units. the psu is an enumeration district (ed), which constitutes a minimum of 100 residences in rural areas and 150 in urban areas. this means that the country was grouped into strata of equal size based on dwellings (eds). based on the psus, a listing of all the dwellings was made, and this became the sampling frame from which a master sample of dwellings was compiled, which in turn provided the sampling frame for the labour force. a total of 620 households were interviewed from urban areas, 439 from semi - urban areas and 935 from rural areas, which constituted 6,783 respondents. the jslc is a modification of the world bank 's living standards measurement study household survey. face - to - face interviews over the aforementioned period were used to collect the data. a structured questionnaire was used and already trained interviewers were then trained again specifically for this task. the questions covered demographic characteristics, household consumption, health status, health care - seeking behaviour, illnesses, education, housing, social welfare and related programmes, and inventory of durable goods. data were stored, retrieved and analyzed using spss - pc for windows version 16.0. cross tabulations were done to examine non - metric dependent and independent variables, which provided the percentages. percentages were computed for dichotomous health statuses (i.e., very poor or poor health status, and the other very poor to moderate health status) ; these were employed for calculating the odds ratio in each dichotomisation of self - reported health status. among men aged 46 - 54 years, 37.7% of those who reported an illness rated their health status as very poor or poor, as compared to 7.3% of those who did not indicate an illness. hence, the odds ratio of very poor - to - poor health status was 7.7 [(37.7/62.3)/(7.3/92.7) ] indicating that men who reported an illness also have 8 times as high odds of reporting very poor or poor health status than those who did not report a dysfunction. in age cohort 46 - 54 years, the percentage of men who reported very poor, poor or moderate health status was 81.4% compared to 39.9% of those who did not report an illness. hence, the odds ratio of very poor, poor or moderate health status versus non - very poor to moderate health status was 9.6 [(81.4/18.6)/ (31.2/68.8) ]., which examined some of the methodological challenges in self - reported data in finland. 's study in a number of respects, such as : 1) their work used age cohort 35 - 64 years while this study used 45 - 85 + years ; 2) self - reported illness was included among the covariates in the examination of self - reported (rated) health status ; and 3) social class and access (or lack of access) to material resources play a critical role in directly and indirectly influencing health, and so this was added to this paper. although higher education plays a vital role in health status, 2% of the sample had tertiary level education and of this, 0.2% was older than 45 years. most of the sample had at most primary level education (87.3%), which means that the role of tertiary education would contribute marginally to this sample. hence, the researcher excluded it from the covariate analysis of self - reported health status. self - reported illness status is a dummy variable, where 1 = reporting an ailment or dysfunction or illness in the last 4 weeks, which was the survey period, 0 = no self - reported ailments, injuries or illnesses. while self - reported ill - health is not an ideal indicator of actual health conditions, because people may underreport, it is still an accurate approximation of ill - health and mortality. self - reported health status (or health status) was measured by the question : generally, how would you describe your health currently ? the options were : very good, good, moderate (or fair), poor, and very poor. age group was classified as children (aged less than 15 years), youth (aged 15 through 25 years), and other age cohorts ranging in 5 year intervals from 26 - 30 years, et cetera. medical care - seeking behaviour was taken from the question : has a health care practitioner, healer, or pharmacist been visited in the last 4 weeks ? medical care - seeking behaviour, therefore, was coded as a binary measure where 1=yes and 0= otherwise. social class is measured using income quintile where it ranges from poorest 20% to wealthiest 20%. the distribution of the different age cohorts for each sex based on self - reported health status is given in figures 1a and 1b. figures 1a and 1b will be used to argue the case for a cut - off point for the dichotomisation of self - reported health status in jamaica. health status (in %) by age cohort controlled for sex (i.e. male) health status (in %) by age cohort controlled for sex (i.e. female) it is well established in biomedical literature that there is a strong negative correlation between health and age ; the current study using self - reported health status by different age cohort controlled for sexes revealed that good health decreases as the individual ages and that more women beyond 80 years old reported very good health status compared to men in the same age cohorts. health status, therefore, can be simply explained by age cohorts, and the aforementioned findings show that sex must be taken into consideration among the covariates in order to comprehend the effects of particular demographic variables on the statistical interpretations of health data. the other covariates must include education level, marital status, area of residence, and social class. the issue of dichotomising self - reported health status continues to be debated in jamaica as researchers continue to grapple with whether to use very poor - to - poor health status versus moderate - to - very poor health status. the issue of using moderate health in poor or good health status is critical as this will aid researchers in understanding whether there should be a cut - off point and where it should be, as this is the crux of the interpretation of the logistic regression model. based on figure 1, the very poor - to - poor health status is marginal at ages below 46 years, and so for the purpose of dichotomisation, ages 46 years and older will be used. of the sample (6783), 48.7% was male ; 51.3% female ; 69.2% never married ; 14.9% reported having an illness in the survey period (4-week) ; 49.0% dwelled in rural areas ; 82.2% reported at least good health and 4.8% reported at least poor health status (table 1). concomitantly, 61.8% indicated no formal education ; 2.0% reported tertiary level education ; 20.4% was classified as in the wealthiest 20% and 19.7% was in the poorest 20%. continuing, the mean age of the sample was 29.9 years (sd = 21.8 years) with 25 percent of the sample being 12 years old ; 50 percent being 26 years old and 75 percent being 44 years old ; 2.1% of the sample was at least 81 years old. furthermore, socio - demographic characteristic of sample, n = 6,783 very poor or poor and moderated - to - very poor self - reported health status of sexes (in %) interpretation of the odds ratios comparatively, for ages 46 - 54 years, the odds ratio for reporting an illness when an individual is a male who self - reported that he had very poor - to - poor health status was 7.7 times compared to a male who did not report an illness. for women of the same age cohort, those who reported an illness who had reported a health status of very poor - to - poor was 3.3 times more likely to report an illness compared to a female of the same age cohort who did not report a dysfunction. the findings revealed that the odds ratio of an 85 + -year - old male reporting an illness when he had indicated very poor - to - poor health status was 7.9 times more than for one who had not indicated a dysfunction. however, the odds ratio of reporting an illness declined for jamaican males (table 3). on the other hand, the odds of a female of the same age who reported an illness indicating that she had very poor - to - poor health status was greater at 85 + years than a 46 - 54-year - old female (table 3). odds ratios for very poor or poor and moderate - to - very poor self - reported health of sexes by particular variables generally, using the odds ratio, males benefited more by being married (table 3) than females (table 3). concomitantly, the variance from adding moderate - to - poor or very poor health status marginally change the odds ratios over very poor - to - poor health status to very moderate - to - very poor self - reported health status. a substantial disparity in the odds ratios occurred in social standing for males, while it was relatively the same for females. table 3 revealed that by adding moderate self - reported health status to very poor or poor self - reported health status for males, the odds ratios at older ages (i.e., 75 + years) increased exponentially over very poor - to - poor self - reported health status. using odds ratios, the cut - off point for poor health status (excluding moderate health) increased over the age cohorts. however, when the cut - off point included moderate health status, the odds ratios from ages 46 years to 84 years showed that as respondents age within this age cohort, their likeliness of reporting poor health increased ; this declined for ages beyond 85 + years. concurrently, the odds ratios are exponentially higher for the latter dichotomisation than the former (table 4). of the sample (6783), 48.7% was male ; 51.3% female ; 69.2% never married ; 14.9% reported having an illness in the survey period (4-week) ; 49.0% dwelled in rural areas ; 82.2% reported at least good health and 4.8% reported at least poor health status (table 1). concomitantly, 61.8% indicated no formal education ; 2.0% reported tertiary level education ; 20.4% was classified as in the wealthiest 20% and 19.7% was in the poorest 20%. continuing, the mean age of the sample was 29.9 years (sd = 21.8 years) with 25 percent of the sample being 12 years old ; 50 percent being 26 years old and 75 percent being 44 years old ; 2.1% of the sample was at least 81 years old. furthermore, socio - demographic characteristic of sample, n = 6,783 very poor or poor and moderated - to - very poor self - reported health status of sexes (in %) interpretation of the odds ratios comparatively, for ages 46 - 54 years, the odds ratio for reporting an illness when an individual is a male who self - reported that he had very poor - to - poor health status was 7.7 times compared to a male who did not report an illness. for women of the same age cohort, those who reported an illness who had reported a health status of very poor - to - poor was 3.3 times more likely to report an illness compared to a female of the same age cohort who did not report a dysfunction. the findings revealed that the odds ratio of an 85 + -year - old male reporting an illness when he had indicated very poor - to - poor health status was 7.9 times more than for one who had not indicated a dysfunction. however, the odds ratio of reporting an illness declined for jamaican males (table 3). on the other hand, the odds of a female of the same age who reported an illness indicating that she had very poor - to - poor health status was greater at 85 + years than a 46 - 54-year - old female (table 3). odds ratios for very poor or poor and moderate - to - very poor self - reported health of sexes by particular variables generally, using the odds ratio, males benefited more by being married (table 3) than females (table 3). concomitantly, the variance from adding moderate - to - poor or very poor health status marginally change the odds ratios over very poor - to - poor health status to very moderate - to - very poor self - reported health status. a substantial disparity in the odds ratios occurred in social standing for males, while it was relatively the same for females. table 3 revealed that by adding moderate self - reported health status to very poor or poor self - reported health status for males, the odds ratios at older ages (i.e., 75 + years) increased exponentially over very poor - to - poor self - reported health status. using odds ratios, the cut - off point for poor health status (excluding moderate health) increased over the age cohorts. however, when the cut - off point included moderate health status, the odds ratios from ages 46 years to 84 years showed that as respondents age within this age cohort, their likeliness of reporting poor health increased ; this declined for ages beyond 85 + years. concurrently, the odds ratios are exponentially higher for the latter dichotomisation than the former (table 4). of the sample (6783), 48.7% was male ; 51.3% female ; 69.2% never married ; 14.9% reported having an illness in the survey period (4-week) ; 49.0% dwelled in rural areas ; 82.2% reported at least good health and 4.8% reported at least poor health status (table 1). concomitantly, 61.8% indicated no formal education ; 2.0% reported tertiary level education ; 20.4% was classified as in the wealthiest 20% and 19.7% was in the poorest 20%. continuing, the mean age of the sample was 29.9 years (sd = 21.8 years) with 25 percent of the sample being 12 years old ; 50 percent being 26 years old and 75 percent being 44 years old ; 2.1% of the sample was at least 81 years old. furthermore, socio - demographic characteristic of sample, n = 6,783 very poor or poor and moderated - to - very poor self - reported health status of sexes (in %) interpretation of the odds ratios comparatively, for ages 46 - 54 years, the odds ratio for reporting an illness when an individual is a male who self - reported that he had very poor - to - poor health status was 7.7 times compared to a male who did not report an illness. for women of the same age cohort, those who reported an illness who had reported a health status of very poor - to - poor was 3.3 times more likely to report an illness compared to a female of the same age cohort who did not report a dysfunction. the findings revealed that the odds ratio of an 85 + -year - old male reporting an illness when he had indicated very poor - to - poor health status was 7.9 times more than for one who had not indicated a dysfunction. however, the odds ratio of reporting an illness declined for jamaican males (table 3). on the other hand, the odds of a female of the same age who reported an illness indicating that she had very poor - to - poor health status was greater at 85 + years than a 46 - 54-year - old female (table 3). odds ratios for very poor or poor and moderate - to - very poor self - reported health of sexes by particular variables generally, using the odds ratio, males benefited more by being married (table 3) than females (table 3). concomitantly, the variance from adding moderate - to - poor or very poor health status marginally change the odds ratios over very poor - to - poor health status to very moderate - to - very poor self - reported health status. a substantial disparity in the odds ratios occurred in social standing for males, while it was relatively the same for females. table 3 revealed that by adding moderate self - reported health status to very poor or poor self - reported health status for males, the odds ratios at older ages (i.e., 75 + years) increased exponentially over very poor - to - poor self - reported health status. using odds ratios, the cut - off point for poor health status (excluding moderate health) increased over the age cohorts. however, when the cut - off point included moderate health status, the odds ratios from ages 46 years to 84 years showed that as respondents age within this age cohort, their likeliness of reporting poor health increased ; this declined for ages beyond 85 + years. concurrently, the odds ratios are exponentially higher for the latter dichotomisation than the former (table 4). the findings of the current study show that the choice of cut - off for the dichotomisation of self - reported health status marginally matters for age, marital status, and area of residence. the odds ratios for males at the different social classes, when moderate heath status is added to poor health status, changed substantially. this suggests that the dichotomisation of self - reporting for males will not shift and will produce a different result from if only poor or very poor were the cut - offs for self - reported health status. the findings of the study showed that the poor or poorest 20% of males benefitted exponentially when moderate self - reported health status is added to the cut - off point in dichotomising poor health status (including very poor). another important finding of this study, which was not examined by finnas., is the validity of using self - reported illness to measure the health status of people. even though the likelihood of a person with an illness reporting very poor - to - poor health status is greater than one, it should be noted that that likelihood falls at older ages for males and increases at older ages for females. for men, when the cut - off point includes moderate health status, the impact of assessing self - reported illness with poor or very poor health status is higher than if the cut - off was only poor or very poor health status. embedded in this finding is the vast difference that is created by merely changing the cut - off point from poor health status to moderate - to - very poor health status for males. while this disparity does not emerge for females, health researchers who use sex as a covariate must be aware of this reality when dichotomising self - reported health status. the cut - off point for dichotomising self - reported health does not matter if one is examining the health status of only females, as the marginal difference in odds ratio is insignificant and would not create a classification disparity in interpreting the final results. however, the same can not be said about males, particularly those of older ages. therefore, with regards to using self - reported health status, combining people from broad age groups should not be done, as this will not capture the challenges identified in health data on males in jamaica. studies have shown that health deteriorates with age[2230 ], indicating the critical role that age plays in the understanding health of people. therefore, in an examination of poor health status, cautioned must be used by the researcher(s), as people are less likely to report very poor - to - poor health at ages 15 - 30 years. on examination of self - reported health status for jamaicans, the researcher became aware of this fact and so the study of dichotomisation of poor health did not use that age cohort., that it was decided that these should be used as covariates. within the context of the current study, which revealed that small percentages of particular age cohorts are likely to report very poor - to - poor health status, the researcher chose age cohorts that are more likely to report very poor - to - poor health status as this was critical to study. unlike finnas. 's work, which cuts off at age 64 years, this study extended as far as to study respondents up to 85 + years. in 2007, 3.8% of jamaicans were 75 + years (i.e., 101,272) ; 1% were older than 84 years (26,821), and given that people at these ages are more likely to report poor or very poor health, the researcher believes that stopping the study at age 64 would have excluded a critical proportion of those who are likely to be reporting poor health status. among the social determinants of health are social class and area of residence[163133 ]. people are not only defined by their ages, but by where they live and the social class in which they belong. the current study revealed that rural jamaican women indicated the greatest percentage of very poor - to - poor health status, while this was not the case for men. however, the inclusion of moderate health status to poor or very poor health status across the age cohorts by area of residence revealed marginal differences as was the case without the inclusion of moderate health status. among men of 85 + years, the odds ratio of reporting very poor - to - poor health approximately doubled over the previous age cohort (75 - 84 years) and this was marginally the same when moderate health was included in the dichotomisation of very poor - to - poor health. for women, this was not the case as the odds ratios were mostly the same for the two dichotomisations. health literature has shown that the poor had the lowest health status. among men, the effect of social class on health showed no consistent pattern and this was the same for women. however, when moderate health status is included in the cut - off for very poor - to - poor health status, significant changes were observed over the age cohorts. for men, exponential increases occurred with the inclusion of moderate health status to the cut - off point, while this was not the case for women. the current study revealed that the dichotomisation of self - reported health status fundamentally increased the odds ratio, suggesting that the moderate - to - very poor exponentially takes in more men based on how self - reported health status is dichotomised in jamaica at older ages (75 + years). embedded in the finding is the disparity between the percentages of sexes who reported moderate health at older ages for men more than women. 's work, and the findings indicated that cut - off point for dichotomisation of health status was somewhat changed for women, but exponentially changed for men. the findings revealed that women ages 85 + years when self - reported health status was dichotomised using very poor - to - poor health had the highest odds of reporting poor health status. when poor health status was expanded to include moderate health status, the younger ages recorded greater odds of indicating moderate - to - very poor health status. this indicates that at longer ages using the latter dichotomisation approach the odds were age - dependent. men of 85 + years recorded the least odds ratio of very poor - to - poor and moderate - to - very poor health status. there was no clear pattern of age - dependence of self - reported illness for men. embedded in the findings is the greater likelihood of men to report moderate health than poor health at higher ages (85 + years). this suggests that they are under - reporting their true very poor - to - poor health status at higher ages. it follows that the narrower categorisation of age was able to capture this which was lost in a wider categorisation. marital status as a covariate indicated that marriage benefits jamaicans men more than it does women. among men, the odds of reporting very poor - to - poor status are less than for those who were unmarried, across the age cohorts. interestingly, beyond 84 years, the odds ratio of very poor - to - poor health status of men declines, suggesting that the benefits of marriage at this age increases compared to earlier ages. when the cut - off point included moderate health status for men, the odds were relatively the same except for men above age 75. the odds ratios of reporting poor health (i.e., including moderate health status) for those of 75 + years fell substantially, which means that health status for men over 75 + years increased with marriage. among women, the odds ratio for those under 55 years who were married was the same as for their unmarried counterparts. it was found that marriage becomes beneficial for women when they are older than 75 + years, compared to unmarried women of the same age. when the dichotomisation of poor health included moderate health, marginal disparities in odds ratios were found among women in different areas of residence compared to when poor health status excluded moderate health. embedded in this finding is the fact that poor health is weakly age - dependent, as there were not clear patterns for the sexes. however, owing to narrowing age groups, this is a new finding which has emerged in health research literature for jamaica that marriage substantially benefits women at older ages (75 + years) than their younger counterparts. one of the critical findings of this study is that a narrower definition of poor health status (excluding moderate health status) had odds ratios that were closer across the age groups, suggesting that it would be better to exclude moderate health status from very poor - to - poor health status on dichotomising health status. however, if researchers decide to include moderate as a part of the dichotomisation of poor health status, they should be aware of some of the methodological implications of their choice, and how this will impact on the interpretation, in particular for men, within the different social classes. in summary, the odds ratios vary substantially for men in different social classes as well as for self - reported illness based on the dichotomisation cut - off point for poor health. among women, there was no clear age dependency based on the cut - off point of poor health ; the vast disparity that was present for men was not found for women in the different social classes. like the study conducted by finnas., this paper agrees that the choice of cut - off point in dichotomising poor health status can not be made primarily on variables such as age, because sex and social class must also play a factor in this choice, as well as the nature of the study. 's work in that with a narrower classification of poor health, the effect of marital status and area of residence were not found to be highly age - dependent. the current study found that dichotomising poor health status is acceptable assuming that poor health excludes moderate health status, and that it should remain as is and ordinal logistic be used instead of binary logistic regression. | background : caribbean scholars continue to dichotomise self - reported health status without empirical justification for inclusion or exclusion of moderate health status in the dichotomisation of poor health.aims:this study will 1) evaluate which cut - off point should be used for self - reported health status ; 2) assess whether dichotomisation of self - reported data should be practiced ; 3) ascertain any disparity in dichotomisation by some covariates (i.e., marital status, age cohort, social class) ; and 4) examine the odds of reporting poor or moderate - to - very poor self - reported health status if one has an illness.materials and methods : the current study used cross - sectional survey data for 2007. the survey used stratified probability sampling techniques to collect the data from jamaicans. the sample consisted of 6,783 respondents, with a focus on participants aged 46 + years (n=1,583 respondents). self - reported health status was a 5-item likert scale question. the dichotomisation was poor health status or otherwise and poor (including moderate) self - reported health. odds ratios were calculated in order to estimate the effect of the covariates.result:when moderate self - reported health status was used in poor health status, the cut - off revealed moderate effect on specified covariates across the age cohorts for women. however, for men, exponential effects were used on social class, but not on area of residence or marital status across the different age cohorts.conclusions:the cut - off point in the dichotomisation of self - reported health status does not make a difference for women and must be taken into consideration in the use of self - reported health data for jamaica. |
burns are one of the most common and devastating forms of trauma and a major public health concern in all around the world 1. the burn patients have unique predisposition to different infections which are linked to impaired resistance from disruption of the skin 's mechanical integrity and generalized immune suppression. the skin barrier is replaced by a protein rich, avascular environment that provides a favourable niche for microbial colonization and proliferation. additionally migration of immune cells is hampered, which contributes to septic process 2 - 6. in spite of considerable advances in the last 60 years in antimicrobial it was shown that approximately 73 per cent of all death within the first five days post - burn has been caused by sepsis 7 - 9. also the worldwide emergence of antimicrobial resistance among bacterial pathogens, limits the available therapeutic options for effective treatment of infections 10, 11. thus, the aim of the current study was to determine the microorganisms and their susceptibility patterns which were isolated from burn wounds of patients at van training and research hospital in van, turkey. this study was conducted retrospectively at a 10-bed paediatric and adult burn unit located in a 400-bed tertiary referral hospital. the burn unit is the reference burn center in van province (with one million inhabitants), turkey. consequently patients hospitalized in this unit come from the emergency of the hospital as well as from transfers from other hospitals. wound swaps were obtained twice weekly to monitor colonisation and when infection was suspected in the burn unit. positive wound swap culture results during a 3-year period (from january 2009 to december 2011) were reviewed and two hundred fifty non - duplicate bacterial species isolated from one hundred seventy - nine patients ' wound swabs, were included in the present study. no routine systemic and topical antimicrobial (e.g. mafenide, silver sulfadiazine) used in our burn unit. all samples were inoculated on 5% sheep blood agar and eosin methylene blue overnight at 37c. identification of isolates was done by conventional biochemical methods according to standard microbiological techniques 12. after determining mainly morphologic criteria of bacteria, panels of automized identification device phoenix automated microbiology system (becton, dickinson - usa) was used in order to determine the certain identification and anti - microbial susceptibility rates. duplicate isolates defined as repeated isolation of the same bacterial species for the same patient with the same profile of antibiotic susceptibility were excluded. pseudomonas aeruginosa and acinetobacter spp were accepted as multidrug - resistant if the microorganism was resistant against at least three antimicrobials groups among antipseudomonal cephalosporins, -lactam--lactamase inhibitor combination, antipseudomonal fluoroquinolones, antipseudomonal carbapenems or aminoglycosides. the antimicrobial susceptibilities were determined according to the clinical and laboratory standards institute (clsi). information on all bacterial isolates including their antibiotic susceptibility pattern were extracted and processed by spss 17.0 software package. this study was conducted retrospectively at a 10-bed paediatric and adult burn unit located in a 400-bed tertiary referral hospital. the burn unit is the reference burn center in van province (with one million inhabitants), turkey. consequently patients hospitalized in this unit come from the emergency of the hospital as well as from transfers from other hospitals. wound swaps were obtained twice weekly to monitor colonisation and when infection was suspected in the burn unit. positive wound swap culture results during a 3-year period (from january 2009 to december 2011) were reviewed and two hundred fifty non - duplicate bacterial species isolated from one hundred seventy - nine patients ' wound swabs, were included in the present study. no routine systemic and topical antimicrobial (e.g. mafenide, silver sulfadiazine) used in our burn unit. all samples were inoculated on 5% sheep blood agar and eosin methylene blue overnight at 37c. identification of isolates was done by conventional biochemical methods according to standard microbiological techniques 12. after determining mainly morphologic criteria of bacteria, panels of automized identification device phoenix automated microbiology system (becton, dickinson - usa) was used in order to determine the certain identification and anti - microbial susceptibility rates. duplicate isolates defined as repeated isolation of the same bacterial species for the same patient with the same profile of antibiotic susceptibility were excluded. pseudomonas aeruginosa and acinetobacter spp were accepted as multidrug - resistant if the microorganism was resistant against at least three antimicrobials groups among antipseudomonal cephalosporins, -lactam--lactamase inhibitor combination, antipseudomonal fluoroquinolones, antipseudomonal carbapenems or aminoglycosides. the antimicrobial susceptibilities were determined according to the clinical and laboratory standards institute (clsi). information on all bacterial isolates including their antibiotic susceptibility pattern were extracted and processed by spss 17.0 software package. a total of 250 bacterial isolates were obtained from 179 patients ' wound swap over a 3-year period. the most predominant bacterial isolate was acinetobacter baumannii (a. baumannii) (23.6%) followed by coagulase negative staphylococci (13.6%), pseudomonas aeruginosa (p. aeruginosa) (12%), staphylococcus aureus (s. aureus) (11.2%), and escherichia coli (e. coli) (10%) as shown in table 1. the susceptibility of the organisms to different antibiotics varied depending on the isolate. although all tested acinetobacter spp. isolates were sensitive to tigecycline (n=37) and colistin (n=43), fifty - five (93%) of isolates were multidrug - resistant. meropenem, amikacin, ciprofloxacin and cefepime were found to be most active antimicrobial agents against p. aeruginosa. all tested e. coli isolates were susceptible to amikacin (n=17), imipenem (n=19) and meropenem (n=21). besides these three antimicrobial agents, gentamicin was also found to be in vitro active against klebsiella pneumonia (k. pneumonia) and other enterobacteriaceae spp. extended - spectrum beta - lactamases (esbl) were found to be 13/25 (52%) and 7/18 (39%) among e. coli and k. pneumonia isolates respectively. among the s. aureus isolated from patients within the burn center, the incidence of methicillin - resistant s. aureus (mrsa) was 19% and the most active antimicrobial agents were found to be vancomycin and linezolid against s. aureus isolates respectively. burn injuries remain a huge public health issue in terms of morbidity and long - term disability throughout the world 13, 14. thermal injury impairs the skin its normal barrier function, thus allowing microbial colonization of the burn wounds. severe dysfunction of the immune system, a large cutaneous colonization, the possibility of gastrointestinal translocation, a prolonged hospitalization and invasive diagnostic and therapeutic procedures, all contribute to infections 13, 15. patient factors such as age, extent of injury, and depth of burn in combination with microbial factors such as type and number of organisms, enzyme and toxin production, and motility determine the likelihood of invasive burn wound infection. although any organism is a potential pathogen in burned patients, coagulase - negative staphylococci and s. aureus and enterococcus spp. were the most common gram positive pathogens and p. aeruginosa, e. coli, k. pneumoniae and acinetobacter spp. the high prevalence of acinetobacter baumannii in our centre differs markedly from most other studies from europe, the usa and south america 19 - 21. highly prevalent in singapore and explaned this situation by constant introduction of acinetobacter spp. carried on human skin (endemic to tropical climate) with every patient admitted in their settings 22. might be more prevalent in warm climates, with corresponding increase in colonization and nosocomial infection 23 - 25. although our center is placed in eastern part of turkey where the climate is dry and cold, this hypothesis could be an explanation for our results also and should be further studied. our finding concerning the frequency of p. aeruginosa (12%) was much lower than many previous reports where this organism was held responsible for the majority of invasive burn wound infections in burn - treatment facilities 26 - 29. s. aureus was the third in the list of microbial isolates recovered in our study. this is contrary to many previous reports indicating a much higher frequency of isolation of this organism 30 - 31. in our study, e. coli was the fourth most frequently recovered organism. the results of the antimicrobial resistance pattern give serious cause for concern because the predominant bacterial isolates were highly resistant to the commonly available antimicrobial agents in turkey. acinetobacter baumannii and p. aeruginosa were found to be multidrug - resistant. despite the increased knowledge of the pathogenesis and antibiotic resistance mechanisms, multidrug - resistant isolates of a. baumanni and p. aeruginosa are special concerns in burn care units 33. the incidence of a. baumanni resistant to imipenem (86%) was high, in contrast to other studies 34 - 36. its easy transmissibility and ability to remain viable in a hospital environment due to its multidrug - resistant status and several other factors have been implicated in the increased incidence of nosocomial infections due to this organism. as reported in other studies 22 - 25 multidrug - resistant acinetobacter spp. have emerged as a significant cause of wound infection in our burn unit. the incidence of p. aeruginosa resistant to ceftazidime (32%), piperacillin / tazobactam (31%) and imipenem (46%) was much higher in our study in contrast to other studies 37, 38. this is consistent with those reported from other countries 37, 38 and supports the fact that there was increasing evidence that mrsa has become a significant problem. we found that vancomycin, linezolid, and ampicillin were still active drugs for the treatment of enterococcus fecalis. the emergence of esbl producing strains among enterobacteriaceae (e. coli, k. pneumoniae, e. cloacae and p. mirabilis) is a special concern 39. guggenheim have showed that imipenem and meropenem were the most active antimicrobial agents for esbl producing strains 40.. the main limitations of our study are the retrospective design and use of only a single burn center 's data. culture isolates were unavailable for additional testing or molecular analysis to determine if isolates were acquired through nosocomial transmission. additionally data obtained from electronic patient records makes it difficult to distinguish infection from colonization. although it is known that the widespread use of broad spectrum antimicrobials in burn units would provide a fertile ground acquisition of resistance and transformation to form new strains 22, detailed treatment regimens were unavailable in our study and it is unknown what impact antibiotic use had on culture data. in conclusion, the growth of multidrug - resistant organisms should be considered as a serious risk in burn units. aggressive infection control measures should be applied to limit the emergence and spread of multidrug - resistant pathogens. | the risk of infection in burns is well - known. in recent decades, the antimicrobial resistance of bacteria isolated from burn patients has increased. for this reason, a retrospective study was conducted at van training and research hospital to analyze the bacterial isolates from the wounds of patients admitted to the burn unit and to determine the susceptibility patterns of the commonly cultured organisms over a 3-year period, january 2009 to december 2011.a total of 250 microorganisms were isolated from burn wounds of 179 patients. our results revealed that the most frequent isolate was acinetobacter baumannii (23.6%), pseudomonas aeruginosa (12%), staphylococcus aureus (11.2%), escherichia coli (10%) respectively. multidrug - resistance has emerged as an important concern in our burn unit. tigecycline, and colistin were found to be the most active drugs against acinetobacter baumannii. carbapenems and amikacin, were found to be the most active drugs against other gram negative bacteria. vancomycin and linezolid were active against gram positive bacteria.aggressive infection control measures should be applied to limit the emergence and spread of multidrug - resistant pathogens. |
a chronic subdural hematoma (csdh) is a slowly growing encapsulated collection of blood and its breakdown products between the dura mater and the arachnoid.12) csdh is a condition mostly present in older people. one rationale for male dominance could be that men generally have a greater exposure to injuries.10,17) although men sustain nearly two to three times as many brain injuries as women,6) it could not enough to explain the reason for male predominance on csdh. it is well documented that mean brain size (weight or volume) is 9 - 12% larger in men than in women.16) there is evidence that some brain regions show age - associated volume decline and that men undergo more accelerated cerebral aging than women.8) the purpose of this study is to find out whether there were any differences in the anatomy of cranium, which may contribute the pathogenesis or risk factors of csdh. we treated 56 patients with csdh by burr - hole drainage from 2011 to 2013. the number of female patients was few to compare the anatomical difference according to the gender. we also included consecutive 100 patients with transient ischemic attack (tia) to compare the morphology and size of the cranium. the study population was consisted of 87 patients with csdh and 100 patients with tia. in all cases, we excluded patient with conservatively treated csdh, since differentiation of subdural hygroma from csdh is often impossible by ct alone. we also excluded patients with tia who had a history of surgical or medical treatment for brain disease. we classified into four groups ; group a (csdh male 47), group b (csdh female 40), group c (tia male 50), and group d (tia female 50). first, we selected a ct axial image where the third ventricle was best visualized. finally, we measured the length of cranium from the glabella to the inion (lap), the width of cranium (wc), and the maximal radius from midline to the right (rr) and left (rl) ends (figure 1). we obtained the ratio of cranium (rc), dividing the width by the length (wc / lap) and difference of cranium (dc), difference between the right and left radiuses. although measurement of the exact cranial asymmetry requires reconstruction 3d program, gross asymmetry could be assured on the conventional ct axial image. the average length and width of male cranium were larger than those of female cranium (table 2). the radius of the left side was slightly larger than that of the right side in all groups except group a (csdh male). although the length and width of the male groups were larger than those of the female groups, the rc was quite similar in all groups. however, the dc was significantly higher in patients with csdh (group a and b)(p=0.03). we also compared with gender (a+c vs. b+d) and disease (a+b vs. c+d) groups. the mean dc was 3.02 mm in male group (group a and c) and 2.52 mm in female group (group b and d). although the mean dc of male group was larger than that of female group, this difference was not statistically significant (by t - test, p=0.24)(table 3). the mean dc was 3.49 mm in csdh group (group a and b) and 2.14 mm in tia group (group c and d). the mean dc of csdh group was significantly larger than that of tia group (by t - test, p<0.05)(table 4). the radius of the left side was usually larger than that of the right side due to cerebral dominance. although the rc was quite similar in all groups, the dc was significantly higher in patients with csdh either male or female. csdh occur in the dural border cell (dbc) layer, located between the dura mater and the arachnoid and the dissection of these layer creates a subdural cavity.9) patients with extensive brain atrophy (elderly and alcoholics) or conditions resulting in intracranial hypotension (ventriculoperitoneal shunt) are vulnerable to developing csdh.1) traversing veins are being stretched by the shrinking brain until only a minor additional force is sufficient to cause the rupture of the bridging veins and create the hematoma.1) this is followed by fibrin deposition, organization, enzymatic fibrinolysis, and liquefaction of the clot. an inflammatory reaction occurs, and neomembranes (inner or visceral and outer or parietal membranes) are formatted with the growth of neocapillaries and enzymatic hyperfibrinolysis.15) csdh tend to gradually enlarge because repeated micro - hemorrhage may lead to clinical signs and symptoms of increased intracranial pressure or compression brain structures.12) the pathogenesis of csdhs remains complex and hypothetical.14) risk factors are not well understood yet, especially in elderly patients.2,3) kim.11) assumed that medical history did not significantly influence the recurrence rate. in addition, the dural blood vessel increases the fragility, and the hemostasis inside the hematoma is not sufficient, because of the simultaneous activation of coagulation and fibrinolytic cascade, with an increased expression of tissue - type plasminogen activator.14) in general, advancing age has been associated with decreased brain tissue and increased cerebrospinal fluid (csf). some reported that in patients over 50 years of age, the mass of the brain is reduced by approximately 200 g, which results in an increased extracerebral volume of up to 11%.13) so we want to validate a theory that the morphologic difference between csdh and normal group is the aggravating factor of development of csdh. if it became an authenticated case, it is helpful to study the pathogenesis of csdh. in the present study, we found a statistically significant difference in the maximal radius between csdh female and tia female group. it means that the asymmetry of women 's cranium with csdh is significantly higher than with tia. we also found a statistically significant difference in the maximal radius between all patients with csdh and all patients with tia, regardless of the sex difference. we could assume that the bulging - headed person is prone to develop a csdh under trauma. we selected patients with tia as a control group, since their age - distribution was similar. so the newly developed dead space after brain atrophy is gradually filled with csf or others, according to monro - kelly 's doctrine. the negative pressure from brain atrophy is constant on each side of the cranium, according to boyle 's law which the pressure of gas tried to hold at a constant temperature change by inverse with the volume of the gas. pv = k ; where p is the pressure of gas, v is the volume of the gas, and k is a constant. since diffuse brain atrophy decreases the left and right hemispheres in the same ratio, the negative pressure of the both hemispheres is the same. as the degree of atrophy is constant, the decreased length of brain diameter on the large hemisphere is longer than on the small hemisphere. so, the distance from the skull to the cerebral cortex in the shrunken hemisphere is longer on the side of large hemisphere. the bridging vein is more stretched on the large hemisphere, so it may be tore by a trivial injury. if the distance exceeds the length of the arachnoid trabeculae, separation of the dbc layer will occur at the large hemisphere, which will create so - called the subdural space. separation of the dbc layer is easy in a large cranium with severe atrophy, which could explain the male prevalence. the gender differences in normal brain structure and function have been carried out.12) male brain is larger than female brains in all locations, especially most prominent in frontal and occipital lobe, bilaterally.10) and the anterior regions of corpus callosum decrease in width at earlier age in men than women.8,10) age - specific changes in brain size were significantly greater in men than women.5,7) with increasing the brain atrophy, the csf in subdural space increase, so it is result in csdh. age - specific changes in brain size were significantly greater in men than women for csf volume, the sylvian fissure csf volume, and the parietooccipital region area.4) this can be another reason for the high prevalence of csdh in males. the male prevalence is related to the cranial size and asymmetry along with great exposure of males to injury, estrogen effect on the capillaries, or fewer medical visit of females.10) identification of this relationship may be useful to elucidate the origin and pathogenesis of csdh. | objectivechronic subdural hematoma (csdh) is a condition mostly present in older people. men are more commonly affected than women. several theories about male predominance could not enough to explain the reason for male predominance on csdh. the purpose of this study is to find out whether there were any differences in the anatomy of cranium, which may contribute the pathogenesis or risk factors of csdh.methodsthe study population was consisted of 87 patients with csdh and 100 patients with transient ischemic attack (tia) from 2006 to 2013. we classified into four groups ; group a (csdh male 47), group b (csdh female 40), group c (tia male 50), and group d (tia female 50). we measured the size of the cranium in the computed tomography scans, retrospectively. we define the difference of cranium (dc), which is difference between the right and left radiuses.resultsthe dc was significantly higher in patients with csdh (group a and b)(p=0.03). the mean dc was 3.49 mm in csdh group (group a and b) and 2.14 mm in tia group (group c and d). the mean dc of csdh group was significantly larger than that of tia group (by t - test, p<0.01).conclusionsize and asymmetry of the cranium may be a risk factor of csdh. gender differences in the anatomy of cranium may contribute pathogenesis of csdh. |
electrical stimulation is a popular treatment used by physiotherapists for muscle strengthening, endurance, spasticity management, pain control, circulation promotion and edema control1,2,3,4,5. transcutaneous electrical nerve stimulation (tens) and interferential current (ifc) are widely used for relief of chronic and acute pain6,7,8,9. tens machines are usually relatively inexpensive, portable, battery operated devices, while ifc machines tend to be more expensive, are not portable, and require an electrical power source10. experimental pain models provide a useful representation of clinical pain and make it possible to control variables such as pain intensity and duration. clinical trials can be expensive and time consuming, while the intensity, location, and duration of clinical pain can be difficult to control. experimental pain models can thus guide subsequent clinical trials, potentially saving time and money. different experimental pain models exist utilizing different types of pain stimulus such as cold, mechanical, electrical, and ischemic pain11. the frequencies of 50 hz and 100 hz have a high analgesic effect12,13,14,15. experimental pain models using the same frequencies for tens and ict analgesic effects have been compared, but the analgesic effects are unclear16,17,18. thus far, there have been few studies that have compare the analgesic effects of tens and ifc with these two frequencies. the ischemic pain model is a method commonly used to validate analgesic effect. the modified version of the submaximum effort tourniquet technique (sett) can cause pain that is similar to clinical pain10, 19. therefore, this study used the ischemic pain model to compare the analgesic effects of 50 hz tens, 50 hz ifc, 100 hz tens and 100 hz ifc. the subjects were 36 university student volunteers (18 male, 18 female) without known pathology. their mean age was 24.5 years (sd 2.2) (table 1table 1.general characteristics of the subjectsvariablemeansdage (yr)24.52.2height (cm)170.312.5weight (kg)64.715.3sd, standard deviation). all subjects who expressed interest in participating in the study were briefed on the experimental procedure (both verbally and in written form) and were screened for contraindications to the experimental procedure or electrotherapy. these contraindications included any illness or pathology such as peripheral vascular abnormalities, hypertension, hypotension, peripheral neuropathies, recent trauma, and menstruation problems. subjects who were taking any medication or who were likely to take any medication during the period of study were excluded. sd, standard deviation the investigator also checked each subject s nondominant arm for signs of previous trauma and recorded the subject s blood pressure from the nondominant arm (because the effectiveness of tens and ifc is dependent on normally functioning nerves in the skin) using a sphygmomanometer. outcome measurements were recorded from the nondominant arm so that subjects could use the dominant arm when completing visual analog scales (vass). all subjects who expressed an interest in the study met the criteria and agreed to participate. subjects were required to sign a consent form and were reminded that they had the right to withdraw from the experiment at any time. all subjects gave informed consent according to the methods outlined by sahmyook university s institutional review board. this study protocol and procedures were also approved by sahmyook university s ethics committees. each subject attended our research laboratory on four separate occasions with a 24-, 48-, or 72- to 96-hour interval between visits. during each visit, self - reported pain intensity was recorded at 1-minute intervals during the ischemic pain test using a vas in which 0 cm represented no pain and 10 cm represented worst pain imaginable. analgesia was induced by one of four treatments : 50 hz tens, 50 hz ifc, 100 hz tens or 100 hz ifc. at the end of the fourth testing session, participants were asked, which of the two modalities, if either, seemed more effective ? and which of the two modalities, if either, felt more comfortable ? a sphygmomanometer cuff is usually applied above the subject s elbow and inflated to 200 mm hg. during pilot studies in our laboratory, we found that most subjects experienced widespread paresthesia in the arm rather than pain. thus, we modified the sett by applying the sphygmomanometer cuff to the forearm 5 cm below the elbow crease, because this placement of the sphygmomanometer cuff produced a dull aching pain that was localized to the area of the cuff in all subjects. before the start of the experiment, maximal grip force was determined using a dynamometer (martin vigorimeter) fitted with a medium bulb. seventy - five percent of the maximal grip force was calculated and identified on the dynamometer scale. subjects raised their nondominant arm vertically above their head for 1 minute to decrease the volume of blood the limb. the sphygmomanometer cuff (15 cm in length) was then inflated to above 200 mmhg at a rate of 40 mmhg per second. full cuff inflation was taken as time 0, and subjects rated the intensity of pain in their raised arm using the vas. the forearm was then returned to rest in the horizontal position on a polystyrene box that was designed to support the forearm and hand without applying pressure to the sphygmomanometer cuff. subjects then performed 20 hand - gripping exercises at 75% of their maximal grip force for a period of one minute (squeeze for two seconds and rest for two seconds). pain intensity was recorded on completion of these exercises and at one minute intervals for the remainder of the experiment. the cuff was deflated over a period of two minutes to allow the volume of blood to increase in the limb, and the final pain intensity rating was taken one minute after cuff deflation. no signs of trauma were observed in the arms of any subjects following the ischemic pain test10, 19. on each visit to the laboratory, subjects were randomly allocated to one of four treatments : 50 hz tens, 50 hz ifc, 100 hz tens and 100 hz ifc. a single - blind experimental approach was used whereby the subjects were not aware of which treatment they were being given. four self - adhesive electrodes (each electrode 4.5 cm) were applied before the start of the experiment, and treatment was switched on 10 minutes and 40 seconds before the arm was raised above the head. we were concerned that afferents under the cuff might be unable to fire due to pressure block from the cuff. however, all subjects reported that they experienced a strong but comfortable electrical paresthesia, suggesting to us that afferents remained active. electrodes were applied in a quadripolar manner to the anterior and posterior aspects of each subjects forearm so that electrical currents would intersect at the midpoint of the cuff. the distal electrode for channel a was attached to the anterior surface of the forearm 5 cm proximal to the first wrist crease. the distal electrode for channel b was attached to the posterior surface of the forearm directly beneath the distal electrode for channel a. proximal electrodes were applied directly above the cuff. subjects in the ifc group were told that to produce an effect, the intensity of the stimulator must be maintained at a strong but comfortable level at all times. initially, when the ifc device was switched on for the first time, the comfortable level was obtained by increasing the current amplitude so that the subjects reported either that the currents were uncomfortable or that the motor threshold had been reached. high analgesic effects of bursts at 50 hz and 100 hz generated by 4-khz sinusoidal waves were applied20, 21. the tens in our study was delivered via four electrodes using a dual - channel device in order to standardize the amount of current administered by the two modalities. electrodes were applied to the anterior and posterior aspects of the each subjects s forearm in an identical manner to that for ifc. to minimize interference of currents from the two channels, both distal electrodes were attached to channel a of the tens device, and both proximal electrodes were attached to channel b. the comfortable level was obtained using the same procedure as that described for ifc. the high analgesic effect of a 125-microsecond phase duration at a frequency of 50 hz and a 200-microsecond phase duration at a frequency of 100 hz were applied20, 21. data were analyzed by calculating the change in pain intensity rating during the treatment between measurements. treatment effects were determined by a two - way repeated measures analysis of variance (anova) of the change in pain intensity during treatment for vas ratings 4 through 9 and all vas ratings. after completing the four interventions, there were significant effects for time, which were attributed to a significant reduction in pain intensity during treatment, for vas ratings 4 through 9 and all vas ratings (vas 112) (p<0.05). there were no significant effects for groups or group - time interaction in pain intensity during treatment for vas ratings 4 through 9 and all vas ratings. there were significant time effects that could be attributed to the increase in pain intensity in all groups during cuff inflation and hand - grip exercises (vas 13) and a decrease in pain intensity immediately upon cuff deflation (vas 1012). this result means pain intensity was significantly increased by the treatment in all groups (p<0.05). but there was no significant difference between the groups (table 2table 2.pain intensity rating for treatments during the ischemic pain testcuffinflatedhandgripexercisestarthandgripexerciseendcuff inflatedcuffdeflatingcuffdeflatingcuffdeflated-20 svas 10 minvas 21 minvas 32 minvas 43 minvas 54 minvas 65 minvas 76 minvas 87 minvas 98 minvas 109 minvas 1110 minvas 1250 hz tensmeansd0.40.81.51.22.41.82.81.63.41.64.01.64.11.74.51.84.81.95.12.22.62.31.32.050 hz ifcmeansd0.50.61.30.62.11.22.41.23.01.33.61.93.92.14.22.24.52.44.32.62.52.51.62.3100 hz tensmeansd0.30.31.30.52.20.92.51.12.91.43.61.53.71.33.91.44.11.63.91.92.21.81.41.2100 hz ifcmeansd0.40.41.10.61.90.82.21.02.81.53.41.73.61.54.01.54.41.44.12.02.31.71.71.1values are expressed as the mean sd. tens, transcutaneous electrical nerve stimulation ; ifc, interferential currents ; vas, visual analog scale). tens, transcutaneous electrical nerve stimulation ; ifc, interferential currents ; vas, visual analog scale at the end of the fourth session, participants were asked which of the four types of intervention felt most comfortable. twenty subjects (56%) reported that 50 hz ifc was the most comfortable, six (17%) felt that 100 hz - ifc was the most comfortable, six (17%) felt that 50 hz tens was the most comfortable, and four (10%) felt that 100 hz tens was the most comfortable. at the end of the second session, nineteen subjects (53%) reported that 50 hz ifc was the most effective, nine (25%) felt that 100 hz ifc was the most effective, five (14%) felt that 50 hz tens was the most effective, and three (8%) felt that 100 hz tens was the most effective. the effects of tens and ifc stimulation at frequencies of 50 hz and 100 hz on pain relief were analyzed to investigate the usefulness of electrical stimulation on ischemic - induced pain. in this study, johnson and tabasam used 100-hz stimulation and found a greater effect with tens than ifc in a cold pain model, but the difference between treatments was not significant. the measured difference was not statistically significant, but the study only had seven participants in each, group so the lack of significance could have been due to low statistical power22. with methods similar to this study, shanahan. they found that tens at a frequency of 100 hz had a greater analgesic effect than premodulated ifc at a beat frequency of 100 hz. the balance of evidence thus indicates that ifc is less effective than tens. cheing and hui - chan reported no significant difference between treatments in a heat pain model23. johnson and tabasam reported no significant difference between treatments in an ischemic pain model20. johnson and tabasam reported a statistically significant result, with 100 hz tens being more effective than premodulated 100 hz ifc23. remarked that the controversy over the effect of pain relief between tens and ifc resulted from lack of statistical power in published papers due to inadequate sample sizes21. found that the effects of tens and ifc at 50 hz on the pain threshold was significantly increased and that there was no significant difference between tens and ifc in a cold pain model21. johnson. found that higher frequencies of pulsed current (above 50 hz) resulted in a lesser hypoalgesic effect17. the between - frequency differences in the study by johnson. were not statistically significant, possibly due to a small sample size, but a downward trend was clearly evident above 40 hz17. speculated that a short burst duration prevents or severely restricts multiple firing of sensory nerve fibers and instead produces one action potential per burst21. thus 50 hz ifc and 50 hz tens are equally effective in alleviating cold - induced pain in healthy subjects21. therefore, in previous studies, there was a debate that not significant difference between the different types of electrical stimulation. the results of our study were similar to those of previous studies and also showed a statistically significant difference between the different types of electrical stimulation, but analgesia was significantly increased during electrical stimulation in all groups. reported that subjects experienced the most analgesia from electrical stimulation and the greatest comfort at a frequency of 50 hz with ifc, as was the case in our study21. the current results and previous tens and ifc comparative studies strengthen the conclusion drawn that low - duty - cycle, burst - modulated ac is more comfortable than conventional low - frequency pulsed current21. that 50 hz ifc is an acceptable treatment is likely a consequence of the lesser discomfort. we conclude that there were no differences in the analgesic effects of the four interventions under the present experimental conditions. however, 50 hz ifc is more comfortable than other interventions and is likely to be better accepted and tolerated by patients. | [purpose ] this study compared the analgesic effects of transcutaneous electrical nerve stimulation (tens) and interferential currents (ifc) on induced ischemic pain in healthy volunteers. [subjects ] the subjects were 36 volunteers (18 male, 18 female) without known pathology that could cause pain. their mean age was 24.52.2 years. [methods ] a single - blind and parallel - group method was used. subjects were randomly allocated to receive each 50 hz tens, 50 hz ifc, 100 hz tens, and 100 hz ifc. this study experimentally induced ischemic pain in otherwise pain - free subjects using a modified version of the submaximal effort tourniquet technique. subjects completed twelve cycles of the ischemic - induced pain test. the primary outcome measure was the change in self - reported of pain intensity during one of four possible treatments. [results ] there were significant effects for time, which were attributed to a significant reduction in pain intensity for all groups. there were no significant effects for groups or group - time interaction. the 50 hz ifc treatment was more comfortable than the other treatments in the present study, and it is likely to be better accepted and tolerated by patients. [conclusion ] we conclude that there were no differences in the analgesic effects of the four treatments under the present experimental conditions. the 50 hz ifc treatment is more comfortable than the other treatments. |
dental trauma remains one of the important oral health problems in childhood and can cause much pain and distress. it can vary from a minor enamel chip to extensive maxillofacial damage involving the supporting structures and displacement or avulsion of teeth. there is perhaps no single dental disturbance that has greater psychological impact on the parents and the child than the loss or fracture of a child 's anterior teeth. primary and permanent anterior teeth are not only important for aesthetics but also essential for phonetics, mastication, integrity of supporting tissues, psychological and mental wellbeing. epidemiological studies of dental trauma have shown that most dental accidents in children occur at home, followed by school. the most important factor determining the prognosis of a replanted tooth is the viability of the periodontal ligament left on the root prior to replantation. the clinical management can be complex and involves the use of numerous diagnostic aids and treatment modalities. correct intervention can play an important role to improve the prognosis of a traumatized tooth. the prognosis highly depends upon correct and prompt emergency management and proper advice, which may frequently be the responsibility of lay people available at the accident site. therefore, it is crucial to ascertain the knowledge and practice of the personnel at home and in school who are in close contact with young children. many international reports have indicated the lacking knowledge of adults likely to be present at the emergency site regarding immediate management of dental trauma. parents can play an important role in improving the prognosis of avulsed permanent teeth of children if they are informed about the first - aid steps to be taken at the time of an accident. before planning information campaigns, it is important to assess the knowledge level of parents. the purpose of this study was to evaluate parents awareness of the emergency management of traumatized primary and permanent teeth by means of a questionnaire in a sample of 1500 parents from rural / urban backgrounds and with different education levels. this study was carried out in the outpatient department (opd) of pedodontics and preventive dentistry, post graduate institute of dental sciences, rohtak, haryana, india. the awareness of the emergency management of dental trauma among 1500 parents attending opd was assessed. a questionnaire [table 1 ] the questionnaire was divided into three parts, part i contained questions on personal information, part ii contained questions based on an imaginary case of trauma and part iii contained questions related to attitude towards dental trauma management education. before distributing the questionnaire, a brief explanation about the objective of the study was given to the parents. the questionnaires were analyzed to observe the correct and incorrect responses for part ii of the questionnaire. in addition, chi - square test was employed to evaluate the effect of residing area, age, level of education and previous dental trauma experience on each question. the demographic characteristics of the participating parents as shown in table 2 ; indicate that 58.5% of parents were from rural area and 65.2% of parents belonged to > 30 years age group. also, 54.3% of parents were educated below graduate level and 36.7% of parents had previous experience of trauma to self or others. demographic characteristics of participating parents tables 36 show the number of correct and incorrect responses for part ii of questionnaire along with the p values. distribution of parents based on residing area distribution of parents based on age distribution of parents based on level of education distribution of parents based on previous experience for question 1, 67.3% (n = 1010) of the participants were not able to recognize the damaged front tooth in an 8-year - old child. for question 2, 57% (n = 855) would not search for lost tooth and 43% (n = 645) would search for it. for question 3, 64.4% (n = 966) would seek professional advice immediately and 35.6% (n = 534) would seek professional advice within 30 min, few hours and before next day. for question 4, 67.5% (n = 1013) would not go for a professional advice if the child does not have any pain. for question 5, 72.7% (n = 1091) would take the child to dental hospital after dental trauma and 27.2% (n = 409) would go to general hospital. for question 6, 31.8% (n = 477) would replant the tooth in its socket and 68.2% (n = 1023) would not replant the tooth. for question 7, 46.4% (n = 697) would rinse the tooth under tap water for cleaning it before replantation, six parents would scrub the tooth gently with the tooth brush, 11 parents would place the tooth straight back to the socket without doing other things, 770 parents did not know how to clean and 16 parents would choose other methods like rinsing it with antiseptic solution or wiping it with wet cloth. for question 8, milk was chosen as transport media by 24.3% (n = 365) of parents, 35 parents would use ice, seven parents chose child 's mouth, 566 parents would use paper tissue or handkerchief, 500 parents would take the tooth in plastic wrap and 27 parents would use antiseptic or alcohol as transport media. for question 9, follow - up by dentist chi - square test indicated that there was no significant difference in the number of correct answers for any question in relation to age, residing area, education level and previous experience. for question 10, 92.36% of parents felt that it is important to have an educational program in management of dental trauma. for question 11, 93.45% of parents were not satisfied with their knowledge on the management of dental trauma. for question 12, 95.13% of parents would like to attend an educational program on management of dental trauma. the study included 1500 parents attending opd of pedodontics and preventive dentistry, post graduate institute of dental sciences, rohtak, haryana, india who were assessed with the help of a questionnaire for their knowledge and awareness regarding emergency management of dental trauma in a child. the age, gender, education level, residing area and previous experience of dental trauma were recorded in part i of questionnaire. in part ii of questionnaire, an imaginary case of dental trauma was presented and questions were designed to test the parents knowledge. lastly, in part iii, attitude of parents towards education of dental trauma management was assessed. out of 1500 parents, only 490 (32.6%) recognized that maxillary incisor is a member of the permanent dentition in an 8-year - old boy. this indicated that many parents do nt realize or notice the age of transition from deciduous teeth exfoliation to permanent incisor eruption. also, this might be related to make sure that the tooth is not swallowed or inhaled by the child. a delay in providing emergency dental treatment may jeopardize the prognosis of an avulsed tooth. more than half of the parents (63.17%) recognized the urgency of seeking professional help as immediately for avulsion injury. however, 36.83% of the parents did not realise the importance of seeking immediate professional help and were concerned primarily with bleeding and pain control measures. pain constitutes one of the major reasons for seeking professional help. many parents (67.5%) did not feel the need to see a doctor if the child has no pain. so parents should be asserted about significance of seeking professional advice after dental trauma irrespective of pain. regarding the important question of replantation, 31.8% of the participants would have tried to put the tooth back in its socket ; although 68.2% of the parents were not confident about undertaking the tooth saving procedure or may not know how to do it. the reasons for reluctance to replant avulsed teeth could be related to lack of knowledge, hurting the child or to the felt urge to stop the bleeding, which is perceived by most people as life threatening. in cases with multi - injury trauma, the replantation of an avulsed tooth may require a low priority but in case of an isolated dental trauma, a simple procedure of replantation could make a huge difference not only in the prognosis of the tooth, but also influence the facial growth, function, esthetics and psychological impact on the patient. six parents chose to clean a soiled avulsed tooth by scrubbing with a toothbrush which might disrupt the periodontal ligament cells on the tooth surface and severely decrease the chance of successful replantation. seven hundred and seventy parents (51.3%) did not have any clue what to do and how to clean the tooth before replantation. there seems to be an urgent need to educate the public and correct the misconceptions. storing the avulsed tooth in a solution compatible with cell viability until replantation is a critical procedure however dry storage selection was prevalent among parents. for transport of an avulsed tooth, paper tissue or handkerchief was the favoured medium for 37.7% (n = 566) of parents followed by plastic wrap by 33.3% (n = 500). in a study on school teachers by chan., a large number of respondents chose ice or iced water as the preferred storage medium ; this may be related to the popular use of ice for transportation of human organs and accidentally detached limbs often reported in the mass media. although patient 's mouth may function well as storage medium, only seven parents were aware of that. the concept of dry storage among parents indicated the lack of knowledge on how avulsed teeth should be handled after an accident. they were not aware that dry storage during transport could seriously prejudice the normal healing process and the prognosis is related to injury to periodontal membrane during the time the tooth is out of its socket. dental trauma sequele like pulp necrosis and root resorption may present later sometimes and therefore successive visits are instrumental in timely detecting and treating such problems. however, more than half (51.6%) parents did not understand the value of follow - ups. the results of this study indicated low level of knowledge regarding tooth avulsion and replantation procedures to be followed in emergency. the residing area and age of parent did not affect their knowledge and awareness. moreover, well - educated parents also had very little or no information about dental trauma first - aid. the lack of significance in correct answers between those with and without such experience indicated that past experience did not seem to have increase the knowledge of the correct emergency procedures. this is because very little or no information about tooth avulsion and replantation had been given to most of them. providing information is a way to increase knowledge of dental first - aid. it would be beneficial if instructions regarding how to manage dental injuries are more widespread in society efforts should be made through population based preventive measures to ensure uniform knowledge about dental trauma. considering the frequency of dental injuries, it is remarkable that they are not included in general first - aid information. 95% of the participants in our study were willing to attend an educational program on dental trauma. most healthcare providers and educators would agree that prevention of injury is a more desirable course of action than dealing with the consequences, which usually means the treatment of injury. many dentoalveolar injuries can be prevented by the use of well - fitted properly constructed mouthguard in any sports in which there is a risk of sudden impact to the face however, the use of mouthguard is limited due a lack of perceived need for them and lack of information provided to the parents of children who participate in contact sports. unfortunately, even in the dental field, the prevention of accidental trauma to the teeth is perhaps overshadowed by the tremendous interest worldwide in the prevention and control of other dental diseases. dental injury prevention and management should be recognized as a major public health issue and adequate resources to be allocated for research in this area along with the development of prevention programmes. the basal findings of this study suggest that there is a lack of proper knowledge on emergency dental first - aid among the study participants. in india, no attempt has been made by the government or other dental organizations to educate people on the management of dental trauma. our findings emphasize a need for educational campaigns to broaden the knowledge of parents regarding emergency management of traumatic dental injuries apart from information on dental caries and brushing methods. mounting posters, leaflets at public places along with media campaigns will educate people for managing avulsed permanent teeth. | aim : traumatic dental injuries frequently occur in society and may occur at home. the ultimate prognosis of an avulsed tooth occurring in a child may depend on the parents knowledge of appropriate emergency measures. this study is aimed at evaluating the awareness level of a sample of indian (rohtak, haryana) parents in the management of dental trauma.materials and methods : a total of 1500 parents were surveyed using a self - administered structured questionnaire. the questionnaire was divided into three parts. the tabulated data were statistically analyzed using the chi - square test.result:this study indicated a low level of knowledge regarding tooth avulsion and replantation procedures to be followed in emergency. the residing area and age of parent did not affect the knowledge and awareness of parents. moreover, well - educated parents also had very little or no information about dental trauma first - aid. the lack of significance in correct answers between those with and without such experience indicated that past experience did not seem to have increase the knowledge of the correct emergency procedures. very little or no information about tooth avulsion and replantation had been given to most of them.conclusion:dental injury prevention and management should be recognized as a major public health issue and adequate resources to be allocated for research in this area. educational programs to improve the knowledge and awareness among the parents have to be implemented. |
the limited supply of fossil fuels and rising energy demands have encouraged research into renewable energy sources, with photovoltaics proving to be a suitable candidate. interest in organic and hybrid solar cells has grown due to their ability to produce flexible lightweight devices with low manufacturing costs and an abundant supply of environmentally friendly materials. small - scale single - junction organic solar cells now achieve power conversion efficiencies over 10%. the best performance is achieved using a bulk heterojunction where an electron donor (usually a conjugated polymer) and an acceptor (usually a fullerene) are blended to obtain a large interfacial area for charge carrier generation. progress has been spurred on by the synthesis of novel polymers and optimization of processing conditions. one of the most efficient blends is of the polymer ptb7 and the soluble fullerene pc71bm, and it achieves high efficiency using a high - boiling - point solvent additive 1,8-di - iodooctane (dio) for processing the active layer. the influence of dio on the morphology of ptb7:pc71bm blends has previously been investigated in detail, which indicated that the blends prepared without additive show pure fullerene clusters of 2060 nm in size which form large agglomerates embedded in a polymer - rich matrix containing about 30 wt % of fullerene. the addition of dio to the casting solvent improves the miscibility of pc71bm with ptb7, dramatically shrinks the size of the clusters to several nanometers, and forms interpenetrating polymer - rich and fullerene - rich phases of tens of nanometers in size. fluorescence quenching was found to be of similar efficiency in blends prepared with additive and without, suggesting that the increase of power conversion efficiency is not due to more efficient charge generation. this is consistent with the power conversion efficiency enhancements occurring in both the polymer and fullerene absorption regions, suggesting that the improvement results from reduced carrier recombination. the origin of reduced recombination is not known ; possible explanations include improved charge separation, higher carrier mobility, or reduced charge trapping. it is therefore clear that a detailed understanding of the free carrier generation, transport, and extraction in this important high - performance blend is lacking. in this work, we combine transient photocurrent measurements with ultrafast optical probes of carrier mobility to investigate charge separation and motion dynamics in ptb7:pc71bm solar cells in order to understand the reasons behind the increased efficiency of devices prepared with the additive dio. we demonstrate that the increase of photocurrent in devices prepared with dio occurs because of a higher dissociation efficiency of photogenerated charge pairs into free carriers. the enhancement of the carrier extraction rate is observed at a low built - in electric field and can contribute to a higher fill factor of dio - prepared solar cells. we measured time - dependent carrier mobility in devices prepared using different blending ratios and show that carrier transport is limited by connectivity of fullerene / polymer domains in addition to trapping at low energy sites. these results give new insights into the influence of morphology on charge separation and transport, providing a full picture of the dynamics of these processes helping to understand the operation mechanisms and the ways to improve the performance. ptb7 with a molecular weight of 92000 and a polydispersity of 2.6 was obtained from 1-material, inc. pc71bm of 99% purity was obtained from solenne and dio from sigma - aldrich. ptb7 and fullerene were dissolved in chlorobenzene (hplc grade from sigma - aldrich) at ratios of 90:10, 40:60, 20:80, and 10:90 by weight and stirred at 50 c for 45 h. we will call these blends [p90:f10 ], [p40:f60 ], [p20:f80 ], and [p10:f90 ], respectively, where the first number denotes the relative amount of the polymer. for the samples prepared with dio, 3% of it by volume was added to the solution which was then stirred for a further 5 min. the blends were spin - coated on a 40 nm layer of pedot : pss which was spin - coated on an indium tin oxide coated glass substrate. the thickness of the ptb7:fullerene layer was 115 nm. the layers of calcium (20 nm) and aluminum (100 nm) were subsequently deposited by vacuum sublimation. the current voltage characteristics were measured under am1.5 conditions using a solar simulator from sciencetech and the intensity calibrated with an oriel reference cell with kg5 filter. an aperture of the same size as the pixel was used to avoid contribution from stray light outside the device area. to measure the carrier - drift dynamics, we have used the time - resolved electric - field - induced second - harmonic generation (trefish) technique, which was described in detail elsewhere. briefly, a pulsed p - polarized probe light is shone on a cell at 45 incidence angle. an applied electric field f breaks centrosymmetry of the blend for incident light so that the second harmonic of the probe light (sh) is generated and its intensity is proportional to f. an optical pump pulse which overlaps spatially with the probe light generates charge pairs which drift in response to electric field, and so they shield the internal electric field and reduce the sh intensity. in this way, the intensity of sh can be used to directly monitor carrier drift in the cell by changing the time delay between pump and probe pulses. the solar cell acts as a parallel plate capacitor ; thus, f = (uappl + w / e)/l where uappl is the applied reverse bias, w = 0.8 ev is the difference of work functions of the electrodes, l is the distance between the electrodes, and e is the elementary charge. the sh dependence on the applied voltage without a pump pulse was measured first and used later to obtain the strength of the internal electric field from the trefish signal. probe pulses at 800 nm and 1 khz repetition rate were generated by a regenerative amplifier (quantronix integra - c), and sh was detected using a photomultiplier tube. the pump pulses were generated in an optical parametric amplifier topas - c (light conversion) and excited the cell at 500 hz, i.e., at every other probe pulse. the pump wavelength was 680 nm, which is the absorption maximum of the polymer. the integral - mode photocurrent transients were measured simultaneously using the same pump pulses and recording the voltage on a 10 k load resistor connected in series with the solar cell using a 500 mhz bandwidth agilent technologies oscilloscope dso5054a. the pump pulse energy was adjusted to give photocurrent transients which were independent of pump energy, indicating negligible nongeminate recombination of charge pairs. the measurements were performed using pump pulse energy densities of 80, 120, 160, and 500 nj cm in the solar cells prepared with the ptb7:pc71bm blending ratios of 40:60, 20:80, 10:90, and 90:10 by weight, respectively. figure 1 shows the current voltage characteristics of the solar cells at the optimum blend ratio [p40:f60 ], prepared with and without dio measured under am1.5 illumination conditions. the device prepared with dio shows about two times higher short - circuit current (jsc) and fill factor (ff). the open circuit voltage (voc) does not depend on the solvent additive. the power conversion efficiency (pce) of the device prepared with dio was 5.4% at am1.5 conditions. it is important to note that our device stack has identical active layer properties (blend ratio, solution concentration, spin - coating deposition parameters, thickness, deposition onto pedot) as the best reported devices but suffers from lower overall performance owing to nonoptimized contacts, giving us efficiencies comparable to those which others report with similar nonoptimized contacts. nonetheless, we still see the same dramatic improvement in overall device performance with the addition of dio and can use this as a basis for understanding in this article how the changes in morphology that are known to be occurring influence the electronic properties of the device. current voltage characteristics of the ptb7:pc71bm [40:60 ] solar cells under 1 sun illumination. figure 2 shows the photoinduced voltage drop u in the devices with 60 wt % of pc71bm which have the highest power conversion efficiency. it is measured by trefish in the time range from 0 to 3 ns using u = f(t)l where f(t) is a decrease of the electric field strength due to carrier displacement and l is the sample thickness. the combination of the two techniques enables us to monitor carrier drift kinetics from subpicoseconds to tens of microseconds, starting from their photogeneration up to extraction from the devices. the photoinduced voltage drop is proportional to the amount of extracted charge in a time interval t1where c is the sample capacitance and i(t) is photocurrent. the voltage drop at long times is proportional to the total amount of extracted charge from the device. at 1000 ns, it is two times bigger in devices prepared with dio, which agrees well with the approximately two times higher photocurrent observed in current voltage characteristics at the short - circuit condition and at reverse bias (figure 1). in order to more clearly compare the extraction kinetics, figure 2 presents the voltage kinetics for the sample without dio normalized to that for the sample with dio. they show that charge extraction at the 3 v bias is only marginally faster from the cell prepared with dio. this indicates that the extraction efficiency of free carriers with a strong internal field is not particularly sensitive to morphology and suggests that the increased short circuit photocurrent in devices prepared with dio is mainly due to more efficient charge separation rather than extraction. combining this with previous observations that the charge generation efficiency is similar in blends prepared with additive and without, we conclude that the increase of photocurrent in devices prepared with dio occurs mainly because of a higher dissociation efficiency of photogenerated charge pairs. the charge extraction at 0 v bias is faster in a device prepared with dio as the time taken to extract a half of the charge decreases from 60 ns without dio to 40 ns with dio. in this case, the internal electric field in the cell is lower than at the short circuit condition because the total voltage drop on the load resistor comes to nearly a half of the built - in voltage. a faster charge extraction from dio - prepared devices at low built - in field reduces recombination losses and can explain the higher fill factor of solar cells prepared with dio. photoinduced voltage drop in devices with 60 wt % of pc71bm at 3 v reverse bias (a) and at the built - in field only (b) as a function of time after the pump pulse. the results are derived from trefish measurements up to 3 ns and from the integral - mode photocurrent at longer times (note log scale after the axis break). black dotted curves show the kinetics from devices without dio which are scaled to match the voltage drop in devices with dio at late times. pump density was 4 10 absorbed photons / cm at the peak of polymer absorption (680 nm). in order to explore the influence of electric field, blend ratio, and the use of dio on the charge separation efficiency, we studied the dependence of the amount of extracted charge in 1 s on the applied external voltage (figure 3). the amount of extracted charge from the cell with 10 wt % fullerene shows approximately linear dependence on the reverse bias with no saturation even at 4 v. at its maximum, u from this cell is still about 20 times lower than from the cells with high fullerene content. this indicates that in the blend with low fullerene content the charge pairs are strongly bound and can only be separated by strong electric fields. it is interesting to note that the additive dio decreases the amount of extracted charge from the polymer - rich blend, which is opposite to what is observed in the fullerene - rich blends, and the reduction factor is independent of the applied bias. in contrast, the amount of charge extracted from other three blends is much higher and shows a weak dependence on the bias between 1 and 4 v indicating that a weaker electric field is sufficient to drive charge separation in fullerene - rich blends. this result is consistent with previous observations of efficient free carrier generation in fullerene - rich blends and much higher initial electron mobility in the fullerene than the hole mobility in the electron donors observed by means of thz spectroscopy. the amount of charge extracted from the blends with 80 and 90 wt % fullerene increases by about 30% with the increase of the negative bias between 1 and 4 v (figure 3 c, d). we attribute this to carrier generation from excitons deep inside pc71bm domains, a process which is assisted by an electric field. at high pc71bm concentrations the absorption by fullerene at the excitation wavelength (680 nm) is comparable to that of the polymer and many excitons are generated deep inside the fullerene phase. the three - dimensional exciton diffusion length in pc71bm can be estimated as where d is exciton diffusivity and is the exciton lifetime. using the reported d and values, we estimate the exciton diffusion length of about 7 nm in the bulk pc71bm. with polymer concentrations at only 1020 wt % not all excitons created in fullerene domains reach the heterojunction. charge carriers can be generated inside the fullerene domains with the assistance of an electric field as observed previously which can explain why the free carrier yield is bias - dependent. this suggests that carrier generation mechanism in the fullerene - rich blends could be different, depending if photon is absorbed by the polymer or by pc71bm. photoinduced voltage drop at 1 s from the integral - mode photocurrent measurements vs an applied external voltage in devices with different blend ratios for the excitation density of 4 10 absorbed photons / cm. dotted black and blue curves correspond to the devices without and with dio, respectively. in order to understand the role of electron and hole motion in charge separation and the effect of morphology we studied the time - dependence of carrier extraction from devices prepared with different blend ratios (figure 4). the blends with high fullerene content show an enhancement of the amount of extracted charge when prepared with dio. the enhancement factor is slightly lower than that observed in a blend with 60 wt % fullerene (figure 2). all fullerene - rich blends show a fast extraction phase within 10 ns which is followed by a slow phase on the 10100 ns time scale. the fast phase gets faster with increasing fullerene content. in the blend with 60 wt % fullerene a half of the mobile carriers are extracted in 10 ns at a 3 v reverse bias (figure 2a), whereas in the blend with 80 wt % fullerene this happens in 2 ns (figure 4b). even faster carrier extraction is observed in the blend with 90 wt % fullerene, which is complete in 0.5 ns. this is similar to previous observations of very fast electron extraction from neat pc61bm films. in contrast, carrier extraction from the polymer - rich blend is much slower and occurs on a 1001000 ns time scale. this trend allows us to assign the fast extraction phase in fullerene - rich blends to electrons and the slow phase to holes. the additive dio enhances the electron extraction rate from the fullerene - rich blends, but slightly slows down carrier extraction from the polymer - rich blend. photoinduced voltage drop for the pump density of 4 10 absorbed photons / cm in ptb7:pc71bm devices with very low and high fullerene content at 3 v reverse bias. black dotted curves show the kinetics from devices without dio which are scaled to match the voltage drop in devices with dio at long times. in order to evaluate the electron and hole mobilities and their dependence on the blend ratio, we have fitted the photoinduced voltage drop to a sum of integrated electron and hole photocurrents2 here, e is the electron and hole charge, f is the electric field strength, a is the area illuminated by the pump pulse, c is the cell capacitance, ne, h(t) = n0(1 e, h(t)/l) are the average electron and hole concentrations in the cell with an account for carrier extraction, e, h are their mobilities, n0 is the density of generated charge pairs, and e(t) and h(t) are the average electron and hole drift distances e, h(t) = f 0te, h(t) dt. the model assumes homogeneous carrier generation within the sample thickness. we approximated the electron and hole mobilities by functions e, h(t) = 0e, ht to account for their time dependences. because of very low excitation densities, the losses to nongeminate carrier recombination are negligible. figure 5 presents the fits to the voltage kinetics for blends without dio as well as the electron and hole contributions to it. the simulations reproduce the experimental results well for the [p40:f60 ] and [p20:f80 ] samples, while the fits for the two extreme blend ratios are much worse. it is apparent that the power law functions can not adequately describe the time dependences of the carrier mobilities at very low donor or acceptor concentrations. this is not surprising because these concentrations are at the percolation limit, and one type of carriers will inevitably encounter dead ends of charge transporting domains. as figures 2 and 4 show, normalized carrier motion dynamics in blends with and without dio are very similar, giving very similar mobility values and kinetics ; therefore, they are not presented and we will not specify it in discussion. the simulated electron and hole contributions to integrated current show that just about 50% of electrons are extracted in 1 s from the blend with 10 wt % of fullerene (figure 5a) and only about 2% of holes from the blend with 90 wt % of fullerene (figure 5d). this indicates that the extraction of the rest of carriers takes much longer time than 1 s in these blends with extreme ratios ; thus, carrier extraction is strongly unbalanced. in solar cells with the optimal blend ratio of 60 wt % of fullerene electrons are extracted in 40 ns at a 3 v bias, whereas the hole extraction takes 200 ns. the insets show the simulated electron and hole mobilities. in the blends with the fullerene content of 60, 80, and 90 wt %, the electron mobility in the first 10 ps after the pump pulse is > 0.1 cm v s. high electron mobility drives fast dissociation of generated charge pairs into free carriers. the blend with 90 wt % of fullerene shows a weak e t time dependence which can be explained by electron trapping at low energy sites. for comparison, the electron mobility in blends with 60 and 80 wt % fullerene shows a stronger time dependence e t, suggesting that there is another mechanism that slows down the electron mobility in addition to relaxation to low energy sites. it is natural to assume that the electron mobility decreases when electrons reach the boundaries of fullerene clusters and fullerene - rich domains. this assumption is supported by an about 10 times lower initial electron mobility in the blend with 10 wt % fullerene and its rapid decrease with time as e t, both showing that the electron transport is at a percolation limit in this blend. the highest initial hole mobility is observed in the optimized blend with 40 wt % of polymer and not in the blend with 90 wt % of polymer, indicating that the slow dissociation of bound electron hole pairs limits the hole mobility in the polymer - rich blend. the time - dependence of hole mobility is very similar to that of electrons and follows h t in the polymer - rich blend, which changes to h t in the blend with 10 wt % of the polymer. our results suggest comparable hole and electron mobilities of around 10 cm v s in the blends with 60 wt % fullerene at 100 ns. this is in good agreement with the previously reported electron and hole mobilities for space - charge - limited current in blends of ptb7 with the fullerene pc61bm with the same blend ratio. the same study has reported a sharp decrease of hole mobility when the fullerene concentration increased beyond 83 wt %, which also agrees with our results. this suggests that approximation of the time - dependent carrier mobility using the power law functions give a realistic separation of the integrated electron and hole photocurrents. our results also suggest that the mobility of both types of carriers is controlled by low energy and spatial traps in the blends. the initial values of hole mobility, however, are not strongly dependent on the blend ratio. this can be explained by the faster hole transport along the conjugated polymer chains as compared to transport between chains. modeling results of the carrier extraction kinetics from the cells prepared with different blend ratios without dio at 3 v reverse bias. black lines are the experimental data, green lines are the modeled kinetics, blue and red lines show electron and hole contributions, respectively. insets show time - dependent electron (blue lines) and hole (red lines) mobility obtained from the fits. let us discuss our experimental findings by taking into consideration the results of previous studies of blend morphology and the influence of the solvent additive. in the most efficient blend of [p40:f60 ] with dio, an optimum morphology of a finely interpenetrating network of ptb7 and pc71bm is formed and the total amount of extracted charge is double that of a blend prepared without dio. here, we have been able to show that this enhancement is based on a higher dissociation efficiency of generated charge pairs into free carriers which is independent of the applied electric field. an internal quantum efficiency of > 0.9 has been demonstrated in ptb7:pc71bm solar cells prepared with dio, implying that the free carrier generation efficiency is close to unity in optimized blends. since the charge - generation efficiency was found to be similar in blends prepared with additive and without, this indicates that about a half of generated charge pairs in the blends prepared without dio never dissociate into free charge carriers. previous studies of morphology and photophysics of ptb7:pc71bm blends prepared without additive showed large pure fullerene clusters of 2060 nm in size which were embedded in a polymer - rich phase with about 30 wt % of fullerene mixed in it. based on our observations of the pair dissociation efficiency being low in polymer - rich blends, we suggest that the geminate charge pairs generated between polymer and dispersed fullerene molecules in the polymer - rich phase never dissociate, as illustrated in figure 6. this happens because the hole mobility in ptb7 is inherently low and fullerene molecules in the polymer - rich phase are too far from each other. ptb7:pc71bm blends prepared using the additive dio have been shown to consist of interpenetrating polymer - rich and fullerene - rich domains of tens of nanometers in size, and large pure domains were no longer observed. these polymer- and fullerene - rich domains imply that there is a concentration gradient of donor and acceptor molecules between these domains. in such a morphology, charge separation can be thermodynamically driven as the entropy increases with carrier motion from the lower to higher concentration of acceptor molecules. for efficient charge separation all fullerene molecules have to be well connected to provide unperturbed electron motion. an insight into the development of a well - connected fullerene network in blends of ptb7 with a fullerene pc61bm processed with dio has been given by in situ measurements of the grazing incidence small - angle x - ray scattering during solvent evaporation. these studies showed rapid formation of crystalline ptb7 aggregates driven by rapid evaporation of chlorobenzene but spontaneous phase separation was not observed presumably because of very slow evaporation of dio which is a good solvent for fullerene molecules. shrinkage of the fullerene domains in ptb7:fullerene blends with addition of dio is different from dio s role in altering the morphology in other photovoltaic blends where it promotes phase separation of two intimately mixed materials. morphology optimization increases the free carrier yield, the short circuit current and the fill factor, whereas the open circuit voltage (voc) is not affected by it (figure 1). the contacts in these cells are nonselective (no charge blocking layers) ; therefore, voc is determined not only by the built - in potential and charge transport but is also reduced by surface recombination (extraction to wrong contacts by diffusion). we speculate that in our case the voltage loss to surface recombination is entirely determined by contacts which can explain why voc is not different. according to previous morphology studies, the blends prepared without additive form pure fullerene clusters of 2060 nm in size which are embedded in a polymer - rich phase with about 30 wt % fullerene mixed in it. the charge pairs generated at the dispersed fullerene molecules do not dissociate because of low hole mobility, and only charge pairs generated at the fullerene clusters give photovoltaic response. the addition of dio to the casting solvent improves the miscibility of pc71bm with ptb7 and forms interpenetrating polymer - rich and fullerene - rich phases of tens of nanometers in size. charge separation in these blends is driven by fast electron motion in the fullerene - rich phase. small - scale phase separation also improves charge transport at low built - in field. the increase of free carrier yield with dio is also observed in blends with 80 and 90 wt % fullerene, but the effect is less pronounced presumably because the fullerene molecules are better connected at higher concentration even without dio. we also found that dio increases the carrier extraction velocity at weak electric field which can be explained by improved connectivity of nanostructured polymer - rich and fullerene - rich domains providing faster percolation channels for extraction of both types of carriers. the dio enhances the performance of all fullerene - rich blends, but it reduces the amount of extracted charge from the polymer - rich blend. even though the charge separation in the polymer - rich blend shows strong electric field dependence, the reduction factor of extracted charge with dio is field - independent, suggesting that dio changes the morphology of this nonoptimum polymer - rich blend, too. transient photocurrent and ultrafast optical measurements of carrier drift enabled us to measure charge - separation dynamics and electron and hole extraction in ptb7:pc71bm solar cells with different blend ratios. we find that the short circuit current is determined by the separation of charge pairs into free carriers. this separation is found to be efficient in fullerene - rich blends and inefficient in the polymer - rich blends, suggesting that high mobility of one type of carriers is essential for efficient charge separation. the free carrier yield is higher by a factor of 2 in devices prepared with dio than without at the optimal blend ratio. half of the generated charge pairs in the blends prepared without dio never dissociate into free charge carriers, which we attribute to charge pairs generated in the polymer - rich domains with molecularly dispersed fullerene molecules. the carrier extraction rate slightly increases with morphology optimization with dio, but only at a low built - in electric fields. these findings explain the higher photocurrent and fill factor of devices prepared with dio. our results also show that carrier extraction is unbalanced in optimized devices with electron extraction being about 10 times faster than the hole extraction. | charge separation and extraction dynamics were investigated in high - performance bulk heterojunction solar cells made from the polymer ptb7 and the soluble fullerene pc71bm on a broad time scale from subpicosecond to microseconds using ultrafast optical probing of carrier drift and the integral - mode photocurrent measurements. we show that the short circuit current is determined by the separation of charge pairs into free carriers, which is strongly influenced by blend composition. this separation is found to be efficient in fullerene - rich blends where a high electron mobility of > 0.1 cm2 v1 s1 is observed in the first 10 ps after excitation. morphology optimization using the solvent additive 1,8-diiodooctane (dio) doubles the charge pair separation efficiency and the short - circuit current. carrier extraction at low internal electric field is slightly faster from the cells prepared with dio, which can reduce recombination losses and enhance a fill factor. |
yeast vectors pgbad - b and pact2-b were obtained from d. markie (university of otago, nz) (16). the plasmids ldr and cf used for the fkbp - frb dimerization system were gifts of t. meyer (stanford university) (17). unless otherwise stated, the pkd2 plasmids used in this work have been previously reported (18, 19). n - terminal ha - tagged full - length and mutant (l703x) pkd2 constructs were created by replacing an xbai and sacii fragment of a wild - type pkd2 plasmid (gift of s somlo, yale university) with the same fragment excised from the previously described ha - l224x plasmid (19). a c - terminal ha - tagged pkd2 mutant construct, r742x, was generated by pcr using the wild - type pkd2pk plasmid as a template including the ha epitope tag sequence and in - frame stop codon in the reverse primer. the missense pkd2 mutation, d511v, was created by site - directed mutagenesis in the pkd2pk plasmid template using a previously published protocol (19). the n - terminal myc - tagged l224x plasmid was generated by pcr and subcloned into the xbai and hindiii sites of pcdna3.1 (-). the plasmids cfp - pkd2-(1177) and cfp - pkd2-(1223) were generated by fusing the n - terminal sequences of pkd2 in - frame with the cfp and fkbp cassette in the vector, cf. immunoblotting and immunoprecipitation hek-293 and mimcd3 cells were cultured in dulbecco 's modified eagle 's medium supplemented with 10% fetal bovine serum. transient transfection was carried out on cells cultured to 90% confluency using lipofectamine 2000 (invitrogen) according to the manufacturer 's instructions. immunoblotting and immunoprecipitation (ip) was performed as previously described using epitope - specific antibodies (10). the pkd2 antibody, p30, generated to the c - terminal 258 amino acids of human pc2, has been previously reported (18). yeast two - hybrid assays yeast two - hybrid assays were performed in the yeast strain ah109 containing ade2, his3, and lacz reporter genes under the control of the gal4 upstream activating sequences (uas) by co - transforming bait and prey constructs of the entire intracellular n terminus of human pc2 (nt2) or its truncations into yeast cells using a published protocol (20). full - length nt2-(1223) was subcloned into bait (bd) vector pgbkt7 and prey (ad) vector pgadt7 (clontech), respectively, by pcr and dna ligation. similarly, the following nt2 truncations, nt2-(1207), nt2-(1198), nt2-(1177), nt2-(1117), nt2-(161), and nt2-(178223) were constructed in both bait and prey vectors. after incubation at 30 c for 34 days, positive co - transformants (containing both bait and prey plasmids) were picked from selective medium s.d./-lt (lacking tryptophan and leucine) and restreaked onto selective media s.d./-lth (lacking tryptophan, leucine, and histidine), s.d./-lth with 2 mm 3-amino-1,2,4-triazole (3-at), and s.d./-ltah (lacking tryptophan, leucine, histidine, and adenine), respectively, to activate the reporter genes his3 and ade2. 2 mm 3-at was used in the selective medium to suppress minor autoactivation of nt2-(1198) and nt2-(1177) in the pgbkt7 vector. the constructs pgbkt753 (p53) and pgbadt7-t (sv40 t - antigen) were used as a pair of positive controls. truncations of the pc2 c terminus (ct2) and pc1 c terminus (ct1), which mimic two naturally occurring mutants lacking their respective interaction domains i.e. pkd2-r742x and pkd1-r4227x, respectively, were generated as a pair of negative controls : pgbad - b - ct1 (41074227) and pact2-b - ct2 (680742). at least three individual colonies were chosen from each plate to quantify growth. live cell imaging imcd3 and hek-293 cells were transiently transfected using lipofectamine 2000 (invitrogen) with the following cdnas : cfp - fkbp - l223 (cf - pkd2223), cfp - fkbp - l177 (cf - pkd2177), and lyn11-frb (ldr). translocation of the fusion proteins to the cell surface was induced 24 h later by the addition of 10 m rapamycin (calbiochem) to the culture media. in some experiments, co - expression of a surface - localized yfp fusion protein (c1 domain of pkc-1, yfp - c1 (pkc)) was used to outline the plasma membrane. live cell measurements were performed on an olympus imaging systems inverted ix-71 microscope with a cfp and yfp filter set to capture cellular fluorescence images with a ccd camera (hamamatsu), driven by simple pci software (c imaging systems). changes in the mean fluorescence intensity over a given region of the cytosol and nucleus were quantified in individual cells (n = 6) using imagej (nih) (21). electrophysiology the whole cell voltage - clamp configuration was used in the perforated mode to measure transmembrane currents in single cells. briefly, patch - clamp recordings were obtained from single cells at 37 c using a warner pc-505b amplifier (warner instrument corp., hamden, ct) and pclamp 8 software (axon instrument, foster city, ca). glass pipettes (plain, fisher scientific) with resistances of 58 m were prepared with a pipette puller and polisher (pp-830 and mf-830, respectively, narishige, tokyo, japan). after the whole cell configuration was achieved, cell capacitance and series resistance were compensated before each recording period. current measurements in time course experiments were made by applying a 100-ms pulse from a holding potential -60 mv to -100 every 10 s for 5 min. current amplitude was extracted at -100 mv and plotted as function of time (min). i - v curves were derived from a voltage step protocol as follows : from a holding potential of -60 mv, voltage steps were applied from -100 to 100 mv in 20-mv increments with 200 ms duration at 3-s intervals. current traces were filtered at 1 khz and analyzed off - line with pclamp 8. the pipette solution contained (in mm) : 0.3 amphotericin b, 110 potassium aspartate, 30 kcl, and 5 hepes, ph 7.2. the bath solution contained (in mm) : 130 kcl, 1 mgcl2, 10 hepes, 0.1 cacl2, and 5 glucose (ph 7.4). zebrafish experiments wild - type zebrafish ab strains were maintained and staged according to standard protocols (22). embryos were treated with 0.003% 1-phenyl-2-thiourea (sigma) in 0.5 e2 solution to reduce pigmentation. at the one - cell stage, embryos were microinjected with pkd2atgmo or control mo (gene tools llc) to block zebrafish pkd2 translation as described (23) at final mo concentrations estimated at 100 nm. in vitro - transcribed capped mrna encoding human pkd2-l177, -l223, and d511v were synthesized using a mmessage mmachine t7 kit (ambion), and 50 pg were injected or co - injected at the one - cell stage. injection solution conditions have been described in a previous report (19). rt - pcr total rna was isolated from 48 hpf embryos by using the rnaqueous-4pcr kit (ambion). nested rt - pcr primers were designed from human pkd2 sequence to confirm expression in zebrafish embryos. rt - pcr was performed using the superscript iii one - step rt - pcr system with platinum taq high fidelity (invitrogen) followed by a second pcr using phusion high - fidelity dna polymerase (new england biolabs). the human pkd2 primers used were : 148f1 : ggcctggagatcgagatg, 332r1 : accaccattccgccttct, 149f2 : gcctggagatcgagatgc, 320r2 : ccttcttccccttccacct. after rehydration, embryos were washed in pbs plus 0.5% triton x-100 and blocked in pbs - dtr (pbs plus 1% dmso, and 0.5% triton x-100) with 10% normal goat serum (ngs) at room temperature for 1 h. anti - polycystin-2 (1:800 ; gift of i. drummond) (24) was incubated in pbs - dtr 2% ngs for 4 c overnight. embryos were washed in pbtx (pbs/0.5% triton x-100) for 2 h and then incubated in 1:1000 cy3-labeled goat anti - rabbit igg (jackson immunoresearch) in pbs - dtr 2x ngs at room temperature for 2 h. after rinsing the embryos in pbtx for 2 h, they were dehydrated, embedded in jb-4 (polyscience), and sectioned at 10-m thickness. sections were stained with 1:1000 dapi (kpl) solution for 3 min at room temperature, the slides rinsed once with pbs and then mounted with fluorescent mounting medium (kpl). section images were taken using a nikon e500 microscope and analyzed by the spot v. 4.4 program. histochemistry48-days postfertilization (dpf) embryos were fixed in 1.5% glutaraldehyde (electron microscopy sciences), 1% paraformaldehyde (electron microscopy sciences), 70 mm napo4 ph 7.4, 3% sucrose at 4 c overnight, dehydrated, embedded in glycolmethacrylate (jb4 ; polyscience), and sectioned at 4-m thickness using hm325 (richard - allan scientific). slides were stained with hematoxylin and eosin (bbc biochemical) according to the protocol from jennifer r. panizzi and lilianna solnica - krezel.4 evidence for a second dimerization domain in pkd2we initially generated three naturally occurring pkd2 mutants, one with a single missense change in the third transmembrane domain (d511v) and two others lacking the c - terminal homodimerization domain, l703x and r742x (fig. the d511v mutation has previously been shown to completely abolish pc2 channel activity whereas l703x and r742x have altered channel properties in some expression systems (12, 25). relevant to the present study, d511v has been demonstrated to function as a dominant negative interfering allele in mimcd3 (9) and llc - pk1 cells (26). as previously shown for endogenous pc2 (10), full - length pkd2 could be detected mainly as monomers under normal reducing conditions on sds - page but as oligomers under non - reducing conditions (fig. apart from prominent dimers, several other high molecular weight bands were detected under non - reducing conditions. these could represent homophilic binding between pc2 subunits or heterophilic interactions with other proteins. pkd2-d511v, showed a similar pattern of oligomerization under these conditions (data not shown). unexpectedly we found that pkd2-l703x, could still form dimers under non - reducing conditions (fig. as predicted, pkd2-d511v bound to wild - type pkd2, but unexpectedly, so did pkd2-r742x in co - immunoprecipitation (co - ip) assays (fig. 2, b and c). to exclude the effect of the epitope tags in the interaction, we repeated these experiments with different pkd2 full - length (myc - tagged) and mutant (c - terminal ha - tagged) constructs and obtained similar results (data not shown). we therefore hypothesized that a proximal interacting domain could lie upstream of the previously described distal c - terminal domain, possibly in the n terminus. indeed, the isolated pc2 n - terminal domain (nt2-(1223)) could interact with itself in hek293 cells (fig. figure 1.schematic diagram of epitope - tagged pkd2 constructs used in the study with their predicted domains. apart from wild - type pkd2, a site - specific mutant (d511v), two mutants with truncations of the c terminus (r742x and l703x), and the isolated n - terminal domain (l224x) were generated for study. schematic diagram of epitope - tagged pkd2 constructs used in the study with their predicted domains. apart from wild - type pkd2, a site - specific mutant (d511v), two mutants with truncations of the c terminus (r742x and l703x), and the isolated n - terminal domain (l224x) were generated for study. defining the dimerization domain using yeast two - hybrid assays to characterize and refine this domain further, yeast two - hybrid assays were performed. 3, yeast co - transformants containing nt2-(1223)-pgbkt7 (bait) and nt2-(1223)-pgadt7 (prey) were able to grow on selective media requiring the activation of two reporters i.e. his3 and ade2. nt2 did not interact with the c terminus of pc2 (ct2, data not shown). further studies were carried out to refine the minimal interacting region using deletion constructs. as showed in fig. 3, the truncation nt2-(1207) could still interact with nt2-(1223), while truncations nt2-(1177) and nt2-(1198) lost the ability to interact with nt2-(1223). shorter sequences of nt2 such as nt2-(161) and nt2-(1117) were unable to interact with full - length nt2-(1223) (not shown). these results indicate that the region from codons 199207 is an essential part of the n - terminal interacting domain. the sequence nt2-(178223) showed a weaker interaction than nt2-(1223) in our assay (data not shown) suggesting that flanking sequences might be important in determining binding affinity. 3c shows the high sequence conservation of this region between human, mouse, and zebrafish pc2. between codons 190 and 207, 12 of 17 amino acids (70.6%) are identical or similar compared with human / mouse pc2. this contrasts with the minimal sequence conservation between human and zebrafish pc2 in the preceding sequence of nt2 (codons 119189). figure 2.biochemical evidence for a proximal dimerization sequence in the n terminus of pc2. a, detection of epitope - tagged wild - type and mutant pc2 expressed in hek293 cells by immunoblotting under reducing (r) and non - reducing (nr) conditions in three individual samples. note that both forms of pc2 are detected predominantly as monomers under reducing sds - page but that there are prominent slower migrating species visible under non - reducing states. for full - length pc2, apart from prominent dimeric species (220 kda), there are also higher bands suggestive of more complex oligomeric structures (e.g. tetramers). for l703x, the monomeric species are more notable than for wild - type pc2 under nr conditions but there is also detectable dimer formation (150kda). b, coimmunoprecipitation of epitope - tagged pc2-d511v (d511vpk) and wild - type pc2 (ha - pkd2). c, pc2 mutant lacking both c - terminal dimerization domains (ha pkd2 r742x) associates with wild - type pc2 (pkd2pk). the p30 and pk antibodies only recognize c - terminal epitope in full - length pc2. however, the ha antibody, which only recognizes the mutant r742x was able to co - ip wild - type pc2 (pkd2pk) suggesting an interacting sequence proximal to the truncation. 1 mg of total cell lysate was used for ip with ha or nis (non - immune serum) and 0.1 mg (1/10) for ip with p30 as indicated. 30 g of lysate were loaded as a positive control. the converse experiment showed that p30 or pk antibodies could pull - down r742x. d, co - immunoprecipitation of ha - tagged n - terminal pc2 protein (nt2) containing the first 223 amino acids (l224x) with co - expressed myc - tagged l224x. a, detection of epitope - tagged wild - type and mutant pc2 expressed in hek293 cells by immunoblotting under reducing (r) and non - reducing (nr) conditions in three individual samples. note that both forms of pc2 are detected predominantly as monomers under reducing sds - page but that there are prominent slower migrating species visible under non - reducing states. for full - length pc2, apart from prominent dimeric species (220 kda), there are also higher bands suggestive of more complex oligomeric structures (e.g. tetramers). for l703x, the monomeric species are more notable than for wild - type pc2 under nr conditions but there is also detectable dimer formation (150kda). b, coimmunoprecipitation of epitope - tagged pc2-d511v (d511vpk) and wild - type pc2 (ha - pkd2). c, pc2 mutant lacking both c - terminal dimerization domains (ha pkd2 r742x) associates with wild - type pc2 (pkd2pk). the p30 and pk antibodies only recognize c - terminal epitope in full - length pc2. however, the ha antibody, which only recognizes the mutant r742x was able to co - ip wild - type pc2 (pkd2pk) suggesting an interacting sequence proximal to the truncation. 1 mg of total cell lysate was used for ip with ha or nis (non - immune serum) and 0.1 mg (1/10) for ip with p30 as indicated. the converse experiment showed that p30 or pk antibodies could pull - down r742x. d, co - immunoprecipitation of ha - tagged n - terminal pc2 protein (nt2) containing the first 223 amino acids (l224x) with co - expressed myc - tagged l224x. induction of zebrafish pronephric cyst formation by co - injection of pkd2-l223 mrna we have previously established the zebrafish as a relevant model system to study human adpkd (19, 24). disruption of zebrafish pkd2 expression with morpholinos (mo) results in cyst formation in the glomerulus and pronephric tubules in conjunction with changes in body axis curvature and hydrocephalus. all of these features were rescued by co - injection of human pkd2 mrna (19, 24). because of sequence conservation between humans and zebrafish, we reasoned that the zebrafish model could be used to test the functionality of the n - terminal domain of pc2 by a dominant negative mechanism. these results are summarized in table 1. to establish if a dominant negative effect could be observed in this system, we initially tested the effect of injecting human pkd2-d511v synthetic mrna into one - cell stage zebrafish embryos. 4e) induced the same phenotypic changes seen in pkd2atgmo - injected embryos (fig., co - injection of pkd2-d511v mrna with pkd2atgmo could not rescue the pkd2atgmo induced phenotype (fig. 4f) unlike wild - type pkd2 mrna. therefore, these results established a a dominant negative mechanism for pkd2-d511v in zebrafish and fully supported previous data using the same construct in mimcd3 cells (9). table 1zebrafish phenotypes after injection of capped pkd2 mrna and/or pkd2 morpholino at the one - cell embryo stagetotal embryos injectednormal embryoscysts and body axis curvature a. control mo 303 303 (100%) 0 (0%) b. human pkd2-l177 254 250 (98%) 4 (2%) c. human pkd2-l223 252 20 (8%) 232 (92%) d. pkd2atgmo 222 12 (5%) 210 (95%) e. human pkd2-d511v 296 6 (5%) 281 (95%) f. pkd2atgmo + human pkd2-d511v 103 2 (2%) 101 (98%) zebrafish phenotypes after injection of capped pkd2 mrna and/or pkd2 morpholino at the one - cell embryo stage figure 3.dimerization of the polycystin-2 n terminus (nt2) detected by yeast two - hybrid assays. a, growth of yeast co - transformants cultured on selective media s.d./-lth with 2 mm 3-at and s.d./-ltah to activate his3 and ade2 selection markers, respectively. the pairs of nt2 sequences tested are numbered from 14 and their sequences indicated. constructs pgbkt753 (p53) and pgbadt7-t (sv40-t antigen) were used as a pair of positive controls while pgbad - b - ct1 (41074227) and pact2-b - ct2 (680742) were used as a pair of negative controls. b, truncations of the n terminus of polycystin-2 (nt2) marked by the number of starting and ending amino acid residue and their interaction with nt2. the fragments on the left column are in bait (bd) constructs, while the fragments in the right column are in prey (ad) constructs. the numbers in the left column indicate the pairs of nt2 constructs tested and correspond to those shown in a. positive (interaction) and negative results are indicated by + + +, + +, +, and - for the appearance of positive growth on selective medium within 24 h, 48 h, and 37 days or no positive growth in 2 weeks, respectively. c, multiple sequence alignment of pc2 orthologues from different species overlapping with the sequence of human nt2-(190238). amino acids which show perfect conservation down to zebrafish are indicated by an asterisk () in the bottom line. in the region between amino acids 190 and 223, this contrasts with the region between amino acids 119 and 189 where only 1 of 70 amino acids (1.4%) show identity from human to danio. the accession numbers for each sequence are as follows : q13563 (homo sapiens), o35245 (mus musculus), q4gzt3 (bos taurus), q5zm00 (gallus gallus), and q6ivv8 (danio rerio). dimerization of the polycystin-2 n terminus (nt2) detected by yeast two - hybrid assays. a, growth of yeast co - transformants cultured on selective media s.d./-lth with 2 mm 3-at and s.d./-ltah to activate his3 and ade2 selection markers, respectively. the pairs of nt2 sequences tested are numbered from 14 and their sequences indicated. constructs pgbkt753 (p53) and pgbadt7-t (sv40-t antigen) were used as a pair of positive controls while pgbad - b - ct1 (41074227) and pact2-b - ct2 (680742) were used as a pair of negative controls. b, truncations of the n terminus of polycystin-2 (nt2) marked by the number of starting and ending amino acid residue and their interaction with nt2. the fragments on the left column are in bait (bd) constructs, while the fragments in the right column are in prey (ad) constructs. the numbers in the left column indicate the pairs of nt2 constructs tested and correspond to those shown in a. positive (interaction) and negative results are indicated by + + +, + +, +, and - for the appearance of positive growth on selective medium within 24 h, 48 h, and 37 days or no positive growth in 2 weeks, respectively. c, multiple sequence alignment of pc2 orthologues from different species overlapping with the sequence of human nt2-(190238). amino acids which show perfect conservation down to zebrafish are indicated by an asterisk () in the bottom line. in the region between amino acids 190 and 223, this contrasts with the region between amino acids 119 and 189 where only 1 of 70 amino acids (1.4%) show identity from human to danio. the accession numbers for each sequence are as follows : q13563 (homo sapiens), o35245 (mus musculus), q4gzt3 (bos taurus), q5zm00 (gallus gallus), and q6ivv8 (danio rerio). next, we injected pkd2-l223 in zebrafish embryos and tested whether it could result in a phenotype similar to the phenotype obtained by injection of pkd2atgmo or pkd2-d511v. 4c shows that pkd2-l223 induced body axis curvature, pronephric cyst formation and hydrocephalus whereas injection of human pkd2-l177 mrna lacking the dimerization domain did not (fig. 4b). all injected embryos with pkd2-l177 exhibited normal histology as compared with embryos injected with control mo (fig. 4a). expression levels of pkd2-d511v, pkd2-l223 and pkd2-l177 were shown to be equivalent in injected embryos by nested rt - pcr using human - specific primers (fig., these data suggest that pkd2-l223 must have interfered with wild - type pc2 and/or its interacting partners. however, because the n - terminal domain of pc2 has been shown not to interact with known c - terminal binding partners such as pkd1 or trpc1, it is highly likely that pkd2-l223 mediated its effect by direct binding to wild - type pkd2. 4c) mrna all showed a reduction of endogenous zebrafish pc2 expression similar to that seen with pkd2atgmo (fig. these results raised the possibility that pkd2-d511v or pkd2-l223 may have bound to wild - type pkd2 and/or marked it for degradation. an alternative possibility was that the acute binding of the mutant protein could have directly inhibited surface channel activity resulting in cysts with degradation occurring as a later event. inducible translocation of pkd2-(223) to the plasma membrane inhibits endogenous and transfected pkd2 surface currents to address whether pkd2 - 1223 could acutely inhibit surface pc2 channels, we exploited a novel ligand - induced (rapamycin) chemical dimerization system (summarized in fig. 5f) based on the rapamycin - induced dimerization between fkbp and frb (17). the frb (fkbp - rapamycin binding) domain was fused to a plasma membrane targeting sequence of the rho gtpase lyn (ldr) while cfp - tagged fkbp (fk506- and rapamycin - binding protein) was fused to the n terminus of pkd2-(1223) to generate cf - pkd2-(223). as a control, we utilized pkd2-(1177) to generate cf - pkd2-(177). in the absence of rapamycin, on addition of rapamycin in the presence of the frb domain (ldr plasmid), there was a rapid translocation of both fusion proteins to the plasma membrane in hek293 (not shown) and mimcd3 cells (fig. 5) as shown by the loss of cytoplasmic cfp fluorescence in individual cells over time. moreover, the decrease in fluorescent intensity (f) over baseline intensity (f0) triggered by rapamycin was significantly altered for cytosolic cfp compared with the nuclear signal in the presence of ldr (n = 6) (fig. the translocated cfp signal clearly overlapped with the yfp signal of a co - expressed surface - localized protein, yfp - c1 (pkc). the original cf (cfp - fkbp - inp54p) construct served as a positive control (data not shown) (21). figure 4.human pkd2-l223 and d511v induce pronephric cysts in the zebrafish and downregulate zebrafish polycystin-2 expression. a, 48 hpf zebrafish injected with a control mo have a normal body ; histology section of 48 hpf embryos showing a glomerulus (glm) in the midline and pronephric tubules connected to bilateral pronephric ducts. endogenous zebrafish pc2, detected with a specific antibody that does not cross - react with human pc2, is distributed in the basolateral membranes and apical cilia in the anterior pronephric ducts (see also h). b, 48-hpf human pkd2-l177 mrna - injected embryos show normal whole mount histology cross - section and zebrafish pc2 expression. c, human pkd2-l223 mrna - injected embryos showing pronephric cysts, body axis curvature, and reduced zebrafish pc2 expression. d, pkd2atgmo - injected embryos showing pronephric cysts, body axis curvature, and hydrocephalus. pkd2atgmo blocked endogenous zebrafish pkd2 translation leading to a reduction in pc2 expression. e, human pkd2-d511v mrna - injected embryos also developed body axis curvature, cyst, and hydrocephalus. f, co - injection of 50 pg of human pkd2-d511v was unable to rescue the pkd2atgmo phenotype and induced more severe body axis curvature, cysts, and hydrocephalus than pkd2atgmo alone. g, rt - pcr analysis for human pkd2 (upper panel) and -actin (lower panel) mrna expression. endogenous zebrafish pc2 expression is clearly down - regulated by co - injection of pkd2-l223 (c) and pkd2-d511v (e) mrna to a similar level as pkd2atgmo (d). human pkd2-l223 and d511v induce pronephric cysts in the zebrafish and downregulate zebrafish polycystin-2 expression. a, 48 hpf zebrafish injected with a control mo have a normal body ; histology section of 48 hpf embryos showing a glomerulus (glm) in the midline and pronephric tubules connected to bilateral pronephric ducts. endogenous zebrafish pc2, detected with a specific antibody that does not cross - react with human pc2, is distributed in the basolateral membranes and apical cilia in the anterior pronephric ducts (see also h). b, 48-hpf human pkd2-l177 mrna - injected embryos show normal whole mount histology cross - section and zebrafish pc2 expression. c, human pkd2-l223 mrna - injected embryos showing pronephric cysts, body axis curvature, and reduced zebrafish pc2 expression. d, pkd2atgmo - injected embryos showing pronephric cysts, body axis curvature, and hydrocephalus. pkd2atgmo blocked endogenous zebrafish pkd2 translation leading to a reduction in pc2 expression. e, human pkd2-d511v mrna - injected embryos also developed body axis curvature, cyst, and hydrocephalus. f, co - injection of 50 pg of human pkd2-d511v was unable to rescue the pkd2atgmo phenotype and induced more severe body axis curvature, cysts, and hydrocephalus than pkd2atgmo alone. g, rt - pcr analysis for human pkd2 (upper panel) and -actin (lower panel) mrna expression. endogenous zebrafish pc2 expression is clearly down - regulated by co - injection of pkd2-l223 (c) and pkd2-d511v (e) mrna to a similar level as pkd2atgmo (d). 6 shows that addition of rapamycin induced a time - dependent reduction in whole cell current amplitude of mimcd3 cells transiently transfected with ldr+cf - pkd2-(223) cells from -23.7 0.9 pa / pf to -14 0.6 pa / pf at -100 mv (fig. 6, a and d). pa / pf to -22.7 1.2 pa / pf at -100 mv) (fig. pa / pf to -22.3 1.4 pa / pf at -100 mv) (fig. it should be noted that constitutive overexpression of pkd2-d511v in these cells suppressed whole cell currents from -24.3 3.4 pa / pf (n = 9) to -10.3 2.28 pa / pf (n = 7) under identical recording conditions (9) suggesting a similar mechanism of whole cell current density inhibition by pkd2-d511v or pkd2-l223. the small difference in whole cell current density (4 pa / pf) between pkd2-d511v and pkd2-l223 may be explained by the higher affinity of pkd2-d511v than pkd2-l223 for binding to wild - type pkd2 or binding of pkd2-d511v to other interacting partners of pkd2 such as trpc1, which was shown to be required for basal activity of native pkd2 in these cells (9). in contrast, pkd2-l223 should not associate with pkd1 or trpc1 (5, 15) and therefore its effect on whole cell current density should be specific to wild - type pkd2, at least based on existing data. to further confirm the specificity of cf - pkd2-(223) on pkd2, we overexpressed full - length pkd2 and tested the effect of cf - pkd2-(177) or cf - pkd2-(223) on transfected pkd2. overexpression of pkd2 resulted in an increase in overall whole cell current density from -23.6 1.2 pa / pf to -45.4 1.8 pa / pf at -100 mv (fig. 6, b and f, black plot), consistent with the formation of active channels at the plasma membrane (9). addition of rapamycin to the bath induced a time - dependent reduction in whole cell currents in pkd2-, ldr-, and cf - pkd2-(223)-cotransfected cells from -43.5 1 pa / pf to -21.8 1 pa / pf at -100 mv (fig. these data provide direct evidence for a dominant negative effect of cf - pkd2-(223) on native or transfected pkd2 surface channel activity. in this system, binding of pkd2-l223 resulted in acute inhibition of channel activity because the effect was observed almost immediately following induced translocation of pkd2-l223 to the plasma membrane. d, cfp fluorescent images in live mimcd3 cells co - transfected with the plasmids cf - pkd2-(177) or cf - pkd2-(223) in the presence or absence of ldr. the left hand panels represent baseline cfp (blue), the middle panels are cfp signals (blue) 545 s following the addition of rapamycin (rap, 10 m) to the medium and the right panels, yfp fluorescence (green) of the fusion protein, yfp - c1-(pkc), which is constitutively localized at the plasma membrane. the translocation of both cfp - pkd2 fusion proteins induced by rap in the presence of ldr can be seen graphically by the rapid reduction in the cytoplasmic cfp signal within the time frame shown (545 s). in contrast, nuclear expression of both fusion proteins is present at baseline but does not change following rap. e, change in cytosolic cfp fluorescence intensity (f) expressed as a ratio of baseline cfp fluorescence (f0) was significantly altered compared with nuclear cfp fluorescence following rap in the presence of ldr (n = 6). f, schematic diagram of the rapamycin - induced chemical dimerization strategy used to translocate cfp - pkd2 fusions to the plasma membrane (pm). the frb (fkbp - rapamycin binding) domain was fused to a plasma membrane targeting sequence of the rho gtpase lyn (ldr), while cfp - tagged fkbp (fk506- and rapamycin - binding protein) was fused to the n terminus of pkd2 (1177 or 1223) to generate cf - pkd2-(177) and cf - pkd2-(223), respectively. addition of rap induces rapid heterodimerization between the pm - anchored frb and fkbp fusion proteins, thus bringing the cf - pkd2 fusions into close proximity of pm - located pkd2 channels. d, cfp fluorescent images in live mimcd3 cells co - transfected with the plasmids cf - pkd2-(177) or cf - pkd2-(223) in the presence or absence of ldr. the left hand panels represent baseline cfp (blue), the middle panels are cfp signals (blue) 545 s following the addition of rapamycin (rap, 10 m) to the medium and the right panels, yfp fluorescence (green) of the fusion protein, yfp - c1-(pkc), which is constitutively localized at the plasma membrane. the translocation of both cfp - pkd2 fusion proteins induced by rap in the presence of ldr can be seen graphically by the rapid reduction in the cytoplasmic cfp signal within the time frame shown (545 s). in contrast, nuclear expression of both fusion proteins is present at baseline but does not change following rap. e, change in cytosolic cfp fluorescence intensity (f) expressed as a ratio of baseline cfp fluorescence (f0) was significantly altered compared with nuclear cfp fluorescence following rap in the presence of ldr (n = 6). f, schematic diagram of the rapamycin - induced chemical dimerization strategy used to translocate cfp - pkd2 fusions to the plasma membrane (pm). the frb (fkbp - rapamycin binding) domain was fused to a plasma membrane targeting sequence of the rho gtpase lyn (ldr), while cfp - tagged fkbp (fk506- and rapamycin - binding protein) was fused to the n terminus of pkd2 (1177 or 1223) to generate cf - pkd2-(177) and cf - pkd2-(223), respectively. addition of rap induces rapid heterodimerization between the pm - anchored frb and fkbp fusion proteins, thus bringing the cf - pkd2 fusions into close proximity of pm - located pkd2 channels. in the present study, we have identified and functionally characterized a new dimerization domain in the n - terminal cytosolic region of pc2. this domain is shown to have a physiologically relevant role in zebrafish development as it phenocopied known loss - of - function constructs of pc2. we propose that the identification of this domain has important implications in type 2 adpkd pathophysiology. the tendency of native pc2 to oligomerize led us initially to investigate how pc2 homodimerization could be regulated. unexpectedly, we found that two naturally occurring pc2 mutants lacking the c - terminal homodimerization domain (l703x, r742x) could still form oligomers and bind to full - length pc2 in mammalian cells. these findings led us to demonstrate the existence of a more proximal dimerization domain within the n - terminal domain and its functionality in two assays of pc2 activity i.e. nephrogenesis in zebrafish embryos and channel activity in mimcd3 cells. overall, our data would support a direct acute inhibitory effect of the mutant protein (pkd2-l223) on the pc2 channel itself, which also leads to subsequent degradation of pc2. recently, it was reported that the transgenic expression of pkd2-l703x in rats gave rise to a cystic phenotype by an undetermined mechanism (27). we believe that our findings of an n - terminal dimerization domain support a dominant negative mechanism as a plausible explanation of the phenotype in this model. the existence of both n- and c - terminal dimerization domains in pc2 provide supportive evidence that pc2 is likely to form functional homotetramers, a possible model is shown in fig. 7. this model does not require the binding of pc1 or that of other trp subunits (such as trpc1) both of which have been shown to interact with pc2 via distinct sequences or amino acids in the c - terminal domain (5). the evidence that pc2 homodimers can function independently in vivo is indirect : pc2 can function independently of pc1 at the embryonic node in the determination of lr asymmetry in the axial body plan (28). nevertheless, an important question is what regulates the assembly of pc2 monomeric subunits into homotetramers or alternatively heterotetramers with pc1 or other trp subunits. in addition, we do not know if pc2 truncation mutants (e.g. l703x), which retain the putative pore region and which can still dimerize via the n - terminal domain are still functional. in some assays, there is evidence for altered pc2 localization (e.g. increased cell surface expression) and for altered channel properties (e.g. loss of calcium responsiveness, voltage - dependence) of this mutant (12, 29). time - dependent inhibition of native (a) or transfected pkd2 (e) channel activity by rapamycin (rap)-induced translocation of a cfp fusion of the pc2 n terminus (nt2, 1223) to the plasma membrane. mimcd3 cells were transiently transfected with ldr plus cf - pkd2-(177) or cf - pkd2-(223) in the absence (a) or presence (e) of transfected wild - type mouse pkd2. translocation of cf - pkd2-(177) or cf - pkd2-(223) to the plasma membrane was induced by the addition of 10 m rapamycin to the bath solution. current densities at -100 mv were obtained by 100-ms pulses from -60 mv to -100 mv applied every 10 s. arrows indicate time points at which voltage steps were applied to derive i - v curves shown in b, c, d, f, g, and h. i - v curves derived from native (b), ldr plus cf - pkd2-(177) (c), or ldr plus cf - pkd2-(223) (d)-transfected mimcd3 cells before (black) or after (red) the addition of rapamycin in the bath solution are shown. i - v curves derived from pkd2 (f), pkd2, ldr, and cf - pkd2-(177) (g), or pkd2, ldr, and cf - pkd2-(223) (h)-co - transfected mimcd3 cells before (black) or after (red) the addition of rapamycin to the bath solution are shown., time - dependent inhibition of native (a) or transfected pkd2 (e) channel activity by rapamycin (rap)-induced translocation of a cfp fusion of the pc2 n terminus (nt2, 1223) to the plasma membrane. mimcd3 cells were transiently transfected with ldr plus cf - pkd2-(177) or cf - pkd2-(223) in the absence (a) or presence (e) of transfected wild - type mouse pkd2. translocation of cf - pkd2-(177) or cf - pkd2-(223) to the plasma membrane was induced by the addition of 10 m rapamycin to the bath solution. current densities at -100 mv were obtained by 100-ms pulses from -60 mv to -100 mv applied every 10 s. arrows indicate time points at which voltage steps were applied to derive i - v curves shown in b, c, d, f, g, and h. i - v curves derived from native (b), ldr plus cf - pkd2-(177) (c), or ldr plus cf - pkd2-(223) (d)-transfected mimcd3 cells before (black) or after (red) the addition of rapamycin in the bath solution are shown. i - v curves derived from pkd2 (f), pkd2, ldr, and cf - pkd2-(177) (g), or pkd2, ldr, and cf - pkd2-(223) (h)-co - transfected mimcd3 cells before (black) or after (red) the addition of rapamycin to the bath solution are shown., the existence of n- and c - terminal dimerization domains would facilitate the assembly of four pc2 monomers into a homotetramer. by contrast, a mutant pc2 monomer lacking the distal c - terminal dimerization domain (such as l703x) would still be capable of forming dimers via the proximal n - terminal domain : these mutant dimers could be functional. the incorporation of one mutant pc2 monomer with 3 normal pc2 monomers would permit the formation of a tetrameric structure but is likely to be non - functional. a tetrameric model of pc2 channel assembly. the existence of n- and c - terminal dimerization domains would facilitate the assembly of four pc2 monomers into a homotetramer. by contrast, a mutant pc2 monomer lacking the distal c - terminal dimerization domain (such as l703x) would still be capable of forming dimers via the proximal n - terminal domain : these mutant dimers could be functional. the incorporation of one mutant pc2 monomer with 3 normal pc2 monomers would permit the formation of a tetrameric structure but is likely to be non - functional. our results also raise the possibility as to whether cyst formation in pkd2 patients could arise by a dominant - negative mechanism as shown for the d511v mutant in addition to two - hit and haploinsufficiency models (30). if pc2 forms an oligomeric structure, the association of a mutant protein with wild - type subunits would result in the generation of non - functional multimeric complexes (fig. 7). for a tetrameric model, potentially 15 of 16 possible combinations between mutant and wild - type subunits could be affected. therefore, although there will be 50% reduction in the wild - type protein, the reduction in function could approximate 100%. this mechanism would only be effective assuming that a stable mutant mrna transcript and protein are produced in patients at levels that would bind in a stoichiometric fashion to wild - type monomers. nonetheless, given the lack of specific inhibitors to pc2, the dominant negative strategy we have described could be a useful way to study the function of the endogenous protein in different systems. | autosomal dominant polycystic kidney disease (adpkd), the most common inherited cause of kidney failure, is caused by mutations in either pkd1 (85%) or pkd2 (15%). the pkd2 protein, polycystin-2 (pc2 or trpp2), is a member of the transient receptor potential (trp) superfamily and functions as a non - selective calcium channel. pc2 has been found to form oligomers in native tissues suggesting that it may form functional homo- or heterotetramers with other subunits, similar to other trp channels. our experiments unexpectedly revealed that pc2 mutant proteins lacking the known c - terminal dimerization domain were still able to form oligomers and co - immunoprecipitate full - length pc2, implying the possible existence of a proximal dimerization domain. using yeast two - hybrid and biochemical assays, we have mapped an alternative dimerization domain to the n terminus of pc2 (nt2 - 1 - 223, l224x). functional characterization of this domain demonstrated that it was sufficient to induce cyst formation in zebrafish embryos and inhibit pc2 surface currents in mimcd3 cells probably by a dominant - negative mechanism. in summary, we propose a model for pc2 assembly as a functional tetramer which depends on both c- and n - terminal dimerization domains. these results have significant implications for our understanding of pc2 function and disease pathogenesis in adpkd and provide a new strategy for studying pc2 function. |
a 40-year - old woman presented with a 1-year history of afterimages in both eyes after having dengue fever. her best - corrected visual acuity (bcva) was 20 / 20 in both eyes, and her intraocular pressure was normal. retina examination revealed hemorrhaging in the retina inner layer and rpe atrophy in both eyes. icg showed late hypofluorescence due to choroidal filling disorder, and infrared (ir) imagery showed hypofluorescence that included both foveae. oct imagery revealed disruption of the inner segment / outer segment junction (is / os) junction and outer retina thinning at the hypofluorescence site of the ir imagery (figs. 1 and 2). a 40-year - old woman presented with an upper visual field defect in the left eye after contracting dengue fever 1 month prior. bcva was 20 / 30 in the left eye, and no other abnormal findings were found in the anterior segment. fluorescein angiography (fa) of the left eye showed a patch choroidal perfusion delay and a window defect due to rpe atrophy. oct imagery revealed disruption of the is / os junction and outer retina thinning at the hypofluorescence site of the ir imagery. a 29-year - old woman treated for dengue fever 1 year prior presented with a visual field defect in the left eye. all symptoms and test were normal in the right eye. fa revealed a faint window defect in the left eye, and ir imagery revealed hypofluorescence in the fovea area. oct imagery revealed disruption of the is / os junction and outer retina thinning at the hypofluorescence site of the ir imagery. a 40-year - old woman presented with a 1-year history of afterimages in both eyes after having dengue fever. her best - corrected visual acuity (bcva) was 20 / 20 in both eyes, and her intraocular pressure was normal. retina examination revealed hemorrhaging in the retina inner layer and rpe atrophy in both eyes. icg showed late hypofluorescence due to choroidal filling disorder, and infrared (ir) imagery showed hypofluorescence that included both foveae. oct imagery revealed disruption of the inner segment / outer segment junction (is / os) junction and outer retina thinning at the hypofluorescence site of the ir imagery (figs. 1 and 2). a 40-year - old woman presented with an upper visual field defect in the left eye after contracting dengue fever 1 month prior. bcva was 20 / 30 in the left eye, and no other abnormal findings were found in the anterior segment. fluorescein angiography (fa) of the left eye showed a patch choroidal perfusion delay and a window defect due to rpe atrophy. oct imagery revealed disruption of the is / os junction and outer retina thinning at the hypofluorescence site of the ir imagery. a 29-year - old woman treated for dengue fever 1 year prior presented with a visual field defect in the left eye. all symptoms and fa revealed a faint window defect in the left eye, and ir imagery revealed hypofluorescence in the fovea area. oct imagery revealed disruption of the is / os junction and outer retina thinning at the hypofluorescence site of the ir imagery. the incidence of ocular manifestations in dengue fever is rare and usually occurs during the acute phase of disease. the main fundus changes are macula and retinal hemorrhages, peripapillary hemorrhage, roth 's spot, diffuse retinal edema, vitreous cells, blurring of the optic disc margin, serous retinal detachment, choroidal effusions, and nonspecific maculopathy. it has been reported that approximately 10% of all dengue fever patients suffer from maculopathy with central vision loss. in our study, all 3 patients had observable rpe atrophy on the fa ; disruption of the is / os junction and outer retina thinning were also observed on the ir imagery. the pathogenesis of ocular manifestations of dengue infection is not completely understood, but is thought to be caused by either a direct viral invasion or an immune - mediated reaction. the mean interval between the onset of visual symptoms and the systemic manifestation of dengue fever reported by wen. was 7.26 days (range, 2 to 15 days) and that reported by lim. was 6.8 days (range, 6 to 7 days), which suggests an immune - mediated process rather than a direct viral infection. this immune - mediated process likely comprises a complex series initiated by direct viral infection. viral invasion of endothelial cells, dendritic cells, monocytes, and hepatocytes cause apoptosis and cellular dysfunction, which may be followed by a transient aberrant immune response, resulting in cd4/cd8 ratio inversion and cytokine overproduction. in addition, overproduction of interleukin-6 triggers the formation of autoantibodies against platelets and endothelial cells, resulting in further immune - mediated damage [9 - 11 ] and leading to endothelial dysfunction, platelet destruction, and consumptive coagulopathy. ultimately, the aggressive immune response may cause increased capillary permeability, plasma leakage, and hemorrhagic diathesis. this hemorrhage bleeding tendency can occur in both the retina and choroid causing retina hemorrhage and choroidal circulatory disorders. the outer retina layer is fed by choroidal circulation making it vulnerable to ischemic diseases. in our study, we noted a decrease in the outer retina layer thickness and a choroidal circulation disorder in all three cases. in case 1, similar choroidal changes can be seen in wegner 's granulomatosis, where ischemic atrophy of the outer retina by choroidal ischemia has been reported. this similarity also supports the possibility that an immune - mediated complex is the cause of the choroidal changes in dengue fever rather than direct viral invasion. henoch - schonlein purpura, essential cryoglobulinemia, serum sickness vasculitis, lupus vasculitis, and hepatitis b microscopic polyarteritis have been reported as vasculitis diseases caused by immune complexes that mostly invade the capillaries, venules, and arterioles. in cases of large vessels immune complex - mediated vasculitis, complexes are removed quickly by mps cells, thus causing minimal damage to the vessels. south korea is not an endemic region for dengue fever, and therefore, acute stage patients are rarely observed. frequently used test such as the hemagglutination inhibition test and immunoglobin g or immunoglobin m enzyme immunoassays were not performed in our study and this is a limitation for this study. in the future, complementary measurements in the serum of dengue fever patients will be needed to further understand the disease process. in conclusion, thinning of the retina outer layer conversely, is / os junction disruption is reported to be reversible, so follow - up studies using oct are necessary to evaluate visual acuity and visual field changes in acute choroidal ischemia in order to improve vision in dengue fever patients with ocular manifestations. | dengue fever is a viral disease that is transmitted by mosquitoes and affects humans. in rare cases, dengue fever can cause visual impairment, which usually occurs within 1 month after contracting dengue fever and ranges from mild blurring of vision to severe blindness. visual impairment due to dengue fever can be detected through angiography, retinography, optical coherence tomography (oct) imaging, electroretinography, event electroencephalography (visually evoked potentials), and visual field analysis. the purpose of this study is to report changes in the eye captured using fluorescein angiography, indocyanine green, and oct in 3 cases of dengue fever visual impairment associated with consistent visual symptoms and similar retinochoroidopathic changes. the oct results of the three patients with dengue fever showed thinning of the outer retinal layer and disruption of the inner segment / outer segment (is / os) junction. while thinning of the retina outer layer is an irreversible process, disruption of is / os junction is reported to be reversible. follow - up examination of individuals with dengue fever and associated visual impairment should involve the use of oct to evaluate visual acuity and visual field changes in patients with acute choroidal ischemia. |
classification and identification of petroleum fuels are a crucial challenge in the scientific investigation of arson and have high importance in cases of commercial, industrial, and forensic criminal acts. the crime of arson may be defined as a willful and malicious act to setting fire to a property and is considered one of the easiest crimes to commit and one of the hardest to investigate [1, 2 ]. the liquids commonly used as accelerants in the criminal acts are gasoline, kerosene oil, or diesel due to their easy availability and low price [2, 3 ]. regulations established for the analysis of ignitable liquid residues provide a common language to the field of forensic science for describing the characteristics of the different inflammable liquids as well as to classify them [46 ]. the quality control of the fuels is guaranteed by means of establishing some technical specifications that vary in different parts of the world (i.e., en 228 in europe, astm d48 14 in the usa, jis k2202 in japan, and is 2796 in india). in particular, commercial or industrial adulteration with lower - price fuels is one of the most difficult situations to detect and is a serious problem in the petrochemical industry because of the similar composition of the adulterant fuels used. the contents of oxygen and hydrocarbons such as aromatics and olefins vary in different samples that produce various kinds of petroleum derivatives with specific characteristics belonging to a particular refinery [1, 8 ]. the chemical composition of the crude oil depends on various factors that must be taken into account each time when performing analysis of these samples and collecting reference materials. these factors include the origin (oilfields and country), refining procedures, and the location of the distributors. apart from these elements, another important factor is the contamination introduced in the fuels due to the residual level of the storage tank. in many situations where fuels are used in criminal acts, factors like air flow and high temperature affect the composition because the more volatile compounds could evaporate. for these reasons, the analysis of the samples could become more difficult. however, there are sites on the crime scene such as the porous materials where the accelerant liquids stay unaffected and retain their original composition that can be recovered for the analysis [1, 8, 9 ]. several methods have been proposed to identify and discriminate fuel samples, based on gas chromatography - mass spectrometry (gc - ms) [13, 813 ], as well as the determination of additives by normal - phase high - performance liquid chromatography (np - hplc) combined with gc - ms, and various spectroscopic methods such as nuclear magnetic resonance (nmr), near infrared (nir) spectroscopy and attenuated total reflection - fourier transform (atr - ftir) spectroscopy [7, 1519 ]. although these analytical techniques are widely used in determination of the composition and the classification of the fuels, there are some drawbacks because they are time consuming and expensive for the analysis. raman spectroscopy has also been used for the classification of gasoline ; nevertheless, there are not sufficiently large differences in the raman spectra for unambiguous identification. detection and identification of fuels using gc analysis the main reason for the application of gc methods to ignitable liquid residue analysis is the need for adequate separation of components comprising petroleum - based fuels. most of the discriminating information is contained in the fraction with a volatility ranging from approximately n - pentane to n - octane. many of these studies have focused on determining the fuels using chemometric procedures such as principal component analysis (pca), linear discriminant analysis (lda), soft independent modeling of class analogy (simca), and quadratic discriminant analysis (qda) [1, 3, 79, 1521 ]. although these studies provided acceptable results, in some cases, correct identification rate of fuels falls below 85%. error rates in many of them are too high to classify a particular gasoline or if there are false negatives or false positives in the results. moreover, studies where the neural networks (nn) chemometric method has been used, only the trainings of the models are made, not performing test - sets or blind samples to validate the accuracy and robustness of the method. furthermore, when the unknown samples are used, methods such as lda are completely unable to make a correct classification. unfortunately, many of these authors do not take into account or are unaware of the pitfalls in their results. therefore, there is a need for more systematic studies that include new approaches to identifying and classifying brands of fuels accurately. another studies have been performed combining nn with another techniques using different types of samples. the satisfactory results, obtained in this methodology using nn, gave us the approach to trying to analyze samples of fuels [24, 25 ]. specifically, the aim of this work was to improve the recognition capacity by developing a method capable of identifying extremely similar samples that have few differences between them. therefore, nn algorithms were combined with gc as they are capable of dealing with large and multivariate information increasing the classification capacity, due to their simple implementation and ability to generalize that makes them useful for qualitative analysis and to improve the accuracy and precision of the classification process. it was intended to develop a methodology based on gc and nn to determine exactly the point of purchase of the gasoline used in a criminal act. five brands of fuels (repsol, shell, british petroleum (bp), cepsa, and galp) collected from fifteen different local petrol stations were studied. for each brand, all samples were stored in a glass recipient at 20c in a freezer to avoid the loss of volatile compounds. for each sample of diesel and gasoline, five chromatograms were acquired, respectively ; therefore, a total of 150 chromatograms were recorded. additionally, three blind samples were collected randomly from these service stations, labeled as experimental conditions were maintained the same as used for the references, recording three chromatograms for each blind sample. all analyses were performed using an acme6000 gas chromatograph connected to a flame ionization detector (fid). the gc was equipped with a 100% polydimethylsiloxane capillary column zb1 (30 m 0.25 mm 0.25 m). the sample injection volume was 1 l, delivered by syringe with a split ratio of 50 : 1. samples were analyzed using the following chromatographic conditions : diesel samples were measured using split injection (2 : 1) at 300c ; the oven temperature was programmed from 70c (1 min isothermal) to 300c at 10c / min (held for 3 min). gasoline samples were performed using split injection (2 : 1) at 300c ; the oven temperature was ramped from 40c (1 min isothermal) to 100c at 15c / min, followed by a ramp to 300c at 50c / min (hold time of 3 min), giving a total run time of 28 min of each chromatogram. injector pressure was 30 psi and detector temperature was 350c. in this work, as a pure sample was used, the chromatographic conditions of diesel and gasoline are different due to the different time of retention of the compounds. diesel samples are more volatile than gasoline samples and it was necessary for obtaining optimum chromatograms to use different gradients of temperature. home - made neural networks software specifically designed to deal with the discrimination of different brands of fuels was developed. the nn models were based on a multilayer perceptron, feedforward, supervised network that consisted of several neurons (information processing units) arranged in two or more layers receiving information from all of the neurons of the previous layer. the connections are controlled by a weight that modulates the output from the neuron before inputting its numerical content into a neuron in the next layer. the nn topology consists of three layers (input, hidden, and output), which is widely used to model systems with a similar level of complexity. in particular, the input layer consisted of 1440 nodes (intensity of chromatogram within selected range of retention time). the number of neurons in the hidden layer was set to 20. the output layer was comprised of j neurons (where j is number of reference samples used) for estimating the similarity between the reference sample (reference chromatogram) and the testing sample chromatogram, denoted hereafter as test - set. the process that optimizes the weights, that is, the learning or training process was based on a back - propagation algorithm [2729 ]. the inputs from each neuron are added by an activation function, and the result is transformed by a transfer function that limits the amplitude of the neuron output. in this work, every nn model was estimated using matlab software (mathworks, 2010a). because the nn is a supervised method, in order to optimize the weight matrix, it is necessary to use input and output data that adequately characterized the system to be modeled. the chromatographic data of the training library was randomly divided as part of the training process into two subsets : 75% for training and 25% for self - validation of the model. once the training and self - validation process was carried out, the models were validated with test - set, with chromatograms being not used in the training process. the identification process was based on the ability of the nn to detect the degree of similarity between the unknown chromatogram and each of the reference chromatograms used in the training process. during the training process, each brand of fuel was associated with an identification number in the output layer. thus, a perfect identification is obtained if the output from the nn model for the test samples of the same brand of fuel matched the identification number assigned to the brand used to train the model. nn training was achieved by applying the back - propagation algorithm based on the conjugate gradient method, one of the general - purpose second - order techniques that helps minimize the goal functions of several variables. second order indicates that such method uses the second derivatives of the error function, whereas a first - order technique, such as standard back propagation, uses only the first derivatives. to determine when the training should be stopped, an early stopping criteria based on performance improving (error rate) of the validation set the number of epochs was not relevant in this case as verification mean square error (mse), as defined in (1), was taken into account to avoid an overfitting of the nn model. for this purpose, the learning process was repeated until mse was decreased : (1)mse=1nknrkyk2,where n, rk, and yk are the number of input data, target output, and the response from each output neuron, respectively. a detailed description of the calculation process is provided in the literature [28, 31 ]. validation of each nn model was performed to evaluate their ability to discriminate brands of fuels, using test - sets comprising the chromatograms not used in the training process. the models performance was evaluated by its classification success rate that is the percentage of correct discrimination of brands within the classified samples. the main difference between the chromatographic profiles is the variability in the number of the peaks. gasoline chromatogram presents lower number of peaks due to the large amount of volatile compounds, while the diesel has a higher number of peaks corresponding to long chain hydrocarbons. as the first minute and the last 3 min (in diesel) and 11 min (in gasoline) of all chromatograms did not contain relevant information, these parts were removed prior to statistical analysis. in all cases, data pretreatment was necessary to correct the variations in retention time and normalization methods (relative to highest peak) were applied to take into account the injection volume and to avoid data variations. the factors that could affect the classification process are instrument variations, shifts in temperature, degradation of stationary phase, variation of pressure, and so forth. small unavoidable differences related to the instrumentation and chemical interactions between the samples and the equipment make necessary a previous alignment of chromatogram for a correct chemometric analysis [1, 3, 22, 32 ]. many authors have studied the chromatogram alignment using different algorithms, but they have some difficulties such as cost - effectiveness of computer time, detection of significant peaks, or knowledge of the components in the sample [22, 3338 ]. in this work, a simple and fast algorithm for alignment based on piecewise linear correlation optimized warping (cow) was used. this algorithm uses two input parameters (stretching and shrinking) estimated from the width of the peaks observed in the chromatograms. the chromatograms before and after alignment for samples of repsol brand are shown in figures 2(a) and 2(b). after the alignment and normalization, four chromatograms were used for training the nn model and the fifth was included in the test - set. the nn training was done using a library with 60 chromatograms (12 per brand), while the test of the nn model was done using 15 chromatograms (3 per brand) in test - set, not included in the training. the larger the information (representative data) used in the training of the nn model, the better the predictive capability of the model. therefore, the whole set of variables that constitute the chromatogram is important in the classification process performed by the nn model. the chromatograms used for the test - set were individually measured with respect to those used for training and unknown to the model. the nn model has the ability to classify correctly the test library of the samples used in the training. this characteristic was used as internal validation and was not usually a result with other chemometric methods (see). despite the fact that there are not significant variations in the chromatogram to easily discriminate fuels, from the mathematical point of view, each sample can be discriminated based on its complete fingerprint. as discussed above, the possible chemical differences in the samples that allowed the identification and discrimination of the brands of fuel, could be the differences in the contents of oxygen and hydrocarbons profiles such as aromatics and olefins, producing various kinds of petroleum derivatives. also, several studies have researched about the influence of the adding of additives, such as detergents, dispersants, antiknock agents and antioxidants, which are compounds added to improve fuel performance, avoid motor problems and minimize the amount of pollutants emitted to the atmosphere. another chemical difference that could allow such discrimination is the introduction of different trace species during the refining and blending processes that may introduce detectable differences between batches of finished fuels. this fact constitutes the basis of nn ability to carry out the discrimination between the brands of fuels with high tolerance for instrument variations and the presence of outliers. our aim is to identify exactly the point of purchase of the gasoline used in a punctual criminal act and collect samples around an established area. in both type of samples, diesel and gasoline, all the chromatograms of each brand, included in the test of the nn model were correctly classified (3/3) over all samples, showing that the gc - nn methodology developed was able to correctly classify all of the samples to their corresponding brands. a perfect classification was achieved for all the test chromatograms giving a 100% of success rate. in addition, no false positives or false negatives were observed in any of the models studied that shows the high robustness of methodology used. even though the samples have been collected from different service stations, which could entail differences in transport fuel, as well as storage tanks where an external contamination is possible, nn is able to perform the classification of each of the samples in their different brands. in order to check the accuracy and robustness of the method, three blind samples were collected randomly from these service stations, labeled as a, b, and c by a third person. three chromatograms were recorded for each blind sample and aligned with all five brands ; therefore, 15 chromatograms for each blind sample were generated and used in the testing and classification process to determine the corresponding brand. data treatment of chromatograms was carried out of the same way as reference samples. although the case of sample a classified as bp has a low classification success rate, most of the chromatograms for this sample were assigned correctly, and only four deviated from the expected value. the discrepancy observed in only 4 out of 15 chromatograms not only is more than acceptable, but also is essential for taking into account the classification success rate. b and c samples were classified correctly with 80.0% and 100% of success rate, respectively. in addition, the computation time for testing each sample was below 5 s, although the test - set matrix was considerably large. other studies have based the classification on refineries, fuel characteristic properties, or type (normal, regular, or premium) without discrimination of brands [8, 9, 15, 16, 20 ]. therefore, classifying each fuel with a specific brand, as well as performing test - set and blind sample to validate the methods, is an important factor in the results of this study. a method based on gas chromatography (gc) and neural networks (nn) algorithms was developed and applied to achieve rapid identification and classification of different brands of fuels based on their characteristic fingerprints. five brands of fuels in spain, that is repsol, shell, british petroleum (bp), cepsa, and galp were used. an important result was that no false positives or false negatives were observed neither in the test nor in blind samples. the combination of gc with a supervised nn method was able to perform the classification of brands due to the reliability and robustness of the estimated nonlinear classification model. a great advantage offered by this methodology was that it did not require a detailed analysis of the fuel composition and the chromatographic fingerprint provided sufficient information to perform the classification of each brand of fuels. also, it may provide an accurate, faster, and useful classification analysis in cases of commercial, industrial, and forensic investigations. | the detection of adulteration of fuels and its use in criminal scenes like arson has a high interest in forensic investigations. in this work, a method based on gas chromatography (gc) and neural networks (nn) has been developed and applied to the identification and discrimination of brands of fuels such as gasoline and diesel without the necessity to determine the composition of the samples. the study included five main brands of fuels from spain, collected from fifteen different local petrol stations. the methodology allowed the identification of the gasoline and diesel brands with a high accuracy close to 100%, without any false positives or false negatives. a success rate of three blind samples was obtained as 73.3%, 80%, and 100%, respectively. the results obtained demonstrate the potential of this methodology to help in resolving criminal situations. |
outcome measures after surgery for adolescent idiopathic scoliosis (ais) have focused mainly on objective parameters, such as radiographic changes. however, radiographic outcomes following surgical treatment of ais have been only weakly correlated with patient - reported outcomes measuring functional status and symptoms.1,2 use of disease - specific, validated questionnaires provides significant information regarding factors that are important to patient quality of life following fusion.3,4,5 a common concern among surgeons performing scoliosis procedures is whether spinal fusion will reduce function in young patients. also, results presented are not conclusive regarding effects on back pain and its correlation to a fusion extending into the lower lumbar spine. this is why outcome studies, focusing on mid - term and long - term impairments as primary effect variables, are critically needed. most of the long - term results in the literature were obtained using the harrington rod system, there are however few, if any, such studies featuring segmental instrumentation systems. this was our main reason for deciding to study pain and disability using segmental hybrid instrumentation for patients with ais. to describe the prevalence of pain following fusion for ais and to identify factors associated with greater pain or disability inclusion criteria : all subjects (n = 142) with ais who underwent spinal fusion between january 1, 1997, and december 31, 2007, at our hospital were potentially eligible. exclusion criteria : patients who had anterior fusion (n = 11) or were older than 17 years (n = 5) were excluded, leaving 126 consecutive patients eligible. patient population and selection (fig. 1) : from 126 consecutive patients who were eligible, 104 were available for analysis. of the 22 patients lost to follow - up, the patient s address was unknown (n = 9), patient refused to participate (n = 4), or traveling distance was excessive (n = 9). choice of fixation device was made based on surgeon preference and was not based on type or severity of deformity. outcomes : questions 1 and 9 of the srs-22 were used to determine prevalence of pain (in the past 6 months) and disability, respectively. the srs pain domain score (pds) was evaluated as an outcome to facilitate comparison with other published studies and further evaluate factors associated with pain. risk factors and potentially confounding factors evaluated : demographic factors : gender, age at surgery, age at follow - up, follow - up time clinical factors : curve type procedural factors : surgical approach, types of instrument, lowest instrumented level complications as risk factors for pain : reoperation, infection, implant failure, dislodging of hook, correction loss, pseudarthrosis, number of levels fused analysis : to evaluate associations between any pain or disability and potential risk factors, responses were dichotomized to reflect no pain and no disability (response of five to the questions) and any pain or disability (responses 14) and chi - square test or fisher exact test (if cells contained fewer than five individuals) were performed. the kruskall - wallis test using the srs pds as a continuous outcome variable was done to compare medians among two or more groups for the categorical variables. all statistical tests were two - tailed. stratified analysis was explored to control for potential confounding when feasible and appropriate. inclusion criteria : all subjects (n = 142) with ais who underwent spinal fusion between january 1, 1997, and december 31, 2007, at our hospital were potentially eligible. exclusion criteria : patients who had anterior fusion (n = 11) or were older than 17 years (n = 5) were excluded, leaving 126 consecutive patients eligible. 1) : from 126 consecutive patients who were eligible, 104 were available for analysis. of the 22 patients lost to follow - up, the patient s address was unknown (n = 9), patient refused to participate (n = 4), or traveling distance was excessive (n = 9). choice of fixation device was made based on surgeon preference and was not based on type or severity of deformity. outcomes : questions 1 and 9 of the srs-22 were used to determine prevalence of pain (in the past 6 months) and disability, respectively. the srs pain domain score (pds) was evaluated as an outcome to facilitate comparison with other published studies and further evaluate factors associated with pain. risk factors and potentially confounding factors evaluated : demographic factors : gender, age at surgery, age at follow - up, follow - up time clinical factors : curve type procedural factors : surgical approach, types of instrument, lowest instrumented level complications as risk factors for pain : reoperation, infection, implant failure, dislodging of hook, correction loss, pseudarthrosis, number of levels fused analysis : to evaluate associations between any pain or disability and potential risk factors, responses were dichotomized to reflect no pain and no disability (response of five to the questions) and any pain or disability (responses 14) and chi - square test or fisher exact test (if cells contained fewer than five individuals) were performed. the kruskall - wallis test using the srs pds as a continuous outcome variable was done to compare medians among two or more groups for the categorical variables. most patients (87.5%) were female, the mean age at time of surgery was 14.9 years (range, 1217 years) and mean age at follow - up was 19.6 years (range, 1327 years). the most common curve types were lenke types 1 (48.1%) and 3 (40.4%). the universal spine system (uss synthes, paoli, pa, usa) was used most frequently with a mean of 11 levels fused (table 1). (30.8%) with only seven patients (6.7%) reporting moderate to severe or severe pain in the previous 6 months (fig. 72.1% of participants reported a current work / school activity level of 100% normal ; 20.2% reported current level of 75% normal (fig. the association between presence of any pain in the previous 6 months and gender was not significant (p =.067). superficial infection, which occurred in five patients (with four patients marking the response any pain on q1) was not significantly involved with complaints of pain (p =.074). furthermore, there was no association between pain in the previous 6 months and any other covariable (table 2). instrumentation type was the only factor that was significantly associated with a positive pds in a single implant system (p =.022) (table 3). the mean scores for individual srs domains were all above 4.0 (of a possible 5.0) (table 4). frontal plane correction loss > 15 less than 10% of values were estimated to be missing. amount of pain in the last 6 months(which best describes the amount of pain during the past 6 months ?) pseudarthrosis (includes patients who had implant failure [n = 4 ]) and correction loss [n = 7 ]) occurred in 10.6% of participants. pseudarthrosis (includes patients who had implant failure ([n = 4 ] and correction loss [n = 7 ]) occurred in 10.6% of participants. we believe that describing the prevalence of pain is more relevant than evaluation of the pain domain score because it is a frequent question that patients and their families want to know before deciding whether to undergo scoliosis surgery in terms of impairment after spinal fusion. the prevalence of low back pain after surgery for scoliosis has been reported from 7%77% in previous studies.6,7,8 in our study, 61.5% of ais patients surgically treated for ais reported back pain at 4.8-year follow - up. this prevalence is similar to the rates reported in some mid - term studies.7,9 however, the prevalence of pain found in our study is lower than the rates (73%77.7%) reported in long - term studies with a minimum follow - up of 10 years.6,10,11 the more favorable results of our study and others7,9 may be related to the shorter follow - up or the method of quantification. we agree with most of the studies that back pain after scoliosis surgery is often mild and does not produce disability in most patients.6,8,9,12,13,14 in support of these findings, danielsson and nachemson6 described that surgically treated ais patients showed no decrease of activity and functioned at the same level compared with controls without scoliosis. tsutsui compared the isolated effects of spinal fusion and deformity magnitude on quality of life in three cohorts of patients with ais (preoperative, postoperative, and nonoperative). in contrast to our results, they found that spinal fusion had an isolated negative effect on ais patients quality of life mostly due to a decrease in scores of the activity domain. a possible explanation is that surgically treated patients usually have more fear of injury than nonoperative patients, thus inhibit their propensity toward engaging in physical activities. we found no correlation of the curve size and curve correction with pain and function scores. this lack of correlation has been reported in some other studies.6,10,13,16 niemeyer reviewed 41 patients with ais treated with spinal fusion and harrington instrumentation with a mean follow - up of 23 years. takayama found that positive sagittal balance at the latest follow - up affected degree of lbp. this finding emphasizes the importance of carefully ensuring sagittal plane alignment in the treatment of spinal deformity. the effect of long versus short fusion has been much discussed. in the present study, we do not find any correlation between pain and length of spinal fusion or the caudal level of fusion as in other studies.6,9,13,17 however, in a study of 180 patients who underwent surgery for ais (harrington rod), fabry described an increase of back pain with fusions extending to l4 or l5. they followed - up 48 patients who underwent surgery for an average of 11 years and found a higher prevalence of low back pain in patients receiving fusion to l4 (61%). also, bartie found that low back pain intensity was slightly higher in those fused to l4 compared with those fused to l2 or l3 the patients reported in these studies received harrington instrumentation, which applies distractive forces on the frontal and lateral planes, and flattens the physiological lumbar lordosis when applied to the lumbar spine. this loss of lordosis probably causes the early degenerative changes and consequent lumbar pain noted in these studies.11,18,19 in 2000 prez - grueso reported on 35 patients fused to l3, l4, or l5 using cotrel - dubousset (cd) instrumentation with a minimum 10-year follow - up. they found that cd instrumentation maintained the physiological sagittal contour and there were no differences between patients and control group insofar as pain or general function was concerned. instrumentation type was the only variable that was significantly associated with elevated pain domain scores (pds) in our study. helenius compared long - term functional and radiographic outcomes using harrington and cotrel - dubousset instrumentation in ais. they found a substantially lower prevalence of low back pain in both groups but the prevalence tended to be lower in the cotrel - dubousset instrumentation group than in the harrison instrumentation group (11% versus 13% reported that they felt back pain often or very often). there seems to be a need for comparison and evidence of superiority of the different types of scoliosis instrumentation. strengths of this study include the use of validated outcomes measures and systematic exploration of factors that may have influenced pain. we also were able to obtain a sizeable long - term follow - up cohort given the timespan of observation. the significant ceiling effect for the pds (and on question one) on one hand may indicate that the highest percentage of patients had mild pain or no pain. it may, however, point to limitations of the scores ability to discriminate across levels of pain. a marked ceiling effect for low odi scores was noted in preliminary analysis, suggesting that overall patients had good function. this ceiling effect (and small numbers of patients in categories, such as specific curve type) precludes meaningful statistical analysis of this measure. the small sample size and possible lack of sensitivity of the odi may have contributed to this. since there are no presurgical scores for srs to compare with those at follow - up, conclusions regarding the extent to which surgical intervention improved patient pain and function can not be made and no conclusions regarding the proportion of individuals who attained a minimal clinically important improvement can be made. selection of instrumentation type was based on surgeon preference, not deformity severity or type. although an association between instrument type and the prevalence of pain at 6 months and the pds was seen, unmeasured factors that contributed to the decision of what instrument to use may confound the relationship. limitations of our study include the following factors : it is a retrospective study that left us unable to compare postoperative results with preoperative evaluations ; we encountered a wide range of follow - up times ; there were small numbers of patients in certain categories. there is likely limited power to detect statistical associations between some variables and the pds due to small numbers of individuals in a given category (eg, only one person had a type 5 curve) or relatively rare events, such as deep infection. given the large proportion of persons reporting normal activity, there were inadequate numbers of patients for further analysis of factors associated with disability. overall, the high percentage (> 69%) of individuals reporting no pain or mild pain in the past 6 months may suggest a certain level of surgical success. it may however point to limitations of srs score to discriminate between various levels of intensity of levels of pain. most patients experienced no severe impairment of their function after spinal fusion (72.1% of participants reported a current work / school activity level of 100% normal and 20.2% reported current level of 75% normal). although degenerative changes in the lumbar spine below the fusion have been a great concern among scoliosis surgeons, there was no association between pain and level of fusion in our study. this mid - term follow - up of spinal fusion for ais showed no important impairment of health - related quality of life. it is obvious that bas and colleagues have put a lot of thought into this study and there are some good - quality data available. this study has a lot of potential for describing factors that may be associated with pain in the ais population. the strength of this study is the incorporation of validated functional measures as the outcome (srs-22 and odi). the authors do not present much analysis of the odi outside of a listing in table 4 and focused on srs-22 instead, raising the question of what role odi played in the analysis. the article goes a long way assuring patients with ais and their families that there is a functional life to be had after fusion for scoliosis and that pain and disability are rare. as is the case with so many articles dealing with multiple variables and potentially confounding factors, this study raises a number of questions as well. what studies were undertaken to understand the potential causes of pain in those patients with poor outcomes ? can we be sure that these patients had no nonunions, malalignment, and prominent hardware or were suffering from indolent infection ? did bas and colleagues have a chance to investigate the psychosocial background of patients with poor results ? frequently, it is the insights gained from selective study of factors associated with good or insufficient outcomes that lead us to new insights. this opportunity was likely lost in this retrospective study due to the types of data gathered. all things considered, they have substantially advanced our understanding of ais surgery and its implications on patient outcomes and deserve our praise. it is obvious that bas and colleagues have put a lot of thought into this study and there are some good - quality data available. this study has a lot of potential for describing factors that may be associated with pain in the ais population. the strength of this study is the incorporation of validated functional measures as the outcome (srs-22 and odi). the authors do not present much analysis of the odi outside of a listing in table 4 and focused on srs-22 instead, raising the question of what role odi played in the analysis. the article goes a long way assuring patients with ais and their families that there is a functional life to be had after fusion for scoliosis and that pain and disability are rare. as is the case with so many articles dealing with multiple variables and potentially confounding factors, this study raises a number of questions as well. what studies were undertaken to understand the potential causes of pain in those patients with poor outcomes ? can we be sure that these patients had no nonunions, malalignment, and prominent hardware or were suffering from indolent infection ? did bas and colleagues have a chance to investigate the psychosocial background of patients with poor results ? frequently, it is the insights gained from selective study of factors associated with good or insufficient outcomes that lead us to new insights. this opportunity was likely lost in this retrospective study due to the types of data gathered. all things considered, they have substantially advanced our understanding of ais surgery and its implications on patient outcomes and deserve our praise. | study design : retrospective prognostic study.objectives : to describe the prevalence of pain following fusion for adolescent idiopathic scoliosis and to identify factors associated with pain and disability.methods : from 126 consecutive patients surgically treated for scoliosis between 1997 and 2007, 104 (82.5%) completed srs-22 and odi questionnaires at a last follow - up (mean, 4.8 years ; range 111.2 years). prevalence of pain and disability were determined from srs questions 1 and 9 respectively, with any pain or decrease in activity considered clinically significant. srs pain domain scores (pds) were also evaluated.results : most participants reported no pain (38.5%) or mild pain (30.8%) and 72.1% of participants reported a current work / school activity level of 100% normal. an association between instrument type and the presence of any pain in the previous 6 months was noted (p =.022). instrument type was the only factor that was significantly associated with the pds (p =.0052).conclusions : the high percentage of patients reporting no pain or mild pain may suggest overall success of the procedures. although an association between instrument type and pain was seen, unmeasured factors that contributed to the decision of what instrument to use may confound the relationship. from these data a causal inference can not be made.final class of evidence - prognosisstudy designprospective cohortretrospective cohortcase controlcase seriesmethodspatients at similar point in course of treatmentf / u 85%similarity of treatment protocols for patient groupspatients followed up long enough for outcomes to occurcontrol for extraneous risk factorsoverall class of evidenceiiithe definiton of the different classes of evidence is available on page 55.potentially confounding factors were systematically explored and considered for stratified analysis as appropriate. |
d.b., a.k.) in accordance with the recommendations of the cochrane collaboration, using pubmed, embase, web of science, cochrane central register of controlled trials, and scopus databases through march 25, 2015. our study was a systematic review and meta - analysis and thus did not require institutional review board approval. the inclusion was limited to the studies (1) that compared cf with cc in radiofrequency catheter ablation of atrial fibrillation, (2) that included an adult population aged > 18 years only, and (3) that provided data on outcomes of interest. we excluded noncomparative trials, case reports, editorials, letters, replies, and reviews. we also excluded any study that included other ablation technologies (eg, cryoablation or robotic navigation) that could affect our results and increase bias. we used the newcastle - ottawa scale to further assess the quality of the observational studies. studies were judged on 3 broad perspectives : (1) selection of the study groups ; (2) comparability of the groups ; and (3) ascertainment of either the exposure or outcomes of interest for case control or cohort studies, respectively.14 the quality of the randomized studies was evaluated based on the 5-point scale outlined by jadad, with the following criteria : randomization with proper concealment of the allocation sequence, blinding of the patient and investigator to treatment allocation with description of the blinding method, and completeness of follow - up.15 three reviewers (m.s., d.b., a.k.) independently extracted the data from published sources ; disagreements were resolved by discussion and, as necessary, in consultation with a third person (e.c. secondary outcomes included procedure and ablation times, total fluoroscopic time, and complication rates. whenever possible, direct communication with the authors of the papers was undertaken in an attempt to obtain the data of interest if presentation in the manuscript was incomplete. the following outcomes were identified as relevant measures to compare for the studied groups : (1) rate of recurrence, defined as any symptomatic or asymptomatic atrial arrhythmia recurrence after ablation (density of monitoring and cutoff for duration is manuscript specific) ; (2) major complications, including embolic events, cardiac tamponade or pericardial effusion requiring intervention, phrenic nerve palsy, pulmonary vein stenosis, atrial esophageal fistula, and death ; (3) minor complications, including pericardial effusion (not requiring intervention) or vascular access complications (including hematoma, arteriovenous fistula, or aneurysm) ; (4) procedural parameters, defined as total procedural time, ablation time, and fluoroscopy time according to the individual study protocols. although the assessment of outcomes across the trials was not standardized, the same criteria were applied equally to the groups within each trial. the software package revman (version 5), provided by the cochrane collaboration, was used for combining outcomes from the individual studies and for statistical analysis. outcomes were pooled using a random - effects model described by dersimonian and laird.16 summary estimates and 95% ci were reported for dichotomous variables as odds ratio (or) and for continuous variables as standardized mean difference. the heterogeneity between studies was assessed using the cochran q test and i. an i > 50% was considered to represent significant heterogeneity.17 statistical significance was set as p 18 years only, and (3) that provided data on outcomes of interest. we excluded noncomparative trials, case reports, editorials, letters, replies, and reviews. we also excluded any study that included other ablation technologies (eg, cryoablation or robotic navigation) that could affect our results and increase bias. we used the newcastle - ottawa scale to further assess the quality of the observational studies. studies were judged on 3 broad perspectives : (1) selection of the study groups ; (2) comparability of the groups ; and (3) ascertainment of either the exposure or outcomes of interest for case control or cohort studies, respectively.14 the quality of the randomized studies was evaluated based on the 5-point scale outlined by jadad, with the following criteria : randomization with proper concealment of the allocation sequence, blinding of the patient and investigator to treatment allocation with description of the blinding method, and completeness of follow - up.15 three reviewers (m.s., d.b., a.k.) independently extracted the data from published sources ; disagreements were resolved by discussion and, as necessary, in consultation with a third person (e.c. secondary outcomes included procedure and ablation times, total fluoroscopic time, and complication rates. whenever possible, direct communication with the authors of the papers was undertaken in an attempt to obtain the data of interest if presentation in the manuscript was incomplete. the following outcomes were identified as relevant measures to compare for the studied groups : (1) rate of recurrence, defined as any symptomatic or asymptomatic atrial arrhythmia recurrence after ablation (density of monitoring and cutoff for duration is manuscript specific) ; (2) major complications, including embolic events, cardiac tamponade or pericardial effusion requiring intervention, phrenic nerve palsy, pulmonary vein stenosis, atrial esophageal fistula, and death ; (3) minor complications, including pericardial effusion (not requiring intervention) or vascular access complications (including hematoma, arteriovenous fistula, or aneurysm) ; (4) procedural parameters, defined as total procedural time, ablation time, and fluoroscopy time according to the individual study protocols. although the assessment of outcomes across the trials was not standardized, the same criteria were applied equally to the groups within each trial. the software package revman (version 5), provided by the cochrane collaboration, was used for combining outcomes from the individual studies and for statistical analysis. outcomes were pooled using a random - effects model described by dersimonian and laird.16 summary estimates and 95% ci were reported for dichotomous variables as odds ratio (or) and for continuous variables as standardized mean difference. the heterogeneity between studies was assessed using the cochran q test and i. an i > 50% was considered to represent significant heterogeneity.17 statistical significance was set as p 20 g were free from af recurrence at 12 months, whereas all patients treated with an average cf of 5 g is associated with adequate lesion formation with impedance fall during 60 seconds of ablation.36 in addition, cf varies widely in certain anatomic locations.37 our study provides important information regarding the optimal average cf of 175 g with acceptable recurrence and complication rates, especially the risk of pericardial tamponade or effusion requiring intervention. there was significant reduction in procedure and ablation times not only in comparison to point - by - point manual radiofrequency ablation but also similar to what is seen in single - shot devices such as the cryoballoon.38 using cf feedback as a main determinant of adequate lesion formation would minimize ineffective lesion formation and thus achieve pulmonary vein isolation faster without the need for additional ablation lesions to confirm isolation. radiation safety remains a major concern in invasive electrophysiology. in this analysis, fluoroscopic time was reduced by 8 minutes in cf compared with cc groups, with an average reported fluoroscopy time of 28 minutes in cf versus 36 minutes in cc groups. this reduction is largely related to the continuous monitoring of the catheter while using cf, enabling operators to manipulate and advance the catheter without the need of excessive fluoroscopic guidance. in contrast, more frequent visualization with fluoroscopy is typically used with ccs in an effort to prevent perforation or to assess contact by the tip of catheter appearance and movements. attention to radiation exposure and a statistically significant decrease in exposure have clinical relevance for both operators and patients undergoing ablation procedures.39,40 this meta - analysis has proven the enhanced safety of using the cf technology with acceptable rates of minor and major complications and reduced risk of cardiac perforation (although it did not reach statistical significance). this is related mainly to the ability to continuously monitor the catheter while manipulating it in the cardiac chambers, with real - time instant feedback of the catheter tip tissue contact. moreover, avoiding ablation at suboptimal cf would reduce the need for excessive ablations and subsequent related complications. some studies were of limited quality, given their retrospective and single - center designs. factors like different levels of experience among operators, catheters used, instrumentation, ablation energy and duration, magnitude of ablation performed, antiarrhythmic drugs, and incomplete data may have altered our conclusions. some operators performed, in addition to pulmonary vein isolation ablation, more complex lesion sets (eg, complex atrial fractionated electrogram, left atrial roof line, mitral isthmus line), and that may have affected the outcomes of these procedures compared with only pulmonary vein isolation procedures. we could not address this issue, given the heterogeneity of ablation protocols among different operators. some of the outcomes had high i representing significant heterogeneity such as procedure, ablation, and fluoroscopic times. that said, outcomes like recurrence rate, major complications, cardiac tamponade, and minor complications had insignificant heterogeneity that could reflect some similarities among studies. some studies were of limited quality, given their retrospective and single - center designs. factors like different levels of experience among operators, catheters used, instrumentation, ablation energy and duration, magnitude of ablation performed, antiarrhythmic drugs, and incomplete data may have altered our conclusions. some operators performed, in addition to pulmonary vein isolation ablation, more complex lesion sets (eg, complex atrial fractionated electrogram, left atrial roof line, mitral isthmus line), and that may have affected the outcomes of these procedures compared with only pulmonary vein isolation procedures. we could not address this issue, given the heterogeneity of ablation protocols among different operators. some of the outcomes had high i representing significant heterogeneity such as procedure, ablation, and fluoroscopic times. that said, outcomes like recurrence rate, major complications, cardiac tamponade, and minor complications had insignificant heterogeneity that could reflect some similarities among studies. this meta - analysis suggests that the use of cf technology results in a significant reduction of af recurrence rate after af ablation in comparison to the cc group. cf technology is able to significantly reduce procedure and fluoroscopic times without compromising the complication rate. dr di biase is a consultant for biosense webster, boston scientific, stereotaxis and st jude medical, and has received speaker honoraria / travel from medtronic, atricure, epiep and biotronik. | backgroundcatheter tissue contact is essential for effective lesion formation, thus there is growing usage of contact force (cf) technology in atrial fibrillation ablation. we conducted a meta - analysis to assess the impact of cf on clinical outcomes and procedural parameters in comparison to conventional catheter for atrial fibrillation ablation.methods and resultsan electronic search was performed using major databases. outcomes of interest were recurrence rate, major complications, total procedure, and fluoroscopic times. continuous variables were reported as standardized mean difference ; odds ratios were reported for dichotomous variables. eleven studies (2 randomized controlled studies and 9 cohorts) involving 1428 adult patients were identified. cf was deployed in 552 patients. the range of cf used was between 2 to 60 gram - force. the follow - up period ranged between 10 and 53 weeks. in comparing cf and conventional catheter groups, the recurrence rate was lower with cf (35.1% versus 45.5%, odds ratio 0.62 [95% ci 0.450.86 ], p=0.004). shorter procedure and fluoroscopic times were achieved with cf (procedure time : 156 versus 173 minutes, standardized mean difference 0.85 [95% ci 1.48 to 0.21 ], p=0.009 ; fluoroscopic time : 28 versus 36 minutes, standardized mean difference 0.94 [95% ci 1.66 ; 0.21 ], p=0.01). major complication rate was lower numerically in the cf group but not statistically significant (1.3% versus 1.9%, odds ratio 0.71 [95% ci 0.291.73 ], p=0.45).conclusionsthe use of cf technology results in significant reduction of the atrial fibrillation recurrence rate after atrial fibrillation ablation in comparison to the conventional catheter group. cf technology is able to significantly reduce procedure and fluoroscopic times without compromising complication rate. |
the single most important prognostic indicator in breast cancer patients is the presence of metastatic dissemination to axillary lymph nodes. the sentinel lymph node (sln) is the lymph node that primarily drains the tumour site and sln biopsy (slnb) is considered the standard of care for clinically node - negative disease. however, the role and utility of high throughput evaluation of slns and the finding of minimal metastases continue to fuel debate. the pathological assessment of the slns with multilevel evaluation and immunohistochemistry (ihc) is common practice and isolated tumour cells (itcs) have been separated from micrometastases in international staging manuals for breast cancer. however, recent studies with up to 6 years of follow - up have demonstrated that both patients with negative sln and patients with sln micrometastases had low recurrence of axillary metastases and they report no statistical differences in overall survival between patients with negative sln and sln micrometastases [5, 6 ]. the upstaging potential of slnb and the workload imposed by a systematic search for low - volume sln involvement has stimulated pathologists to move toward establishing alternative procedures such as rt - pcr to detect minimal amounts of mrnas of specific breast cancer markers [713 ]. the majority of the studies [7, 10, 12, 13 ] have compared rt - pcr analysis of fresh nodal tissues with h&e and ihc analysis of formalin - fixed paraffin embedded slns. few studies [14, 15 ] have directly compared rt - pcr and step - sectioning and ihc results on frozen sections. most mrna extracted for rt - pcr is obtained from fresh sln tissues for the purpose of determining sln involvement and of guiding an immediate formal axillary dissection. a recent study by langer. demonstrated that with intraoperative frozen sections at three levels with a cutting interval of 150 m, nearly all macrometastases can be detected and as a consequence, the number of second operations for axillary dissection was low. in our breast unit, we use ultrasonographically guided fine needle aspiration of suspect lymph nodes as a pre - operatory staging procedure. using this procedure, the percentage of metastatic sln is markedly reduced and metastatic slns are confined to micrometastases and itcs. therefore, we use a protocol for management of patients that includes day surgery for breast cancer and sln excision (without any examination of intraoperatory frozen sections), followed by step - sectioning of sln on formalin - fixed tissue. however, formalin, a cross - linking agent, induces rna chemical changes and fragmentation, impairing quantification of gene expression. recently, new tissue fixatives providing preservation of both tissue morphology and rna / dna / proteins have been proposed [1921 ]. methacarn is a non - cross - linking organic solvent consisting of a solution of methanol, chloroform and acetic acid. it not only preserves optimal morphology and antigenicity for ihc, but it also allows for the analysis of mrna and protein expression levels and the mutational analysis of target genes from microdissected tissue samples of paraffin embedded tissues, with yields close to those achievable using unfixed frozen tissues. thus, in the present study, we fixed a series of slns in methacarn with the aim to evaluate and compare the reliability of conventional histopathology or ihc versus rt - pcr to detect nodal metastatic foci on the same sln. as a pre - operatory staging procedure, all patients with invasive breast cancer are submitted to ultrasound (us) examination of the axilla followed by ultrasonographically guided fine needle aspiration (us - fna) of suspect lymph nodes. patients with negative us or negative fna of axillary lymph node proceed to sln biopsy and cancer excision in day surgery without any intraoperatory frozen section examination. the criterion for sln biopsy was early - stage (t12 n0 m0) invasive breast cancer (table 1). using the pre - operatory staging procedure and the sln biopsy criterion, from march 2006 to december 2006, a total of 74 sln biopsies were performed in 62 patients out of 137 breast cancer cases treated in our institution. characteristics of the 58 breast cancer patients 9 cases with macro- and micro - metastases in sln, 2 cases with itc and in 2 cases with negative sln (pn0(i)). during the study period, all slns were immediately fixed in methacarn (60% methanol, 30% chloroform and 10% glacial acetic acid). this regional guideline was developed to help ensure that none of the macro or micrometastases are missed during tissue processing, since in the local surgical protocol, an axillary dissection is performed only in the presence of metastases larger than 2 mm. however, each patient s management was discussed at multidisciplinary meetings and therapeutic decisions could be modified depending on other clinical and pathological parameters such as presence or absence of lymphovascular invasion. after 6 hrs of fixation gross sections were placed in one or two blocks depending on the size of the sln and kept in their correct position with the aid of a sponge. during embedding in paraffin wax, care was taken to preserve the original sequence of slices ; the top cut - surface of a gross section and the bottom surface of the next section represented contiguous areas of the sln (fig. (a) sentinel nodes gross sectioning at 2 mm and (b) sentinel node haematoxylin and eosin stained slides of case 10. sections of level 13 without metastases, 14 and 20 with metastases (circled) are shown at higher magnification (20) (arrows) ; (c) rt - pcr : mrna for ck19, cea and mammaglobin (mmg) not detected on sections from levels 4 to 5 and (d) detected on sections from levels 17 to 20 corresponding to the site of metastases. each sln was step - sectioned at 100 m intervals with a standard microtome until the extinction of the wax blocks. the first two consecutive sections (4 m thickness) of each level were used for h&e staining and for ihc analysis. six sections (7 m thickness) were cut from between levels 4 and 5 and were collected in an rnase - free tube for molecular analysis. to reduce the risk of contamination, new blades were used for sln cutting during both gross and microscopic sectioning. similar to the slns, the non - slns were isolated from the fat tissue of the axillary dissection specimen and grossly sliced at 2 mm intervals if larger than 0.5 cm. if the sln was found to be negative on h&e staining, the sections collected for ihc analysis were stained using an automated immunostainer (benchmark autostainer, ventana medical systems, tucson, az, usa) using ae1/ae3 cytokeratin antibodies (clones ae1/ae3 & pck26, pre - diluted, ventana - diapath, tucson, az, usa). metastatic deposits were measured in two dimensions and categorized according to the new american joint committee on cancer staging system as isolated tumour cells / clusters (itcs ; pn0(i+)) when they were 0.2 mm or less in the larger dimension and as micrometastases (pn1mi) when they measured more than 0.2 mm, but not greater than 2.0 mm ; sln metastases larger than 2 mm were categorized as having pn1a category nodal involvement. mrna was extracted from the six sections collected from between step - sectioning levels 4 and 5. in one case, the tissue for rt - pcr was also obtained from seven 4-m - thick sections previously collected for ihc. rna was isolated using the masterpure purification kit (epicentre, madison, wi, usa) ; a dnase i treatment step was included. rna concentration was measured with a spectrophotometer (biophotomer eppendorf ag, hamburg, germany). one microgram of each sample rna was reverse transcribed to cdna with superscript ii enzyme (invitrogen corporation, carlsbad, ca, usa) using oligo - dt according to the manufacturer s instructions. cdna samples were subsequently amplified for the target sequences by using published primers for cea (ceacam5, nm_004363.2) and mammaglobin (scgb2a2, nm_002411.2) and semi - nested and single step pcr, respectively. to optimize ck19 (krt19, nm_002276.4) mrna detection in methacarn - fixed tissue, we developed a semi - nested rt - pcr with a first round pcr using primers previously reported, followed by a second pcr using an inner primer designed with the primer 3 program (http://frodo.wi.mit.edu/cgi-bin/primer3/primer3_www.cgi) from the sequence of the human ck19 gene. mrna for beta-2 microglobulin (b2 m, nm_004048.2) was amplified for each sample for quality control of rna integrity and absence of dna polymerase inhibitors. the cea gene was amplified with a touchdown (td) pcr program. in selected cases, rt - pcr for mammaglobin was performed on rna extracted from six sections (7-m thick) of the corresponding primary tumour, using the same extraction protocol and the same quality and quantity controls reported above. in this case, the histological tissues were formalin fixed and paraffin embedded (ffpe) ; therefore, we optimized mammaglobin molecular analysis by targeting a smaller amplicon with primers designed with the primer 3 program (http://frodo.wi.mit.edu/cgi-bin/primer3/primer3_www.cgi) on the same human mammaglobin gene sequence. forward and reverse primer sequences, the size of the corresponding amplicon and the annealing temperature are reported in table 2. every reaction included 5 l cdna (corresponding to 100 ng), 1.5 mm mgcl2, 0.2 mm each dntp, 1 u of taq dna polymerase, 5 pmoles of each primer and pcr buffer 1x. for mammaglobin expression analysis on cdna from ffpe primary tumours, 25 pmoles of each primer and 300 ng of cdna were used. nested and semi - nested pcrs were performed with 5 l of the first step pcr product in 50 l of final volume with the same starting conditions. sentinel nodes of six patients with cutaneous melanoma who underwent lymphadenectomy (reactive lymph nodes by histology without evidence of malignancy) were used as negative controls. both mrna extracted from a pool of mcf-7 and skbr3 breast cancer cell lines and from a primary breast cancer fixed in methacarn and embedded in paraffin served as positive controls. all pcr products were visualized by electrophoresis on 2% agarose gels, containing 0.5 g / ml ethidium bromide. forward and reverse primer sequences, corresponding amplicons and annealing temperature of cea, ck19, mammaglobin and 2 microglobulin for mammaglobin rt - pcr analysis using rna from methacarn - fixed paraffin embedded tissues. for mammaglobin rt - pcr analysis using rna from formalin - fixed paraffin embedded tissues. to reduce the risk of contamination from previously amplified products, separate areas were used for rna isolation, amplification and electrophoresis. as a pre - operatory staging procedure, all patients with invasive breast cancer are submitted to ultrasound (us) examination of the axilla followed by ultrasonographically guided fine needle aspiration (us - fna) of suspect lymph nodes. patients with negative us or negative fna of axillary lymph node proceed to sln biopsy and cancer excision in day surgery without any intraoperatory frozen section examination. the criterion for sln biopsy was early - stage (t12 n0 m0) invasive breast cancer (table 1). using the pre - operatory staging procedure and the sln biopsy criterion, from march 2006 to december 2006, a total of 74 sln biopsies were performed in 62 patients out of 137 breast cancer cases treated in our institution. characteristics of the 58 breast cancer patients 9 cases with macro- and micro - metastases in sln, 2 cases with itc and in 2 cases with negative sln (pn0(i)). during the study period, all slns were immediately fixed in methacarn (60% methanol, 30% chloroform and 10% glacial acetic acid). this regional guideline was developed to help ensure that none of the macro or micrometastases are missed during tissue processing, since in the local surgical protocol, an axillary dissection is performed only in the presence of metastases larger than 2 mm. however, each patient s management was discussed at multidisciplinary meetings and therapeutic decisions could be modified depending on other clinical and pathological parameters such as presence or absence of lymphovascular invasion. after 6 hrs of fixation gross sections were placed in one or two blocks depending on the size of the sln and kept in their correct position with the aid of a sponge. during embedding in paraffin wax, care was taken to preserve the original sequence of slices ; the top cut - surface of a gross section and the bottom surface of the next section represented contiguous areas of the sln (fig. (a) sentinel nodes gross sectioning at 2 mm and (b) sentinel node haematoxylin and eosin stained slides of case 10. sections of level 13 without metastases, 14 and 20 with metastases (circled) are shown at higher magnification (20) (arrows) ; (c) rt - pcr : mrna for ck19, cea and mammaglobin (mmg) not detected on sections from levels 4 to 5 and (d) detected on sections from levels 17 to 20 corresponding to the site of metastases. each sln was step - sectioned at 100 m intervals with a standard microtome until the extinction of the wax blocks. the first two consecutive sections (4 m thickness) of each level were used for h&e staining and for ihc analysis. six sections (7 m thickness) were cut from between levels 4 and 5 and were collected in an rnase - free tube for molecular analysis. to reduce the risk of contamination, new blades were used for sln cutting during both gross and microscopic sectioning. similar to the slns, the non - slns were isolated from the fat tissue of the axillary dissection specimen and grossly sliced at 2 mm intervals if larger than 0.5 cm. if the sln was found to be negative on h&e staining, the sections collected for ihc analysis were stained using an automated immunostainer (benchmark autostainer, ventana medical systems, tucson, az, usa) using ae1/ae3 cytokeratin antibodies (clones ae1/ae3 & pck26, pre - diluted, ventana - diapath, tucson, az, usa). metastatic deposits were measured in two dimensions and categorized according to the new american joint committee on cancer staging system as isolated tumour cells / clusters (itcs ; pn0(i+)) when they were 0.2 mm or less in the larger dimension and as micrometastases (pn1mi) when they measured more than 0.2 mm, but not greater than 2.0 mm ; sln metastases larger than 2 mm were categorized as having pn1a category nodal involvement. mrna was extracted from the six sections collected from between step - sectioning levels 4 and 5. in one case, the tissue for rt - pcr was also obtained from seven 4-m - thick sections previously collected for ihc. rna was isolated using the masterpure purification kit (epicentre, madison, wi, usa) ; a dnase i treatment step was included. rna concentration was measured with a spectrophotometer (biophotomer eppendorf ag, hamburg, germany). one microgram of each sample rna was reverse transcribed to cdna with superscript ii enzyme (invitrogen corporation, carlsbad, ca, usa) using oligo - dt according to the manufacturer s instructions. cdna samples were subsequently amplified for the target sequences by using published primers for cea (ceacam5, nm_004363.2) and mammaglobin (scgb2a2, nm_002411.2) and semi - nested and single step pcr, respectively. to optimize ck19 (krt19, nm_002276.4) mrna detection in methacarn - fixed tissue, we developed a semi - nested rt - pcr with a first round pcr using primers previously reported, followed by a second pcr using an inner primer designed with the primer 3 program (http://frodo.wi.mit.edu/cgi-bin/primer3/primer3_www.cgi) from the sequence of the human ck19 gene. mrna for beta-2 microglobulin (b2 m, nm_004048.2) was amplified for each sample for quality control of rna integrity and absence of dna polymerase inhibitors. the cea gene was amplified with a touchdown (td) pcr program. in selected cases, rt - pcr for mammaglobin was performed on rna extracted from six sections (7-m thick) of the corresponding primary tumour, using the same extraction protocol and the same quality and quantity controls reported above. in this case, the histological tissues were formalin fixed and paraffin embedded (ffpe) ; therefore, we optimized mammaglobin molecular analysis by targeting a smaller amplicon with primers designed with the primer 3 program (http://frodo.wi.mit.edu/cgi-bin/primer3/primer3_www.cgi) on the same human mammaglobin gene sequence. forward and reverse primer sequences, the size of the corresponding amplicon and the annealing temperature are reported in table 2. every reaction included 5 l cdna (corresponding to 100 ng), 1.5 mm mgcl2, 0.2 mm each dntp, 1 u of taq dna polymerase, 5 pmoles of each primer and pcr buffer 1x. for mammaglobin expression analysis on cdna from ffpe primary tumours, 25 pmoles of each primer and 300 ng of cdna were used. nested and semi - nested pcrs were performed with 5 l of the first step pcr product in 50 l of final volume with the same starting conditions. sentinel nodes of six patients with cutaneous melanoma who underwent lymphadenectomy (reactive lymph nodes by histology without evidence of malignancy) were used as negative controls. both mrna extracted from a pool of mcf-7 and skbr3 breast cancer cell lines and from a primary breast cancer fixed in methacarn and embedded in paraffin served as positive controls. all pcr products were visualized by electrophoresis on 2% agarose gels, containing 0.5 g / ml ethidium bromide. forward and reverse primer sequences, corresponding amplicons and annealing temperature of cea, ck19, mammaglobin and 2 microglobulin for mammaglobin rt - pcr analysis using rna from methacarn - fixed paraffin embedded tissues. for mammaglobin rt - pcr analysis using rna from formalin - fixed paraffin embedded tissues. to reduce the risk of contamination from previously amplified products, separate areas were used for rna isolation, amplification and electrophoresis. the mrna extraction from fixed tissue was successful only in 93% of cases, corresponding to 69 slns from 58 patients. the total rna extracted from each sln varied from 4 to 140 g (see fig. clinical - pathological characteristics of these 58 patients are detailed in table 1 distribution of the amount of total rna (g) extracted from the 69 slns studied by rt - pcr. by h&e, seven of these cases were classified as macrometastases and two as micrometastases. in one sln, a macrometastasis of 3.3 mm infiltrating the capsule and the pericapsular fat tissue was observed only in the sections cut from the last seven consecutive levels (fig. itcs were detected by ihc staining and in three of these, the results were confirmed by retrospective examination of h&e stained slides. cell line mrna and/or mrna from the primary methacarn - fixed breast cancer used as positive controls showed strong combined expression of cea, ck19 and mammaglobin. none of the slns from the 6 melanoma patients used as negative controls were amplified by rt - pcr using the 3 breast cancer mrna markers. overall, 33 out of 69 (47.8%) slns were positive for at least one gene with rt - pcr performed on sections obtained from between levels 4 and 5 (table 3). specifically, 6 out of 7 cases positive for metastases and 1 out of 2 micrometastases were rt - pcr positive with all three markers. the specificity of this kind of positivity was indirectly shown by the negative results of rt - pcr performed on sections obtained in early levels of the sln affected by macrometastases in the last 7 levels of sectioning (table 4, case 10, fig. the sections collected for ihc analysis of these last levels were thus used for rt - pcr and this time the tissue was positive for all 3 markers (fig. analogously, rt - pcr performed on tissue obtained between levels 4 and 5 was negative for 3 markers in one sln affected by a micrometastases detected by h&e staining of sections from levels 1 through 4 (table 4, case 9). in this same case, none of the slns affected by itc were positive for mammaglobin by rt - pcr. in the set of itcs, one sln was positive for cea alone (table 4, case 4) and two were positive for ck19 and cea (table 4, case 5 and 7). of the 23 slns negative by both h&e and ihc, 8 were both ck19- and cea - positive ; 8 were only cea - positive and 7 were ck19-positive only. results of rt - pcr markers in rt - pcr positive slns mapping of the results obtained by h&e / ihc and rt - pcr in consecutive levels of 16 sln with isolated tumour cells (itc) (0.2 mm), micrometastases (> 0.2 2 mm) and macrometastases (2 mm, using either morphological or molecular procedures. in a previous geometrical model, cserni showed that incomplete sectioning of the slns in extreme situations would almost completely miss metastases > 2 mm, which would be identified at best as itc. a step - sectioning protocol with levels of 250 m or 200 m would be adequate for detecting nearly all micrometastases as defined by the current tnm definitions (i.e. metastases > 0.2 mm). in the present study, using a step - sectioning protocol with intervals of 100 m, we demonstrated that both ihc and rt - pcr are of limited value to detect all itcs. in particular, we confirmed that itcs are randomly distributed in the lymph node and that some of them may be missed regardless, or alternately, that they might be found by chance. these data should be taken into account when dealing with studies considering the predictive and prognostic value of itcs, such as a recent study that concluded that nanometastases (defined identically to itcs ; i.e. as tumour cell deposits 0.2 mm) detected by ihc in axillary lymph nodes predict a worse survival than larger micrometastases which do not influence the outcome. in addition, recent data suggest that patients with occult micrometastases in axillary lymph nodes not receiving adjuvant systemic therapy have a prognosis similar to that of patients without micrometastases. ideally, for comparing different methodologies, sections obtained from all cutting levels should be analysed by rt - pcr, but the labour - intensive nature of the process makes this prohibitive. using the protocol described herein, we could precisely map the distribution of metastases and indirectly prove that rt - pcr analysis using mammaglobin would be specific enough to find all metastases and micrometastases, but that it is relatively insensitive for submicroscopic disease. the expression of mammaglobin in the primary tumours that gave rise to itc in mammaglobin negative slns reinforces this hypothesis. on the contrary, a recent study that tested both mammaglobin a and b isoforms in sln using rt - pcr reported a high sensitivity for these markers. on the other hand, the expression by rt - pcr of ck19 and/or cea in morphologically negative sln could be more effective just in detecting itcs considering that the thickness of the tissue analysed by rt - pcr in our study was 0.042 mm (6 sections of 7 microns). however, one case with lympho - vascular invasion and sln negative at histology, but positive for cea by rt - pcr, had non - sln metastases, suggesting that sporadic itc could have been missed in the histological examination but recognized by rt - pcr. previous studies utilizing epithelial mrna markers to detect breast cancer micrometastases by rt - pcr [711, 14, 26 ] have outlined the risk of false - positive results, whereas tumour mrna markers are more specific, but are not uniformly expressed in all cancers causing the risk of false - negative results. thus, a combination of both epithelial and tumour markers could potentially yield the most reliable results. recently, new assays for quantitative measurement of mammoglobin and/or ck19 expression [3335 ] have been described and proposed for rapid molecular diagnosis on sln. in addition, one of these assays indicates specific cut - off values for ck19 mrna to distinguish micrometastatic from metastatic slns and to define pn0 sln (including itcs) with an overall concordance rate between the molecular assay and histopathology of 98.2%. however, in the sixth edition of the ajcc cancer staging manual, metastatic lesions identified only by rt - pcr are classified as pn0(mol+) because there are insufficient data to determine whether such lesions are clinically significant. prospective studies that would analyse the whole sln using quantitative mrna measurement and a pool of markers would probably validate molecular procedures in substituting the high - throughput histopathological analysis of the sln. in conclusion, the main goal of our study was to show that the sampling procedures of the tissue to be submitted to rt - pcr or conventional histopathological processes are the main reason for discrepancies between molecular and morphological analysis. | the optimal pathological assessment of sentinel nodes (slns) in breast cancer is a matter of debate. currently, multilevel histological evaluation and immunohistochemistry (ihc) are recommended, but alternative rt - pcr procedures have been developed. to assess the reliability of these different procedures, we devised a step - sectioning protocol at 100 micron - intervals of 74 slns using methacarn fixation. mrna was extracted from sections collected from levels 4 to 5. mammaglobin, cea and ck19 were used for rt - pcr. mrna extraction was successful in 69 slns. of these, 7 showed macrometastases (> 2 mm), 2 showed micrometastases (< 2 mm) and 7 showed isolated tumour cells (itc) by ihc. rt - pcr was positive for the three markers in 6 of 7 macrometastases and in 1 of 2 micrometastases. in the 2 rt - pcr negative cases, metastases were detected only on sections distant from those analysed by rt - pcr. cea and/or ck19 were positive by rt - pcr in 3 of 7 itc and in 23 morphologically negative slns. in conclusion, the main goal of our study was to show that the use of alternate sections of the same sample for different procedures is the key reason for the discrepancies between molecular and morphological analyses of sln. we believe that only prospective studies with quantitative mrna analysis of specific metastatic markers on the whole lymph node can elucidate the utility of molecular assessments of sln. |
nasal deformities associated with unilateral cleft lip are characterized by asymmetry, which gradually progresses with severity of the cleft, and nostril structures with morphological changes.1 2 3 4 the main supporting structure of the nasal wing, the nasal lower lateral cartilage, in patients with cleft lip is concave with a depressed nasal tip, and separates from the opposite side, not the cleft, resulting in depression and collapsed nasal tip asymmetry,5 which is very common, even after surgical treatment of cleft lip.6 the creation of a symmetrical nose is a big challenge, and it is also difficult to evaluate an outcome after surgery. subjective evaluation by expert surgeons in cleft lip surgery is the standard ; however, a simple objective measure would be important if it could faithfully reflect this pattern.3 use of anthropometric techniques for preoperative quantitative evaluations of morphological changes in soft and stiff tissues of the cleft face is essential to objectively determine the facial anatomy and surgical treatment result.7 understanding how the nostril is modified after the surgical treatment of cleft lip may contribute to the clinical practice of health professionals who work on the nose. this knowledge may influence the surgical program focused on the specific need of each patient and the rehabilitation process of lip mobility and facial expression, which are closely linked to the muscles of the superior lip and nose base. considering that few studies have investigated this theme, the present study aims to review the literature on the condition of the nostril morphometry in patients with cleft lip before and after cleft surgery, as well as identify the issues involved in assessing these changes in this population. a literature review was performed from the databases of the medical literature analysis and retrieval system online (medline), and the data search occurred in january 2012. a specific strategy was developed for crossing the descriptors (mesh)keywords for retrieving subjects from literature and scientific terms not found in mesh terms but of relevance to the research. in medline, using the pubmed search engine we performed a search strategy using the syntax : cleft lip (mesh) ; cleft lip (mesh) and nostril ; cleft lip (mesh) and morphometry ; anthropometry (mesh) and nostril ; nostril and morphometry. inclusion criteria were original articles (excluding editorials and case reports) reporting on individuals with unilateral cleft lip or cleft lip and palate who underwent anthropometric measurements of the nose or nostril before and after cleft lip surgical correction. exclusion criteria were measurements performed on bony structures or other facial structures that did not include the nostrils or the nose ; studies that compared surgical techniques ; articles written in oriental languages (mandarin, japanese) ; and articles not found by switching bibliographic system (comut). we excluded those that clearly did not fit any of the criteria for inclusion in this study. then, we read remaining abstracts and excluded those that clearly did not fit any of the inclusion criteria previously established. in the third stage, all the studies that were not excluded in these first two steps were read in entirety for the selection of articles to be included in this review (fig. revew of literature stages. in medline via pubmed, crossing the free terms nostril and morphometry found three articles, of which all were excluded by the title. crossing the keyword morphometry found seven articles that were excluded by the title. crossing the keyword anthropometry and the free term nostril found 71 articles, of which 61 were excluded by the title, 8 were excluded after reading the abstract, and 2 were selected for full reading. crossing the keyword cleft lip and the term free nostril found 177 articles, of which 131 were excluded by the title, 41 were excluded after reading the abstract, and 5 articles were selected for full reading. crossing the keywords cleft lip and anthropometry found 1,085 articles, of which 923 were excluded by the title and 147 were excluded after reading the abstract, leaving 15 articles selected for full reading. of the 22 articles selected for full reading, we excluded 5 papers that were repeated, 1 written in mandarin, and another not found by switching bibliographic system (comut). considering inclusion and exclusion criteria, of the 15 fully read articles, ten articles were excluded for different reasons : 3 because they did not perform morphometric comparison between pre- and postoperative nostril, 2 because they were not performed in patients with clefts, 2 because they were performed without analyzing the nose or nostrils, 1 because a morphometric study was not performed, 1 because it used cephalometric radiographs for analysis, 1 because it conducted analysis and validation of measurement method without evaluating and interpreting results of the evaluations. there were no sufficient data to conduct a meta - analysis because the articles ' heterogeneity did not allow grouping for statistical analysis (table 1). thus, the results of this study are in the form of systematic review without meta - analysis. according to cochrane in situations where meta - analysis is not practical, the researcher should feel encouraged by the line of research on building a field for randomized clinical trials. for a better presentation of the results, the following variables of the selected articles were considered : author and year, country, number of patients, measurement method, patient age, follow - up, associated procedures, cleft type, and measurements and differences after surgery (table 2). the studies are from the end of the 1990s (one article) and the 2000s (four articles). although study of cleft lip deformities began at the late 19th century,8 it is clear that the association with anthropometric studies of the face only occurred beginning in the 1960s ; however, measurements were performed with very simple criteria.9 10 we believe that the absence of studies that compared pre- and postoperative anthropometric measures in that period arises from difficulty in standardizing measures in the early days of anthropometry, as well as the difficulty of techniques using direct measurements, which are difficult to obtain in children.11 studies using standardization emerged in the 1980s,6 12 13 noting the concern of choosing anthropometric points.14 the manner in which anthropometric data are collected should be considered when comparing the values obtained and the parameters reported in the literature, due to the small variation between the values acquired directly versus those acquired indirectly.15 recent research shows concern about the use of modern techniques for measurement. a example of this are two articles found in this study reporting the use of photogrammetry5 16 and three studies using three - dimensional measurements.17 18 19 digital photogrammetry is a noninvasive, inexpensive, and common method to investigate pre- and postoperative changes and provides a permanent record of patients. additionally, data can be stored and managed in a digital format that takes measurements using software.20 21 advancement in technology has allowed three - dimensional images,22 such as computed tomography, that require expensive equipment and has limited ability to determine the characteristics of soft tissues.23 moreover, ethical considerations limit the use of radiation for studies, especially in children. for these reasons, techniques for three - dimensional surface imaging such as laser scanning and stereophotogrammetry studies based on three - dimensional images appear to be the best alternative for assessing children with clefts both pre- and postoperatively ; these methods provide more information than two - dimensional methods. according to harris and smith, most deductions that occur in studies are derived from statistical analysis and, in addition to concerns about accounting adequately for known sources of variation within the research project, a major source of variability is measurement error.24 with the increasing access to data collection methods, computers have improved the ease of incorporating repeated measures on statistical models, with increased chance of finding biologically true differences when they exist. one study was in a japanese population,17 three in a taiwanese population,5 16 18 and one study occurred in cambodia.19 according to dixon,25 cleft lip and palate affects 1/700 live births, and in general, asian and amerindian populations have the highest prevalence, often as high as 1/500. european populations have intermediate prevalence rates of 1/1,000, and africans have the lowest prevalence rates of 1/2,500. these data justify the predominance of studies in eastern countries, but we emphasize that population - based studies to estimate the true incidence of cleft lip and palate are scarce. sporadic reports suggest that there has been some decrease in the incidence of cleft deformities ; the reasons for this decline are multifactorial.26 these studies have small numbers of patients, from 1 to 57.16 17 according to harris and smith, researchers commonly infer characteristics about populations from relatively small samples of study that can lead to errors in the evaluation of the results.24 the amount of variability in the numbers of individuals evaluated reflects the lack of homogeneity in the samples. other factors that influence the definition of clinical evidence is heterogeneity in the age of attainment of the first surgery and clinical procedures associated with the surgical procedure, such as the use of presurgical orthopedics or labial adhesion.27 there was variation in periods of postoperative follow - up. some studies cited shorter periods of around 2 months, and others cited up to 6 months, and others even longer periods, up to 3 years.5 16 17 18 19 the medical literature suggests that monitoring children with cleft lip and palate should continue during their growth, including adolescence,27 both for evaluation of results and to monitor facial growth. shorter term - limited follow - up or search for other surgical procedures to be performed on children can sometimes be justified, such as palatoplasty at 9 months of life. we believe following patients longer term allows better characterization morphological evolution of the nostril, which usually has increased asymmetry over the months. thus, control of the growth is accepted as a useful tool in evaluating the natural state of health of an individual.28 numerous methods have been described for repair of cleft lip deformity. repair of unilateral deformity is usually approached by rotation and advancement technique as described by millard29, under the concept of advancement of a flap on the lateral side of the upper lip combined with the rotation of the medial segment. this technique preserves both cupid 's bow and the philtrum.30 procedures associated with surgery, such as nasoalveolar molding or lip adhesion, were performed in three studies.5 16 19 some authors aligned the alveolar segments to create the foundation for the primary lip surgery to obtain good results and, to achieve this, early assessment and initiation of preoperative orthopedics should happen in the first days of life.27 other authors, who disagree with the preoperative procedures, claimed that the secondary deformities on the nose are caused by many factors, but the greatest determinant of nasal appearance after treatment is the primary deformity,3 and cited that associated procedures generate higher costs and are very dependent on the cooperation patient and family for a good result.2 among the results, we noticed that patients who used a nostril mold had improvement of asymmetry even before surgery. he performed a retrospective study to correlate the width of the cleft with the severity of unilateral nasal deformity in patients with cleft lip and palate before repair primary lip and found that the width of the nose, nasal length, and width of the lip were larger in patients with complete clefts.31 the preoperative facial asymmetry in patients with unilateral complete cleft is obvious and is widely established, and deficit of transverse tissue is generally more severe in complete clefts.6 at preoperative evaluation of unilateral complete cleft, the width of the cleft is a reliable guide to the severity of the other parameters.4 these data show that unilateral complete cleft represents a greater deformity compared with an incomplete fissure and indicate the need for uniformity of the population when comparing studies ; only four articles described patients with unilateral complete cleft, and in only one work were these patients the majority of the group.5 17 18 19 some authors only measured width, height, and length of the nose without including measurements of the nostrils.17 the majority of authors surveyed agreed that these measures should be included, but only allowed the observation, already established, that the cleft nose was wider preoperatively than postoperatively.3 6 32 experts are certainly capable of subjectively grading patients according to degree of nasal deformity, and two anthropometric measures that are easily obtained objectively nostril width and columellar angle can correlate with the expert 's ranking.3 four studies performed more elaborate measurements,5 16 18 19 based on anthropometric points described in the literature, allowing them to apply a more detailed analysis of the entire nasal morphometry to compare the possibility of a greater number of variables and use different ratios in assessment of results.6 visual judgment is influenced by the most impressive disproportions and can not determine the factors causing the disproportions ; the consensus among researchers is that the quality of facial morphology results can be estimated only by the proportions shown by quantitative data.7 a standard morphometric assessment outside the nose could be a reliable parameter for comparing rhinoplasty results.33 we believe that measurements should contain the maximum information possible, therefore covering all nostril points, and anthropometric measurements should include the angle of the columella, allowing a richer analysis when comparing the periods evaluated. among the residual deformities after surgery for complete unilateral cleft, asymmetry of the width of the floor of the nostrils was the most common finding, followed by columellar length asymmetry, low nasal bridge, broad nose, flat nasal tip, and low and shorter columella on the cleft side.5 6 we believe the application of this knowledge will allow an individualized approach to the patient for example, if after measurements nostril asymmetry is observed, with a greater breadth of cleft nostril, treatment is aimed at correcting that deformity during surgical procedure (cleft lip surgery). although all studies have established and described inclusion criteria, none of them observed all the criteria for randomization, which encourages future researchers to try to complete new work with greater methodological rigor in the future. the articles concluded that there is an important improvement in nostril asymmetry when comparing preoperative and postoperative measurements. the main changes that occurred after surgery were reduction of columellar angle, reduction of the width of the cleft nostril, and increase in the height of the cleft nostril. | introduction the purpose to this work is to review systematically the morphological changes of the nostrils of patients undergoing surgery for correction of cleft lip and identify in the literature the issues involved in the evaluation of surgical results in this population. review of literature a review was conducted, searching for clinical evidence from medline. the search occurred in january 2012. selection criteria included original articles and research articles on individual subjects with cleft lip or cleft palate with unilateral nostril anthropometric measurements before and after surgical correction of cleft lip and measurements of soft tissues. there were 1,343 articles from the search descriptors and free terms. of these, five articles were selected. discussion most studies in this review evaluated children in eastern countries, using different measurement techniques but with the aid of computers, and showed improved nostril asymmetry postoperatively compared with preoperatively. conclusion there is a reduction of the total nasal width postoperatively compared with preoperative measurements in patients with cleft lip. |
dental research has shifted interest from causes of dental disease to how dental disease affects the quality of life of affected patients. the effects of untreated dental caries may have a long - term impact on the child 's family, schooling, personality, social relationships, sleep patterns, physical growth and development. on the other hand, the extensive work is needed to effectively manage dental caries in children which requires prolonged multiple visits and a level of cooperative behavior that is not usually achievable in infants and preschool children, very often necessitating the use of dental general anesthesia (dga) in order to provide satisfactory dental treatment. due to the potential risks associated with dga this is also related to costs and parental acceptability. however, for a pediatric dentist to decide to expose a child to the calculated risk associated with general anesthesia, he / she is left under the ethical obligation that the outcome of treatment would outweigh the risk. a study by acs. on the effect of dental rehabilitation on the body weight of children with early childhood caries (ecc) concluded that comprehensive treatment under general anesthesia results in catch - up growth, such that children with a history of ecc no longer differed in percentile growth from the control group. the authors followed up with another study in 2001 in which they concluded that children who received dga achieved improvement in their quality of life as well as overall health. white. investigated parental satisfaction of 45 cases of dga and their perception of the impact of the procedure on the physical and social quality of life. their findings indicated that dga for preschool children has a high degree of satisfaction among parents and is perceived to have a positive social impact on their children. in 2004, anderson. also concluded that dga results in an immediate improvement in oral health and aspects of the quality of life for both children and their families. in 2007, amin and harrison investigated 26 parents compliance to long - term (6 months) maintenance of healthy behaviors following dga. they also concluded that parents readiness for change is a detrimental factor in the successful outcome of dga. the conservative nature of saudi arabians and people of gulf countries may have an effect on postoperative communication with caregivers, jeopardizing the quality of postoperative care. this may call for more studies on parental perception regarding the postoperative outcome of dga and how does single - visit full rehabilitation dga might impact the child 's quality of life in saudi arabia. the aim of this study is, therefore, to investigate the possible impact of single - visit full dental rehabilitation dga on aspects of children 's oral health - related quality of life from parents and children 's perspective in saudi arabia over a follow - up period of 2 years. institutional ethical approval was obtained from the relevant committees based on the institution 's corporate social responsibility (csr) standards. informed consent was routinely obtained from parents and guardians following joint commission for international accreditation (jcia) standards. consent forms included information describing the use of data, photos and videos for research and quality improvement purposes, and a statement regarding the compliance of the protocol with the world medical association declaration of helsinki on ethical principles for medical research involving human subjects, october 2001. this is a prospective study of questionnaire data obtained from 352 pediatric patients before and after treatment of ecc with full dental rehabilitation under general anesthesia. questionnaires focused on oral symptoms, functional limitations, and emotional and social well - being before and after dental treatment. the present study was conducted in a saudi private hospital of 600 bed capacity and 90 outpatient clinics. american society of anesthesiologists (asa) i and ii children with ecc whose indication for dental treatment under general anesthesia was due to lack of cooperation and/or the load of required dental treatment were included. children who underwent dga for non - restorative procedures (e.g. tooth transplantation, removal of impacted supernumeraries, surgical orthodontics, traumatic cut wounds or facial fractures) with or without restorative work were excluded from the study. out of a total 473 children who had dga that year, 431 children fulfilled the inclusion criteria and their records were included in the study. the arabic version of the above questionnaire was obtained from a legal translation office authorized by the american consulate in jeddah, ksa. parents were asked to complete this translated composite questionnaire that sought their perception of treatment outcomes to dental rehabilitation. pre- and postoperative parental perception questionnaire pre- and postoperative interviews were carried out with both the children and their caregivers to gain accuracy of children 's self - assessment added to the perspectives of parents as decision makers. also, in cases of child patients with special needs and/or children with limited communication skills, parents / caregivers were the main source of information. the first author and the same two dental assistants carried out all pre- and postoperative interviews. preoperative interviews were conducted on 431 cases ; however, 79 patients did not show up for postoperative care visits during the following 2 years. so, the study was carried out on the remaining 352 cases that attended postoperative care. a standardized dental rehabilitation protocol [table 2 ] was followed for all children undergoing dental rehabilitation under general anesthesia in the medical facility, including the 352 cases investigated in the current study. dental rehabilitation protocol this protocol was thought to minimize postoperative dental needs and subsequent repeat of dga. patients were recalled for an early follow - up visit 710 days after the procedure and subsequently every 6 months for a 2-year follow - up period. preoperative interviews were conducted on the day of initial examination during the recording process of the chief complaint and dental history. after recovery, and before discharge, parents were given a copy of the questionnaire as a reminder of the items related to their child 's outcome of dental rehabilitation, in order to be prepared for postoperative interview at the next visit. due to doubts regarding parent 's compliance with the postoperative care regimen, postoperative interviews were carried out on the first possible postoperative visit during the follow - up period of 2 years. demographic and clinical data were collected from the hospital 's electronic filing system and questionnaires analyzed using microsoft excel. the before and after comparisons of oral health - related quality of life were analyzed using mcnemar test included in analyse - it standard edition software plug - in package for microsoft excel. american society of anesthesiologists (asa) i and ii children with ecc whose indication for dental treatment under general anesthesia was due to lack of cooperation and/or the load of required dental treatment were included. children who underwent dga for non - restorative procedures (e.g. tooth transplantation, removal of impacted supernumeraries, surgical orthodontics, traumatic cut wounds or facial fractures) with or without restorative work were excluded from the study. out of a total 473 children who had dga that year, 431 children fulfilled the inclusion criteria and their records were included in the study. the arabic version of the above questionnaire was obtained from a legal translation office authorized by the american consulate in jeddah, ksa. parents were asked to complete this translated composite questionnaire that sought their perception of treatment outcomes to dental rehabilitation. pre- and postoperative parental perception questionnaire pre- and postoperative interviews were carried out with both the children and their caregivers to gain accuracy of children 's self - assessment added to the perspectives of parents as decision makers. also, in cases of child patients with special needs and/or children with limited communication skills, parents / caregivers were the main source of information. the first author and the same two dental assistants carried out all pre- and postoperative interviews. preoperative interviews were conducted on 431 cases ; however, 79 patients did not show up for postoperative care visits during the following 2 years. so, the study was carried out on the remaining 352 cases that attended postoperative care. a standardized dental rehabilitation protocol [table 2 ] was followed for all children undergoing dental rehabilitation under general anesthesia in the medical facility, including the 352 cases investigated in the current study. dental rehabilitation protocol this protocol was thought to minimize postoperative dental needs and subsequent repeat of dga. patients were recalled for an early follow - up visit 710 days after the procedure and subsequently every 6 months for a 2-year follow - up period. preoperative interviews were conducted on the day of initial examination during the recording process of the chief complaint and dental history. after recovery, and before discharge, parents were given a copy of the questionnaire as a reminder of the items related to their child 's outcome of dental rehabilitation, in order to be prepared for postoperative interview at the next visit. due to doubts regarding parent 's compliance with the postoperative care regimen, postoperative interviews were carried out on the first possible postoperative visit during the follow - up period of 2 years. demographic and clinical data were collected from the hospital 's electronic filing system and questionnaires analyzed using microsoft excel. the before and after comparisons of oral health - related quality of life were analyzed using mcnemar test included in analyse - it standard edition software plug - in package for microsoft excel. a total of 431 cases underwent the preoperative interview, out of which 227 were males and 204 were females. mean age was 3 years 8 months, ranging between 2 years 4 months and 10 years 11 months. the study was completed on a total of 352 (192 males, 160 females) cases due to absence of 79 children for postoperative follow - up protocol. fifty - six percent of these needed pulpotomy (6.2 teeth per child), 37% underwent fillings which accounted to 4.2 teeth per child, and 7% had to be extracted (0.74 teeth per child). the numbers of dental services provided under general anesthesia are presented in table 3. along the 2-year follow - up period, 58.8% of the 352 cases who showed up postoperatively were found to have recurrent caries. dental services provided in the study under general anesthesia caries recurrence was observed in 50% of children who fully complied with postoperative care. the percentage of caries recurrence was 67.8% among children who did not comply with postoperative care schedule, i.e. showed up only because of pain. a weak statistical significance was observed between compliance with postoperative care schedule and caries recurrence (somers d < 0.15). perceived outcomes to dental rehabilitation [table 4 ] demonstrated 92 from 352 (26.13%) children complained of food impaction and 69 out of 352 complained of dental and/or oral pain before treatment. none of them had those complaints after treatment. perceived outcomes after full dental rehabilitation under general anesthesia forty - one children out of 352 (11.64%) had bad breath, out of which only 3 had the same complaint even after treatment. fourteen children out of 352 (3.97%) had mouth sores before treatment ; only 2 of them had the same complaint after treatment. twenty - two children out of 352 (6.25%) complained of bleeding gums before treatment and only 1 (0.28%) had the same complaint after treatment. before treatment, 31.25% had difficulty chewing coarse food, 27.27% complained of discomfort or pain upon drinking hot or cold foods, and 33.8% reported difficulty in eating foods they normally enjoy, and none of them had the same complaint after treatment. forty - seven children out of 352 (13.35%) had special food to be prepared for them separately from that for the rest of their families (restricted diet). disturbed sleeping expressed as interrupted sleep or taking long time to fall asleep was reported in 64 (18.18%) before treatment, whereas only 3 of them reported the same complaint after treatment. before treatment, 15 children (4.26%) reported fear of being less attractive ; none of them had this complaint after treatment. fourteen children (3.97%) were reported to be shy and/or embarrassed because of the appearance of their teeth ; only one of them reported to be shy after treatment. twenty - six children out of 352 (7.38%) reported that they avoided smiling in front of strangers before treatment. eighteen children out of 352 (5.11%) were reported to be reluctant when invited to read or speak in public or in class. twenty - four children out of 352 (6.81%) were embarrassed by being asked about the condition by their classmates. eleven children out of 352 (3.12%) reported frequent absence from school either for toothache or for attending dental treatment. three hundred and forty - nine parents (99.14%) found the whole experience satisfactory [table 5 ]. three hundred and forty - three parents (97.44%) would consider general anesthesia again for treatment when needed. nineteen parents out of 352 (5.39%) would prefer multiple visits under sedation over general anesthesia. three hundred and forty - nine parents (99.14%) found the whole experience satisfactory [table 5 ]. three hundred and forty - three parents (97.44%) would consider general anesthesia again for treatment when needed. nineteen parents out of 352 (5.39%) would prefer multiple visits under sedation over general anesthesia. the current study investigated the parents perception on the outcome of dga in three dimensions. it seems that children suffering from ecc do not necessarily express it in the form of pain. this finding is in agreement with anderson. where preoperative pain was observed in 20% of cases, but less than half of what was reported earlier by low. the difference might be attributed to the authors choice to record only those who were definitely affirming preoperative toothache experience. the three studies are in agreement that the reported preoperative pain experience was not proportional with the preoperative dmf rate. this suggests that even in the presence of aggressive rampant caries, children do develop their ways to adapt with pain rather than to seek dental treatment. the current study demonstrated that single - visit total dental rehabilitation under general anesthesia results in a sudden elimination of oral symptoms of ecc, and also, there is highly significant reduction of bad breath, mouth sores, and bleeding gums. having bad breath in patients postoperatively could be attributed to obligatory or habitual mouth breathing. our study demonstrated that dga has a significant positive impact on children 's eating and sleeping behaviors. there was complete elimination of difficulties children used to encounter when attempting to eat healthy fibrous food, cold foods, and the types of food they would prefer. it is worth noting that 4 cases out of 47 continued to have specially prepared food. sanjad sakati syndrome, a condition confined to bedouin arab tribes that is characterized by hypoparathyroidism and failure to thrive (ftt). the three children continued postoperatively on soft diet due to limited postoperative improvement in their ability to grind and chew hard food. a dramatic change in emotional and social well - being was reported in the study. it is of interest that three children missed school postoperatively due to failure of an anterior tooth restoration, which indicates the impact of restoring upper anterior teeth on the psychology of children. it is a matter of interest that some parents observed marked improvement in their child 's behavior and sleeping habits postoperatively even though they did not report it preoperatively. those parents did not link preoperative aggressive behavior and disturbed sleep to oral health until dental caries was eliminated. in 2012, alkarimi. demonstrated that comprehensive treatment of dental caries on multiple visits for a group of saudi children significantly improved oral health - related quality of life including pain, sepsis, and dissatisfaction with teeth, smile, and poor appetite. the study was conducted on saudi children as in our study, but with a different methodology and scale. the results of both studies are in agreement regarding the oral symptoms, functional limitations, as well as emotional and social well - being. this indicates that even if a single - visit elimination of caries under general anesthesia might result in an impressive impact on postoperative outcome, doing the same service on several visits would have the same result in the long run. patients knowledge and awareness has boomed in the last decade with the information revolution. patient satisfaction may play a major role in the near future of healthcare. for pediatric dentists, patient satisfaction includes the child and his / her decision makers, parents, or caregivers. however, they reported a tendency for parents not to report dissatisfaction in an in - house survey. this might hold true in a public hospital, while in a private investment hospital where our study was done, both the hospital and its patients are sensitive to failure in satisfying patients and patients are eager to report such failures. only 2.55% of parents would not repeat the procedure under general anesthesia when needed due to the complexity of the procedure and difficulty in obtaining insurance company coverage. the relatively higher percentage of parents who would prefer sedation over general anesthesia (5.39%) could be explained on the basis that the postoperative needs of their children became already minimized to the extent of being manageable in a few visits with sedation, if needed. we observed high postoperative no show - up rate [79 (18.32%) cases ]. this might reflect the international nature of the workforce in saudi arabia and gulf countries, with its high turnover and rapidly changing health insurance policies. in contrast, self - funding families had the motivation of getting value for their money and, consequently, they had the best postoperative compliance rate with postoperative care plans. as the postoperative follow - up in the current study was for 2 years, no change in the high satisfaction rate was observed during the 2 years of observation. caries recurrence was observed in 58.8% of the sample, which is higher than that reported by graves. the differences probably are reflections of the complexity of caries etiology as a multifactorial problem. it is of interest that the high caries recurrence rate did not affect parental satisfaction over the 2-year follow - up period. this could be explained on the basis that postoperative caries was discovered early and treated with simple intervention within the cooperation margin of the child ; therefore, the severity of postoperative decay was not extensive to disturb the child. a higher caries recurrence rate was observed among children who did not comply with the postoperative plan of care ; but this did not affect parental satisfaction, which may be because parents acknowledged that their failure to attend proper postoperative care is to be blamed for any unsatisfactory postoperative outcome. studies which seek to investigate satisfaction through interviews and/or questionnaires could be affected by misinformation and acquiescence biased answers. moreover, the child 's immaturity and dependence on adults render him / her unqualified to be the sole source of information. a shy child may be too shy to verbalize his / her shyness and a child in pain does not have the same skill as that of an adult to express the nature of his / her suffering. accordingly, we had to include parents and caregivers in the pre- and postoperative interviews. added limitations to this study are confined to saudi communities. while the mother is generally the primary caregiver, it is not uncommon for a male dentist to interview the mother through the father, which imposes a potential risk that data might be lost or distorted. furthermore, imported labor constitutes half of saudi workforce, so it is common to have a patient whose primary caregiver is a domestic helper recruited from south east asia, which adds a potential language barrier for the accurate communication of data during interviews. attempting to overcome these limitations, we conducted the interviews with both child patients and their caregivers, trying to get the advantage of a child 's self - reporting and adult 's skill of communication and abilities to verbalize realities. we formulated a questionnaire in accordance with guyatt and cook 's recommendations that when considering a measure for use in clinical practice or clinical trials, clinicians and investigators should look for evidence that the outcomes it addresses are in fact important to the target population. within that context, our questionnaire items were selected to address problems that occur most frequently and cause the maximum worry to our child patients, rather than the items that measure health status. for example, anderson. investigated how parents find it difficult to arrange dental appointments for their child without having to take time off work with subsequent loss of income. such an item is hard to investigate in saudi arabia, as women are not allowed to drive and the majority of families rely on recruited private drivers to normalize family activities, and so it is very common to have a child attending a dental visit escorted solely by his / her driver while his / her parents are at work. should a male pediatric dentist interview a mother, a dental nurse has to be around and whenever the principal caregiver is a recruited foreigner, the hospital has assigned interpreters available on 24/7 basis for urdu, bahasa indonesia, farsi, tigrinya, in addition to the pilipino dental assistants covering almost all the possible languages spoken by the patients. response, we recorded that as negative if the questioned item was related to oral symptoms or functional limitations. for emotional and social well - being items, there was a possibility that the response is a positive one and the child is too shy to talk about it. in such cases, parents were further questioned to get a closer answer to reality as possible. it seems that children suffering from ecc do not necessarily express it in the form of pain. this finding is in agreement with anderson. where preoperative pain was observed in 20% of cases, but less than half of what was reported earlier by low. the difference might be attributed to the authors choice to record only those who were definitely affirming preoperative toothache experience. the three studies are in agreement that the reported preoperative pain experience was not proportional with the preoperative dmf rate. this suggests that even in the presence of aggressive rampant caries, children do develop their ways to adapt with pain rather than to seek dental treatment. the current study demonstrated that single - visit total dental rehabilitation under general anesthesia results in a sudden elimination of oral symptoms of ecc, and also, there is highly significant reduction of bad breath, mouth sores, and bleeding gums. having bad breath in patients postoperatively could be attributed to obligatory or habitual mouth breathing. our study demonstrated that dga has a significant positive impact on children 's eating and sleeping behaviors. there was complete elimination of difficulties children used to encounter when attempting to eat healthy fibrous food, cold foods, and the types of food they would prefer. it is worth noting that 4 cases out of 47 continued to have specially prepared food. sanjad sakati syndrome, a condition confined to bedouin arab tribes that is characterized by hypoparathyroidism and failure to thrive (ftt). the three children continued postoperatively on soft diet due to limited postoperative improvement in their ability to grind and chew hard food. a dramatic change in emotional and social well - being was reported in the study. it is of interest that three children missed school postoperatively due to failure of an anterior tooth restoration, which indicates the impact of restoring upper anterior teeth on the psychology of children. it is a matter of interest that some parents observed marked improvement in their child 's behavior and sleeping habits postoperatively even though they did not report it preoperatively. those parents did not link preoperative aggressive behavior and disturbed sleep to oral health until dental caries was eliminated. in 2012, alkarimi. demonstrated that comprehensive treatment of dental caries on multiple visits for a group of saudi children significantly improved oral health - related quality of life including pain, sepsis, and dissatisfaction with teeth, smile, and poor appetite. the study was conducted on saudi children as in our study, but with a different methodology and scale. the results of both studies are in agreement regarding the oral symptoms, functional limitations, as well as emotional and social well - being. this indicates that even if a single - visit elimination of caries under general anesthesia might result in an impressive impact on postoperative outcome, doing the same service on several visits would have the same result in the long run. patients knowledge and awareness has boomed in the last decade with the information revolution. patient satisfaction may play a major role in the near future of healthcare. for pediatric dentists, patient satisfaction includes the child and his / her decision makers, parents, or caregivers. however, they reported a tendency for parents not to report dissatisfaction in an in - house survey. this might hold true in a public hospital, while in a private investment hospital where our study was done, both the hospital and its patients are sensitive to failure in satisfying patients and patients are eager to report such failures. only 2.55% of parents would not repeat the procedure under general anesthesia when needed due to the complexity of the procedure and difficulty in obtaining insurance company coverage. the relatively higher percentage of parents who would prefer sedation over general anesthesia (5.39%) could be explained on the basis that the postoperative needs of their children became already minimized to the extent of being manageable in a few visits with sedation, if needed. we observed high postoperative no show - up rate [79 (18.32%) cases ]. this might reflect the international nature of the workforce in saudi arabia and gulf countries, with its high turnover and rapidly changing health insurance policies. in contrast, self - funding families had the motivation of getting value for their money and, consequently, they had the best postoperative compliance rate with postoperative care plans. as the postoperative follow - up in the current study was for 2 years, no change in the high satisfaction rate was observed during the 2 years of observation. caries recurrence was observed in 58.8% of the sample, which is higher than that reported by graves. the differences probably are reflections of the complexity of caries etiology as a multifactorial problem. it is of interest that the high caries recurrence rate did not affect parental satisfaction over the 2-year follow - up period. this could be explained on the basis that postoperative caries was discovered early and treated with simple intervention within the cooperation margin of the child ; therefore, the severity of postoperative decay was not extensive to disturb the child. a higher caries recurrence rate was observed among children who did not comply with the postoperative plan of care ; but this did not affect parental satisfaction, which may be because parents acknowledged that their failure to attend proper postoperative care is to be blamed for any unsatisfactory postoperative outcome. studies which seek to investigate satisfaction through interviews and/or questionnaires could be affected by misinformation and acquiescence biased answers. moreover, the child 's immaturity and dependence on adults render him / her unqualified to be the sole source of information. a shy child may be too shy to verbalize his / her shyness and a child in pain does not have the same skill as that of an adult to express the nature of his / her suffering. accordingly, we had to include parents and caregivers in the pre- and postoperative interviews. added limitations to this study are confined to saudi communities. while the mother is generally the primary caregiver, it is not uncommon for a male dentist to interview the mother through the father, which imposes a potential risk that data might be lost or distorted. furthermore, imported labor constitutes half of saudi workforce, so it is common to have a patient whose primary caregiver is a domestic helper recruited from south east asia, which adds a potential language barrier for the accurate communication of data during interviews. attempting to overcome these limitations, we conducted the interviews with both child patients and their caregivers, trying to get the advantage of a child 's self - reporting and adult 's skill of communication and abilities to verbalize realities. we formulated a questionnaire in accordance with guyatt and cook 's recommendations that when considering a measure for use in clinical practice or clinical trials, clinicians and investigators should look for evidence that the outcomes it addresses are in fact important to the target population. within that context, our questionnaire items were selected to address problems that occur most frequently and cause the maximum worry to our child patients, rather than the items that measure health status. for example, anderson. investigated how parents find it difficult to arrange dental appointments for their child without having to take time off work with subsequent loss of income. such an item is hard to investigate in saudi arabia, as women are not allowed to drive and the majority of families rely on recruited private drivers to normalize family activities, and so it is very common to have a child attending a dental visit escorted solely by his / her driver while his / her parents are at work. should a male pediatric dentist interview a mother, a dental nurse has to be around and whenever the principal caregiver is a recruited foreigner, the hospital has assigned interpreters available on 24/7 basis for urdu, bahasa indonesia, farsi, tigrinya, in addition to the pilipino dental assistants covering almost all the possible languages spoken by the patients. in managing the response, we recorded that as negative if the questioned item was related to oral symptoms or functional limitations. for emotional and social well - being items, there was a possibility that the response is a positive one and the child is too shy to talk about it. in such cases, children with dental caries do not necessarily express it verbally as pain ; the disease has a lot of other expressions affecting children 's quality of life, including the ability to sleep, thrive, and socializeparental acceptance for general anesthesia as an option for their child 's dental treatment is growingpostoperative preventive care, early diagnosis and treatment of recurrent caries are key factors to maintain favorable postoperative outcome of dgain the arab gulf region, the principal caregiver is not necessarily the mother. care should be taken by the pediatric dentist to identify and include the principal caregiver in planning postoperative preventive care should a successful outcome of dga be achieved and maintainedthe current study contributes to the existing literature that parents prefer single - visit full dental rehabilitation under general anesthesia more than doing the same procedure on multiple visits due to the immediate positive impact on the physical and social well - being of children and their familiesthe positive impact on parental perception observed in this study was not affected by postoperative high recurrence rate of caries. children with dental caries do not necessarily express it verbally as pain ; the disease has a lot of other expressions affecting children 's quality of life, including the ability to sleep, thrive, and socialize parental acceptance for general anesthesia as an option for their child 's dental treatment is growing postoperative preventive care, early diagnosis and treatment of recurrent caries are key factors to maintain favorable postoperative outcome of dga in the arab gulf region, the principal caregiver is not necessarily the mother. care should be taken by the pediatric dentist to identify and include the principal caregiver in planning postoperative preventive care should a successful outcome of dga be achieved and maintained the current study contributes to the existing literature that parents prefer single - visit full dental rehabilitation under general anesthesia more than doing the same procedure on multiple visits due to the immediate positive impact on the physical and social well - being of children and their families the positive impact on parental perception observed in this study was not affected by postoperative high recurrence rate of caries. | purpose : to investigate the perceived clinical outcome and parents satisfaction after dental rehabilitation under general anesthesia over a follow - up period of 2 years.materials and methods : a prospective study of questionnaire data obtained from 352 pediatric patients before and after treatment of early childhood caries with full dental rehabilitation under general anesthesia. questionnaires focused on oral symptoms, functional limitations, and emotional and social well - being before and after dental treatment. cases were followed up for 2 years postoperatively.results:a dramatic disappearance of symptoms was reported from parents perspective. there was a high satisfaction rate (99.14%) also among parents of the children included in the study.conclusion:children with early childhood caries do not necessarily express it verbally as pain. the disease has a lot of other expressions affecting children 's behavior and habits, including the ability to sleep, thrive, and socialize. this study contributes to the existing literature that full dental rehabilitation under general anesthesia [dental general anesthesia (dga) ] has an immediate positive impact on the physical and social quality of life of children suffering from early childhood caries as well as on their families. postoperative preventive care, early diagnosis, and treatment of recurrent caries are key factors to maintain postoperative outcome of dga. |
the compound 4-nitrophenol (4-np) is a nitro - aromatic that is frequently encountered as a product of the degradation or fabrication process of rubber chemicals, lumber preservatives, car exhausts, industrial wastes and also as the main degradation product of organophosphorous pesticides, such as fenitrothion and parathion. due to the characteristics of its biorefractory compounds, it is considered a hazardous pollutant with toxic effects on human health and the environment. as a result, in the last few years, numerous methodologies have been developed for the analytical determination of 4-np in different samples. these procedures have involved the use of high - performance liquid chromatography, gas chromatography, capillary zone electrophoresis, and spectrophotometry with flow - injection analysis. meanwhile, various electroanalytical techniques also have been used with success as alternatives to chromatographic and spectroscopic techniques. for this, different solid surface electrodes, either bare or modified with different organic and inorganic compounds, have been designed to evaluate the analytical responses and to study the redox process mechanisms of the 4-np [68 ]. nevertheless, in this type of electrode, the renovation of the electrodic surfaces can be complicated due to the memory effects associated with the strong adsorption processes of organic compounds, which, in general, result in a decrease in the reproducibility of voltammetric responses. several works are based on processes occurring at hg surfaces, such as in the hanging mercury drop electrode (hmde). however, the highly toxic nature of mercury and other heavy metals makes the search for alternative electrode materials a necessity ; for this, there is an increasing interest in the construction of solid electrodes using metallic amalgams, which are prepared by mixing a fine metal powder (ag, cu, au, bi, sn, or zn) with liquid mercury [911 ]. in general, solid amalgam electrodes are convenient electrodic surfaces because they are practically nontoxic, easily prepared, and mechanically stable.. the solid amalgam electrodes also present a solid and homogeneous surface, allowing easy renewal by mechanical polishing, and shows a high overpotential to the hydrogen evolution reaction [914 ]. finally, as a major characteristic, these electrodes minimize any environmental contamination with mercury because the amount discarded in each analysis is minimal. thus, the main goals of this work were to employ a silver solid amalgam electrode (agsae), to evaluate its efficiency in the study of electrochemical behaviour, and to establish an appropriate methodology for the analytical determination of 4-nitrophenol (4-np). for this, the voltammetric responses obtained on the agsae were compared with similar results obtained using an hmde and a silver electrode (age). with regard to the voltammetric technique, square wave voltammetry (swv) was employed due to its extreme sensitivity in the detection of organic molecules. in addition, swv allows valuable information concerning the electrochemical redox mechanisms to be obtained by the appropriate use of a well - developed theoretical model. all voltammetric measurements were carried out using an autolab model pgstat 30 potentiostat from metrohm - ecochemie, which was controlled by a personal computer with gpes software (general purpose electrochemical system), version 4.9, from metrohm - ecochemie. a denver instrument ultrabasic model ph - meter equipped with a 3.0 mol l ag / agcl / kcl - glass combined electrode was used for adjusting ph values. water of high purity (conductivity 1.0 s cm), purified by means of a gehaka model os20 lx farma, was used to prepare all the solutions. a conventional cell with a three - electrode system was used in all experiments, which incorporated the ag / agcl / cl 3.0 mol l electrode as the reference electrode, a platinum wire as the auxiliary electrode, and a agsae, hmde, or age as the working electrode. a metrohm model e410 hanging mercury drop electrode, with a drop area of 0.052 cm, was used as one of the working electrodes. a new drop was formed by dislodging the old one and extruding more triply - distilled mercury. the age used was constructed from a 0.10 mm diameter silver wire (goodfellow), where the wire was inserted into a piece of glass tube with approximately 0.30 mm internal diameter, which was later filled with epoxy resin. after the epoxy resin hardened, the age was polished using a mechanical polisher and glasspaper of different granulations. after this polishing procedure, the electrode was cleaned with water, and a smooth and homogeneous surface was observed. a 0.1 mol l britton - robinson (br) buffer was employed as the supporting electrolyte, and the ph was adjusted to the desired value by the addition of appropriate amounts of 1.0 mol l naoh stock solution. a 1.0 10 mol l stock solution of 4-np from merck (98% purity) was prepared daily by the dissolution of the appropriate quantity in ultra - pure water ; the solution was stored in a dark flask and maintained in the refrigerator to avoid degradation. before each day of experiments or after every delay longer than one hour, the agsae was activated according to the procedure previously described [913 ]. the optimization of the analytical procedure for swv was then performed following a systematic study of the experimental parameters that affect the responses, such as the ph of the medium, the pulse potential frequency (f) related to the total pulse duration, the amplitude of the pulse (a), and the height of the potential step (e s) or scan increment. the mentioned parameters were optimized in relation to the maximum value of peak current and the maximum selectivity (half - peak width). before each experiment, the solutions were deaerated by bubbling nitrogen, and the electrochemical cell was kept under a nitrogen atmosphere throughout the experiments. to accomplish the abovementioned, the working electrodes were placed in the measuring cell, which was filled with 10 ml of an electrolyte support solution containing a known concentration of 4-np, and the experimental and voltammetric parameters were subsequently studied. in all experiments, all parameters were optimized for the use of the agsae, hmde, and age. the standard deviation of the mean current (s b) measured at the reduction potentials of 4-np for 10 voltammograms of the blank solution in pure electrolytes together with the slope of the straight line of the analytical curves (s) were used in the determination of the quantification and detection limits (ql and dl, resp.), according to guidelines recommended by iupac. these experiments were carried out by adding a known amount of 4-np to the supporting electrolytes followed by standard additions from the 4-np, stock solutions and plotting the resulting analytical curves. the recovery efficiencies (% r) were calculated considering the relation between the value of the concentration obtained by extrapolating the analytical curves of the corresponding spiked samples and the concentration previously added. the precision and accuracy of methodology were tested with different standard solutions of 4-np and the relative standard deviations (rsd) were calculated, considering the standard deviation of the mean current values obtained and the mean peak current values. to attest the applicability of the proposed methodology, interfering effects were studied using water samples collected from two different points of the river monjolinho, a local river in the city of so carlos, brazil. the sampling points, designated as sample 1 and sample 2, were known to show different characteristics with regard to pollution. sample 1, located at the riverhead away from the city, is relatively free from urban or industrial pollution. sample 2, a point halfway along the river length and situated in the city, has domestic and industrial sources of pollution. the samples were previously filtered in filter paper (12.5 cm, vetec) and subsequently used to prepare the supporting electrolyte (br buffer ph 7.0). mol l 4-np solutions, and the recovery curves were constructed by the standard addition method, similar to the procedure used for the pure electrolyte. according to de souza and coworkers, the agsae presents a globular structure with solid and compact surfaces, which enables the easy renewal of the electrode surface via mechanical polishing. additionally, the presence of the prevailing form in the agsae (ag2hg3) with the nominal 30/70 (ag / hg : m / m) composition allows for the acquisition of good reproducibility in responses, even after mechanical polishing. for this, the analytical responses for 4-np on the agsae were evaluated, and the results obtained were compared to similar results obtained using the hmde and age. several works have been previously published with reviews dealing the use of solid amalgam electrodes in the 4-np determinations [1821 ]. despite that agsae has been intensively evaluated in amalgam surfaces, no works, to the best of our knowledge, reporting a comparative study in silver and traditional mercury electrodes and mainly the electrochemical behaviour study in silver and traditional mercury electrodes can be found in the available literature. for this, all voltammetric parameters were preliminary evaluated. preliminary experiments using swv were realized for 4-np using a sample of 0.1 mol l br buffer solution at ph 7.0 with 1.05 10 mol l 4-np solution, with parameters of f = 100 s, a = 50 mv, and e s = 2 mv. these experiments were used to determine the analytical responses to 4-np of the agsae, hmde, and age. the responses obtained are shown in figure 1, where the presence of a single well - defined cathodic peak can be observed at around 0.56 v, 0.96 v, and 0.80 v for hmde, age, and agsae, respectively. swv experiments were also carried out to evaluate the type of redox process by analyses of the forward, reverse, and resultant components of current. the results demonstrated that the electrochemical behaviour of 4-np presented characteristics of an irreversible electrode reaction controlled by the diffusion of the reagent, in close agreement with cyclic voltammetry results. since the direct and reverse components of current are presented in the same direction, this indicates that the reactant and the product are both strongly adsorbed. a study about the effects of ph on the analytical responses for 4-np on the agsae, age, and hmde were also evaluated using the swv technique. for this, a 0.1 mol l br buffer solution with varied ph between 2.0 and 10.0 was used with 1.05 10 mol l 4-np, f = 100 s, a = 50 mv and e s = 2 mv. figure 2 shows the responses obtained for 4-np on the agsae at different ph values, where the insert indicates the responses for i p and e p as a function of the ph values used. in all electrodes, it can be observed that the potential peaks were strongly ph - dependent, and the e p shifts towards more negative values as the ph increases. this shift in potential with ph can be represented by the following equations (1), (2), and (3) for agsae, hmde, and age, respectively, (1)ep=0.3120.066 ph, (2)ep=0.1750.058 ph, (3)ep=0.2300.036 ph, where the potential values are given in volts. equations (1) and (2) represent a straight line with a slope that is very close to the theoretical value predicted by the nernst equation for an electrochemical reduction process involving the same number of protons and electrons (0.059 v / ph at 25c). on the other hand, for use of the age, defined by (3), only one proton for two electrons can be involved in the electrode process. considering the use of the agsae and hmde, the results obtained are in accordance with previous work realized with other electrodic surfaces, such as boron doped diamond electrodes or modified electrodes [68 ]. additionally, the relationships between the peak currents and ph values revealed that the peak currents present the maximum value at ph 4.00 for the hmde and age, and for lower and higher phs, the peak currents decreased sharply. a close analysis of this value shows that this ph is close to the pka value reported in the literature, confirming that the participation of protonation equilibrium precedes the electron transfer reaction. more detailed studies about the electrochemical behaviour of 4-np for the agsae, hmde, and age were realized using cyclic voltammetric experiments. for these experiments, the potential scan rates were varied from 10 to 200 mv s. for the responses obtained, it was observed that the i p increases with the square root of the scan rates in all electrodes used ; in addition, it was confirmed to be a diffusion - controlled process with the characteristics of an irreversible process, including the absence of anodic peaks in the reverse scans. additionally, the relationships between peak currents and the square root of the scan rates allowed the determination of the number of electrons related to the 4-np reduction process for each electrode employed. this was determined using the randles - svecik equation for an irreversible process, according to the following equation : (4)ip=(2.99105)n(na)1/2crdr1/2v1/2. the values used in this equation were 1.05 10 mol cm for concentration of 4-np, a d r of 9.19 10 cm s, of 0.5, and n a calculated by n a = 47.7/(e p e p/2), where e p is the peak potential and e p/2 is the half - height potential. from this, it was possible to evaluate the number of electrons in the determinate step and the total electrons involved in redox process. as a result, the agsae, hmde, and age implicated the transference of one electron in the determinate step, and the total number of electrons calculated was four. these values, for number of electrons, are in accordance with previous works employing other electrodic surfaces [7, 24 ]. the square wave voltammetry parameters were optimized for analytical and redox mechanism proposes for the use of all electrodes. as is well known, a variation in the frequency of application of pulse potential usually exerts a marked effect on the response of swv. this effect thus provides a good criterion for the diagnosis and can be used to indicate any process of adsorption or reaction in solution or the reversibility or irreversibility of the electrochemical process. for the agsae, hmde, and age, it was observed that an increase in the f values was accompanied by an increase in i p. additionally, a linear relationship was obtained between the i p values and the square root of the f values, which, according to the theoretical model proposed by lovric and coworkers for swv, may indicate diffusion - controlled electrode process. this was confirmed by the cyclic voltammetry experiments for all the electrodes, which also presented a linear dependence of the peak current on the square root of the scan rate and had a log i p versus log v relationship with a slope very close to 0.5, a value expected for a diffusion controlled process. moreover, the inclination of the relationships between i p and the square root of the f values supply a direct estimate for the values of the standard reaction rate constant (k) for a redox process, according to as a result, the values of k were 5.55 10 4.40 10, 4.10 10 1.57 10, and 2.63 10 7.90 10 s for the agsae, hmde, and age, respectively. according to these values, it is possible to assume that the transfer kinetic of the process is faster for the agsae than for the hmde or age. the influence of f on the e p values was also evaluated, and the results showed a shift towards more positive values, varying linearly with the logarithmic value of f according to (13), which was developed for an irreversible redox process with adsorption complications : (6)eplog f=2.3rtnf, where r is the gas constant, t is the temperature, is the electron transfer coefficient, n is the number of electrons only in the determining step of the process, and f is the faraday constant. the slope of each curve was experimentally determined to be 29.47, 107.64, and 111.21 mv per decade for the agsae, hmde, and age, respectively. considering = 0.5, using t as the room temperature, and substituting the known values of r, f, and the slope into the right - hand side of (5), n was calculated to be equal to four for the agsae, and one for the hmde and age. based on the previously observed results with the swv parameters, in conjunction with the previous experiments of cyclic voltammetry (cv) and the literature reports [7, 24, 25 ], considering experiments with exhaustive electrolyses and determinations of products of reduction processes by spectroscopy techniques, it can be presumed that the 4-np is irreversibly reduced in a 4-electron step. this process is probably due to a four - electron reduction of the nitro - group (r - no2) to a hydroxylamine - group (r - nhoh), which is typical of the nitro - substituted compounds : (7)r - no2 + 4e+ 4h+ r- nhoh+h2o. considering the results from the hmde and age for the determining steps, evaluated by cv and swv experiments at ph 4.0, the transference of one electron is related to the slow step of the reaction, and three electrons are related to a fast step, defined by (8)r - no2+er - no2 (slow), (9)r - no2+3e+4h+r- nhoh+h2o (fast). upon studying the influence of the variation in amplitude (a), it was experimentally observed that i p varied proportionally to a, up to 50 mv, without any observed shift in e p or in the half - peak width. after 50 mv, the half - peak width showed an increase, suggesting a loss of sensibility of the method, considering the use of three electrodes. following this observation, it was concluded that the best value of a for the detection of 4-np was 50 mv. furthermore, a linear relationship can be observed between i p and the a values, which can be employed to determine the concentration of the adsorbed species on the electrode surface, according to (10)ip=(51)102an2ffesa, where is the adsorbed species concentration, and all terms represent parameters previously presented in this paper. from this, the values calculated were 3.02 10, 6.29 10, and 1.62 10 mol / cm for the agsae, hmde, and age, respectively. these results are indicative that 4-np is less adsorptive for the agsae and that the greatest adsorptive process occurs for the age. this assumption is confirmed by fact that for the use of age, a step to clean the electrodic surfaces is necessary between each experiment, which increases the difficulty for analytical reproducibility. according to the theoretical aspects of the swv developed by lovric and coworkers, for a totally irreversible redox process, the variation in half - peak width (e p/2) of the swv responses is dependent on the n products, for a > 20 mv, according to (11)ep/2=(63.50.5)n. considering that = 0.5, the calculated values for e p/2 are given by 127/n. for 4-np on the agsae, hmde, and age, the e p/2 values observed were 133, 117, and 121 mv, respectively. the difference between the observed and calculated values occurs because the system redox is influenced by the adsorption process and the value is not exactly 0.5. for the scan increment, the increase in this value will also increase the signal and the sensitivity of the technique. however, for large values of e s, a widening of the peaks may occur, thus diminishing the resolution of the analysis. for this reason, e s was evaluated for the reduction of 4-np for the agsae, hmde, and age. the results obtained show that the increase in e s does not have any influence on the peak potentials. in this case, an increase in e s results in an increase in i p, but no linear relationship was observed. as a result, in subsequent experiments, a value of e s = 2 mv was adopted. using the above optimized parameters, linear calibration curves were obtained for 4-np on the three electrodes. for this, aliquots from the stock 4-np solution were consecutively added to the electrochemical cell containing 10 ml of the electrolyte. the swv responses were recorded for a concentration range between 5.51 10 and 4.86 10 mol l for the agsae and hmde and between 1.62 10 and 1.61 10 mol l for the age. for the agsae, hmde, and age, the square wave voltammograms presented linear relationships between the peak currents and the concentrations added. figure 3 shows the square wave voltammograms obtained with the agsae, and the insert presents the analytical curve obtained. the linearity between the densities of the peak currents and the added concentrations of 4-np for the agsae, hmde, and age are described by equations (12), (13) and (14), respectively, (12)i (a / cm2)= 1.99 + 1.674107 c (mol / l), (13)i (a / cm2)= 0.79 + 1.801107 c (mol / l), (14)i (a / cm2)= 1.72 + 1.908106 c (as these curve exhibited a negative interception, the presence of random errors was evaluated with the statistic test, t - test, and the calculated t values indicated that the negative interception was free from random errors. besides, the analytical sensitivities, defined by the inclination of the analytical curves, for the agsae and hmde presented similar values, indicating the similar sensitivities for both electrodes ; meanwhile, for the age, the analytical curve inclination presented lower values, indicating that the age promotes responses with lower sensibilities than those obtained with the agsae and hmde. the detection (dl) and quantification limits (ql) were obtained for the experimental conditions and criteria presented in the experimental section for all electrodes employed, and table 1 shows the experimental results obtained. this table shows the correlation coefficients (r), which determine the degree of linearity of the relationship between the concentration of 4-np and the peak current (without considering the electroactive area), the standard deviations of the arithmetic mean of ten blank solutions (s b), the slope of the working curves (s), the detection limits (dl), and the quantification limits (ql). the values for the dl and ql obtained using the agsae and hmde for the 4-np reduction process presented very similar values, and these values were very close to those obtained previously for the hmde or other electrodic surfaces [6, 7, 19 ]. for the age, the values for the dl and ql indicated that its surface is very inconvenient for the determination of 4-np, due to the low sensitivity and, principally, to the difficulties related to the reproducibility of the analytical responses, which was caused by the strong adsorptive process of 4-np. recovery experiments were also performed using the agsae and hmde in order to evaluate and compare the efficiency of each electrode. for this, pure electrolyte was composed of 0.10 mol l br buffer solutions, with the ph adjusted to 7.0 for the agsae and 4.0 for the hmde ; the pure electrolyte was then spiked with 1.05 10 mol l 4-np, followed by the addition of the standard solutions. the recovery percentages were used to evaluate and quantify the 4-np that was added. in this way, the efficiency of each electrode could be determined. the recovery percentages obtained were around 94.95 0.95% and 97.88 0.89% for the agsae and hmde, respectively, indicating that this methodology can be successfully applied for the analytical determination of 4-np. the precision and accuracy of the analytical responses obtained for all proposed electrodes were evaluated by the experiments for reproducibility and repeatability. the reproducibility was evaluated on different days, using five different solutions containing 1.02 10 mol l 4-np for the agsae and hmde and 1.06 10 mol l 4-np for the age. the rsd value obtained for n = 5 was lower than 2.00% for the agsae and hmde (1.95% and 1.49%, resp.) but larger than 5.00% for the age. the repeatability was evaluated ten times in the same solution containing 1.02 10 mol l 4-np for the agsae and hmde and 1.06 10 mol l 4-np for the age. the rsd value obtained for n = 10 also presented values lower than 2.00% for the agsae and hmde (1.60% and 1.25%, resp.). to test the applicability of the agsae in complex samples, the methodology was employed for the construction of analytical curves and evaluation of recovery efficiencies for experiments using two natural water samples. these two natural water samples contained different level of contamination, which were determined by measurements of the biochemical oxygen demand (bod) and chemical oxygen demand (cod). the water samples were used as collected as the solvent in preparing the br buffer at ph 7.0. this procedure was aimed at identifying any influence of the matrix components on the analyses and detection of 4-np for the agsae. it is important to highlight that prior to the introduction of the first aliquot of 4-np, the electrolyte was free from any signs of 4-np ; consequently, the level of any possible contamination by nitro - compounds was below the detection limit of the proposed methodology. the analytical curves for 4-np on the agsae were evaluated using the natural samples water and were compared to the calibration curves obtained using pure water electrolyte. from these, it was possible to observe a reduction in the value of the slopes, indicating a loss of sensitivity in the analytical methodology, as shown in figure 4. this loss of sensitivity must be related to the amount of organic matter present in the water samples, as indicated by the estimates of bod and cod. the bod and cod parameters are related to the relative oxygen requirements in the biochemical and chemical degradation, respectively, of organic materials and inorganic materials present in the samples, which are susceptible to oxidation. in practice, the cod values are slightly higher than the bod values ; thus, it can be inferred that the higher bod and cod values obtained indicate a greater amount of organic matter present in the sample. recovery experiments were also performed to evaluate the interference of organic and inorganic components of the natural water samples on the reduction of 4-np for the agsae. recovery curves for the samples spiked with 1.0 10 mol l 4-np were obtained by the standard addition method. the results of the present study, shown in table 2, depict the inverse dependence of the peak current on the amount of organic matter present in different samples. these results are shown to be in a suitable range for analytical applications where the acceptable values are in the interval between 70% and 130%. the utilization of agsae minimized any environmental contamination with mercury because the amount discarded in each analysis is practically insignificant. the use of swv was faster and more sensitive than other conventional techniques and to make possible to evaluate the electrochemical redox process that occurred for 4-np on agsae. the memory effects associated with the strong adsorption processes of 4-np were unravelled by surface conditioning potential, which was sufficient to promote a total renewal thus yielding a new and reproducible surface. this extra advantage lowers the time used in the analysis, hence resulting in improvement in analytical sensibility and in the reproducibility of the responses. so, the results presented in this work proved that the agsae can be considered a suitable tool in the analytical determination and in the electrochemical behaviour study of the 4-np, as well as other biological and environmentally interesting compounds that present analytical responses on mercury surfaces. | this work reports a comparative electrochemical behaviour study and p - nitrophenol analytical detection using silver solid amalgam, hanging dropping mercury, and silver electrodes. for this, square wave voltammetry was employed, where the analytical responses and the redox mechanisms could be compared for reduction processes of 4-nitrophenol by analysis of the voltammetric responses. the analytical performance of the electrode was evaluated and detection and quantification limits, recovery percentages, repeatability, and reproducibility for the silver solid amalgam and hanging dropping mercury electrodes presented similar values ; the results presented for the silver electrode indicated worse analytical parameters than the other electrodes. the results indicate that the silver solid amalgam electrode can be considered a suitable tool and an interesting alternative for the analytical determination of 4-nitrophenol, as well as for the determination of other biological and environmentally interesting compounds that present analytical responses on mercury surfaces. |
dichloroacetate (dca) is a xenobiotic of interest to both environmental toxicologists and clinicians. the chemical is a product of water chlorination and of the metabolism of various drugs and industrial chemicals. its accumulation in groundwater and at certain superfund sites is considered a potential health hazard. however, concern about dca toxicity is predicated mainly on data obtained in inbred rodent strains administered dca at doses thousands of times higher than those to which humans are usually exposed. in these animals, chronic administration of dca induces hepatotoxicity and neoplasia. ironically, the dca doses used in animal toxicology experiments are very similar to those used clinically for the chronic or acute treatment of several acquired or hereditary metabolic or cardiovascular diseases. as a medicinal, dca is generally well tolerated and stimulates the activity of the mitochondrial pyruvate dehydrogenase enzyme complex, resulting in increased oxidation of glucose and lactate and an amelioration of lactic acidosis. by this mechanism, the drug may also enhance cellular energy metabolism. dca is dehalogenated in vivo to monochloroacetate and glyoxylate, from which it can be further catabolized to glycolate, glycine, oxalate, and carbon dioxide. it remains to be determined whether important differences in its metabolism and toxicology exist in humans between environmentally and clinically relevant doses.imagesfigure 1figure 2 |
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hind limb ischemia / reperfusion injury may occur clinically after a release of tourniquets during orthopedic surgery, or extrication of a trauma victim who is compressed with a heavy weight for a prolonged period (crush syndrome). several reports have indicated that the pathophysiology of the injury varies according to the duration and grade of the ischemia (13). it is well recognized that sustained ischemia can induce cell death and tissue necrosis, which are mainly caused by energy insufficiency. many studies have shown that tissue injury in many organs occurs not only during the ischemic period but also in the reperfusion period. skeletal muscle ischemia / reperfusion is associated with a systemic inflammatory response and determines the effect on remote organs (liver, lung, kidney, myocardium and testis) structure and function (49). the development of remote organ dysfunction was observed only following reperfusion, which implies that humoral and/or cellular mediators produced locally in the limb were responsible for mediating remote organ injury (1012). ptx through effects of increasing intracellular cyclic amp on red blood cells improve the oxygen delivery to ischemic tissues and also increases the cyclic amp on polymorph nuclear leukocytes and decreases oxygen free radical production (10, 13 - 16). in addition, ptx limit the inflammatory response with reduction in cellular activation, phagocytosis and endothelium adhesion. recent studies have indicated that ptx improves ischemia / reperfusion injury in many organs (1719). however, the effects of ptx on remote testicular injury caused by skeletal muscle ischemia / reperfusion are not clear. the objective of the present study was to investigate the role of ptx on remote testicular injury caused by unilateral hind limb ischemia / reperfusion of rats. for this purpose, the biochemical and pathological effects of skeletal muscle ischemia / reperfusion and ptx in testicular tissues of rats have been investigated. the results of our investigation would help to clarify the potential importance of the use of ptx in situations of oxidative damage. these findings may encourage the use of antioxidants to reduce remote organ injury after skeletal muscle ischemia / reperfusion. the study was conducted on 20 healthy male wistar rats, 12 - 16 weeks old and weighing between 270 - 300 g. all rats of this study were kept according to the norms of the islamic azad university college of veterinary medicine tehran iran laboratory of animal experimentations ; this investigation was approved by the committee of ethics in research with animals of islamic azad university. the study was designed so as to minimize the number of animals required for the experiments. the rats were housed in individual cages under temperature controlled standard conditions, 12h/12h light / dark cycle with free access to standard rodent food and filtrated tap water. the animals were allocated randomly into two experimental groups of ten rats each as follows : ischemia / reperfusion (ir group) and ischemia / reperfusion + pentoxifylline (ir+ptx group). anesthesia was induced with a mixture of ketamine and xylazine (50 mg / kg and 10 mg / kg, respectively) via intramuscular injection. the level of anesthesia was continuously monitored by observing the respiratory patterns and toe pinch reflex. after the induction of anesthesia, fur was completely removed from left hind limbs with an electric shaver to facilitate the measurement of limb perfusion. unilateral hind limb ischemia was induced by placing an orthodontic rubber band at the hip joint. before using the tourniquet, the animals were kept anesthetized throughout the duration of ischemic period with additional anesthetic doses as needed. body temperature was maintained with a heating pad whilst the animals were under anesthesia. in ir+ptx group, pentoxifylline tablets were dissolved in normal saline as explained in previous studies (20). the analgesic nalbuphine hydrochloride after 24h of reperfusion, all animals were anesthetized again with the same mixture and a longitudinal incision of approximately 2 cm was made to the scrotal raphe for bilateral orchiectomies (a procedure involving cord clamping and hemostasis). right testes of seven rats were processed for histopathological examination and left testes were washed with normal saline and stored in a 80c refrigerator for the evaluation of biochemical parameters. then the rats were euthanized with an overdose of intraperitoneal pentobarbital by injection (300mg / kg). the left testicular tissues were washed three times in cold normal saline solution (0.9%). then, the tissues were homogenized in ice - cold tris - hcl buffer solution, within a homogenizer for 2min at 11200g. the levels of mpo were determined in the supernatant, and mda levels were studied in the homogenate. for a further extraction procedure, the supernatant was extracted in ethanol/ chloroform mixture (5/3 v / v). after a second centrifugation at 3500g for 20min, the clear upper layer was taken and used for sod activity determination (21). the study was conducted on 20 healthy male wistar rats, 12 - 16 weeks old and weighing between 270 - 300 g. all rats of this study were kept according to the norms of the islamic azad university college of veterinary medicine tehran iran laboratory of animal experimentations ; this investigation was approved by the committee of ethics in research with animals of islamic azad university. the study was designed so as to minimize the number of animals required for the experiments. the rats were housed in individual cages under temperature controlled standard conditions, 12h/12h light / dark cycle with free access to standard rodent food and filtrated tap water. the animals were allocated randomly into two experimental groups of ten rats each as follows : ischemia / reperfusion (ir group) and ischemia / reperfusion + pentoxifylline (ir+ptx group). anesthesia was induced with a mixture of ketamine and xylazine (50 mg / kg and 10 mg / kg, respectively) via intramuscular injection. the level of anesthesia was continuously monitored by observing the respiratory patterns and toe pinch reflex. after the induction of anesthesia, fur was completely removed from left hind limbs with an electric shaver to facilitate the measurement of limb perfusion. unilateral hind limb ischemia was induced by placing an orthodontic rubber band at the hip joint. before using the tourniquet, the animals were kept anesthetized throughout the duration of ischemic period with additional anesthetic doses as needed. body temperature was maintained with a heating pad whilst the animals were under anesthesia. in ir+ptx group, pentoxifylline tablets were dissolved in normal saline as explained in previous studies (20). the analgesic nalbuphine hydrochloride (2 mg / kg) was used via subcutaneous during observation time. after 24h of reperfusion, all animals were anesthetized again with the same mixture and a longitudinal incision of approximately 2 cm was made to the scrotal raphe for bilateral orchiectomies (a procedure involving cord clamping and hemostasis). right testes of seven rats were processed for histopathological examination and left testes were washed with normal saline and stored in a 80c refrigerator for the evaluation of biochemical parameters. then the rats were euthanized with an overdose of intraperitoneal pentobarbital by injection (300mg / kg). the left testicular tissues were washed three times in cold normal saline solution (0.9%). then, the tissues were homogenized in ice - cold tris - hcl buffer solution, within a homogenizer for 2min at 11200g. the levels of mpo were determined in the supernatant, and mda levels were studied in the homogenate. for a further extraction procedure, the supernatant was extracted in ethanol/ chloroform mixture (5/3 v / v). after a second centrifugation at 3500g for 20min, the clear upper layer was taken and used for sod activity determination (21). the principle of the sod activity determination method was based on the inhibition of nitroblue tetrazolium reduction described by sun. one unit of sod was defined as the enzyme activity causing 50% inhibition in the nitroblue tetrazolium reduction rate. the mda levels in testicular tissues were analyzed by a method based on the reaction with thiobarbituric acid at 95c (24). in the thiobarbituric acid test reaction, mda or mda - like substances and thiobarbituric acid react together to produce a pink pigment with an absorption maximum of 532 nm. the results were expressed as nanomol per gram wet tissue (nmol / g tissue). the principle of the assay is based on determination of the rate constant k (s) of h2o2 decomposition at 240 nm. testicular injury was quantified by measuring testicular mpo activity, the activity of infiltrated polymorphonuclear leukocytes, using a protocol modified from a previous report (26). the wet / dry weight ratio, the tissue edema index, was measured to evaluate testicular injury. briefly, freshly harvested testes were weighed, placed in an oven for 24h at 60c and weighed again when dry (26). the principle of the sod activity determination method was based on the inhibition of nitroblue tetrazolium reduction described by sun. one unit of sod was defined as the enzyme activity causing 50% inhibition in the nitroblue tetrazolium reduction rate. the mda levels in testicular tissues were analyzed by a method based on the reaction with thiobarbituric acid at 95c (24). in the thiobarbituric acid test reaction, mda or mda - like substances and thiobarbituric acid react together to produce a pink pigment with an absorption maximum of 532 nm. the results were expressed as nanomol per gram wet tissue (nmol / g tissue). the principle of the assay is based on determination of the rate constant k (s) of h2o2 decomposition at 240 nm. testicular injury was quantified by measuring testicular mpo activity, the activity of infiltrated polymorphonuclear leukocytes, using a protocol modified from a previous report (26). the wet / dry weight ratio, the tissue edema index, was measured to evaluate testicular injury. briefly, freshly harvested testes were weighed, placed in an oven for 24h at 60c and weighed again when dry (26). the wet / dry weight ratio was then calculated. the tissue specimens were placed in paraffin blocks, sectioned at 5m, and stained with hematoxylin and eosin (h&e) for light microscopic analysis. an experienced pathologist, who was blinded to the experiment and data, examined the samples histopathologically. (27) classification as follows : grade - i : showed normal testicular architecture with an orderly, arrangement of germinal cells ; grade - ii : injury showed less orderly, non - cohesive germinal cells and closely packed seminiferous tubules ; grade - iii : injury exhibited disordered sloughed germinal cells with shrunken pyknotic nuclei and less distinct seminiferous tubule borders ; grade - iv : injury defined seminiferous tubules that were closely packed with coagulative necrosis of the germinal cells. kruskal - wallis test was used to compare the biochemical and histopathological parameters between groups. when kruskal - wallis test results were significant, bonferroni adjusted mann - whitney u test was used in the paired comparison. kruskal - wallis test was used to compare the biochemical and histopathological parameters between groups. when kruskal - wallis test results were significant, bonferroni adjusted mann - whitney u test was used in the paired comparison. one of the most interesting observations at the histopathological examination of the testicular tissues is the presence of sloughed germinal cells within the seminiferous tubules and disorganization in rat testes after the unilateral hind limb ischemia / reperfusion. the testicular injury score increased significantly in the ir group compared with the ir+ptx group (p<0.05) (table-1). in the ir group, coagulative necrosis with loss of seminiferous tubule epithelium, edema and sloughed germinal cells were predominant features in sections (figure-1). however, the rats in the ir+ptx group had essentially normal seminiferous tubule morphology (figure-2). table 1the tissue cat and mpo activities and levels of sod and mda in testicular tissues and score of testicular histological changes and muscle wet / dried weight (w / d) ratio.ischemia/reperfusionischemia/reperfusion + pentoxifyllinesod (u / mg protein)4.0160.288.120.13 cat (k / g protein)81.034.1798.102.73 mpo (u / g tissue)1.030.200.610.08 mda (nmol / g tissue)2.740.331.850.21 histological grading2.300.671.400.51 w / d ratio2.200.131.640.09 p < 0.05 compared with the ischemia - reperfusion group. p < 0.05 compared with the ischemia - reperfusion group. figure 1light microscopic view of testis tissues from ir group showing coagulative necrosis with loss of seminiferous tubule epithelium, edema and sloughed germinal cells. figure 2light microscopic view of testis tissues from ptx treated group showing normal seminiferous tubule morphology with orderly arrangement of germinal cells.. activities of sod and cat in testicular tissues were decreased by ischemia / reperfusion, but administration of ptx increased these levels (p<0.05) (table-1). the tissue levels of mda increased in the ir group in comparison to the ir+ptx group (table-1). mpo activity in testicular tissues in the ir group was significantly higher than in the ir+ptx group (p<0.05) (table-1). the wet / dry weight ratio of testicular tissues in the ir group was significantly higher than in the ir+ptx group (p<0.05) (table-1). the systemic inflammatory response syndrome is a consequence of many conditions, such as surgery, trauma, burn, shock, or bacterial infection (28), that results in the development of potentially fatal complication known as multiple organ dysfunction syndrome. hind limb ischemia / reperfusion has been extensively used in our laboratory as a model of systemic inflammatory response syndrome, which closely resembles the acute traumatic and ischemic insult seen in systemic inflammatory response syndrome patients (29). it has been demonstrated that hind limb ischemia / reperfusion causes cellular injury in remote organs (kidney, lung, and myocardium) and contributes to the development of multiple organ dysfunction syndrome (5, 29). as far as we know, there are only a few reports demonstrating testicular remote injury following muscle ischemia / reperfusion injury (8). the results of takhtfooladi. (8) indicated that skeletal muscle ischemia / reperfusion induces severe testicular damage and n - acetylcysteine has protective effects on testicular injury after hind limb ischemia / reperfusion. their data supported this view that temporary occlusion of the femoral artery induced testicular injury in rats (8). previous studies indicate that inflammatory response and injury to remote organs can be caused by the systemic release of the pro - inflammatory mediators and free oxygen radicals upon reperfusion of ischemic limbs (5, 30). despite decades of research in this area, ischemia / ptx is one of the phosphodiesterase inhibitors that have been reported to increase intracellular cyclic amp and reduce superoxide anion production by both monocytes and polymorphonuclear cells dose dependently in vitro (31). (32) indicated that ptx led to a marked increase in cyclic amp levels, whereas cyclic gmp levels were only marginally elevated in lipopolysaccharide stimulated human monocytes. ptx has received considerable attention with respect to its action on leukocytes in many organs (3335). (19) reported that after reperfusion, myeloperoxidase activity and blood neutrophil count were lower with ptx than with saline, and changes in the filtration coefficient were correlated to the percent changes in blood neutrophils during reperfusion. they suggested that this effect may be mainly caused by a decrease in sequestration of neutrophils in the lung during reperfusion. their group also reported that ptx prevented endothelial injury during ischemia / reperfusion by decreasing neutrophil sequestration in isolated perfused rat and rabbit lungs and in pigs after left lung allotransplantation (33). recently administered ptx was shown to protect against ischemia / reperfusion injuries in local and remote organs and it was suggested that this protective effect may be due to its ability to inhibit of free radical generation (12, 20). there is growing evidence regarding its beneficial effects in ameliorating testicular ischemia / reperfusion injury (39). in an experimental study savas. (39) suggested that the administration of ptx at a dose of 50 mg / kg 15 min. before spermatic cord torsion their results suggest that pentoxifylline treatment attenuates reperfusion damage on both sides, possibly with its effects on blood flow and neutrophils (39). (40), who demonstrated that ptx may be a potential protective agent for preventing the negative changes related to oxidative stress in testicular injury caused by spermatic cord torsion in miniature horse stallions. however, the protective effect of ptx on testes from skeletal muscle ischemia / reperfusion injury has not been studied to date. in the current study, we tested the hypothesis that ptx could protect the testes from remote organ injury after skeletal muscle ischemia / reperfusion. oxidative stress is associated with an increased rate of cellular damage induced by oxygen and oxygen - derived oxidants, commonly known as reactive oxygen species (41, 42). the major targets of reactive oxygen species are membrane lipids, in a process known as lipid peroxidation. it is also acknowledged that testicular tissues and spermatozoa are very sensitive to reactive oxygen species attack and lipid peroxidation. the susceptibility of testicular tissues to oxidation is attributed to the high polyunsaturated fatty acid content of sperm membranes (42, 43). many tissues contain powerful endogenous scavengers that provide protection against free radical damage, including sod, cat, glutathione peroxidase, ascorbic acid and -tocopherol (44). (45) have reported that adequate levels of antioxidants such as sod, cat and possibly glutathione peroxidase and reductase, maintain the scavenging potential in gonads and seminal fluids, which is referred to as oxidative stress status. tissue cat and mpo activities, and sod and mda levels are considered to indicate oxidative stress. in the present study, sod level and cat activity were decreased significantly in ir group compared with ir+ptx group. the tissue mda level and mpo activity in testes were increased significantly in ir group compared with ir+ptx group. according to the observations of this study, the histopathological injury score was significantly decreased in ir+ptx group compared with that of ir group. in the ptx treated group, histopathological features such as edema, congestion, hemorrhage, and necrosis of the germinal cells were markedly less than in ir group. the findings in this study indicate that reperfusion of the ischemic limb leads, within 24h of reperfusion, to a systemic response as demonstrated by the biochemical and histological impairment of the testis. also, data on cat and mpo activity, and sod and mda levels suggested a protective effect of ptx against testicular remote injuries after unilateral hind limb ischemia / reperfusion. these data together with previous findings (39) confirm the potent anti - oxidation capacity of ptx. these data also support the concept that ptx may be an effective therapeutic adjunct to reperfusion injury. the clinical relevance of this manuscript refers to the previous use of ptx in situations requiring procedures ischemia with reperfusion to reduce or prevent distant organs damage. the results of this study showed that unilateral hind limb ischemia / reperfusion induced testicular injury in rats. nevertheless, ptx administration significantly decreased testes injury induced by skeletal muscle ischemia / reperfusion according to our histological and biochemical findings. ptx is already in clinical use for the treatment of vascular diseases and these results suggest the possibility of clinical application of ptx in testicular remote organ injury following skeletal muscle ischemia / reperfusion. | abstractobjectivethe objective of the present study was to investigate the role of pentoxifylline (ptx) on remote testicular injury caused by unilateral hind limb ischemia / reperfusion of rats.materials and methodstwenty healthy male wistar rats were allocated randomly into two groups : ischemia / reperfusion (ir group) and ischemia / reperfusion + pentoxifylline (ir+ptx group). ischemia was induced by placement of a rubber tourniquet at the greater trochanter for 2h. rats in ir+ptx group received ptx (40 mg / kg ip) before the reperfusion period. at 24h after reperfusion, testes were removed and levels of superoxide dismutase (sod), malondialdehyde (mda), catalase (cat) and myeloperoxidase (mpo) activity were determined in testicular tissues. three rats of each group were used for wet/ dry weight ratio measurement. testicular tissues were also examined histopathologically under light microscopy.resultsactivities of sod and cat in testicular tissues were decreased by ischemia/ reperfusion (p<0.05). significantly increased mda levels in testicular tissues were decreased by ptx treatment (p<0.05). mpo activity in testicular tissues in the ir group was significantly higher than in the ir+ptx group (p<0.05). the wet / dry weight ratio of testicular tissues in the ir group was significantly higher than in the ir+ptx group (p<0.05). histopathologically, there was a statistically significant difference between two groups (p<0.05).conclusionsaccording to histological and biochemical findings, we conclude that ptx has preventive effects in the testicular injury induced by hind limb ischemia / reperfusion. |
hypoparathyroidism is an acquired or inherited condition characterized by decreased levels of circulating parathyroid hormone (pth) leading to hypocalcemia and slightly higher phosphate levels. the most common cause of acquired hypoparathyroidism is the damaging, removal and/or devascularization of the parathyroid glands, which may be secondary to a trauma or surgery to the thyroid, parathyroids or the neck. transient hypoparathyroidism is frequent (24.1%) after total thyroidectomy, while chronic hypoparathyroidism is rarer (1.2%). hypocalcemia caused by hypoparathyroidism is one of the most common and morbid complications after total thyroidectomy. hypocalcemia may lead to severe and life - threatening condition, such as nephrocalcinosis, kidney stones, chronic kidney disease and myocardial dysfunction. however, there is an ongoing controversy about the predictive power of post - surgery pth levels for chronic hypoparathyroidism. observed that the pth levels reached a nadir 3h after surgery and that pth measured 3 h after surgery was superior in predicting the risk of transient or persistent hypocalcemia than measurement at the end of the operation. the risk of developing hypoparathyroidism is a challenge for the best care offered to patients, and is controversial. indeed, some studies indicate that pth levels measured during or shortly after surgery have a high predictive value [47 ], while others advocate daily calcium monitoring, calcium monitoring in selected patients only, or calcium monitoring only during the first postoperative 24 hours [1012 ]. another study showed that a serum calcium level 1.9 mmol / l on postoperative day 2 and pth 2.00 mmol / l. these treatments included calcium supplements and, in cases of recalcitrant hypocalcemia, intravenous calcium gluconate. transient hypoparathyroidism was defined as serum pth levels below the normal range during the first day after surgery. in the present study, permanent hypoparathyroidism was diagnosed if : 1) serum pth levels were below the normal range or ca 2.00 mmol / l. these treatments included calcium supplements and, in cases of recalcitrant hypocalcemia, intravenous calcium gluconate. transient hypoparathyroidism was defined as serum pth levels below the normal range during the first day after surgery. in the present study, permanent hypoparathyroidism was diagnosed if : 1) serum pth levels were below the normal range or ca < 2.00 mmol / l for more than 12 months ; or 2) calcium and/or vitamin d supplementation were necessary to treat hypocalcemia - related symptoms for more than 12 months. blood samples were taken from each patient at 6 am, 1 day before the operation, and on the first, second, third, fourth, and fifth postoperative days. follow - up visits were performed at 2 weeks and at 2, 6, and 12 months for patients whose serum pth and/or ca levels remained below the normal or if the patients still needed calcium or vitamin d supplements for hypocalcemia - related symptoms. at 14 days, 113 samples were taken, 78 at 2 months, 69 at 6 months and 53 at 12 months. follow - up was performed for all patients with hypocalcaemia until levels returned to the normal range. serum pth levels were measured using a cobas e601 autoanalyzer (hitachi high - technologies corporation, tokyo, japan), and the normal range was 15.0065.00 serum calcium was measured using an automated analyzer (toshiba medical systems corporation, tochigi - ken, japan), and the normal range was 2.202.70 mmol / l. continuous variables are presented as mean standard deviation (sd) or median (range), and were analyzed using friedman s rank test or student t - test, as appropriate. a total of 438 patients were included in the study including 352 women and 86 men. the indications for surgery and the extent of thyroidectomy are shown in tables 1 and 2. autotransplantation of parathyroid tissue was performed in 39 patients (39 glands), at the surgeon s discretion. figure 1 shows the perioperative kinetics of serum pth and ca in the first 5 days and for up to 12 months after surgery in all 438 patients who underwent total thyroidectomy. perioperative pth levels declined from 41.89 ng / l (100%) before operation to 17.55 ng / l (41.90% of the initial value) on the first day morning after operation (p<0.01). during the first 5 days after surgery, there was a small but statistically significant increase compared with baseline values (p<0.01), up to a value of 20.49 ng / l (48.90% of baseline). at the first follow - up outpatient visit, pth levels reached 26.03ng / l (62.14% of baseline) (figure 1b). we also analyzed the data according to whether the patients had benign or malignant tumors and the serum pth levels are presented in figure 1c. the benign tumor group had a higher preoperative level of serum pth, at 55.90 ng / l, than the malignant tumor group, at 39.91 ng / l, and these remained separated by a similar amount but the overall trend of the decline in pth then small increase as described above for the total cohort was followed by both groups. the preoperative levels of pth in the groups treated with different surgical methods were 40.0413.95 ng serum ca values declined from 2.41 mmo / l (100%) before operation to 1.99 mmol / l (82.8% of baseline) on the first day after surgery, and reached a nadir on the second day after operation (1.97 mmol / l, 81.8% of baseline) (p<0.01). during the first 5 days after surgery, a slight increase was observed, up to 2.03 mmol / l (84.27% of baseline) (p<0.01) (figure 1a). at the first follow - up outpatient visit, the ca values reached 2.22 mmol / l (92.0% of baseline) (figure 1b), a value similar to baseline. however, at this time a number of patients were receiving calcium supplements, which probably interfered with the results. mean serum pth and ca levels were lower than the initial value at follow - up examinations, up to 12 months (figure 1b). this subsequent decline may be due to a negative selection bias : patients who did not feel ill tended to drop out. the risk of postoperative hypoparathyroidism in relation to neck dissection, thyroiditis, and iatrogenic removal of parathyroid glands were analyzed (tables 3 and 4). cnd increased the risk of transient hypoparathyroidism compared with total thyroidectomy alone (p<0.001), but did not increase the risk of permanent hypoparathyroidism (p=0.733). total thyroidectomy and combined cnd and lateral neck dissection (lnd) also increased the risk of transient hypoparathyroidism compared with total thyroidectomy alone (p=0.002), but did not increase the risk of permanent hypoparathyroidism (p=0.514). however, lnd in addition to cnd did not increase the postoperative transient or permanent hypoparathyroidism risk compared with total thyroidectomy and cnd (p=0.699 and p=0.582, respectively). patients with thyroiditis had increased risk of permanent hypoparathyroidism compared with those without thyroiditis (p=0.042), but not an increased risk of transient hypoparathyroidism (p=0.814). iatrogenic removal of parathyroid glands increased the risk of chronic hypoparathyroidism compared with those without iatrogenic parathyroidectomy (p=0.018), but did not increase the rate of transient hypoparathyroidism (p=0.183). on preoperative sampling in patients with no abnormality in pth or calcium homeostasis, there was no correlation between pth and serum calcium levels (r=0.024, p=0.524) (table 5). however, 1 day after surgery, a positive correlation was observed (r=0.345, p<0.001) (table 5). after surgery, this significant (p<0.01) positive correlation was maintained for up to 14 days (table 5). as can be seen from table 5, the correlation was stronger on postoperative day 4 and subsequently gradually weakened. on preoperative sampling in patients with no abnormality in parathyroid secretion or serum phosphate homeostasis, there was no correlation between pth and phosphate levels (r=0.062, p=0.093) (table 5). however, 1 day after surgery, a negative correlation was found (r=0.236, p<0.001) (table 6). after surgery, this significant (p<0.01) negative correlation was maintained for up to 14 days (table 5). as can be seen from table 5, the correlation become stronger in the first 5 days after surgery and subsequently weakened on day 14. twelve (2.74%) of the 438 patients who underwent total thyroidectomy and neck dissection developed chronic hypoparathyroidism. patients with chronic hypoparathyroidism had a pronounced perioperative decline in pth levels, which was more important than in patients with transient hypoparathyroidism (figure 2). however, there was also a smaller decline in pth levels in patients with pth levels within the normal range. accordingly, the correlations shown in tables 5 and 6 were recalculated after excluding patients with transient or permanent hypoparathyroidism. this reanalysis showed that the correlations between pth and serum ca disappeared and that the correlations between pth and serum phosphate were still present during the first 3 days after surgery (table 6). as could be predicted from the decline in pth level, the postoperative decrease in serum ca levels was more important in patients with chronic hypoparathyroidism. in addition, pth and serum ca levels were maintained at the lowest levels without increasing in the first 5 days after surgery in patients with persistent hypoparathyroidism (figure 2). mmol / l on day 1 after surgery had a sensitivity of 58.33% and a negative predictive value of 97.70% for permanent hypoparathyroidism. the specificity (49.77%) was limited and the positive predictive value (3.16%) was poor. thus, a low postoperative ca level predicts neither that a patient will develop persistent hypoparathyroidism, nor a normal ca concentration excludes it. when pth levels on the first day after surgery were examined, all twelve patients who eventually developed persistent hypoparathyroidism had levels < 7 ng / l. < 7ng / l on the first after surgery had a sensitivity of 100% and a negative predictive value of 100% for permanent hypoparathyroidism. the specificity (70.19%) was limited and the positive predictive value (8.63%) was poor. thus, although a postoperative pth level below 7 ng / l does not predict that a patient will develop persistent hypoparathyroidism, pth level above 7 a total of 438 patients were included in the study including 352 women and 86 men. the indications for surgery and the extent of thyroidectomy are shown in tables 1 and 2. autotransplantation of parathyroid tissue was performed in 39 patients (39 glands), at the surgeon s discretion. figure 1 shows the perioperative kinetics of serum pth and ca in the first 5 days and for up to 12 months after surgery in all 438 patients who underwent total thyroidectomy. perioperative pth levels declined from 41.89 ng / l (100%) before operation to 17.55 ng / l (41.90% of the initial value) on the first day morning after operation (p<0.01). during the first 5 days after surgery, there was a small but statistically significant increase compared with baseline values (p<0.01), up to a value of 20.49 ng / l (48.90% of baseline). at the first follow - up outpatient visit, pth levels reached 26.03ng / l (62.14% of baseline) (figure 1b). we also analyzed the data according to whether the patients had benign or malignant tumors and the serum pth levels are presented in figure 1c. the benign tumor group had a higher preoperative level of serum pth, at 55.90 ng / l, than the malignant tumor group, at 39.91 ng / l, and these remained separated by a similar amount but the overall trend of the decline in pth then small increase as described above for the total cohort was followed by both groups. these patients had lower pth levels (14.85 ng / l on the first day after surgery). the preoperative levels of pth in the groups treated with different surgical methods were 40.0413.95 ng serum ca values declined from 2.41 mmo / l (100%) before operation to 1.99 mmol / l (82.8% of baseline) on the first day after surgery, and reached a nadir on the second day after operation (1.97 mmol / l, 81.8% of baseline) (p<0.01). during the first 5 days after surgery, a slight increase was observed, up to 2.03 mmol / l (84.27% of baseline) (p<0.01) (figure 1a). at the first follow - up outpatient visit, the ca values reached 2.22 mmol / l (92.0% of baseline) (figure 1b), a value similar to baseline. however, at this time a number of patients were receiving calcium supplements, which probably interfered with the results. mean serum pth and ca levels were lower than the initial value at follow - up examinations, up to 12 months (figure 1b). this subsequent decline may be due to a negative selection bias : patients who did not feel ill tended to drop out. the risk of postoperative hypoparathyroidism in relation to neck dissection, thyroiditis, and iatrogenic removal of parathyroid glands were analyzed (tables 3 and 4). cnd increased the risk of transient hypoparathyroidism compared with total thyroidectomy alone (p<0.001), but did not increase the risk of permanent hypoparathyroidism (p=0.733). total thyroidectomy and combined cnd and lateral neck dissection (lnd) also increased the risk of transient hypoparathyroidism compared with total thyroidectomy alone (p=0.002), but did not increase the risk of permanent hypoparathyroidism (p=0.514). however, lnd in addition to cnd did not increase the postoperative transient or permanent hypoparathyroidism risk compared with total thyroidectomy and cnd (p=0.699 and p=0.582, respectively). patients with thyroiditis had increased risk of permanent hypoparathyroidism compared with those without thyroiditis (p=0.042), but not an increased risk of transient hypoparathyroidism (p=0.814). iatrogenic removal of parathyroid glands increased the risk of chronic hypoparathyroidism compared with those without iatrogenic parathyroidectomy (p=0.018), but did not increase the rate of transient hypoparathyroidism (p=0.183). on preoperative sampling in patients with no abnormality in pth or calcium homeostasis, there was no correlation between pth and serum calcium levels (r=0.024, p=0.524) (table 5). however, 1 day after surgery, a positive correlation was observed (r=0.345, p<0.001) (table 5). after surgery, this significant (p<0.01) positive correlation was maintained for up to 14 days (table 5). as can be seen from table 5, the correlation was stronger on postoperative day 4 and subsequently gradually weakened. on preoperative sampling in patients with no abnormality in parathyroid secretion or serum phosphate homeostasis, there was no correlation between pth and phosphate levels (r=0.062, p=0.093) (table 5). however, 1 day after surgery, a negative correlation was found (r=0.236, p<0.001) (table 6). after surgery, this significant (p<0.01) negative correlation was maintained for up to 14 days (table 5). as can be seen from table 5, the correlation become stronger in the first 5 days after surgery and subsequently weakened on day 14. twelve (2.74%) of the 438 patients who underwent total thyroidectomy and neck dissection developed chronic hypoparathyroidism. patients with chronic hypoparathyroidism had a pronounced perioperative decline in pth levels, which was more important than in patients with transient hypoparathyroidism (figure 2). however, there was also a smaller decline in pth levels in patients with pth levels within the normal range. accordingly, the correlations shown in tables 5 and 6 were recalculated after excluding patients with transient or permanent hypoparathyroidism. this reanalysis showed that the correlations between pth and serum ca disappeared and that the correlations between pth and serum phosphate were still present during the first 3 days after surgery (table 6). as could be predicted from the decline in pth level, the postoperative decrease in serum ca levels was more important in patients with chronic hypoparathyroidism. in addition, pth and serum ca levels were maintained at the lowest levels without increasing in the first 5 days after surgery in patients with persistent hypoparathyroidism (figure 2). serum ca < 2.0 mmol / l on day 1 after surgery had a sensitivity of 58.33% and a negative predictive value of 97.70% for permanent hypoparathyroidism. the specificity (49.77%) thus, a low postoperative ca level predicts neither that a patient will develop persistent hypoparathyroidism, nor a normal ca concentration excludes it. when pth levels on the first day after surgery were examined, all twelve patients who eventually developed persistent hypoparathyroidism had levels < 7 ng / l. < 7ng / l on the first after surgery had a sensitivity of 100% and a negative predictive value of 100% for permanent hypoparathyroidism. the specificity (70.19%) was limited and the positive predictive value (8.63%) was poor. thus, although a postoperative pth level below 7 ng / l does not predict that a patient will develop persistent hypoparathyroidism, pth level above 7 the aim of the present study was to analyze the kinetics and factors affecting pth levels after total thyroidectomy and cnd. results showed that pth levels declined to 41.90% of its initial value on the first day after operation. after surgery, pth was correlated positively with calcium and inversely with phosphate levels from postoperative day 1 to 14. l on postoperative day 1 was predictive of persistent hypoparathyroidism, with sensitivity and negative predictive value 100%, but poor specificity (70.19%). patients with thyroiditis had an increased risk of permanent hypoparathyroidism compared with those without thyroiditis. iatrogenic removal of the parathyroid glands increased the risk of permanent hypoparathyroidism compared with those without iatrogenic parathyroidectomy. considerable effort has been spent on the prevention of recurrent laryngeal nerve palsy after thyroidectomy [1619 ], but postoperative hypoparathyroidism and its consequences remain widely underrated. total thyroidectomy has been shown to be a significant risk factor for hypoparathyroidism and it occurs more frequently in total thyroidectomy than in near - total or subtotal thyroidectomy. permanent loss of parathyroid function still occurs in 1.2% after total thyroidectomy plus bilateral cnd, and up to 16.2% according to some studies. in the present study, transient hypoparathyroidism was observed in 227 (51.83%) of the 438 patients. during surgery, manipulations in the vicinity of the parathyroid gland may transiently compromise the blood flow to the gland or impair the venous efflux. conversely, the gland may be subjected to mechanical stress, which can result in the transient release of pth. these opposite changes may cancel each other and thus the mean pth levels at the end of surgery may not reflect the real function of the reserved parathyroid.. observed that pth levels reached a nadir 3 h after surgery and that pth measured 3 h after surgery was superior in predicting the risk of transient or persistent hypocalcemia to measurement at the end of the operation. in the present study, the pth levels reached the nadir on the first day morning after surgery (10 to 16 hours after surgery), and in the first 5 days after surgery, there was a small but significant increase. from these results, it appears that the pth levels on the first day morning after surgery could really reflect the function of the preserved parathyroid glands. several studies have addressed the question of when serum pth should be measured to identify patients at risk of developing persistent hypocalcemia, but the early studies were not large enough [4,6,7,2224 ]. one recent large prospective study of 523 patients concluded that subnormal pth values 4 h after surgery did not predict postoperative hypocalcemia. another recent large prospective study of 402 patients concluded that normal pth levels 3 h after surgery and normal serum calcium levels on the first postoperative day ruled out persistent hypoparathyroidism. in the present study, the observation period was sufficiently long (more than 12 months) to differentiate between patients with transient hypocalcemia - related symptoms and those with persistent hypocalcaemia. based on these observations, determination of pth levels on the first day morning after surgery could be used to rule out the possibility of persistent hypocalcemia. in the present study, cnd increased the risk of transient hypoparathyroidism. this conclusion is supported by the findings from roh. and cavicchi. however, in the present study, cnd did not increase the risk of chronic hypoparathyroidism. this phenomenon may be due to the small number of patients who underwent bilateral cnd, which weakened the impact of cnd on parathyroid glands function. however, in the present study, lnd did not increase the risk of transient or permanent hypoparathyroidism. this phenomenon may be due to the efforts spent to preserve the superior thyroid vessel and the middle thyroid veins during lateral neck dissection. indeed, the blood supply of the superior parathyroid glands comes from the superior thyroid vessel, and the middle thyroid veins also play a role in blood efflux of the superior and inferior parathyroid glands. reported that malignant thyroid diseases were a risk factor for the iatrogenic removal of the parathyroid glands, and that iatrogenic parathyroidectomy increased the rate of transient hypocalcaemia after thyroidectomy. in the present study, careful examination of the surgical specimen intraoperatively decreased the incidence of inadvertent parathyroidectomy during thyroidectomy. therefore, it is important to examine the resected specimen for overlooked parathyroid tissue that could be returned by autotransplantation. in the present study, therefore, in the majority of the patients, pth secretion became the driving force for maintaining serum calcium levels. the low serum calcium levels did not trigger an appropriate increase in pth secretion, presumably because the secretory capacity of the parathyroid glands was insufficient. this impaired function was seen for up to 14 days in the entire study population, but was not seen in patients without postoperative hypoparathyroidism. this impaired function was aggravated in the first 4 days after surgery, and attenuated from the fifth day. in addition to its effects on serum calcium, pth is also the predominant regulator of serum phosphate. in humans, the change in serum phosphate levels in response to a change in pth concentration is almost immediately detected, whereas the alteration in serum calcium levels may be delayed. in the present study, there was a negative correlation between phosphate levels and circulating pth concentrations after surgery. this correlation was still observed for up to 3 days in patients without postoperative hypoparathyroidism, and for 14 days if extended to the entire study population. this temporal difference in the response of serum calcium and phosphate to circulating pth makes serum phosphate a potentially valuable predictor of hypocalcaemia post - thyroidectomy. l on postoperative day 1 was predictive of persistent hypoparathyroidism, with sensitivity and negative predictive value 100%, but poor specificity of 70.19%. however, more markers should be measured to achieve a model with a high predictive value. indeed, a previous study showed that combining pth levels (15 pg / ml) on postoperative day 2 with calcium levels (1.9 mmol / l) on day 1 resulted in a sensitivity of 96.3%, specificity of 96.1%, positive predictive value of 86% and negative predictive value of 99%. in the present study, even if the sample size was large, the number of patients who developed persistent hypoparathyroidism was small. in addition, the follow - up study suffered from a selection bias : patients feeling well and with good calcium levels mostly dropped out, leaving the uncontrolled patients. in the present study, ionized calcium was not determined separately because this measurement requires sophisticated instrumentation that was not available routinely. finally, the pth threshold < 7ng / l was determined based on clinical observation since the number of patients was too small to perform reliable roc curve analyses. further prospective multicenter studies are necessary to correctly assess the role of pth levels after thyroidectomy. pth levels < 7 ng / l on the first day after surgery might be associated with persistent hypoparathyroidism. | backgroundpostoperative hypocalcemia caused by hypoparathyroidism is one of the most common morbidities of total thyroidectomy. the aim of this study was to analyze the kinetics and factors affecting pth levels after total thyroidectomy and central neck dissection (cnd).material / methodswe performed a retrospective study in 438 consecutive patients who underwent total thyroidectomy between january 2007 and december 2010. no patient had a history of thyroid or neck surgery. pth and calcium levels were recorded 1 day before the operation, during the first 5 days, and during follow - up (2 weeks and 2, 6, and 12 months).resultspth levels declined to 41.90% of its initial value on the first day after the operation. after surgery, pth was correlated positively with calcium and inversely with phosphate levels from postoperative day 1 to 14. based on clinical observation, using a pth threshold of < 7 ng / l on postoperative day 1 was predictive of persistent hypoparathyroidism, with sensitivity and negative predictive value 100%, but poor specificity (70.19%). cnd increased the risk of transient hypoparathyroidism compared with total thyroidectomy alone. patients with thyroiditis had an increased risk of permanent hypoparathyroidism compared with those without thyroiditis. iatrogenic removal of the parathyroid glands increased the risk of permanent hypoparathyroidism compared with those without iatrogenic parathyroidectomy.conclusionspth declined on the first day after thyroidectomy. pth levels < 7 ng / l on the first day after surgery might be associated with persistent hypoparathyroidism. cnd, thyroiditis, and iatrogenic parathyroidectomy increased the risk of hypoparathyroidism. |
the global incidence of diabetes mellitus (dm) among adults (age 18 years and older) was 9% worldwide in 2014, while its prevalence still shows an increasing tendency due to obvious obesity epidemic and aging of the population [24 ]. in hungary, a total of 865 069 patients (9.5% of the population) suffered from dm in the same age group in 2011, and some degree of diabetic retinopathy (dr) could be observed among 19% of the patients with type 1 dm (t1 dm) and 24% in those suffering from type 2 dm (t2 dm) for 3 or 4 years. dr is the fourth most common cause of blindness in the overall population, but it is in second place among active adults in industrialized countries, accounting for a significant drop in quality of life (qof) and working ability of the patients [8, 9 ]. in a study comparing data from 35 populations, the global prevalence of sight - threatening retinopathy (str) was estimated at 10.2% for all dm patients. known risk factors for developing dr are age, gender, duration, and type of dm, elevated hba1c, high blood pressure, and retinopathy stage, while other correlating risk factors are being investigated. unfortunately, 50% of the people with diabetes are unaware of the characteristics of their disease and the compliance in attending screening programs is poor. since high blood sugar and fat destroy the wall of the arteries, it is not surprising that people with diabetes have 2 to 4 times higher cardiovascular mortality rate and 2 to 4 times higher risk of strokes than patients without diabetes. renal failure is also a common complication with the estimated number of 3040% of the patients with diabetes, while 6070% of the patients develop neuropathy. this is not only an individual problem, but a societal problem as well. according to a 2009 survey, the average annual health expenditure for diabetic patients was $ 1205 per capita and for patients with complications this number was $ 2276 per capita. half of this cost is made up of drugs, but only a quarter of the cost spent on drugs is for antidiabetics. similarly, the treating expenses doubled in germany and america, where $ 174 billion was spent on the treatment of diabetes in 2007. the hungarian data cover only the cost of the national health insurance fund, while there are other economic aspects like time off from work or restricted work due to complications of the disease. proper screening for dr is an important milestone towards achieving early and efficient laser photocoagulation and/or anti - vascular endothelial growth factor (anti - vegf) treatment for preventing visual loss. depending on the severity of dr, four stages can be distinguished in general : preretinopathy (r0), background retinopathy (r1), preproliferative retinopathy (r2), and active proliferative retinopathy (r3a). a further subclassification exists for stable proliferative retinopathy (r3s) in patients who have received panretinal laser photocoagulation (prp) under r3a and then became stable ; these cases are considered safe to keep in a surveillance clinic. once fundus lesions appear as a complication of dm, the patient has an apparent dr with either low, intermediate, or high risk for developing some grade of dr. therefore, the focus should rather be on raising prevention programs and early detection, as well as successful treatment of the basic disease. dr is usually asymptomatic before the appearance of any vision loss, but it is detectable by retinal imaging techniques objectively and by accurately taken best corrected va measurements. much research around the world has been focused on the use of telemedicine tools for fundus imaging and screening, the uk system standing up at the top in terms of reliability, precision, and standardized input and output. the results so far have been very promising, with each study being reported to date pointing out the high sensitivity for detecting several fundus lesions in the initial stages of dr by a standard fundus camera and a grading software. the spectra dr software is designed around the requirements of the uk national health service (nhs) national screening program for dr ; it is highly complex and requires a high level of sophistication in the software to meet its requirements. spectra dr enables patient appointments to be created, data entry, image capture, and grading. a generation of patient results is provided together with a report regarding the patients ' screening prediction via a plug - in algorithm. with the use of nonmydriatic or investigational hand - held portable cameras, a quick and simple dr evaluation process will likely improve the patients ' willingness to participate in future screening tests. in 1980, iceland began regular dr screening for t1 dm patients, which resulted in the reduction of disease - related blindness from 2.4% to 0.5%. the icelandic population being used for the cohort study and development of a commonly used risk calculator (risk medical solutions, iceland) is much more homogenous when it comes to ethnic and socioeconomic differences compared to the population in hungary. nevertheless, with these new screening and telemedicine tools, it is realistic to expect similar results to be achieved in other european countries, including hungary, within 5 to 10 years time. the present research explores how dm patients subjectively experience the telemedicine tools and examination through participation in a free fundus camera screening program conducted in a southeastern county (csongrd) in hungary and obtains feedback on whether they would participate in such an examination in the future. furthermore, demographic factors such as age, gender, economic activity, and socioeconomic status (ses) (level of education, support from family, and subjectively perceived financial status) are examined for their effect upon participation in future screening programs. a free screening test was performed on a random population including 178 eyes from 89 patients with confirmed dm diagnosis. handling of the fundus camera and the image acquisitions were performed by a qualified professional in a darkened room, which were then forwarded through a secure internet connection to a specialist doctor / ophthalmologist (a. f./m. c. m.) or rog (g. r./p. another image was taken after ensuring normal intraocular pressure level and applying cyclopentolate (5 mg / ml) eye drops to achieve mydriasis. the assessment of the fundus images was performed within 10 working days using spectra dr software. the recordings were safely deposited and kept inaccessible to third parties for 10 years at a central server, so that later they can be used in further comparative studies on dr. the images were acquired by an 18-megapixel canon eos digital camera which was connected to a canon cr2 color, nonmydriatic, 45 retinal camera. two pictures were taken of the participants ' each eye : one with the macula and another with the optic nerve in the center this is in line with the uk screening requirements. in case of presence of amblyopia or nontransparent media (e.g. cataract and corneal or visual axis obstructing conditions), the patients were excluded from the study. during image evaluation, the graders (a. f./m. c. m./g. k.) classified the signs and the stages of dr and maculopathy in the standardized uk - based software spectra dr and graded the images in alignment with the uk standard grading protocols. each image was evaluated in two stages : first, the rog (g. r./p. r.) evaluated them, and then a supervisor / ophthalmic consultant confirmed the diagnosis (a. f./m. c. m.). at the end, an expert opinion regarding the grade of retinopathy was sent back to the screening site, that is, stage of retinopathy (r0/1/2/3a / s) and absence or existence of maculopathy (m0/1). other discovered abnormalities were not diagnosed in this study, although they were recorded, as they can provide further information about other symptoms which may have occurred in the past, and therefore may require medical attention over a specified period of time. the classification of the dr was as follows : m0 : no maculopathy was detected ; repeated screening was recommended one year later.m1 : there was a sight - threatening maculopathy ; within one month a medical examination is required.r0 : there was no clinical anomaly ; repeated screening was recommended one year later.r1 : mild nonproliferative phase, microaneurisms, dot- or blot - like hemorrhages, or exudates could be seen ; control examination was recommended one year later.r2 : moderate or severe nonproliferative phase, major bleeding(s), cotton - wool spots, venous looping, and intraretinal microvascular abnormalities (irmas) were visible ; control examination was required within one month.r3a : active proliferative phase, neovascularization of the optic disc (nvd) or elsewhere (nve) or preretinal bleeding(s), vitreous bleeding, preretinal fibrosis, and tractional retinal detachment could be observed ; immediate medical examination was required within two weeks.r3s : stable proliferative retinopathy ; a retinal image showing stable post - prp laser with no signs or reactivation or active referable retinopathy ; only to be determined in the presence of benchmark images taken at the time of discharge for comparison ; screening intervals may be at the discretion of the trained rog.other recorded, but not reported, changes / fundus pathology included age - related macular degeneration (amd), glaucoma changes in the optic nerve, and any other signs of eye disease. m1 : there was a sight - threatening maculopathy ; within one month a medical examination is required. r0 : there was no clinical anomaly ; repeated screening was recommended one year later. r1 : mild nonproliferative phase, microaneurisms, dot- or blot - like hemorrhages, or exudates could be seen ; control examination was recommended one year later. r2 : moderate or severe nonproliferative phase, major bleeding(s), cotton - wool spots, venous looping, and intraretinal microvascular abnormalities (irmas) were visible ; control examination was required within one month. r3a : active proliferative phase, neovascularization of the optic disc (nvd) or elsewhere (nve) or preretinal bleeding(s), vitreous bleeding, preretinal fibrosis, and tractional retinal detachment could be observed ; immediate medical examination was required within two weeks. r3s : stable proliferative retinopathy ; a retinal image showing stable post - prp laser with no signs or reactivation or active referable retinopathy ; only to be determined in the presence of benchmark images taken at the time of discharge for comparison ; screening intervals may be at the discretion of the trained rog. the self - completed questionnaire collected information about the individual 's demographic status such as age, gender, economic activity (full - time, part - time, and retired), ses such as education (primary, secondary, and higher), and marital status (married or lives with a partner, single, separated or divorced, and widowed). the general part of the questionnaire was based on the european health interview survey 2009, and it collected data about dr associated exposure parameters and some other health connected parameters, type of dm, or presence of hypertension, as well as the type of eye diseases. furthermore, data were collected about the frequency of measuring blood sugar levels and also about participation in screening programs, which are important for preventing retinopathy, including the frequency of attending diabetology or ophthalmology specialist clinics. questions regarding the perceived reliability of results (yes / no / maybe), willingness to participate again (yes / no / maybe), comfortability (dissatisfied / satisfied / acceptable) of the tests performed, and the overall perception of the screening examinations as well as whether they would participate in a similar examination next time were being asked / collected as well. some categories underwent merging due to missing data, for example, the intensity of blood sugar measurement (monthly / less than a month, weekly / every few days, and daily / more than once a day). if the participants could not understand or read the questionnaire for whatever reason, they received professional help accordingly. the analysis of the data was performed by descriptive statistical analysis on n number of participants, and percent distribution, median, and interquartile range (iqr) are being shown. the chi - square () and fisher exact tests were used to test differences of the distributions of categorical variables. the relationship between two variables was considered statistically significant when p < 0.05. the graphs were made in graphpad prism 5.01 (graphpad software inc., la jolla, ca, usa). the statistical analysis of the data was performed by using stata (intercooled stata 8.0, stata corporation, college station, tx, usa) and excel software (microsoft corporation, usa). the regional and institutional human medical biological research ethics committee of the albert szent - gyrgyi health centre, university of szeged, approved the study protocol (number 197/2015). the research provided anonymity to the participants. before the beginning of a test, the participants signed a written consent form in which they agreed to permit the use of data for research purposes. 178 eyes of 89 people were examined in the study out of which 30 were men (33.7%) and 59 were women (66.3%). table 1 shows the demographic characteristics of the patients, the median age of whom ranged between 56 and 68 years of age and had median hba1c of 7.2% (ranging between 6.4 and 7.9%). table 2 shows the distribution of the types of dm and the stages of dr in the screened population, based upon and compared to the uk grading system and software (spectra dr). twenty percent of the participants had t1 dm out of which 70.8% had t1 dm diagnosed by a diabetology department, the rest being yet undiagnosed or hidden disease patients. mild nonproliferative dr (grade r1) was detected in 23.0% of the participants, while higher (moderate / r2 and proliferative / r3) grade dr was detected in 1.4% and 1.4% of the subjects, respectively ; maculopathy / m1 was present in 5.4% of the studied group (representative images from these were captured from each grade and processed in the spectra software as shown in figure 1). there was an overall left - shift in the distribution of earlier stages of dr in the uk population compared to the one represented by the hungarian graded images and based upon the spectra dr software, probably due to the existence of a well established screening system in the uk and early detection of the disease. according to the self - perceived satisfaction with the classical pupil dilation versus fundus camera examination, 20.4% versus 83.6% of the participants expressed satisfaction, respectively, the classical pupil dilation versus fundus camera examination was found to be definitely reliable by 75.5% versus 72.0%, possibly reliable by 18.4% versus 16.0%, and unreliable by 6.1% versus 12.0%, respectively. the willingness to participate in a classical pupil dilation versus fundus camera examination was found to be positive by 78.2% versus 67.3%, while 9.1% versus 10.9% responded that they would not participate, and 12.7% versus 21.8% responded as maybe doing it. there was no significant difference between the satisfaction from the examination (p = 0.9) and reliability (p = 0.3), although the willingness to participate significantly differed between the classical versus fundus camera examination (p = 0.01) (table 3). the economic activity significantly affected the participation in a blood sugar screening (p = 0.001). sixty percent of those employed in a part - time job had attended blood sugar screening more than once a day or daily, 20% weekly / every few days or monthly / less than a month. the daily / more than once a day attendance was 33.3% among retired, while the weekly / every few days screening was 55.6%, and the monthly / more than a month was 11.1% in this age / patient group. among the full - time workers, the daily / more than once a day and monthly / less than a month screening was 45.5% versus 54.5% (figure 2(a)). similarly, marital status (being married or living with a partner) significantly impacted the likeliness to attend blood sugar screening (p = 0.04) ; this population had a higher daily / more than once a day blood sugar screening attendance, with a frequency of 50% compared to those living alone (single, separated, or divorced : 30.8% ; widowed : 18.2%) ; the latter two populations had otherwise the highest weekly / every few days attendance (single, separated, or divorced : 53.9% ; widowed : 72.7%). the least frequent or monthly / less than a month screening attendance was the highest among married or living with a partner population (28.6%), while it was the smallest among widowed participants (9.1%) (figure 2(b)). the willingness to participate in the annual fundus camera screening was the highest among the full - time workers (91.7%) and the lowest among part - time workers (20.0%) those who reported maybe versus no attendance were higher among part - time workers (40.0% versus 40.0%, resp.), while the willingness to participate differed significantly between the analyzed economical groups (p = 0.003) (figure 3(a)). the satisfaction with the fundus camera screening also increased significantly with the level of education (primary (69.2%) and secondary (82.8%) ; higher (100%), p = 0.003) (figure 3(b)). among participants with secondary or higher education, the most common argument used against the classical fundus exam was i can not see clearly after. the participants with primary school level education had significantly higher rate of stating dissatisfaction of the pupil dilation examination. this reason was not stated among higher educated patients, although the i can not drive after reason seemed to appear more often in this group of patients. the present study aimed to investigate the patients ' experience with the use of telemedicinal tools for screening of dr and the ability to collect the parameters needed to calculate dr risk (age, gender, type and duration of dm, hba1c, hypertension, and fundus image grading) in a southeastern county (csongrd) in hungary. the justification for using health care tools aimed at screening dr is high, due to the great availability of tools for dr prevention and avoidance of late complications such as str. the population of csongrd county is very plausible for initiating such a study, since it has a known higher prevalence of dm compared to other counties in hungary. in addition, the study followed the progressive trend of dm worldwide and examined the willingness to participate in screening tests, the attitude towards screening examination, and the influence of demographics and socioeconomic factors like education, financial, and marital status. regarding the risk factors, the ses has been already shown to have a very significant impact on the attendance in screening examinations, while occupation has been related to a greater impact on nonattendance in screenings. the screening frequency for blood sugar levels in full - time workers was indeed significantly lower in our study, but the willingness to participate in fundus screening examination was higher in that subpopulation. from the standpoint of dr formation and progression, it is 76.4% of the patients who had high blood pressure which, by itself or as a codisease, gives poorer prognosis for the dm patients due to a predisposition for premature vascular sclerosis. the occurrence of dr in the studied sample population was 25.5%, which is higher than any previous results in hungary, although somewhat expected in csongrd county. although the diabetology guidelines recommend blood glucose levels to be checked several times a day, only a little over a quarter of the participants performed it accordingly. strikingly, 60% of the study participants performed blood glucose testing every few days, if not more rarely. upon diagnosis with dm, the diabetologist or the general practitioner informed the patient of the possible complications from the disease and recommended an annual eye screening test. our results coincide with the international diabetes federation 's (idf 's) observation that 50% of the people with dm are not aware of the characteristics of their disease. in hungary, the number of known patients with diabetes makes nearly 10% of the total population. it would take 100 ophthalmologists (from the total of 968 practicing) working full - time if they want to carry out only the annual screenings by using traditional tools on such a sized population. a new screening program always faces challenges, but previous studies from other countries show promising results. diabetes causing vision loss is successfully confined in countries like iceland, where regular screening was implemented. in our study, only a third of the participants had not visited an ophthalmologist, while 12.4% of them have been diagnosed with dm within a year ; only 56.2% of the participants complied with the one - year recommendation. in the uk, patients compliance in attending traditional screening was 45% and 50% in fundus camera screening in the first year. after using a mobile fundus camera screening unit to reach more patients, the compliance elevated to 80% in the fifth year. compliance is a highly influential factor of cost effectiveness because of the fixed costs (digital imaging camera, computer system, etc.). the response to the subjective experiences perceived during fundus examination did produce satisfactory results : more than three - quarters of the participants were satisfied with the fundus camera examination and one out of five with the traditional method. in both cases, three - quarters of the participants considered the results of the study to be reliable, a significant difference being found between the two screening procedures. there were fewer problems than expected (e.g., subjects being not able to drive after pupil dilation), but it can be a factor which is most likely related to older age of the sampled population. it is interesting to note, however, that during the procedure of pupil dilation, one quarter of the subjects found administering eye drops being irritating or uncomfortable, in particular, those who had lower education. there is a level of contradiction in the assessment of reliability and satisfaction in the study, since significantly more people were willing to participate in the traditional retinal screening method than in the fundus camera test (78.2% versus 67.3%). 83.6% of the participants were dissatisfied with the examination, which raises the suspicion they could have chosen other for their response to having no other comments, and this could have been done out of necessity. among the inconvenience experienced during the test with pupil dilation, the other category was chosen by only 4.1% in which no mention of any reasons for the selection made was stated whatsoever. during the analysis, the economic activity and education appeared to pose an effect on the individual 's willingness to participate in the screening test. the fundus camera test was preferred mostly by the full - time employees, with whom it was presumably important to see well after the test in order to be able to continue their work during the same day. based on the level of education, the few subjects that evaluated the fundus camera test as satisfactory were those who found eye drops to be the most uncomfortable in the traditional test. these data are somewhat contradictory, as mydriatic drops are always required in traditional testing. people with higher education found only the driving restriction and the bad sight after the examination as a negative aspect of the screening ; in this context, they were 100% satisfied with the fundus camera test. the telemedicine part of the study also concerns data safety and patient anonymity preservation which are now guided by an eu law contained in the charter of fundamental rights of the european union, article 8 (2000/c 364/01), as well as the need to safely store and make backup files for high resolution fundus images acquired from the patients and their retention ; these rules were followed in the present study entirely. the issue of having decentralized and near the patient dr screening and fundus imaging services and centralized image reading remains to be evidenced in future telemedicinal studies for screening dr in hungary, the uk grading system being the golden standard for achieving the task properly. in conclusion, the analyzed demographic and socioeconomic factors showed a significant relationship with the future participation in the fundus camera screening for dr. the participants ' age or gender appears not to affect the experience (satisfaction) of the examination (e.g., fundus examination under pupil dilation). however, the level of education appears to have an important role : higher educated patients were more likely to participate in pupil dilation examination using an ophthalmoscope. this is in contradiction to the fact that only slightly more than half of the participants in this group took part in such screening examination within a period of one year. it was also not confirmed that the distribution of dr grades in this study is similar to the results of previous national studies [17, 22 ], as csongrd county is not a representative population comparable to other parts of hungary where the prevalence of dm and dr is lower. further research is therefore needed on a larger or more representative sample from different counties in hungary where the percent of distribution of patients diagnosed with dm varies. in general, the treatment of dm patients is an interdisciplinary task of primary care physicians, diabetologists / dietologists, ophthalmologists / optometrists, and public health specialists. these professionals are responsible for giving lifestyle advice and for directing patients towards more appropriate screening tests. ophthalmic monitoring is required every year after the diagnosis of diabetes and every other year for patients with excellent glycemic control without retinopathy at the previous examination but annually if there are risk factors. furthermore, if retinopathy is manifested to some degree, the screening time should be reduced to half a year (in the case of nonproliferative retinopathy) and three months (for preproliferative retinopathy). in case of proliferative retinopathy patients should go immediately to an ophthalmologist, in order to initiate laser treatment in time and thus save the eye from str. the present state, unfortunately, seems to involve lack of realistic assessment or judgment of the risk from complications by the patients, and therefore a neglect to participate in the recommended screening tests. fast, easy, and accurate fundus camera examination is an alternative to the traditional, time - consuming, and the patients, who tried this method, agreed that this new way is more satisfaction than the one they got used to, while they appreciated its reliability in the same way. indeed, in the uk, due to the systematic screening implemented, dr is no longer the leading course of blindness in the working age population. | introduction. diabetic retinopathy (dr) is a sight - threatening complication of diabetes. telemedicine tools can prevent blindness. we aimed to investigate the patients ' satisfaction when using such tools (fundus camera examination) and the effect of demographic and socioeconomic factors on participation in screening. methods. pilot study involving fundus camera screening and self - administered questionnaire on participants ' experience during fundus examination (comfort, reliability, and future interest in participation), as well as demographic and socioeconomic factors was performed on 89 patients with known diabetes in csongrd county, a southeastern region of hungary. results. thirty percent of the patients had never participated in any ophthalmological screening, while 25.7% had dr of some grade based upon a standard fundus camera examination and uk - based dr grading protocol (spectra software). large majority of the patients were satisfied with the screening and found it reliable and acceptable to undertake examination under pupil dilation ; 67.3% were willing to undergo nonmydriatic fundus camera examination again. there was a statistically significant relationship between economic activity, education and marital status, and future interest in participation. discussion. participants found digital retinal screening to be reliable and satisfactory. telemedicine can be a strong tool, supporting eye care professionals and allowing for faster and more comfortable dr screening. |
mycoplasma pneumoniae (m. pneumoniae) is the only recognized human pathogen that is the main cause of respiratory infections in school - aged children and young adults. m. pneumoniae infections occur worldwide and throughout the year ; the infection is endemic in large communities, with epidemic outbreaks occurring every 4 - 7 years. in small communities, the infections are sporadic, with long - lasting and smoldering outbreaks occurring at irregular intervals.1) community outbreaks largely spread through contact in schools ; however, up to 40% of household members of the infected student also develop mycoplasma infection.2) m. pneumoniae is a common pathogen that causes upper and lower respiratory tract infections, manifesting as pharyngitis, bronchitis, and atypical pneumonia in children and adolescents.3) patients with or without respiratory symptoms can manifest illness involving the skin, central nervous system (cns), blood, heart, gastrointestinal tract, and joints. skin lesions include a variety of exanthemas, most notably maculopapular rashes, erythema multiforme, and stevens - johnson syndrome.4) cardiac involvement, such as acute myocarditis, pericarditis, or myopericarditis, is a rare manifestation of m. pneumoniae infection.5) in 1979, pnk6) reported that among 560 patients with serologically confirmed m. pneumoniae infection, 25 (4.5%) had carditis (19 perimyocarditis and 6 pericarditis). in contrast, in korea, only 4 cases of m. pneumoniae - associated pericarditis have been reported in pediatric patients.7 - 10) clarithromycin and azithromycin are among the drugs of choice for the treatment of respiratory tract infections. it is important to investigate the association between antibiotic use and resistance as the resistance of common respiratory pathogens to macrolides has been increasing.4) in this report, we present a case of severe acute myopericarditis with pneumonia caused by m. pneumoniae in a healthy 6-year - old girl. a previously healthy 6-year - old girl was transferred from a primary clinic due to fever, cough, vomiting and lethargy. her past medical history was unremarkable. on the third hospital day, the patient developed lethargy and the fever increased up to 39. there was no chest pain, but oliguria and dyspnea were presented. blood test results showed hemoglobin levels at 12.5 g / dl ; white blood cell count was 6700/l ; platelets count was 295000/l ; aspartate aminotransferase was 932 iu / l ; alanine transaminase was 449 iu / l ; creatinine kinase (ck) was 17122 iu / l ; ck - mb was 307 ng / ml ; pro - brain natriuretic peptide (pro - bnp) was 16338 pg / ml ; lactate dehydrogenase levels were 2857 iu / l ; erythrocyte sedimentation rate was 15 mm / h ; c - reactive protein was 16 chest radiograph showed signs of bronchopneumonia, mild parapneumonic effusion, and prominent cardiomegaly (fig. echocardiogram showed mild pericardial effusion, prominently decreased left ventricle contractility, enlarged left atrium and ventricle, along with severe mitral regurgitation (fig. in particular, the left ventricular internal dimension increased 4.41 cm, while the ejection fraction decreased 44.89% (fig. previously, the patient was administered azithromycin 10 mg / kg / day, a novel macrolide, for five days while in the private pediatric clinic ; however, there was no response and the symptoms were aggravated. therefore, we administered clarithromycin 15 mg / kg / day on the second hospital day, with intravenous ceftriaxone (60 mg / kg / day), and treated the patient with intravenous immunoglobulin (500 mg / kg / day for 5 days) and diuretics, inotropics such as furosemide, spironolactone 2 mg / kg / day on the second hospital day, and dopamine 5 mg / kg / min. on the tenth hospital day, m. pneumoniae igm ab increased to 464.2 u / ml in a follow - up laboratory test, and the results of the polymerase chain reaction (pcr) test of the m. pneumoniae - containing sputum were positive. thereafter, her symptoms gradually improved and appetite was gradually restored. on the thirteenth hospital day, cardiomegaly signs improved on chest radiograph (fig. 2c), left ventricle and left atrial dilatation was improved, and left ventricular contractility was normalized (fig., the patient was discharged without sequela. on follow - up at the outpatient pediatric department, ck - mb and pro - bnp had improved to 25 ng / ml and 107 pg / ml, respectively (table 1). the myopericarditis is primarily pericarditic syndrome with minor myocardial involvement, which encompasses the majority of combined pericarditis and myocarditis cases encountered in clinical practice.8) cardiac complications associated with m. pneumoniae infection are relatively uncommon. the incidence of cardiac involvement ranged from 1% to 8.5% in persons with serological evidence of infection, and it is more common in adults than in children.9) a smaller study with 20 pediatric patients reported eight with purulent pericarditis, six with collagen vascular disease, presumed viral or idiopathic in four, and two with neoplastic mediastinal masses.10) acute pericarditis is often accompanied by some degree of myocarditis. in clinical practice, both pericarditis and myocarditis co - exist because they share common etiologic agents, which are primarily cardiotropic viruses. well known causes of myopericarditis are viruses including enterovirus, epstein - barr virus, cytomegalovirus, human herpes virus-6, adenovirus, influenza a and b, parvovirus b19, hepatitis b and c, human immunodeficiency virus, and varicella.11) the causative organism, eventually discovered to be m. pneumoniae, was first isolated in tissue culture from the sputum of a patient with primary atypical pneumonia by eaton in 1944, and thereafter it was known as the eaton agent. m. pneumoniae infection is common in children and adolescents, and is often accompanied by extrapulmonary complications. the extrapulmonary complications involving all the main organ systems can occur in association with m. pneumoniae infection as a result of direct invasion and/or autoimmune response.12) extrapulmonary complications of m. pneumoniae, such as cns manifestations and arthritis, appear to occur more frequently in children.2) however, the mean age of the patients with m. pneumoniae - associated carditis appears to be relatively greater. therefore, m. pneumoniae - associated carditis is rare and more commonly described in adults than in children.5)6) hawkins.12) reported that constrictive pericarditis secondary to infection with m. pneumoniae. the following hypotheses have been proposed for m. pneumoniae - associated carditis : direct invasion of the myocardium by the organism via either the lymphatic or circulatory systems or from the lower respiratory tract by contamination, autoimmune mechanism, or increased tendency for intravascular coagulation.5)10) in general, only 7 - 11% of the infected patients develop pneumonia, while 5 - 20% may develop pleural effusions.13) however, paz and potasman5) found that 43% of patients with carditis had pneumonia and 19% had pleural effusions. these rather high values raise the question of whether the more severe respiratory cases are indeed associated with the development of carditis. tachycardia was found in only 43% and chest pain in 38% of the patients studied. contrary to the abovementioned findings, electrocardiographic (ecg) abnormalities were found in 100% of the reported ecgs. typical ecg findings of pericarditis were found in 75% of patients. a large study of adult patients reported effusive - constrictive pericarditis in 15 of 190 patients who underwent pericardiocentesis and cardiac catheterization.14) the best method for identifying pathogenic agents associated with myopericarditis is to obtain tissue directly from the involved area or a pericardial effusion for use in microbiologic cultures.5)11) however, there are significant risks associated with obtaining a sample of tissue from the pericardium. additional methods for diagnosing m. pneumoniae infection have been developed, including serological methods such as the compliment fixation test, cold agglutination test, particle agglutination test, immunofluorescence antibodies, enzyme immunoassay (eia) and enzyme - linked immunosorbent assay, as well as pcr - based methods.1) according to kim.15) a 4-fold or more increase in the igg antibody titer, or a single titer 1 : 640 confirms the diagnosis of m. pneumoniae infection. however, when the pcr with southern hybridization result was combined with the igm - capture test result with the acute - phase sera, the sensitivity of rapid laboratory diagnosis increased to 95%. so, waris.16) explained that m. pneumoniae igm serology test is the single most valuable tool for the diagnosis of m. pneumoniae infection in children of any age. at our hospital, the eia test is performed when m. pneumoniae infection is suspected ; if the result of this test showed > 70 u / ml igm titer and/or pcr (+) for specific deoxyribonucleic acid in nasopharyngeal aspirates, the diagnosis of m. pneumoniae infection is confirmed. diagnosis may be diagnosed through the visualization of echogenic intrapericardial fibrinous strands during echocardiography.17) echocardiogram is also helpful in documenting the physiologic consequences of constrictive pericarditis, including decreased early diastolic mitral valve inflow velocity with a short deceleration time and reciprocal respiratory variation of left and right ventricular inflow.18) clarithromycin (6-o - methylerythromycin) is synthesized by substituting a methoxy group of erythromycin with c-6 hydroxyl group, which produces an acid - stable antimicrobial compound that prevents the degradation of the erythromycin base to the hemiketal intermediate. the increased acid stability of clarithromycin results in improved oral bioavailability and reduced gastrointestinal intolerance. azithromycin (9-deoxo-9a - aza-9a - methyl-9a - homoerythromycin) is formed by inserting a methyl - substituted nitrogen in place of the carbonyl group at the 9a position of the aglycone ring. the resulting dibasic 15-membered ring macrolide derivative is more appropriately referred to as an " azalide ". this structural change increases the acid stability of the compound, significantly increases its serum half - life and tissue penetration, to ultimately result in increased activity against gram - negative organisms and decreased activity against some gram - positive organisms when compared with erythromycin.19) both azithromycin and clarithromycin achieve high extracellular concentrations in respiratory tissue and are commonly used as a first - line empirical treatment for community - acquired respiratory tract infections. however, they differ substantially in their plasma half - life and tissue persistence, which are both very important factors in the selection of macrolide - resistant organisms.13)19) for instance, the time required for the clearance of a drug or reduction in its concentration to below the minimum inhibitory concentration is between 5 and 7 half - lives. therefore, since the plasma half - life of azithromycin is 68 hours, it may persist in vivo for at least 3 - 4 weeks after treatment. in contrast, clarithromycin has a half - life of only 5 - 7 hours, and therefore it exerts little post - treatment effect. this difference explains why azithromycin use is selected for significantly more macrolide - resistant streptococci until about 4 weeks than compared with clarithromycin.20) in conclusion, we report the case of child who was successfully treated with oral clarithromycin, intravenous ceftriaxone, intravenous immunoglobulin and other cardiac supportive medications after confirming m. pneumoniae infection - associated acute myopericarditis with symptoms of upper respiratory infection. m. pneumoniae should be considered in the differential diagnosis of pathogens in acute myopericarditis patients. it is important to administer appropriate treatment to achieve a good outcome for mycoplasma - associated carditis, and furthermore, serological testing for mycoplasma species should be included in the routine workup of carditis of unknown cause. | mycoplasma pneumoniae (m. pneumoniae) primarily causes respiratory tract infections in persons aged 5 - 20 years. tracheobronchitis and bronchopneumonia are the most commonly recognized clinical symptoms associated with m. pneumoniae infection. complications of this infection are unusual ; in particular, cardiac involvement is very rare and is generally accompanied by pneumonia. nonrespiratory illness can therefore involve direct invasion by m. pneumoniae or autoimmune mechanisms, as suggested by the frequency of cross reaction between human antigens and m. pneumoniae. herein, we report a case of severe acute myopericarditis with pneumonia caused by m. pneumoniae in a healthy young child who presented with fever, lethargy, oliguria and dyspnea. she survived with aggressive therapy including clarithromycin, intravenous immunoglobulin, inotropics, and diuretics. the patient was discharged on the 19th day after admission and followed up 1 month thereafter at the outpatient clinic without sequelae. |
clinically, frontal lobe syndromes, frontal network syndromes, frontal systems syndromes, executive dysfunction, and metacognition have all been used to describe disorders of frontal lobes and their extended networks although they are not all synonymous. however, because the frontal lobes network with every other part of the brain, strictly speaking, frontal network syndromes constitute the most accurate neurobiological depiction. the term, frontal network syndromes (fns,) emphasizes the universal connectivity of the frontal lobes with all other brain regions. for example, the stroke literature is replete with fns that have been reported with discreet lesions outside the anatomical boundary of the frontal lobe, such as subcortical grey matter, subcortical white matter, with isolated lesions of the brainstem, cerebellum, temporal, and parietal lobes [18 ]. for the purposes of simplification, five primary syndromes and numerous secondary syndromes may be delineated. impairment in working memory, executive function, abulia, disinhibition, and emotional dyscontrol may be regarded as the elementary deficits of fns. in addition, a number of secondary manifestations may be identified such as a wide array of behavioral abnormalities such as loss of social norms, imitation behavior, compulsions, and obsessions [9, 10 ] (figure 1). to begin to understand the most complex object in the universe, the human brain and in particular the frontal lobes, it is most illuminating to study the evolution of our mind and thereby gain a better understanding of the clinical syndromes we are faced with today. in the words of theodosius dobzhansky, nothing in biology makes sense except in the light of evolution. life on earth evolved approximately 3.7 billion years ago and thereafter continuously shaped by extra - terrestrial and geological events, punctuated by a number of key events. the inclusion of prokaryotes into eukaryotic cells furnished cells with a powerhouse, the mitochondria. some time after snowball earth, when glaciers reached the equatorial regions about 620590 million years ago (mya), with the cambrian explosion of organism diversity, vertebrates (bony fish, amphibians, reptiles, birds, and mammals) formed (~520 mya). formation of the vertebrate skeleton allowed rapid movement, an advanced nervous system, and high degree of encephalization even though 98% of animal species are invertebrates versus 2% being vertebrate. myelination enabled a vastly improved neural transmission, speeding up neural transmission by a factor of 10 (~9 meters per second in unmyelinated fiber versus 50100 meters per second in myelinated fiber), with increased temporal precision, faster communication between the brain and body parts, and ability to react more rapidly to prey and predator [13, 14 ]. with warming conditions, fish evolved lungs and walked on land about 365 mya, mammalian evolution (~200 mya) and subsequent proliferation after dinosaur extinction (~65 mya). east side story event (african rift valley formation leading to a hot and dry east africa) precipitated bipedalism, increase in brain size, and tool making. the emergence of dopamine as a key neurotransmitter was critical in cooling our bodies and brains in a thermally stressed environment and later exapted for executive function. around this time, our frugivorous diet (since ~60 mya) was supplemented with meat and with the advent of marine isotope stage 6 (180120 mya) that may have also served as an important event that highlighted the key dietary changes to sea - food that may have played a factor in advancing our cerebral connectivity that ultimately made us modern humans [16, 17 ]. shell fish (scallops oysters and prawns) are rich in both iodine and essential fatty acids, both of which have been correlated with boosting dopamine activity and intellectual development. as a starting point using the so - called missing link hominid, australopithecus africanus (brain volume approximately 450 ml), there was an increase in size to approximately 1500 ml in neanderthals over a 3 million year period and subsequently a slight decrease again in modern humans homo sapiens sapiens to 1350 ml. during this time, there was a reduction in the size of the visual striate area (ba 17) with a relative increase in the posterior parietal cortices and frontal lobe reorganization at the network, neurotransmitter, and receptor levels. the size of frontal lobes in various mammalian species is frequently cited as steadily progressing allometrically from the so - called lower forms (rats and mice) to dogs and cats with primates and humans having the biggest proportionally. the frontal lobes comprise of 37%39% of the cerebral cortex macroscopically and connect to all other parts of the brain, often in a reciprocal manner. the frontal lobe in humans is as large as that would be calculated for an ape of human brain size overall, not larger as is often reported. however, what sets us apart from other mammals is not so much brain size but reorganization of our brains in terms of connectivity and neurotransmitter changes. these changes may be summarized in the following manner.progressive increase in size.hemispheric asymmetry, also called cerebral torque (right frontal and left occipital petalias).neuropil reorganization.reorganization in terms of neurotransmitter systems.receptor modification. hemispheric asymmetry, also called cerebral torque (right frontal and left occipital petalias). axons, dendrites, and space between the neurons and glial cells constitute the neuropil which is decreased to ba 10 in humans relative to other primates. in broca 's area (ba 44, 45), the cortical architectural units or minicolumns are wider in humans relative to primates. ba 10 is twice as large in terms of overall brain volume compared to any of the other great apes (1.2% in humans versus 0.46%0.74% in great apes). spindle cells or von economo cells appear in layer vb in both the fronto - insular cortex as well as the anterior cingulate cortex, only in humans and african (not asian) great apes and are approximately 30% more numerous in the right hemisphere of these species. as they arose in our last common ancestor about 10 mya, they probably subserve the role of social and emotional processing which arose many millions of years before language. in view of this as an example of convergent evolution, other intelligent species such as the cetaceans have spindle cells. the neuronal density of the important ba 10 and 13 in humans is about half (human ba 10~32000 and ba 13~30000 neurons per cubic millimeters) that of the great apes (chimpanzee ba 10~60000 and ba 13~43000) and is often much less than half (orangutan ba 10~78000 neurons per cubic millimeter). the increase in neuropil particularly of ba 10 is likely related to the connectivity of this region with other tertiary association cortex and the other hemisphere. surprisingly, this cortical region is larger in size in humans than would be predicted for an ape of human brain size. there is a relative increase in the size of white matter, and the ratio of gyral - to - core white matter in the temporal lobes is larger than would be predicated for other hominoids. this relatively enlarged gyral white matter compared to core white matter is interpreted as reflecting greatly increased interconnectivity subserved by short association fibers. of the component amygdaloid nuclei (lateral, basal, and accessory nuclei), the lateral nucleus is relatively larger in humans than would be expected in an ape of human brain size. this has been attributed to the increased interconnectivity with the temporal lobe 's unimodal and polymodal sensory information. in human evolution, there has been a differential expansion and reorganization not only of the temporal lobes and amygdaloid complex, but also of the inferior parietal lobes. this implies that evolution may have acted on neural systems rather than discrete anatomical structures. areas that are critical to social behavior which include the amygdaloid nuclei and limbic component of the frontal cortex are both volumetrically larger and revealed reorganization in their networks. this is in direct contrast to the traditional view that limbic structures are conserved whereas the frontal lobes had enlarged. within the frontal lobes themselves, however, many organizational and network changes have of course taken place. structures and networks that are implicated in social and emotional processing include the orbitofrontal cortex, the amygdala, fronto - insular cortex, and temporal polar cortex with the latter also important in language processing. these represent the so - called mosaic reorganization that has been a feature of human evolution. the quick acting excitatory (glutamate) and inhibitory (gaba) neurotransmitters (nt) act via ion channels with charged ions enabling a relatively quick response in terms of microseconds and seconds. the neuromodulators (nm) such as serotonin (5-ht), dopamine (da), norepinephrine (ne), acetylcholine (ach), and histamine (h) act differently in that they promulgate longer lasting and more diffuse actions via the g - protein cascade system. the ultimate outcome on a network may be excitatory, inhibitory activation of their own presynaptic autoreceptors or interaction synergistically with the other nm systems. different nt 's subserve different higher cortical functions (hcf), and in neurodegenerative disease nt deficits occur in varying combinations. the downside of nt modulatory systems for intellectual advances probably made humans more susceptible to a number of neurodegenerative diseases, unique to humans. within the frontal subcortical circuits, the reasons for this have been proposed as part of a very plausible and well - researched hypothesis by fred previc and their open loop systems ; da and ach became the predominant nt 's in the left hemisphere and ne and 5ht in the right hemisphere. da is considered to have been one of the key factors in the emergence of human intelligence. east side story with east african becoming relatively dry and arid, heat management and combating the deleterious effects of hyperthermia (including so called heat stroke) were a critical factor in survival of the mammals and the newly emerged bipedalist, australopithecus africanus. the function of da in lowering body temperature presumably enabled early hominoids to better tolerate the hyperthermia of chase hunting and catching prey that succumbed to chase myopathy [52, 53 ]. thereafter in an evolutionary sense, da expansion occurred, due to increased calcium metabolism from prolonged aerobic activity as well as the increased tyrosine (a dopamine precursor) consequent to increasing meat supplementation about 2 mya. the clinical sequelae of blocking dopamine (by drugs such as haloperidol, risperidone, and quetiapine) as malignant hyperthermia and neuroleptic malignant syndromes may be therefore interpreted in an evolutionary perspective. da became exapted as the most important nt in our evolving brains, eventually concerned with most of the core frontal functions working memory, cognitive flexibility, motor planning, abstract representation, temporal sequencing, and generativity. da exerts a modulatory effect (affects signal - to - noise ratio) on the pfc g - protein linked receptors on dendritic shafts and spines of glutaminergic pyramidal neurons and dendrites of gaba - ergic neurons. humans and great apes feature da input to all cortical areas, in contradistinction to the paucity of da - ergic innervation of rodents. this was determined by measuring cortical da innervation (axon density) using tyrosine hydroxylase immune reactivity. in addition, there is a regional da - ergic distribution most intense in layer i and v - vi of the association cortices. furthermore, compared to great apes, humans have a generalized increased da - ergic input to the prefrontal cortical regions. the dopaminergic hypothesis in human evolution purports that the expansion of human da - ergic in particular was the most important factor in human tool making, exploration, cultural, and scientific developments. this theory also proposes that the drawbacks are the propensity for hyperdopaminergic syndromes such as schizophrenia, bipolar disease, autism, attention deficit hyperactivity disorders, and neurodegenerative diseases. in humans and great apes, compared to other mammals, there is an overall increase in the cortical output 5-ht efferents. the 14 different serotonergic g - protein - related receptors and one ion channel receptor (5-ht 3) enable the modulation of several different functions simultaneously, including memory, learning, and inhibition. this occurs via receptors on pyramidal cells and dendritic shafts and via interneurons which allows signal modulation from local circuits with reference to extrinsic stimuli. from a clinical point of view, serotonin in the ofc circuitry has been linked to self - control, emotional, processing, and inhibition regulation. in the neuromodulatory axons of humans and great apes exist varicose type axons that are likely to have a role cortical plasticity, associated with advanced traits such as superior learning capability, social learning, advanced tool manufacture, and self - awareness. these effects are mediated via 5 muscarinic receptors (m1m5), all of which are g - protein linked. nicotinic receptors are all ligand gated ion channels. both transmit mediating excitatory and inhibitory effects on gaba interneurons and pyramidal cells. hominoids are able to imitate behavior, and imitation ability of primates and hominoids was crucial to the cultural evolution. the imitation ability may be termed an all purpose learning mechanism. mosaic in that it involves the pfc, parietal, temporal, and cerebellar regions. the core features of memory and attention as well as more specific cognitive domains such as language and tool use are presumed to be based on the mosaic pattern, itself based on the imitation behavior circuitry that may have a visual, auditory, and tactile dimension. the fast acting excitatory (glutaminergic) and inhibitory (gaba) amino acid neurotransmitters are modulated by a number of widely projecting slower acting (most g - protein linked) neurotransmitters. this type of chemical architecture is useful in coordinating many neurons and neuronal circuits in response to a stimulus or threat. there are currently 8 chemical or neuromodulatory tracts (da, 5-ht, ne, ach, h, oxytocin / vasopressin, and orexin) (figure 3) with their nuclei of origins in the brainstem, basal forebrain or hypothalamus, and extensive cortical ramifications, two examples of which are depicted in figures 3 and 4 [1, 2 ]. the main functions of the frontal lobes are motor action and the temporal integration of behavior. frontal lobe evolution may be seen as progressive refinement of pyramidal pathway responses (motor, speech, and behavior) by incorporating cognitive and emotional processes. optimal decision - making requires a flexible system that can incorporate a wide range sensory inputs, at the same time prioritizing and choosing the most effective response in a changing environment. this is achieved through the major cerebral network systems : superior frontal occipital fasciculus, superior longitudinal fasciculus, inferior longitudinal fasciculus, cingulum and uncinate fasciculus, and seven frontal subcortical circuits [63, 64 ]. the major fasciculi are depicted in figure 5 and can also be imaged by diffusion tensor imaging with directional specificity that is color - coded.u fibers (orange).superior occipito - frontal fasciculus (royal blue).superior longitudinal fasciculus (pink).inferior longitudinal fasciculus (dark green).perpendicular fasciculus (dark blue).uncinate fasciculus (purple).arcuate fasciculus (light blue)note two tracts.corpus callosum (brown).cingulum (red).fornix (light green). namely, cortex - caudate - globus pallidus - thalamus cortex (figures 6 and 7). confusion of terminology may exist because of the rather cumbersome terminology related to subcortical structures, best delineated by a diagrammatic representation (figure 8). the neurotransmitters and neuropeptides integrated in this circuitry are mainly excitatory (glutamate) and inhibitory (gaba) being the principal ones but many others involved in a neuromodulatory capacity including the monoamines, enkephalin, neurotensin, substance - p, dynorphin, adenosine, and neuropeptide - y [9, 6567 ]. direct and indirect pathways.connections with the other circuits (corticocortical).connections to areas outside the fsc 's. direct and indirect pathways. glutamate release occurs from frontal cortical regions to the striatum, mostly caudate nucleus, less often to putamen and nucleus accumbens. this releases gaba at internal segment of gp and sn ; gaba from gpi (globus pallidus interna) to thalamus diminishes and in turn the thalamus increases glutaminergic excitation of cortical regions. the striatal neurons within this pathway that project to the gpi are termed striosomes with d1 receptors. the net result is a thalamic disinhibition [9, 65, 66].the indirect pathway balances the direct pathway. here the striatal efferents are termed matrix efferents wirt d2 receptors and project from the globus pallidus externa (as opposed to interna) with a net thalamic inhibition. some of the gpe neurons are cholinergic but most are gaba - ergic with two different types of gaba ergic cell types within the gpe termed gp - ti and gp - ta, which mediate crosstalk between the afferent and efferent circuits of the gpe as well as the direct and indirect pathways.connections to regions outside the fsc 's include (i) dlpfc (ba 46) to parietal (ba 7), (ii) anterior cingulate to temporal lobe (hippocampus, amygdala, entorhinal cortex), (iii) medial ofc to temporal lobe and iv) lateral ofc to heteromodal sensory cortex [9, 69 ] direct pathway. glutamate release occurs from frontal cortical regions to the striatum, mostly caudate nucleus, less often to putamen and nucleus accumbens. this releases gaba at internal segment of gp and sn ; gaba from gpi (globus pallidus interna) to thalamus diminishes and in turn the thalamus increases glutaminergic excitation of cortical regions. the striatal neurons within this pathway that project to the gpi are termed striosomes with d1 receptors. the striatal efferents are termed matrix efferents wirt d2 receptors and project from the globus pallidus externa (as opposed to interna) with a net thalamic inhibition. some of the gpe neurons are cholinergic but most are gaba - ergic with two different types of gaba ergic cell types within the gpe termed gp - ti and gp - ta, which mediate crosstalk between the afferent and efferent circuits of the gpe as well as the direct and indirect pathways. connections to regions outside the fsc 's include (i) dlpfc (ba 46) to parietal (ba 7), (ii) anterior cingulate to temporal lobe (hippocampus, amygdala, entorhinal cortex), (iii) medial ofc to temporal lobe and iv) lateral ofc to heteromodal sensory cortex [9, 69 ] there is a complex interplay of the neurotransmitters, receptors, and circuit plasticity such as enhancement of striatal dopamine release by cholinergic agonists. the importance of recognizing and understanding this circuitry is in the relationship they have to clinical syndromes clinicians appreciate with regard to frontal pathology. these may be conveniently conceived of as three principal syndromes of the following.predominant dorsolateral prefrontal cortex (dlpfc) : temporal organization of information, executive function, working memory, and multitasking. impairments here lead to akinetic mutism and the abulic spectrum of disorders.orbitofrontal (ofc) circuitry : the cortical component of the limbic system where the emotional and other limbic components are integrated into behavioral output. hence, disinhibitory syndromes may occur in response to lesions of the medial as well as the lateral ofc. personality change in the context of frequently normal cognitive (dlpfc) function is usual and a manifold of presentations is encountered principally. the various forms of echopraxia or imitation behavior, utilization behavior, and environmental dependency syndromes (field - dependent behaviors) are sometimes the overriding clinical manifestations of these lesions. from a neurophysiological point of view, the medial ofc attaches emotional valence to events in turn determines the strength of the episodic memory. impaired autonomic and endocrine processing is associated with lesions of the medial ofc.lateral ofc lesions have been correlated with ocd, depression, irritability, mood disorders, and field - dependent behaviors. obsessive compulsive disorders spectrum group of disorders include ocd spectrum itself that comprises of obsessions (intrusive urges thoughts, images), compulsions (repetitive, ritualistic type of activities of a physical or cognitive nature), tourette 's syndrome, kleptomania, risk seeking behavior, pathological gambling, body dysmorphic disorder, and the immune disorder of pediatric autoimmune neuropsychiatric disorders (pandas) [73, 74 ]. predominant dorsolateral prefrontal cortex (dlpfc) : temporal organization of information, executive function, working memory, and multitasking. orbitofrontal (ofc) circuitry : the cortical component of the limbic system where the emotional and other limbic components are integrated into behavioral output. hence, disinhibitory syndromes may occur in response to lesions of the medial as well as the lateral ofc. personality change in the context of frequently normal cognitive (dlpfc) function is usual and a manifold of presentations is encountered principally. the various forms of echopraxia or imitation behavior, utilization behavior, and environmental dependency syndromes (field - dependent behaviors) are sometimes the overriding clinical manifestations of these lesions. from a neurophysiological point of view, the medial ofc attaches emotional valence to events in turn determines the strength of the episodic memory. impaired autonomic and endocrine processing is associated with lesions of the medial ofc.lateral ofc lesions have been correlated with ocd, depression, irritability, mood disorders, and field - dependent behaviors. obsessive compulsive disorders spectrum group of disorders include ocd spectrum itself that comprises of obsessions (intrusive urges thoughts, images), compulsions (repetitive, ritualistic type of activities of a physical or cognitive nature), tourette 's syndrome, kleptomania, risk seeking behavior, pathological gambling, body dysmorphic disorder, and the immune disorder of pediatric autoimmune neuropsychiatric disorders (pandas) [73, 74 ]. personality change in the context of frequently normal cognitive (dlpfc) function is usual and a manifold of presentations is encountered principally. the various forms of echopraxia or imitation behavior, utilization behavior, and environmental dependency syndromes (field - dependent behaviors) are sometimes the overriding clinical manifestations of these lesions. from a neurophysiological point of view, the medial ofc attaches emotional valence to events in turn determines the strength of the episodic memory. lateral ofc lesions have been correlated with ocd, depression, irritability, mood disorders, and field - dependent behaviors. obsessive compulsive disorders spectrum group of disorders include ocd spectrum itself that comprises of obsessions (intrusive urges thoughts, images), compulsions (repetitive, ritualistic type of activities of a physical or cognitive nature), tourette 's syndrome, kleptomania, risk seeking behavior, pathological gambling, body dysmorphic disorder, and the immune disorder of pediatric autoimmune neuropsychiatric disorders (pandas) [73, 74 ]. in addition to these three principal behavioral fsc 's, two others have recently been added. the inferotemporal subcortical circuit may be associated with deficits in visual discrimination, visual scanning, visual hallucinations, and psychosis. in addition, a circuit between the posterior parietal region (ba 7) and the prefrontal region (ba 46) also contributes to processing of visual stimuli of significance and accordingly visuospatial processing. individual lesions or disease processes usually affect more than one to differing degrees and in various combinations. to facilitate the diagnostic components and sometimes treatment options, it is useful to consider the fsc 's and their clinical counterparts in terms of neurophysiological core components and in correlation to the clinical syndromes (figure 2). from a clinical point of view, a vast panoply of symptoms have been associated with frontal lobe lesions but may conveniently grouped under the following primary domains. these include initiation, disinhibition, working memory, attention, monitoring, language, and emotional control. in addition, loss of creativity, curiosity, and initiative may also be present disinhibition. these include (ub, ib, eds) loss of judgment, loss of insight, impairment comportment, inappropriate social behavior, loss of empathy, irritability, aggression, irascible, excessive jocularity, irresponsible behavior, restlessness, hyperactivity, hypersexuality, hyperorality, and incontinence. this include verbal and nonverbal working memory, multitasking, abstract thought, planning ahead, temporal sequence of events. examples of executive functions include learning new information both verbal and visuospatial, searching memory systems, activation of past memories, temporal organization of behavior, attention, and generation of motor activity that includes speech, writing, or limb movement. these involve at least four of the five subprocesses : task setting, initiation of the task, monitoring, error detection, and behavioral self - regulation. these are, therefore, modulated by up to four of the fsc 's with task setting correlating with left dlpfc and monitoring / error detection correlating with right dlpfc activity. initiation of a new task involves both left and right superior medial frontal fsc 's and behavioral regulation disrupted after medial ofc circuit lesions. cerebral lesions distinct from the fsc 's may be associated with fns such as parietal lesions. for example, caudate lesion patients may have poor recall but relative preservation of recognition. thalamic lesion patients on the other hand may have impairment of recall and recognition. attention. under this rubric regarding differentiating attention and working memory ; attention allows certain stimuli be they sensory or cognitive to be given preference over other competing ones. working memory refers to the keeping a limited amount of information usually for a few seconds to allow manipulation or use of that information for another task. selective attention maintain behavioral or cognitive set in context of distracting or competing stimuli. alternating attention mental flexibility that allows individuals to shift their focus of attention and move between tasks. highest level of attention with ability to respond simultaneously to multiple tasks [78, 79 ]. focused attention respond discretely to specific visual, auditory tactile stimuli. selective attention maintain behavioral or cognitive set in context of distracting or competing stimuli. alternating attention mental flexibility that allows individuals to shift their focus of attention and move between tasks. divided attention highest level of attention with ability to respond simultaneously to multiple tasks [78, 79 ]. monitoring. this includes, broca 's aphasia (lh), expressive aprosodia (rh), transcortical motor aphasia, aphemia, and central aphasia. clinical lesions studies have implicated in particular the orbitofrontal cortex as part of the neural network for emotional responses. patients with orbitoprefrontal and medial frontal regions were significantly impaired in both cognitive and affective empathy as compared to parietal patients and healthy controls and those with damage restricted to the prefrontal cortex, no matter which side, resulted in impaired empathy and lesions involving the right parietal lesions. subsequently from a registry analysis a much more widely distributed lesion site network impairs ei, in keeping with the extensive contemporarily appreciated neurobiological emotional network proposed by pessoa (table 6). many different brain lesions may affect ei, including frontal, temporal, subcortical, and even subtentorial stroke syndromes with the strongest relationship (ei scores) pertained to the frontal and temporal regions (table 2). however, they are all frontal network syndromes that just happen to be treated by differing brain - related clinicians for historical reasons. frontal network syndrome phenotypes may comprise of a mixture of the primary and secondary syndromes in various combinations. clinical syndromes are also treated by different disciplines, with considerable overlap by psychiatry, neurology, neuropsychology, speech and language and physical medicine, and rehabilitation. it may be readily appreciated that the construction of many clinical scales we use such as the fab scale, the new consensus criteria for the behavioral variant of fronto - temporal lobe degeneration, and frontal network testing format of the coconuts are all derivatives of these core and secondary syndromes. there are other frontal network presentations that are not readily classifiable under the above system. as a brain lesion may cause both hypo- or hyperfunction of a circuit or a lesion may cause hypo- or hyperfunction remotely (diaschisis) because of the hodological nature of brain function or connectomics. one theory termed the paradoxical functional facilitation proposes that one brain area reverses inhibition in other areas or results in compensatory augmentation, resulting in counter intuitive paradoxical improvement in certain functions function. according to this theory, examples of such syndromes include the following.(i) emergent artistic ability in the setting of neurodegenerative disease has been reported in association with frontotemporal lobe disorders, stroke, alzheimer 's disease, parkinson 's, epilepsy, and migraine.(ii) delusional misidentification syndromes, seen particularly with right frontal stroke.(iii) increased humor, particularly after right frontal lesion such as stroke.(iv) loss of visual imagery in dreaming.(v) savant syndromes that may include the sudden, acquired prodigious, sudden, splinter, or talented subtypes. emergent artistic ability in the setting of neurodegenerative disease has been reported in association with frontotemporal lobe disorders, stroke, alzheimer 's disease, parkinson 's, epilepsy, and migraine. delusional misidentification syndromes, seen particularly with right frontal stroke. increased humor, particularly after right frontal lesion such as stroke. savant syndromes that may include the sudden, acquired prodigious, sudden, splinter, or talented subtypes. the time pressured nature of clinical practice, limited interview time, emergency room evaluation, and patient cooperation all place constraints on the nature of tests and how much testing can be performed. hence it is useful to consider available tests in a time - orientated hierarchical manner. the overall decision on how to deploy more time consuming neuropsychological tests is detailed in the recommendations by the aan neuropsychological testing guidelines. an emerging viewpoint is that subcortical processes, mostly cerebrovascular, may be the most frequent cause of cognitive disorders, particularly in the mild cognitive impairment domain. the cognitive component in question is frontal network systems particularly executive function, attention, and working memory [91, 92 ]. executive interview bedside test (exit)administration time approximately 1520 min. comprehensive cognitive neurological test in stroke (coconuts). cns vital signs. global tests delis - kaplan executive function system (dkef). wechsler adult intelligence scale (wais - iv) components. clinical syndrome orientated, questionnaire based (3) frontal systems behavior scale (frsbe).(4) behavior rating inventory of executive function (brief).(5) frontal behavioral inventory (fbi). frontal behavioral inventory (fbi). working memory / executive function tests (6) trail making tests (comprehensive, trails a and b, color trails).(7) letter and category fluency list generation.(8) wisconsin card sorting test.(9) tower of london test.(10)working memory tests (verbal and nonverbal). trail making tests (comprehensive, trails a and b, color trails). tests of disinhibition / inhibition (13)stroop neuropsychological screening test.(14)iowa gambling test. stroop neuropsychological screening test. brown attention deficit disorder scales. depression (18)ces - d (center for epidemiological studies depression scale).(19)beck depression scale.(20)hamilton depression inventory. behavioral neurological tests (21)faux pas test.(22)reading the mind in the eyes test.(23)hotel task.(24)multiple errands test.(25)ambiguous figures test. other tests that predominantly assess frontal network systems (27)visual search and attention test.(28)rey complex figure test. visual search and attention test. qualitative without normative data (29)the executive control battery. the executive control battery. a novel approach (30)the metacognitive battery incorporating neurological, neuropsychological, and neuropsychiatric syndromes. the metacognitive battery incorporating neurological, neuropsychological, and neuropsychiatric syndromes. aphasia tests useful in motor aphasia, dysnomia, and aphasias in general (31)western aphasia battery.(32)boston diagnostic aphasia evaluation. elementary neurological olfaction (the smell identification test, sensonics gait incontinence primitive reflexes (grasp reflex, palmomental reflex, sucking reflex) volitional eye movements. olfaction (the smell identification test, sensonics primitive reflexes (grasp reflex, palmomental reflex, sucking reflex) volitional eye movements. due to the expansive frontal subcortical circuits and their open connections, it may be readily appreciated that the frontal lobes connect with all other regions of the brain including the cerebellum and brainstem. clinical lesion studies have repeatedly shown that no matter where the brain lesion is, whether subcortical gray matter, subcortical white matter, cerebellum, brainstem, or even parietal and occipital lobe, a greater or lesser degree of frontal systems syndrome is present [17, 134 ]. ftd is relatively confined to the frontal and anterior temporal lobes, similarly herpes simplex encephalitis. however, cognitive vascular disorders (cvd), disorders of white matter such as the leukodystrophies and cadasil, vasculitis tbi, ms, most of the toxic metabolic encephalopathic all tend to affect the frontal subcortical networks in a more diffuse fashion and hence present with inattention, dysexecutive syndrome, and dysmemory as the hallmark signature syndromes, considered the most common presentations of frontal network syndromes. many of these will have varying degrees of neuropsychiatric syndrome admixtures most commonly depression and anxiety and perhaps less often disinhibitory behavior, irritability / aggression, obsessive - compulsive disorders, and adult onset adhd. the serotonin toxidrome may present with barely perceptible symptoms to coma with cognitive, somatic, and autonomic manifestations. the cognitive features may include hypomania, agitation, hallucinations, the autonomic hyperthermia, hypertension, tachycardia, diarrhea, and somatic features : myoclonus, tremor, and hyperreflexia. pathophysiologically, there is an increase in cerebral serotonin or overstimulation of the 5ht 2a receptors, often due to maoi 's in combination with ssri, snri, tca, appetite suppressants, or opioids. the diagnosis remains a clinical one. similarly with the neuroleptic malignant syndrome, cognitive changes, autonomic instability, tremors, muscle cramps, tremors, and elevated creatinine phosphokinase the fever is caused by a hypothalamic dopamine receptor blockade and the muscular effects due to blockade of the d2 receptor and the pathophysiology due to low dopamine or dopamine receptor blockade with sympathoadrenal hyperactivity. with the malignant hyperpyrexia syndrome, exposure to anesthetic drugs such as succinylcholine, a neuromuscular blocking agent, halothane, or desflurane, an abnormal muscular activity is induced with hyperpyrexia and circulatory collapse that can be fatal. the inheritance is autosomal dominant, usually for the ryanodine receptor (gene ryr1) located on the sarcoplasmic reticulum and opens in response to an increase in intracellular calcium which is exaggerated in this condition. both have a similar presentation including cognitive autonomic and muscular symptoms and signs but with some important differences such as tachycardia, mydriasis urinary retention, hyperthermia with the former and diarrhea, urination, hypothermia miosis, and bradycardia with the latter. both also present with psychosis, seizures, hallucinations, delirium, and myoclonus. paids (paroxysmal autonomic instability and dystonia syndrome) is usually seen after significant traumatic brain injury and also presents with a combination of cognitive alterations, autonomic abnormalities, and, in this instance dystonias rather than muscular rigidity and tremor (figure 9). frontal syndromes or frontal network syndromes may come to attention by way of the patients symptoms, a clinical syndrome elicited clinically or primarily by neuroimaging findings bearing in mind that this part of the brain has sometimes been termed clinically silent (tables 35 and figure 10). frontotemporal lobe degeneration as a generic term rather than dementia is recommended because many remain in a category of mci for a long time before frank dementia supervenes. because of the protean manifestations and long duration of decline, sometimes for decades, diagnostic difficulty is the rule. eventually neuroimaging may reveal focal degeneration of frontal insular and temporal lobes although the basal ganglia and spinal motor neurons may also be involved and the presentations may accordingly be initially one of the parkinsonian dementia syndromes (cbd, psp, and even dsdb) and mnd. hence it is useful to consider ftld in the context of other primary dementias (table 6). ftd is more common than ad in those < 60 years and the prevalence is about the same as ad in 6070 group and there are reports of ftd presenting in the third decade. the most common subtype is bv - ftd and a rapidly progressive ftd is seen in association with motor neuron disease with a mean survival of approximately two years. by comparison, the survival of bv - ftd is between 68 years with the language variant subtypes currently demonstrating the longest survival times of 812 years [143145 ]. the symptoms and signs initially are subtle, often undiagnosed for years and frequently misdiagnosed as bipolar disease, mania, obsessive compulsive disorder, personality disorder, and depression. dysexecutive function is not specific to ftd and is also seen with other dementias including ad, whatever the emotional impairment is. when the pathology is left sided, language impairment is a major clue and all present with word finding problems. however, in logopenic progressive aphasia (lpa), the person retains the so - called islands of normal speech without dysarthria. the criteria of neary. have been augmented by the revised international consensus criteria of rascovsky. which are presented in abbreviated form (appendix 2) in. in brief, the major features include (figure 11) the following(i) progressive deterioration of behavior and/or cognition by observation,(ii) early behavioral disinhibition,(iii) early apathy,(iv) early loss of sympathy or empathy,(v) early perseverative, stereotyped, or compulsive / ritualistic behavior,(vi) hyperorality and dietary changes,(vii) executive deficits with relative sparing of memory and visuospatial functions. progressive deterioration of behavior and/or cognition by observation, early behavioral disinhibition, early loss of sympathy or empathy, early perseverative, stereotyped, or compulsive / ritualistic behavior, hyperorality and dietary changes, executive deficits with relative sparing of memory and visuospatial functions. tar dna binding protein 43 (tdp-43) was identified as the major ubiquitinated protein, with positive inclusions in neurons and glia, occurring in ftd and approximately half of ftd patients have tdp 43 inclusions due to mutations in the progranulin gene found in the frontal and temporal neurons and dentate gyrus. most the remainder is due to tau inclusions caused by mutations in the tau gene. mnd association, sv is mostly linked to tdp 43, and hippocampal sclerosis (frequently seen in ftd) and is usually associated with tdp 43. about one quarter of progressive lewy body disease patients may also show tdp 43 inclusions [148152 ]. overall, in ftd the pathology is, therefore, either a tauopathy or tdp 43 proteinopathy with a small number associated with other pathologies (table 7). tau is a microtubule - associated protein that is integral to microtubule assembly and in stabilizing microtubules [153155 ]. histologically, von economo neurons, a relatively unique spindle - shaped cell found in mammals of such as cetaceans, hominoids, and elephants, are found in the anterior cingulate cortex and fronto - insular regions and are thought to be important in social networks. neurochemically, the presentations of ftd may be due to a significant reduction of serotonin receptors in the cingulate, orbitofrontal, and insular regions. overall, 10% of ftd cases are associated with an autosomal dominant pattern of inheritance with the rest sporadic. both tau and progranulin mutations have been linked to chromosome 17, and other genetic loci linked to ftd occur on chromosome 3 and 9. both tau and progranulin mutations cause inclusions in neurons and glia. in general, bv - ftd has the strongest inheritance, sv the less, and pnfa intermediate. (1) behavioral variant.(2) progressive non fluent aphasia (left perisylvian degeneration associated with tau pathology).(3) semantic variant (anterior temporal degeneration associated with tdp 43 proteinopathy but about 10% have ad).(4) logopenic progressive aphasia (angular gyrus of parietal lobe usually associated with ad pathology). progressive non fluent aphasia (left perisylvian degeneration associated with tau pathology). semantic variant (anterior temporal degeneration associated with tdp 43 proteinopathy but about 10% have ad). logopenic progressive aphasia (angular gyrus of parietal lobe usually associated with ad pathology). other neurological deficits may be elicited including peripheral neuropathy, parkinsonism, apraxia, and gaze abnormalities which would support the existence of one of the following syndromes.ftd and parkinsonism and corticobasal - ganglionic syndrome.ftd and als more common with bulbar than limb.ftd and psp present vertical gaze impairment, with early falls axial rigidity. cbd : asymmetric movement disorder with alien hand syndrome and cortical sensory loss. rather than just a primary disorder, cbd may be caused by secondary conditions such as ftd, ad, and creutzfeldt - jakob disease. for example, cbd, psp, and half of people with ftd have mapt (microtubule associated protein tau). ftd and psp present vertical gaze impairment, with early falls axial rigidity. cbd : asymmetric movement disorder with alien hand syndrome and cortical sensory loss. rather than just a primary disorder, cbd may be caused by secondary conditions such as ftd, ad, and creutzfeldt - jakob disease. for example, cbd, psp, and half of people with ftd have mapt (microtubule associated protein tau). it is important to employ specific frontal behavioral batteries such as fbi and frsb as neuropsychological cognitive testing may be normal with a distinct discrepancy in the relative absence of amnesia and visuospatial impairment. structural (mri / ct) may reveal frontal or temporal atrophy and functional (pet, spect) revealing frontotemporal abnormality much earlier with frontal and/or anterior temporal lobe hypometabolism or hypoperfusion, respectively. a study using mr perfusion scanning revealed similar results and may prove to be more practical. csf tau and ratio of tau to a - beta 42 are significantly lower in ftd than in ad with a sensitivity of 79%90% and specificity of 65% to 97%. in brief, pharmacological management may be employed using serotonergic agents for decreasing obsessions disinhibition, overeating, and repetitive behaviors, as exemplified by the randomized control trial of trazodone. atypical antipsychotics for agitated behavior and avoidance of cholinesterase inhibitors is also recommended as the latter may cause agitation. investigational treatments include both tau- and pgrn- based approaches. for example, inhibition of tau kinases to prevent tau hyperphosphorylation may be accomplished using lithium chloride or valproate. prevention of polyubiquitination to decrease tangle maturation by using hsp-90 and so antagonizing tau fibrillation or stabilizing microtubules using paclitaxel or anti - inflammatory agents is another possible avenue. cognitive impairment no stroke brain at risk stage (risk factors only : hypertension, diabetes, dyslipidemia) transient ischemic attacks and cerebral infarct with transient symptoms. brain at risk stage (risk factors only : hypertension, diabetes, dyslipidemia) transient ischemic attacks and cerebral infarct with transient symptoms. cognitive impairment subcortical infarct strategic infarct : caudate, thalamus, basal ganglia leukoaraiosis (figure 11) watershed infarction (figure 12) deep venous system tegmentothalamic lesions (figure 13). strategic infarct : caudate, thalamus, basal ganglia leukoaraiosis (figure 11) watershed infarction (figure 12) deep venous system tegmentothalamic lesions (figure 13). cortical infarct left angular gyrus (figure 14), right temporal lobe, frontal. left angular gyrus (figure 14), right temporal lobe, frontal. cognitive impairment most of the various cerebrovascular syndromes such as small vessel disease, leukoaraiosis, and vasculitis as well as multiple sclerosis and traumatic brain injury affect the brain more diffusely. this is important to consider as many of these patients have a considerably reduced attention span (and some may be irritable or irascible at the same time due to their disease process), and this greatly impacts the mode of testing. extensive neuropsychological testing in such patients is not usually practical and one of the shorter batteries may suffice. however, decline in memory is strongly associated with ad and decline in executive function is strongly associated with cvd. vascular dementia. most of the various cerebrovascular syndromes such as small vessel disease, leukoaraiosis, and vasculitis as well as multiple sclerosis and traumatic brain injury affect the brain more diffusely. this is important to consider as many of these patients have a considerably reduced attention span (and some may be irritable or irascible at the same time due to their disease process), and this greatly impacts the mode of testing. extensive neuropsychological testing in such patients is not usually practical and one of the shorter batteries may suffice. however, decline in memory is strongly associated with ad and decline in executive function is strongly associated with cvd. the origin of cognitive impairment in the context of vascular disease and whether neurodegenerative disease might be implicated ; the following are considered.the pattern and severity of cognitive impairments.the size and location of infarcts including symptomatic infarcts, silent infarcts, and leukoaraiosis.the severity of atrophy patterns in particular hippocampal atrophy and pattern of atrophy of frontal versus parietal.do the vascular lesions adequately explain the cognitive impairment?if the vascular lesions do not adequately explain the cognitive impairment, then neurodegenerative disease is likely present as well. the pattern and severity of cognitive impairments. the size and location of infarcts including symptomatic infarcts, silent infarcts, and leukoaraiosis. the severity of atrophy patterns in particular hippocampal atrophy and pattern of atrophy of frontal versus parietal. if the vascular lesions do not adequately explain the cognitive impairment, then neurodegenerative disease is likely present as well. in studies of neuropsychological series of vascular cognitive impairment and ad, there were no differences in language, constructional abilities, and attention [163, 164 ]. the current definition of dementia is memory centered, requires adl impairment, and does not emphasize the predominant executive dysfunction of the cvd subtypes. for minor cognitive deficits due to vascular disease, the term vci - nd has been recommended (cognitive deficits that do not meet criteria for dementia but impair minor chores). more wm changes or leukoaraiosis and less medial temporal lobe atrophy are seen in cvd when compared to ad although overlap occurs. two types may be discerned ; when the wm lesions are distinct and separated from ventricles, this tends to be more specific for vascular dementia. when the wm hyperintensity is periventricular, this tends to support a neurodegenerative rather than vascular process. a formidable number of conditions can present with white matter hypertensities on mri brain scan (table 8). leukoaraiosis may affect one, some, or all of the fsc and quantification will likely become more important in the future ; apart from neuroimaging software capabilities, the junque classification system (table 9) is a very useful manner of quantifying and measuring over time. in general the leukoaraiosis (la) on mri can be interpreted as follows.(1) mild mri la is correlated with markedly lower scores on episodic memory compared to working memory and is a neuropsychological feature associated with ad.(2) moderate mri la correlates with both amnesia and executive dysfunction.(3) severe mri la correlates with significantly lower scores on working memory and executive dysfunction. mild mri la is correlated with markedly lower scores on episodic memory compared to working memory and is a neuropsychological feature associated with ad. these findings have treatment implications with the milder forms more amenable to cholinergic therapy and the more severe forms might perform better with dopaminergic therapy. cerebral infarctslacunes microinfarcts up to 5 mmwidening of perivascular spacesincomplete infarctionleukoencephalopathy associated with svd.laminar necrosis selective involvement in the third and fifth layers. granular atrophy patches of gray matter between 2 or 3 arterial territorieslobar hemorrhages linked to a - angiopathy and adsmall hemorrhages association htn at corticosubcortical junction (slit hemorrhages) and bg. microinfarcts up to 5 mm widening of perivascular spaces incomplete infarction leukoencephalopathy associated with svd. granular atrophy patches of gray matter between 2 or 3 arterial territories lobar hemorrhages linked to a - angiopathy and ad small hemorrhages leukoaraiosis (la) may interrupt the neurotransmitter modulatory systems such as aminergic or corticostriatal and thalamocortical networks. patients with moderate mri la may respond better to aricept especially with junque la scores of 10 compared to those with minimal mri la. there may be at least moderate mri la in context of a dysexecutive syndrome, which may be a marker for the relative preservation of cholinergic neurons. perhaps what is important in this context is that the cerebral vasculature may be observed in real time by fundoscopy. the following were predictive of lacunar stroke : narrower central retinal arterial artery equivalent, wider central retinal vein equivalent, focal arteriolar narrowing, and arteriovenous nicking. overall, a mixed dementia is the most common type ; one that has been aptly termed a vascular tsunami (hakim). population - based autopsy studies indicate that less than 50% of patients have pure ad and many people diagnosed with ad may in fact have vcd. the neuropathology of ad and vci may coexist and influence each other and 8 of 10 of the traditional vascular risk factors also pertain to ad such as hypertension, hyperlipidemia, hyperhomocysteinemia, apoe2, and apoe 4. importantly, treatment of vascular risk factors is associated with slower decline in ad with no cvd [167, 171 ]. ad is normally regarded as disease process afflicting the posterior brain, namely, parietotemporal regions with a clinical correlate of dysmemory, visuospatial impairment, geographical disorientation, and only much later behavioral abnormalities that are characteristic of fns. the usual ad variants include a primary progressive aphasia and visuospatial and posterior cortical atrophy syndrome subtypes or variants. reports indicate that about 14%17% of ad patients have nonmemory presentations as an atypical subtype. in addition, executive function is not considered as a component of ad at least in mild - to - moderate disease. however, a subpopulation of ad patients may present with an fns early on including all three principal frontal syndromes of abulia, dysexecutive, and disinhibition. the neurobiology may be a disturbance of the frontal subcortical systems but the underlying etiology remains to be determined. one study found a 10-fold greater neurofibrillary tangle pathology in ba 8 of the frontal lobes. other explanations may include white matter disease, coexistent lewy body disease, and other cerebrovascular pathologies that influence the clinical presentation of the ad as a frontal variant [175, 176 ]. as a pathophysiological process with a few or numerous subcortical plaques, ms is a process that may impair one or more of the fsc 's. recent studies cite a data indicating that 43%72% of patients with ms are considered to have cognitive impairment. ms plaques frequently lie adjacent to the ventricles and so tend to interrupt the long association fibers in the cerebrum such as the fronto - occipital fasciculus, the superior and inferior longitudinal fasciculi. a recent study underscoring the important neurobiology of the frontal subcortical tracts employed frontal neuropsychological as well as behavioral neurological metric tests in comparison to diffusion tensor imaging (dti) of the frontal subcortical systems. tests sensitive to the orbitofrontal and cingulate regions of the frontal lobes were used including the hotel task test, iowa gambling test, faux pas test, multiple errands test, and reading the mind in the eyes test. dti evaluations were performed in the frontomedial, frontolateral, orbitofrontal anterior cingulate and a significant difference was found with lower fa values in the fm and fl in the ms patients compared to controls. a significant correlation was also found with loss of fiber integrity in the frontolateral regions and an impairment on the hotel task test and multiple errands test. when cognitive impairment in ms is evaluated, the symptoms elicited are principally inattention, abulia (apathy), dysexecutive syndrome, dysmemory (working memory), and disinhibition (inappropriate jocularity).. stimulant therapy with dopaminergics that pertain to the first four and sodium valproate or carbamazepine is often effective for treating disinhibition syndromes. tbi is associated with a complex syndrome characterized by cognitive (memory, attention and executive problems), elementary neurological symptoms (headache dizziness, vertigo, imbalance), and neuropsychiatric impairment (anxiety, depression, irritability, irascibility, mania, disinhibition, impulsivity). symptoms may be relatively mild to severe and may be present with normal neuroimaging and even anatomical pathology leading to frequent misdiagnosis and under appreciation of the severity of the syndrome. key to understanding the complexity of these syndromes is the current understanding of the pathophysiology (table 10). the dramatic advances in neuroimaging and alterations in the biochemistry and vascular system might best be described as a networktopathy with neurotransmitter and vascular perturbations that often escape anatomical imaging. the realization that vasospasm is a frequent accompaniment during concussion and tbi has led to the transcranial doppler study initiatives as a novel way of noninvasive monitoring. default mode network imaging may prove to be the most sensitive diagnostic tool yet in diagnosis. the frontal lobes are particularly susceptible to the effects of alcohol, as revealed by recent magnetic resonance spectroscopy studies. in addition, all of the following alcohol related cerebral conditions can affect cognitive functioning in particular frontal systems of working memory, attention, and executive function.alcohol intoxication coma, pathologic intoxication, alcoholic blackouts / memory loss.abstinence or withdrawal syndromes.nutritional disease (wernicke korsakoff).cerebrovascular infarcts and hemorrhages. cardiomyopathy.cerebellar degeneration.marchiafava-bignami disease.central pontine myelinolysis.alcoholic dementia.cerebral atrophy.neurologic conditions secondary to liver cirrhosis, portal shunts.traumatic brain lesions during intoxication. alcohol intoxication coma, pathologic intoxication, alcoholic blackouts / memory loss. abstinence or withdrawal syndromes. nutritional disease (wernicke korsakoff). traumatic brain lesions during intoxication. previously characterized as a subcortical dementia, this syndrome is still diagnosed by the clinical triad of cognitive, gait impairment and urinary incontinence. the predominant cognitive presentation is a frontal syndrome characterized by dysmemory, dysexecutive syndrome, inattention, and speed of information processing slowing. the frontal lobe syndrome or fns is much more profound than that seen with ad and the memory disturbance is relatively mild with nph, being much more severe with ad. the pathophysiology includes damage to fsc, corpus callosum, thalamus, basal ganglia, and hippocampus. the cognitive impairments respond to appropriate shunt procedures but are usually less so than the improvement in gait impairment.. recently the importance of specific assessment of executive function assessment has been proposed as this can be differentially improved by shunting relative to other cognitive impairments. neuroimaging with pet (or spect which measures perfusion rather than metabolism) has been shown to be the best diagnostic tool and a frontal hypometabolism (in nph) as opposed to a posterior parieto - occipital hypometabolism (in ad) has been shown to differentiate nph from ad better than cisternography. a reappraisal of autoimmune conditions associated with cognitive impairment and at times frank dementia has led to the concept of immunotherapy responsive dementias and encephalopathies. in addition to being treatable and reversible causes of dementia, they may account for up to 20% of the so - called young dementia patients (< 45 years). the cognitive profile includes the usual combination of fsc impairments in speed of information processing, dysmemory, and behavioral abnormalities typically of a fluctuating nature as well as agitation, hallucinations, and seizures. the finding that 1 alpha dendrotoxin antibodies against vgkc noted in patients with morvan 's disease, limbic encephalitis, and neuromyotonia completely changed the understanding from limbic encephalitis being a rare paraneoplastic condition with poor outcome and associated with anti - hu, anti - ma2- or cv2/crmps antibodies. autoimmune dementia syndromes (aid) in general are disorders with antibodies against synaptic proteins such as nmda, ampa, and gaba - b receptors and are treatable (table 11). the identification of a number of neural specific autoantibodies such as voltage gated potassium channel (vgkc) antibodies has increased the number of phenotypic presentations of aid. the diagnosis is made by establishing cognitive impairment or an encephalopathic state with clinical, radiologic, or serologic evidence of autoimmune etiology. other causes of dementia require exclusion and a beneficial response to an immunotherapy trial [30, 187 ]. in addition to the specific antibody tests, cerebrospinal fluid analysis is useful with the following parameters regarded as support for aid, raised protein especially over 100 mg / dl, pleocytosis, oligoclonal bands, and igg index elevation. cancer screening is important with computerized chest, abdomen, and pelvis required in all patients, mammography in women and prostate specific antigen and testicular ultrasound in men. treatment options for aid include intravenous methylprednisolone (1000 mg 35 days then weekly for 68 weeks), ivig if seropositive for gad 65 or ia2 autoantibodies with plasma exchange can not tolerate steroids or ivig. long - term therapy is indicated in responders that may require steroid sparing inhibitor such as azathioprine, mycophenolate, cyclophosphamide, or methotrexate [29, 187 ]. these conditions share several features in that they involve the brain diffusely often with small or microscopic lesions, have relatively covert onset and often subtle signs and syndromes, and may often be overlooked as consequence. the fsc 's are affected as well as the open - ended connections to the posterior parts of the brain, the brainstem, and cerebellum. in addition to standard anatomical brain imaging, csf analysis is required and very often, functional imaging with spect or pet brain scanning, at times magnetic resonance spectroscopy and less often brain biopsy. the challenge is usually in considering or entertaining these disorders in the first place as antibody testing, csf analysis, cerebral catheter angiography and at times brain biopsy as indicated are often the diagnostic. many pathological processes may have as their earliest manifestations some degree of cognitive impairment, most commonly involving the frontal network systems with systemic and meningeal irritation manifestations either absent or appearing much later in the illness. hence frontal network syndromes may be the first sign of a potentially treatable disorder, which if missed, is devastating. with csf analysis of dementia, currently a nonroutine investigation, it is possible to ascribe a slowly dementing state to the most common disorders such as ad or ftd and miss a treatable condition such as chronic cryptococcal meningitis. this has indeed been reported several times and may present a tip of the ice berg phenomenon [188190 ]. others seen much less frequently include candida, aspergillus, blastomyces, coccidioides, and histoplasma (table 12). in the largest series to date (n = 101) of cerebral vasculitis, cognitive impairment in general was present in over three quarters of the patients with altered cognition in 50% and aphasia in 28% of patients. as with other subcortical and more diffuse brain processes, inattention, dysexecutive function, and working memory problems are the most common cognitive disturbances. laboratory (esr, crp), cerebral angiography (often four vessel catheter cerebral angiography) and at times brain biopsy are required for diagnosis but the most important clinical error is not to entertain the diagnosis in the first place in the appropriate clinical context (table 13). conditions that may present with focal or diffuse arterial narrowing and in the differential of vasculitis radiotherapy vasospasm : acute hypertension, migraine, benign reversible cerebral angiopathy, or posterior reversible encephalopathy syndrome (pres) lymphoma of the central nervous system intracranial dissection : traumatic, spontaneous, and fibromuscular dysplasia intracranial atherosclerosis recanalizing embolus moyamoya disease and syndrome tumor encasement due to pituitary adenoma or meningioma sickle cell anemia neurofibromatosis [192, 193 ]. vasospasm : acute hypertension, migraine, benign reversible cerebral angiopathy, or posterior reversible encephalopathy syndrome (pres) lymphoma of the central nervous system intracranial dissection : traumatic, spontaneous, and fibromuscular dysplasia intracranial atherosclerosis moyamoya disease and syndrome tumor encasement due to pituitary adenoma or meningioma neurofibromatosis [192, 193 ]. in the context of fsc syndromes, the first step is to exclude emergency neurological conditions such as cerebral infarct, hsv-1, meningitis, subdural hematoma, and mass lesions using anatomical imaging with ct and multimodality mr imaging. thereafter the pursuit of underlying etiological processes is attended often requiring and complemented by functional imaging usually such as dti, f - mri, fluorodeoxyglucose position emission tomography (fdg - pet) brain, single - photon emission computed tomography (spect), brain pet pittsburgh compound b (pib), and pet receptor (dopa) imaging (tables 14 and 15). fdg- pet brain in particular has been an important tool in the early diagnosis of mild cognitive impairment (mci) and in differentiating types of dementia, with frontotemporal disorders (ftd) and alzheimer 's disease (ad). functional imaging is increasingly able to detect pathology, long before the clinical state emerges with pet brain imaging being the most accurate diagnostic method for most common dementia categories. pet brain scan patterns reliably differentiate the major dementia subtypes including the ad variant, posterior cortical atrophy syndrome (benson syndrome), ftd (frontotemporal hypometabolism), and ad (temporal, parietal, posterior cingulate hypometabolism) being relatively easily identified. there are also overlap syndromes such as ad and cognitive vascular disorders (cvd), the frontal variant of alzheimer 's disease, and bv - ftd which can not be differentiated easily clinically. other conditions that present predominantly as an fns syndrome include autoimmune dementias, toxic dementias, hiv dementia, and the prefrontal atrophy secondary to chronic stimulation of the pain matrix (chronic pain syndrome) [198201 ]. positron emission tomography (pet) [202204 ] and functional magnetic resonance imaging (fmri) [205, 206 ] implicate the anterior cingulate cortex (acc) and posterior cingulate cortex (pcc) having key roles in processing of pain perception. mr perfusion scanning gives similar information to pet brain scanning, and, being based on mri techniques, lack of radiation may give this modality preference in the near future. the default mode network (dmn), for example, reflects the basal or default mode activity of the brain (without activation procedures). regions metabolically active include the posterior cingulate, the precuneus, lateral parietal, lateral temporal, and medial frontal areas. dmn connectivity disruption has been documented in ad, ftd, epilepsy, autism, schizophrenia and depression (table 16). interestingly, the distribution of the dmn impairment is similar to the fibrillar amyloid deposition seen with ad (amyloid pet scanning). dmn disruption was accurate in identifying major depression with a 94% correct classification with the amygdala, anterior cingulate cortex, parahippocampal gyrus, and hippocampus exhibiting the highest discriminative power in classifying major depression. using fc - mri of the dmn and other rsn such the salience (for ftd), as well as attentional networks, allows rsn patterns to differentiate ad and ftd. in ad, for example, cholinergic (nicotinic receptors) and dopaminergic systems measurements have revealed increased c nicotinic binding sites associated with cognitive improvement after rivastigmine for 3 months. in parkinson 's disease using c methyl-4-piperidyl acetate (mp4a), dopaminergic system imaging with f fluorodopa (fdopa) showed decreased uptake in the striatum. dti has become the imaging modality of choice to objectively quantify the anatomical pathology which predominantly affects the fiber tracts that occur with traumatic brain injury and multiple sclerosis, for example, often with normal standard mri scans [211, 212 ]. there are a number of pathomechanisms that are associated with brain injury and an understanding of these may lead to avenues of improved brain function after injury. in the endeavor of promoting improvement after brain injury, consideration needs to be given to the following : augmenting and supporting mechanisms of spontaneous recovery, avoiding interventions (particularly medications) that may worsen the condition, pharmacotherapy mainly the ascending monoaminergic systems, behavioral therapies using the top down influence of the prefrontal cortex, overcoming inhibitory influences after injury (mvf - type therapy). augmenting and supporting mechanisms of spontaneous recovery, avoiding interventions (particularly medications) that may worsen the condition, pharmacotherapy mainly the ascending monoaminergic systems, behavioral therapies using the top down influence of the prefrontal cortex, overcoming inhibitory influences after injury (mvf - type therapy). pharmacotherapy is mainly concerned with the neuromodulatory systems, which are mainly concerned with adjusting signal - to - noise ratios and so influence processing. neuromodulation may be associated with augmenting, diminishing, or prolonging signaling in neuronal networks. there is also a top down regulatory control over the ascending modulatory systems from the pfc to the brainstem neuronal cells groups of ne, da, 5ht, and ach [214, 215 ]. information gleaned mostly from animal models have revealed the cellular and molecular responses to brain injury. currently known processes that are involved in spontaneous recovery in the stroke model, for example, include the following. cellular increased angiogenesis increased synaptogenesis increased dendritic branching and spine density increased neuronal sprouting. increased angiogenesis increased synaptogenesis increased dendritic branching and spine density increased neuronal sprouting. receptor gaba downregulation increased n - methyl - d - aspartate receptor binding. increased n - methyl - d - aspartate receptor binding. molecular increased growth factors increased cell cycle proteins increased growth associate proteins increased inflammatory markers hyperexcitability with long - term potentiation facilitation. increased growth factors increased cell cycle proteins increased growth associate proteins increased inflammatory markers hyperexcitability with long - term potentiation facilitation. future treatment strategies have been proposed for the stroke model but these may be equally applicable to other brain pathologies (table 17). an important principal revealed by basic science animal models has been the realization that neurotransmitter systems function in phasic as well as tonic modes. this applies to the modulatory ascending systems and correlate with the concept of a bell - shaped curve or the yerkes - dodson inverted u - shaped response seen in animals and humans. this psychological concept relates to the task performance with the horizontal axis representing level of arousal and vertical axis represents a persons particular performance with the peak or top of the bell being the site of optimal performance. this implies that level of monoaminergic function optimal for one particular task may not be so for another task and may be either be sub- or supraoptimal. for example, different levels of norepinephrine (ne) may affect different ne receptors, with moderate levels of ne release affecting high affinity alpha 2 a receptors, and even higher ne levels as encountered during stress, involve alpha 1 adrenoreceptors and beta adrenoreceptors. these receptors have opposing functions in the pfc, the former improving and the latter impairing pfc function. the same applies to d1 and d2 receptors where different levels of presynaptic dopamine levels may either improve components of cognition or impair others [220, 221 ]. both ne and da are also considered to have complementary actions affecting cognition function in the pfc as has been reported with respect to spatial wm function, for example. the specific mechanisms of the monoamines of regulation of working memory have implicated the hyperpolarization activated cyclic nucleotide gate cation channels (hcn channel) which are localized on heads and necks of dendritic spines near incoming synapses in the superficial layers of monkey pfc. the other functions of the monoamines on the pfc include excitatory and neuroplasticity effects [224, 225 ]. the ne component has been associated with sustained attention when in its phasic mode and distractibility in its tonic mode in nonhuman primates performing a go - no - go visual attentional task. in addition, single unit recordings of the locus coeruleus was associated with optimal performance on a go - no - go visual target detection paradigm and in rhesus monkeys was correlated with phasic firing of ne cells. these include alterations in gene encoding molecules associated with glutamate signaling, cortical development, and the ascending monoaminergic systems as follows. disc 1 (disrupted in schizophrenia 1) : major susceptibility gene for mental illness including schizophrenia, bipolar disease, and depression. disc1 interacts with phosphodiesterase 4b (pde4b) and impaired disc 1 function likely leads to overactivation of camp - hcn signaling and weakening of the pfc network connections [223, 227 ]. rgs4 (regulator of g - protein signaling 4) : one of the regulatory proteins acting as gtpase activating proteins that drive g - alpha subunits into inactive gdp form, decreasing their activity. reduced rgs4 leads to an excess of pkc signaling and impaired pfc cognitive function. dgkh (diacylglycerol kinase isoenzyme) : one of the lipid kinases catalyzing conversion of diacylglycerol (dag) to phosphatidic acid with an overall reduction in dag which is a cofactor in the activation of pkc. loss of dgkh leads to an increase in pkc signaling and mutations are linked to bipolar disease and mania. of note is that the treatments with valproate, lithium, and tamoxifen inhibit pkc signaling. disc 1 (disrupted in schizophrenia 1) : major susceptibility gene for mental illness including schizophrenia, bipolar disease, and depression. disc1 interacts with phosphodiesterase 4b (pde4b) and impaired disc 1 function likely leads to overactivation of camp - hcn signaling and weakening of the pfc network connections [223, 227 ]. rgs4 (regulator of g - protein signaling 4) : one of the regulatory proteins acting as gtpase activating proteins that drive g - alpha subunits into inactive gdp form, decreasing their activity. dgkh (diacylglycerol kinase isoenzyme) : one of the lipid kinases catalyzing conversion of diacylglycerol (dag) to phosphatidic acid with an overall reduction in dag which is a cofactor in the activation of pkc. loss of dgkh leads to an increase in pkc signaling and mutations are linked to bipolar disease and mania. of note is that the treatments with valproate, lithium, and tamoxifen inhibit pkc signaling. dlpfc syndrome and dopaminergic and noradrenergic therapy. using executive function tasks such as word fluency and trails tests, these have been associated with a positive response to clonidine in schizophrenia and korsakoff 's patients [232, 233 ]. medications augmenting da and ne systems have been shown to improve executive function in tourette 's syndrome and attention deficit hyperactivity disorder. a number of medications have been used including tricyclic antidepressants, guanfacine, clonidine, and deprenyl [234, 235 ]. in another example, idazoxan has been correlated with improved executive function in a frontotemporal lobe degeneration patient. based on animal and human data, dopamine agonists such as apomorphine and bromocriptine appear effective in treatment of the condition akinetic mutism spectrum of syndromes, for example. on the other hand, presynaptic dopaminergic agents (methylphenidate and carbidopa / levodopa) seem ineffectual [237239 ]. midbrain infarction with damage to the dopaminergic neurons also causes akinetic mutism and is responsive to da agonists. in patients where there is damage to the anterior cingulate gyrus with da receptor damage, however, it has been speculated that da agonists may fail. hence loss of dopaminergic input from cortical structures such as the anterior cingulate gyrus as opposed to the striatum may be a factor in determining the type of da treatment. however, other clinical studies in patients with apathy various psychiatric conditions, stroke, wilson 's disease, and hiv dementia have revealed benefit from array of da - ergic medications including bromocriptine, amantadine, methylphenidate, buproprion, and selegiline [241245 ]. behavioral disinhibition correlates with a central serotonergic deficiency and serotonergic treatment has been reported to be beneficial in treatment of aggression. one hypothesis regarding aggression is that it may be due to a downregulation of 5-ht2 receptors in the striatum and nucleus accumbens where they occur in abundance [247, 248 ]. serotonergics such as fluoxetine and clomipramine may also be useful in disinhibited, impulsive, aggressive behavior. serenics (5ht 1a agonists) that bind to postsynaptic 5ht 1a receptors have been successful in treating aggression in animals. propranolol, pindolol, and buspirone are examples. in behavioral syndromes that may include mania and noradrenaline overactivity, adrenergic therapy may be beneficial in certain syndromes associated with bilateral inferior orbitofrontal contusions and respond to clonidine. other agents that have been useful include carbamazepine, sodium valproate, propranolol, clonidine, and lithium. aided recently by improved neuroimaging, namely, functional neuroimaging studies have delineated increased activity, either metabolism or blood flow in the orbitofrontal cortex, the head of caudate nucleus, and anterior cingulate gyrus. in general, serotonin reuptake inhibitors as well as clomipramine have been the most advocated pharmacotherapeutic approaches, and right caudate head glucose metabolism (pet brain scan) was reduced with successful fluoxetine therapy for obsessive compulsive disorder (ocd). the interaction in cerebral da and 5 ht may account for the improvement seen in some ocd with neuroleptics. in cases where hypometabolism (pet brain scan) occurs in the anterior cingulate region and right ofc, this too has been correlated with an improved response to clomipramine therapy. in refractory cases, sumatriptan (5ht 1d agonist) has improved both depression and ocd. both cognitive behavioral therapy and ssri therapy have been shown effective in ocd treatment and their combination potentiated. in a landmark international study, randomized, double - blinded, placebo - controlled trial of inpatient rehabilitation patients (n = 184) with minimally conscious or vegetative state were given amantadine 100 mg bid and increased to 200 mg bid by week 4. outcome determined by the disability rating scale (drs) and recovery was faster in the amantadine group as recorded by 0.24 units difference in the drs per week over the period week 4 to week 16, in the drs (p = 0.007). the beneficial effects of amantadine were attributed to presynaptic release facilitation and postsynaptic reuptake blockade thereby augmenting dopaminergic transmission in the mesolimbic, nigrostriatal, and mesocortical circuitry that subserve attention, conation, and arousal. in one of the few randomized - controlled trials, the core frontal component of attention was found to significantly improve speed of information processing in 40 participants with moderate - to - severe tbi receiving methylphenidate at a dose of 0.3 mg / kg twice daily. in a meta - analysis, ftld patients were presumed to have predominant serotonergic deficit as well as dopaminergic deficit with little evidence for ach and ne related impairment. a double blinded, placebo - controlled, crossover trial of trazodone with 300 mg daily revealed a significant improvement using the neuropsychiatric inventory score. trazodone is a selective serotonin reuptake inhibitor with ssri, a 5ht1a, 5ht1c, and 5ht2, with the active metabolite being a direct serotonin receptor agonist as well as a adrenergic (alpha 1, alpha 2) and histamine (h1) blocking agent. the effects were noted in the domains of behavior rather than cognition [261, 262 ]. in the flame (fluoxetine for motor recovery after acute ischemic stroke) trial patients with ischemic stroke and moderate - to - severe motor deficit, the early use of fluoxetine in combination with physiotherapy enhanced motor recovery at a 3 month evaluation. the mechanism of action is suggested to be a modulation of spontaneous brain plasticity by drugs attributed to brain - derived neurotrophic factor. partly preempted by the advent of the new dsm - v criteria, there is increased emphasis toward a neurobiological models of disease, a renewed neuroimaging focus and using dimensional scales as opposed to categorical diagnoses only (dsm - iv r) on traditional neuropsychiatric conditions. the neuropsychiatric disorders include a diverse collection of syndromes affecting behavior, emotion, executive function, and other core frontal network functions that primarily affect emotion, executive function, higher cognition and their circuitry. with a distinct paucity of biomarkers for these syndromes, similar to the approach in neurology, it seems essential to integrate basic neuroscience, neuro - genetics, epigenetics, and neuroradiology in order to establish a foundation for diagnostic based on pathophysiology and presumed etiology. the current psychiatric classifications (dsm iv) have had the effect of dichotomizing disease when they are better configured dimensional traits overlapping with normality which is also in accordance with the polygenic mode of inheritance. many patients diagnosed according to the in dsm iv receive multiple diagnoses that is termed comorbidity, probably reflective of a diagnostic artifact due to symptom splitting and lumping. both antipsychotic and antidepressant agents, for example, are used to treat many different psychiatric disorders [264, 265 ]. thinking in terms of symptom clusters and the core components of the frontal subcortical network circuitry may help construct a more neurobiological and pathophysiological relevant approach. this does, however, combine neurological, cerebrovascular, psychiatric, neuropsychological, general medical, and neuroradiological information and consequently the disciplines to advantage (table 18). serotonergic agents, electroconvulsive therapy, transcranial magnetic stimulation physical exercise, and cognitive behavioral therapy have all been shown to benefit major depressive disorder [266268 ]. an important study using pet brain scanning before and after treatment with cbt and a comparison group with the serotonergic agent paroxetine revealed changes in brain metabolism. the cbt group had increased activity in the hippocampal and dorsal cingulum and decreased in the frontal cortex activity whereas the paroxetine group had increased metabolism in the prefrontal cortex and decreased activity in the subgenual cingulate and hippocampus. these findings have been interpreted to suggest that cbt has a top down effect on the medial frontal and cingulate cortex and the pharmacological group work in a bottom up manner [269, 270 ]. interpersonal psychotherapy (itp) compared to venlafaxine similarly showed activation of the right posterior cingulate and right basal ganglia and of the right posterior temporal lobe and right basal ganglia in the venlafaxine group ; this time assessed by spect scans. similarly, the psychosurgical treatment, namely, anterior cingulotomy, reserved for severe treatment resistant depression revealed a decrease of metabolism measured by pet brain scanning in the left subgenual pfc and left posterior thalamus, from the preoperative values. stimulants such as methylphenidate increase ne and to a lesser extent da in the pfc while producing lesser effects in the subcortical regions. atomoxetine also increases ne and da with less effect on striatal da and may have a beneficial effect on impulsivity as well. atomoxetine is an important new treatment option for adults with adhd and is particularly so for those who are at risk for substance abuse. atomoxetine is effective and a well - tolerated nonstimulant and the first adhd treatment approved specifically for adult use administered as a single daily dose and is not a controlled substance.. involuntary emotional expression disorder (ieed) is a more frequently diagnosed condition especially poststroke, traumatic brain injury, multiple sclerosis, and neurodegenerative diseases. recently, pharmacological treatment has been successfully demonstrated with the efficacy of the dextromethorphan - quinidine combination (nuedexta) [274, 275 ]. although in its infancy, one example of a regimen to improve one 's emotional style comprising outlook, self - awareness, attention, resilience, social intuition, and sensitivity to context has been detailed by davidson and begley [276278 ]. currently there is support for aphasia therapy, attentional training, rehabilitation of unilateral spatial neglect, and compensatory strategies for apraxia. using information from randomized controlled trials, case series and single case reports to classify recommendations for the following forms of cognitive rehabilitation modalities has been made. this rationale of this mode of therapy proposes that in some patients there is a kind of learned nonuse of the paretic or paralyzed hand or arm after stroke or tbi. a phase iii trial of constraint therapy of 2 weeks of such therapy resulted in significant gains that endured for approximately 2 years. this was subsequently analyzed further to report that comparing early (39 months) and later (1521 months) initiation of cimt after stroke resulted in both groups achieving similar level of significant arm motor function 24 months after enrollment. depending on the pulse frequency, either hypofunction or improved function may ensue due to the inhibitory or excitatory effects on cortical function. this may have application in those instances where inhibitory cortical circuits are operative and if diminished function may return. a randomized trial of treatment - resistant depression with tms has established this as a therapeutic component. in controlled case series studies, mvf has been effective in treating poststroke paresis (arm or leg), phantom limb pain, and complex regional pain syndromeand anxiety. there is also evidence that the modality can modulate pain and reverse objective signs such as inflammation and paralysis [283, 284 ]. patients with arm paresis treated with mvf (n = 17, controls n = 19) with significant values were reported for hand fim and arm and brunnstrom scores at baseline, 4 weeks and at 6 months. in a study of 40 patients with leg weakness after stroke, compared to best rehabilitation therapy and placebo - controlled with opaque glass, a statistically significant improvement was documented. proposed postulated mechanisms included visuomotor tract restoration leading to an unlearning of the learned paralysis this mechanism may also be attributed to a function of the mirror neuron system that involve interactions between the motor, vision, and proprioception modalities. limb weakness after stroke may be both fiber tract damage, as well as - so - called learned paralysis whereby neurons and their fiber tracts are inhibited and that this can be unlearned using a mirror. also called action observation treatment, this therapy is based on the premise that circuits are activated by observation, similar to those that perform the movement. initial results from observational studies as an add - on therapy appear promising [287, 288 ]. | frontal lobe syndromes, better termed as frontal network systems, are relatively unique in that they may manifest from almost any brain region, due to their widespread connectivity. the understandings of the manifold expressions seen clinically are helped by considering evolutionary origins, the contribution of the state - dependent ascending monoaminergic neurotransmitter systems, and cerebral connectivity. hence, the so - called networktopathies may be a better term for the syndromes encountered clinically. an increasing array of metric tests are becoming available that complement that long standing history of qualitative bedside assessments pioneered by alexander luria, for example. an understanding of the vast panoply of frontal systems ' syndromes has been pivotal in understanding and diagnosing the most common dementia syndrome under the age of 60, for example, frontotemporal lobe degeneration. new treatment options are also progressively becoming available, with recent evidence of dopaminergic augmentation, for example, being helpful in traumatic brain injury. the latter include not only psychopharmacological options but also device - based therapies including mirror visual feedback therapy. |
both cutaneous and visceral leishmaniasis are present in iran and cutaneous leishmaniasis (cl) has a vast distribution, being found in almost all provinces, especially in fars, isfahan, and kerman [26 ]. the laboratorial diagnosis of cl is mainly based on parasitological method which searches for the parasite in the lesions or culture of the lesion material. although these methods are specific, they suffer from the low sensitivity, the possibility of culture contamination, and also the difficulties of parasite growth. along with parasitological method, molecular techniques have been used for the diagnosis of cl. however, the technical requirements, the relatively high cost, and also the persistence of parasite in the lesion after the treatment hindered its routine applicability [710 ]. serological assays have been extensively evaluated for diagnosis of visceral leishmaniasis (vl) and considered as a routine method for diagnosis of vl while these methods are rarely used for diagnosis of cl. in recent years, serological approaches have been evaluated for diagnosis of cl in few studies [1117 ]. among the serological tests, indirect immunofluorescence (ifa), direct agglutination test (dat), and enzyme immunoassay (eia) are the most frequently evaluated tests for diagnosis of mainly american cutaneous leishmaniasis [12, 14, 16 ]. however, such approaches have not been properly evaluated for diagnosis of zoonotic cutaneous leishmaniasis (zcl). lack of such information, especially in iran, justified the conductance of the current study which aimed to determine the efficacy of two serological assays, elisa and ifa, for diagnosis of cutaneous leishmaniasis in iran. sera samples were collected from sixty - one parasitologically confirmed cl patients referred to shiraz hospitals. for parasitological diagnosis, a sample was taken from the skin lesion of each suspected case. with each case, the border of the skin lesion was slit with surgical lancet and a tissue scraping from the slit was smeared on a clean glass slide, fixed with methanol, and then stained with giemsa 's stain. each such smear was carefully examined by light microscopy under oil immersion and the diagnosis of cl was confirmed by the positive smear for leishman bodies. furthermore, samples prepared from the lesion of the patients were analyzed by pcr to find out the species of the parasite. control samples (n = 50) were obtained from healthy individuals with no history of cl. ethical approval of the study was given by the ethics committee of shiraz university of medical sciences, and consent was obtained from the participants. the parasites were washed three times (1500 g for 20 min) with pbs. protease inhibitors were added and the parasites were resuspended in pbs and submitted to freeze - thawing followed by sonication. the lysed material was centrifuged (1500 g for 20 min at 4c) and the supernatant was removed ; the protein content was estimated and the extracted antigen was stored at 20c until the use in elisa. moreover, the promastigotes of l. major (same strain as above) were propagated in rpmi media. antigen slides for ifa were prepared by washing the promastigote cells thoroughly with pbs and by loading 20 l of cell suspension at a density of 5 10 cell / ml onto each spot of a multiple spot teflon slide. the slides were allowed to dry at room temperature and stored at 20c until use. to detect anti - leishmania antibodies in sera of cl patients, collected sera were evaluated in an elisa system. elisa was carried out in flat - bottom 96-well microplates (nunc, nalgene, nunc international, roskilde, denmark). the plates were sensitized with 5 g / ml of amastigotes antigens (100 l / well) in coating buffer (0.05 m carbonate - bicarbonate buffer, ph 9.6) and incubated at 4c overnight. excess antigen was removed by washing the plate five times in phosphate buffered saline - tween 20 (pbst, ph 7.4 containing 0.05% tween 20). blocking was made with 3% skimmed milk in pbst for 2 hours. the wells were washed as before and 100 l of serum samples (1/100 dilution in pbst) from cl patients along with samples from healthy subjects, as negative controls, and sera from non - cl patients were applied to the plates and incubated for 1.5 hour at room temperature. the plates were washed as before and 100 l of horseradish peroxidase (hrpo)-conjugated antibody against either human igg or igm (abcam) or hrpo - conjugated mouse anti - human igg1 or igg4 (gibco) in pbst was added to the plates and incubated for 1 hour at room temperature. after washing as before, the plates were incubated with chromogen / substrate (100 l / well of opd, 0.025% h2o2 in 0.1 m citrate buffer, ph 5). the absorbance at 490 nm was checked with an elisa microplate reader. for each antigen, the cut - off value, which differentiates positive from negative results, was set by defining the cutoff as the mean value of the normal serum group plus three standard deviations. the serum samples were diluted 1 : 16 in pbs - skimmed milk 2% for preliminary screening and the positive samples were serially diluted up to 1 : 1024. ten microliters of each diluted serum was placed in the well of the slides and incubated in a humid chamber at 37c for 30 minutes. slides were washed in pbs (three times, each 10 minutes), dried, and incubated for 30 minutes at 37c with fluorescent - conjugated rabbit anti - human igg (sigma), diluted 1 : 1000, and evans blue solution, diluted 1 : 10000. finally, the samples were observed under immunofluorescent microscope and the titers of 1 : 16 and above were considered as positive. to determine the cutoff that best discriminates the sera of cl patients from the others, sera from cl patients, non - cl patients, and healthy individuals were analyzed and the cut - off point chosen was 1 : 16 titer. cl patients consisted of 41 males and 20 females. mean age of the patients was 37 (aged between 9 and 83 years). most of patients (37%) had cl lesion in their hands whereas the rest had lesion in their feet (21.3%), faces (13.1%), or trunks (6.4%). using elisa for detecting of total - igg against l. major, 51 cases of cl patients (83.6%) had a positive reaction in elisa while 10 cases (16.4%) of healthy controls and 19 cases of non - cl patients were also positive by elisa. accordingly, sensitivity and specificity of elisa for diagnosis of cl were 83.6% (95% ci = 71.4%91.4%) and 62.7% (95% ci = 52.9%71.6%), respectively. when the system was used for detecting anti - leishmania igm, a sensitivity of 84.7% (95% ci = 72.5%92.3%) and specificity of 54.3% (95% ci = 44.2%64.6%) were obtained for the assay. the elisa system was also used for detection of anti - leishmania igg subclasses (igg1 and igg4) and a sensitivity of 64% and 85% was found for igg1 and igg4, respectively. using ifa, a sensitivity of 91.6% (95% ci = 80.8%96.8%) and a specificity of 81% (95% ci = 71.6%87.8%) was found for ifa when the cutoff was set at 1 : 16. however, when the cut - off point was set as 1 : 128, fourteen cases (27.5%) of cl patients remained positive while none of healthy controls or non - cl patients had a positive reaction. accordingly, the assay showed a low sensitivity but a specificity of 100% when the cutoff was set at 1 : 128 titer. table 1 shows the details of the performance of elisa and ifa in diagnosis of cl in this study. statistical analysis of the data showed a fair agreement (kappa = 0.4) between elisa (for detecting total igg) and ifa. parasitological diagnosis, which relies on detecting of leishmania parasite in lesion or on cultivation of tissue samples of cl patients, still remains as a gold standard for diagnosis of cl [8, 9, 19 ]. although the direct microscopic identification of the parasite is simple, its low sensitivity of less than 60% is problematic, particularly in chronic cases where parasitemia is low. cultivation of tissue samples is a useful parasitological method since it allows the isolation of the parasite identification of the species, but it is time - consuming and culture may get infected by fungi or bacteria. different serologic assays such as elisa, ifat, direct agglutination test (dat), and immunoblotting have been evaluated and used for routine diagnosis of visceral leishmaniasis [2022 ]. the use of these serologic tools in diagnosis of cl has also been evaluated in a few of studies [2325 ]. the principal objective of the current study was to evaluate the performance of two common serological methods, elisa and ifa, in diagnosis of zcl in iran. findings of the study demonstrated an appropriate performance (sensitivity of 91.6%) for ifa in diagnosis of cl. previous studies which evaluated the efficacy of serological methods in diagnosis of cl have generated variable results. zeyrek. reported a sensitivity of 78.4% and specificity of 69.3% for an elisa system for diagnosis of cl in turkey. reported a sensitivity of 89% for an elisa system with antigen of leishmania mexicana and sensitivity of 71% with antigen of leishmania braziliensis. jensen. reported an elisa test with a sensitivity of 67% in 33 l. major infected cl patients, by using a sequence specific peptide antigen. in our study lower sensitivity has been reported by mosleh (81%) and also by monroy - ostria (85.4%) for ifa [23, 26 ]. in these studies, the cut - off point has been set on 1 : 16 titer and we used a same cutoff for ifa. in our study, when the cutoff raised to 1 : 124, none of control samples remained positive by ifa while more than 20 percent of cl patients were strongly positive by this and also by higher titers (up to 1 : 1028). considering this cutoff, the specificity of the test is 100% while the sensitivity lowered to 23%. in the current study, attempt was made to detect anti - leishmania igg subclasses by elisa in cl patients. however, the results were not any better than that of the detection of total igg. in cl patients, antibody levels this is not the case in vl where antibodies may reach exceedingly high levels leading to hypergammaglobulinemia which is a feature of the diseases. in addition, seropositivity may also depend on the duration of the disease and also the number of skin lesions. thus in cl, lower sensitivity of serologic tests can be expected due to low antibody titers. in our study in cl patients, correlation between the positive serological test and duration of the disease was significant (p < 0.05) while no association was found between the number of lesions and seropositivity. taken together, results of this study demonstrated that serological tests, especially ifa, have an appropriate performance for diagnosis of cl and these tests, along with the parasitological methods, can be used for proper diagnosis of cl. | serological assays have been extensively evaluated for diagnosis of visceral leishmaniasis (vl) and considered as a routine method for diagnosis of vl while these methods are not properly evaluated for diagnosis of cutaneous leishmaniasis (cl). this study aimed to assess the performance of indirect immunofluorescent - antibody test (ifa) and enzyme - linked immunosorbent assay (elisa) for serodiagnosis of cutaneous leishmaniasis in iran. sixty - one sera samples from parasitologically confirmed cl patients and 50 sera from healthy controls along with 50 sera from non - cl patients were collected. antigen was prepared from promastigotes and amastigotes of leishmania major. ifa was used to detect anti - leishmania igg while elisa was used to detect anti - leishmania igm, total igg, or igg subclasses (igg1 and 4). elisa, for detection of total igg and igm, showed sensitivity of 83.6% and 84.7% and specificity of 62.7% and 54.6%, respectively. sensitivity and specificity of elisa for detecting igg1 and igg4 were 64%, 75% and 85%, 49%, respectively. sensitivity and specificity of ifa were 91.6% and 81%. conclusion. findings of this study demonstrated that serological test, especially ifa, can be used for proper diagnosis of cl. |
growing in culture many cell types, particularly those of me - senchymal origin, display prominent bundles of filamentous actin (f - actin) associated with myosin ii, -actinin and several other cytoskeletal proteins. these structures, known as stress fibers (sfs) occur in several distinct forms. frequently, they are associated at one or both ends with adhesions to the underlying matrix, known most commonly as focal adhesions (fas). for over 40 years there has been considerable interest in the functions of these structures, their role in cell migration, and how they assemble and disassemble. much evidence has indicated that these structures are mechanosensitive [15 ] and it was concluded earlier that mechanical tension contributes to their assembly. however, several recent studies have challenged this view and demonstrated a more complex situation [811 ]. here, we consider the role of mechanical force in the assembly of sfs and fas. it is often forgotten, even by those who study fas and sfs, that these structures are not needed for cell migration. many cells (e.g. leukocytes) do not develop fas or sfs but migrate highly effectively. indeed, the presence of fas can hinder cell migration due to excessive adhesion. nevertheless, many migratory cells do display fas and sfs. in these cells there must be a dynamic coupling of adhesion strength and traction force for cells to move forward. it is important that both adhesion and traction at the front are stronger than at the rear, and so mechanisms must exist for modulating these. force at the front of migrating cells derives from both retrograde actin flow and myosin - generated tension. for many years, the term stress fiber was most commonly applied to the large bundles of f - actin that traverse much of the cell and that are anchored at both ends by fas. however, it was readily apparent that a variety of structures were often referred to as stress fibers. in 1998, small and colleagues distinguished 2 types of sf : ventral sfs, which are anchored at each end by a fa, and dorsal sfs, which are anchored only at one end by a fa close to the cell front. they also discussed arcs, convex bundles of f - actin that form behind the leading edge of migrating or spreading cells and which move rearwards below the dorsal surface. subsequent studies have included arcs, often referred to as transverse arcs, as a form of sf. arcs contain many of the same proteins, but unlike other types of sf, they are not directly anchored by adhesions to the matrix. however, arcs can give rise to ventral sfs and will be considered here as a form of sf. one complicating factor in the relationship between mechanical tension and the assembly of fas and sf is that the three types of filament bundle collectively referred to as sfs differ in their genesis, behavior, and relationship to fas. most studies have examined the assembly and disassembly of these structures as cells spread and migrate on coverslips coated with extracellular matrix (ecm) (most commonly fibronectin) [4,1417 ]. this system is well suited to analyzing adhesion dynamics and actin organization as cells migrate. the second experimental model for examining fa and sf assembly was pioneered by ridley and hall. they exploited the observation that some cells lose their sfs and fas when deprived of serum to become quiescent. upon re - addition of serum or other factors that activate rhoa, fas and sfs it should be noted that these cells are usually in a non - migratory state and often confluent. this system was used to identify rhoa as a key regulatory protein controlling the assembly of these structures. it was also the system used to show that rhoa - induced assembly of fas and sfs was blocked by a variety of inhibitors of myosin activity and contractility. this led to the conclusion that rhoa - stimulated myosin activity drives the assembly of sfs and fas. the bundling of f - actin to form a sf was attributed not only to the tension generated by myosin but also to myosin s crosslinking of f - actin. however, subsequent studies using blebbistatin and y27632, which inhibit the activities of myosin ii and rock respectively, have also shown that inhibiting myosin activity blocks the formation of most fas and sfs. similarly, in one study this was found for both myosin iia and iib, whereas in another this was dependent on knockout of myosin iia but not iib this second model system (stimulation of quiescent cells with serum or rho - activating factors) was well suited to microinjection of constitutively active or dominant negative constructs. in addition, it has the advantage of allowing synchronous assembly of fas and sfs to be studied in many cells. however, many cell types are resistant to serum - starvation ; they either maintain fas and sfs, or show only a slight decrease in these structures when deprived of serum. another disadvantage is that this system does not recapitulate the events that occur as cells migrate and engage the ecm at new sites. cell migration involves a series of transitions that affect both the adhesions and organization of the actin cytoskeleton. as the lamellipodium extends, driven by arp2/3 complex - mediated actin polymerization, initial adhesions form as integrin receptors engage the underlying matrix. maturation of adhesions that are not disassembled occurs at the transition between the lamellipodium and lamella, where retrograde actin flow changes from being driven by actin polymerization to myosin - based contraction. whereas actin is organized in the lamellipodium as a branching dendritic network, in the lamella it is often bundled into the different sf types. the maturing adhesions elongate in the direction of retrograde actin flow and retard the rate of rearward movement of actin. consistent with this clutch - like function, it was observed that in stationary cells fas are often pulled toward the nucleus, whereas in migrating cells they are stationary. however, the different types of adhesion are poorly defined and it is often difficult to distinguish one type from another. in general, fas are dependent on rhoa activity, whereas focal complexes are dependent on active rac1 or cdc42. hotulainen and lappalainen used live cell imaging to analyze assembly of the different sf types in migrating osteosarcoma cells. a subsequent study using higher resolution imaging concluded that little if any myosin ii is incorporated into dorsal sfs. transverse arcs arose behind the lamellipodium from the combination of short myosin filaments plus actin filaments generated at the leading edge by the arp2/3 complex. in this system, ventral sfs developed most commonly from the fusion of each end of an arc with a dorsal sf. the annealing of two dorsal sfs growing from opposite sides of a cell also gave rise to ventral sfs, again anchored at each end by fas. 1. burnette and colleagues used the same cells to explore the factors that maintain the lamella flat as cells migrate. they determined that dorsal sfs, which they found contained little to no myosin ii, acted as struts connecting the ventral adhesions with the dorsal contractile actin meshwork. their analysis revealed that contraction of transverse arcs generated tension on dorsal sfs, and this caused the dorsal sfs to pivot, thereby flattening the lamella. besides the evidence that blocking rho - mediated myosin activity inhibited ventral sf and fa assembly in quiescent cells, support for mechanical tension stimulating assembly comes from several observations. for example, shear stress at levels equivalent to that experienced in arteries induced endothelial cells in culture to develop sfs. direct evidence for mechanical tension stimulating growth of fas came from riveline and colleagues who applied force directly to individual cells with a glass rod. they observed growth of adhesions by irm optics and incorporation of fluorescently labeled fa proteins. adhesion growth was blocked by inhibiting rhoa activity, but this could be rescued by expression of the constitutively active formin, mdia1, which is normally activated by rhoa. it was concluded that fa growth requires both tension and actin polymerization, with each being driven by active rhoa. supporting this idea that growth of fas reflects the tension applied to them, geiger s group used traction force microscopy to reveal a close correlation between the size of fas and the force that is transmitted across them. if mechanical force promotes the growth and maturation of fas, one prediction is that new components will be recruited to fas in response to force. this is indeed what was seen both when individual components such as vinculin were examined, as well as when the fa proteome was analyzed. examining the composition of isolated fas, many proteins were found to be recruited to fas in the presence, but not the absence of active myosin. the idea that tension stimulates fa growth and maturation is also supported by experiments using beads coated with integrin ligands to apply force to cells. reinforcement of the adhesion made to beads has been observed in multiple studies [31,3437 ]. fa proteins are recruited to the bead adhesion sites under tension, again consistent with force contributing to adhesion assembly. what is the basis for the stiffening or reinforcement that occurs when tension is applied exogenously on integrins ? one of the signaling pathways activated in response to tension on integrins is the rhoa pathway [3638 ]. not only will this lead to increased actin polymerization via the rhoa - activated formin mdia1, but it will also promote the stability of actin filaments by inhibiting the severing activity of cofilin (via rock4 limk4 phosphorylation of cofilin). additionally, the activation of myosin ii by rock will lead to a positive feedback loop increasing tension. just as importantly, activated myosin ii that has assembled into bipolar filaments is a multivalent cross - linker and bundler of f - actin. additionally, f - actin reveals enhanced affinity for myosin ii when under tension. similarly, in response to tension, f - actin binds less cofilin and is more resistant to severing by cofilin. one way may involve exposure of tension - sensitive cryptic protein binding sites (illustrated in fig. originally demonstrated for fibronectin, this has also been shown for proteins at the cytoplasmic face of fas, such as talin. applying tension to single talin molecules exposed similarly, other proteins may expose sites that become modified, thereby facilitating new interactions. for example, mechanically stretching p130cas exposed multiple tyrosines that could be phosphorylated by src. first demonstrated when cells experience cyclic stretch or shear stress, zyxin shuttles from fas to zones of tension within sfs and triggers local recruitment of -actinin and vasodilator - stimulated phosphoprotein (vasp) that thickens and reinforces sfs. zyxin - dependent sf reinforcement has also been observed in cells that are not subjected to external mechanical stress. this repair mechanism serves to limit sf elongation and breakage at sites of excessive strain. although the mechanism involved is not fully understood, multiple models have been proposed and hinge on tension - dependent conformational changes in sf - associated proteins to expose new docking sites for zyxin. little is known about how the bonds between particular proteins in fas respond to mechanical tension. however, in some cases catch bonds, which strengthen in response to tension, have been identified, for example, for the fibronectin - binding integrin 51. we anticipate that some of the signaling associated with mechanical tension will modify protein interactions, converting slip bonds into catch bonds, thereby facilitating tension - induced stabilization. conversely, the conversion of catch bonds into slip bonds may contribute to the disassembly of adhesions at the rear of migrating cells. in contrast to the above studies, several have challenged the importance of myosin - generated tension in fa and sf assembly. some of the different conclusions may reflect that different types of sf and fas assemble through different mechanisms. so, for example, in the study by hotulainen and lappalainen, inhibiting myosin activity rapidly blocked formation of transverse arcs, but had little initial effect on dorsal sfs and the small adhesions that anchor them. tension was also important in the conversion of transverse arcs into ventral sfs, following their fusion with dorsal sfs. both structures consist of narrow bundles of f - actin anchored either in a tip complex (filopodium) or nascent adhesion (dorsal sf). the filaments within these bundles all appear initially to have a single polarity, with polymerization occurring at the membrane attachment site, i.e. the filopodial tip or matrix adhesion. because myosin ii can not generate contractile force on bundles of f - actin with a single polarity, whenever myosin ii incorporates into these structures (such as when dorsal sfs are fusing with transverse arcs), then the polarity of the actin filaments must be broken so that the bundle contains f - actin of opposite polarities. little is known about how this polarity transition occurs, although some possibilities have been suggested. tension generated within stress fibers is transmitted across fas as demonstrated by studying traction forces at a subcellular scale [29,5457 ] and shown most directly using a vinculin tension sensor. as mentioned above, geiger and his colleagues correlated the magnitude of force generated at a fa with the cross - sectional area of the fa. a very different conclusion was reached by beningo and colleagues who found that small adhesions at the front of a migrating cell developed greater tractional force than the larger adhesions further away from the front. a key difference in these studies is that these last two groups examined fas in migrating cells, whereas the study reporting a positive correlation between fa size and tension used stationary cells. another study found that for larger fas their size was proportional to the force being transmitted, but for small adhesions the correlation broke down. since small adhesions are usually at the cell front, a potential explanation is that the additional force experienced by these small adhesions arises from retrograde actin flow and from transverse arcs that transmit tension to the matrix via dorsal sfs. however, this does not explain why these small adhesions do not enlarge in response to the high force being applied, which would be predicted by the riveline study. initial adhesion assembly occurred within the lamellipodium independently of myosin ii on a template, the dorsal sf. the subsequent maturation of these nascent adhesions into fas was blocked by knockdown of myosin iia expression. strikingly, the cells depleted of myosin iia could be rescued by inactive motor mutants of myosin iia that were unable to generate force. it was concluded that myosin s crosslinking function is more important than its tension - generating function for maturation of nascent adhesions. some of the strongest arguments against tension driving assembly of fas have come from gardel and her colleagues. they demonstrated that in migrating cells up to 60% of the force exerted on the substratum is generated by the lamellar actin network rather than by sfs. using blebbistatin wash - out as a trigger for inducing myosin activity, they showed that the assembly of sfs lagged temporally behind the development of traction forces. they also noted that fa growth rate remained constant under a range of different tensions. titrating the level of myosin activity by varying the concentration of inhibitors led them to conclude that tension is necessary but not sufficient for fa maturation. waterman s group found that fa growth was fast in cells lacking vinculin but that these cells exerted low traction on the substratum. they concluded that vinculin when present couples retrograde actin flow to integrins and acts as a molecular clutch. their finding that vinculin promotes the transmission of force to the substratum but results in slower growth of fas runs counter to the idea that force itself contributes to fa growth. some of the differences may come from the use of the different systems, migrating cells versus quiescent stationary cells. other differences surely derive from grouping together the different types of sf and adhesion that are seen in migrating cells. the organization and behavior of dorsal sfs is different from transverse arcs and ventral sfs. the former seem largely independent of myosin - generated contractility whereas myosin is important for both transverse arcs and ventral sfs. however, myosin is needed for the maturation of adhesions, particularly its crosslinking function as shown by choi and colleagues. in the original model of tension driving sf and fa assembly, the pulling together and alignment of pre - existing filaments, already attached to integrins, was more important than nucleation of new filaments. this was supported by limited actin polymerization as sfs and fas assembled in the quiescent cell model. however, it is clear that actin polymerization is essential for the development of sfs and fas in migrating cells. one possibility is that mechanical tension may stimulate formin - mediated actin polymerization at fas. such a mechanism was suggested in the rive - line study, in which they observed that tension - dependent adhesion growth was dependent on mdia. consistent with this, schiller and colleagues recently observed that actomyosin contractility was necessary to recruit mdia to adhesions. using a proteomic approach, they discovered that 1 and v integrins cooperate to activate rhoa during adhesion to fibronectin but that downstream of rhoa the pathways diverged. unexpectedly, 1 integrins were coupled to rock and activation of myosin ii, whereas v was coupled to activation of mdia and actin polymerization. however, in response to myosin ii activity and high tension, v integrin was recruited along with mdia to the adhesions. they concluded that cooperation between these fibronectin - binding integrins and their rhoa signaling pathways (fig. it has long been recognized that sfs are more prominent in cells growing on plastic or glass surfaces than they are in the same cells in their native tissue environment or growing in 3d systems. is the prominence of sfs and fas in cells growing in vitro due to the two dimensional nature of the substratum, to its rigidity, or both ?. fibroblasts will actively contract free - floating gels, but if the gels are anchored to prevent contraction to a smaller volume, the cells develop isometric tension and develop sfs [6365 ]. release of the gels from their attachments to the culture dish results in rapid contraction of the gels and disassembly of the sfs [6365 ]. the role of substrate rigidity in sf and fa assembly was examined directly by pelham and wang, who discovered that assembly of these structures was inhibited on soft substrata. how might this occur ? with the recognition that active rhoa drives formation of these structures, this raised the question whether adhesion to a rigid substratum stimulates rhoa activity. measuring rhoa activity revealed that rhoa is activated in cells grown on rigid substrata. experimentally applying tension to engaged integrins was also found to activate rhoa. dissecting the pathway, guilluy and colleagues identified both gef - h1 and larg as guanine nucleotide exchange factors (gefs) responding to tension applied to fibronectin - coated beads. gef - h1 was activated downstream from fak, ras and the mek / erk pathway, whereas larg was activated via the tyrosine kinase fyn. keely s group also found gef - h1 was activated in cells adhering to rigid substrata, but in their case this was attributed to the release of gef - h1 from microtubules. using proteomic approaches two groups have observed both gef - h1 and mdia being recruited to adhesions in response to actomyosin contractility, suggesting that mechanical tension activates a gef - h1/rhoa / mdia pathway that may promote adhesion growth. high resolution traction force microscopy revealed that traction generated at one fa was often independent of the traction being generated by an adjacent fa within the same cell. the force transmitted by an adhesion was either stable over time or it fluctuated. in situations where it fluctuated, this tugging at fas was dispensable for their maturation, as well as for directional migration in response to chemotactic or haptotactic cues. the authors concluded that fluctuating tension at fas provides a mechanism by which a cell senses the stiffness of the substratum. it has long been known that tension exerted on the cell surface is transmitted to the nucleus and that multiple connections link the nucleus to the cytoskeleton. indeed, mechanical force exerted on cells has profound effects on gene transcription [7174 ], and tension exerted via sfs impacts nuclear structure and function. the nesprin family of proteins span the outer nuclear membrane, connecting the different filament systems to proteins spanning the inner nuclear membrane such as sun 1 and 2. these proteins connecting the nucleus with the cytoskeleton have been referred to as the linker of nucleoskeleton and cytoskeleton (linc) complex. investigating the relationship of the centrosome and nucleus to signals driving directional fibroblast migration, gundersen s group observed that the nucleus was pulled rearwards by actin filaments, such that the centrosome came lie in front of it. pursuing this further, they identified bundles of actin filaments (dorsal actin cables) moving rearward over the nucleus and forming aligned arrays with the linc complex. these transmembrane actin - associated nuclear lines (tan lines) appear closely related to arcs that arise near the cell front and move backwards via retrograde flow. however, once these arcs pass over the nucleus, they engage and align the linc complex. the linc complex disrupts tan lines and prevents nuclear movement in response to migration signals. depletion of emerin, an inner nuclear membrane protein involved in the interaction of the linc complex with the nuclear lamins, caused the tan lines to slip over immobile nuclei. examining the relationship between cell and nuclear shape, wirtz s group identified a subset of ventral sfs that wrap over the nucleus. these sfs connect to the nucleus via the linc complex and form a perinuclear actin cap. these sfs were implicated in determining nuclear shape, which was observed to be elongated in the direction of cell migration in moving cells, but round in stationary cells. like other ventral sfs, the sfs of the actin cap are anchored by fas at each end. however, these fas and their associated sfs are distinctive in several respects. the fas have a larger area and are more elongated than the fas associated with other ventral sfs. together with the sfs of the actin cap, they are more susceptible to disassembly by the actin depolymerizing drug latrunculin b. they are also more dynamic, with several fa components having shorter half - lives. strikingly, the fas associated with the actin cap were the first to disassemble after adhesion to soft substrata, leading to the conclusion that these fas are more mechanosensitive than most fas. one has to wonder whether these actin cap - associated fas are the same as those described by waterman s group, i.e. fluctuating in tension to sense the stiffness of the substratum. persistent migration was associated with an actin cap, whereas this disassembled when cells paused to change direction. experimentally disrupting the above results raise the question of what the role of the nucleus is in cell migration, a topic beyond the scope of this brief review. however, numerous studies have observed that disrupting the linc complex and hence the connections between the nucleus and the cytoskeleton have major effects on cell migration. whereas the nucleus appears important in cell migration, it should also be remembered that cells lacking a nucleus are capable of migrating. our knowledge of the structure and organization of sfs and fas has increased greatly in the last few years. although there are still components that need to be identified, much of the analysis is moving toward the biophysical and attempting to understand how these structures that generate and transmit tension are organized by the forces that they are experiencing. one area that will be important in the future is characterizing which protein interactions within sfs and fas are governed by catch bonds. another area concerns how cells respond to different strain rates and the effect of different force regimes. for example, fletcher s group found that the rate of application of force impacts a cell s response. a step displacement causing the height of a 12 mm tall cell to increase by 1 mm resulted in immediate increase in tension followed by a rapid viscoelastic relaxation to a new baseline above the previous steady - state level. however, when the same change in height was imposed at a rate of 0.1 mm / min, there was just a gradual increase in steady - state tension to a new level that was below the value eventually achieved by the step increase in height. when the height was increased ten times faster (by 1 mm / min), there was a much greater increase in steady - state tension, but again no viscoelastic relaxation. high rates of strain have also been found to lead to fluidization of the cytoskeleton. reconciling the different responses to force, i.e. reinforcement versus fluidization will be important. although this may partly reflect differences in experimental conditions, ultimately it will involve understanding how the different protein interactions respond to different force regimes and whether slip or catch bonds are involved. | stress fibers and focal adhesions are complex protein arrays that produce, transmit and sense mechanical tension. evidence accumulated over many years led to the conclusion that mechanical tension generated within stress fibers contributes to the assembly of both stress fibers themselves and their associated focal adhesions. however, several lines of evidence have recently been presented against this model. here we discuss the evidence for and against the role of mechanical tension in driving the assembly of these structures. we also consider how their assembly is influenced by the rigidity of the substratum to which cells are adhering. finally, we discuss the recently identified connections between stress fibers and the nucleus, and the roles that these may play, both in cell migration and regulating nuclear function. |
kluyvera spp. are gram negative bacilli that had been initially thought to be benign saprophytes. water, sewage, soil, milk, hospital sinks, and cows have been reported as environmental sources, suggesting that kluyvera spp. the biochemical profile is similar to that of other enterobacteriaceae. a member of the enterobacteriaceae, although initially described in 1936, the genus kluyvera was not well characterized until 1981 by farmer.. previous to 1981, the organism has also been referred to as cdc enteric group 8 and as api group 1. currently the genus kluyvera has four species, k. cryocrescens, k. ascorbata, k. georgiana and k. cochleae. k. cryocrescens is known to be an opportunistic pathogen and its infection is considered to be rare,,,,. we report k. cryocrescens bacteremia in an adult and review the literature in order to highlight the clinical features, antimicrobial susceptibilities and treatments used in recent clinical reports. an 81-year - old japanese man with a history of interstitial lung disease and 3 years of home oxygen therapy made regular clinic visits and took a blood test every month. anemia was detected and he was admitted to the hospital to treat the anemia. on admission, blood test findings included hemoglobin 6.6 g / dl, serum iron 16 g / dl and serum ferritin 4.2 ng / ml. the fecal occult blood test was negative. he had no other significant clinical history including injection drug use, human immunodeficiency virus infection and treatment with immunosuppressive agents. a proton pump inhibitor and a blood transfusion were administered. on admission day 3, upper gastrointestinal endoscopy was carried out and multiple superficial gastric ulcers without bleeding (forrest classification : iii) were found around cardia. administration of the proton pump inhibitor was continued. on admission day 4, fever of 39.3 c developed, along with an altered level of consciousness and hypotension. laboratory findings revealed elevated serum c - reactive protein and serum and urine white blood cell counts. two samples of blood and one sample of urine were cultured and k. cryocrescens was detected from the blood samples. piperacilin / tazobactam (4.5 g every 6 h) was initiated on hospital day 5. it was suspected that the k. cryocrescens bacteremia was related to the peripheral venous catheter because the urine culture was negative and there were no significant findings to suggest an alternative source, though peripheral catheter tip was not cultured. k. cryocrescens isolates from blood were susceptible to extended spectrum cephalosporins, ampicillin / sulbactam, piperacillin / tazobactam, fluoroquinolones, aminoglycosides, tetracycline, and carbapenems, and resistant to ampicillin and 1st and 2nd generation cephalosporins. on admission day 11, two blood samples were cultured and no microorganisms were detected. on admission day 16, administration of the antimicrobial agent was terminated. however, on admission day 19, there was an acute and fatal exacerbation of the interstitial pneumonia. we present a case of bacteremia due to k. cryocrescens in a patient with interstitial lung disease. clinical features of k. cryocrescens bacteremia remain unclear because there are few pertinent published reports. we reviewed the previously published cases, and the results, along with those of the current case, are summarized in table 1. in contrast, there were some similarities of clinical backgrounds : 8 of 9 patients had severe comorbidities and another was a premature infant, suggesting that k. cryocrescens can cause severe infections such as septicemia in immunosuppressed patients. in all, 8 of the 9 cases were nosocomial infections, consistent with previous descriptions of k. cryocrescens as an opportunistic microorganism. primary infection sites were determined in only 2 cases, which were both central catheter - related blood stream infections. although primary infection sites were undetermined in the remaining 7 cases, all of these had peripheral and/or central intravenous catheters. results of catheter tip culture or culture of blood drawn through the catheter were not described in any of those 7 cases, suggesting that catheter - related blood stream infections might have been present in some of these. intravenous catheter use might be a major risk factor. through production of beta - lactamases, | kluyvera cryocrescens infection has been considered rare ; clinical features of k. cryocrescens bacteremia remain unclear because few reports have been published. we report a case of k. cryocrescens bacteremia in an adult male patient and review the literature.our case was one with nosocomial bacteremia in a patient with interstitial lung disease. the primary infection site was undetermined, although he had an indwelling peripheral intravenous catheter and a urinary catheter.piperacilin/tazobactam was administered for 2 weeks and the bacteremia resolved. unfortunately, there was acute exacerbation of the interstitial lung disease, which was fatal. according to our review, including our case, k. cryocrescens bacteremia tends to occur in immunocompromised hosts, and indwelling catheters might be risk factors. extended spectrum cephalosporins, carbapenems, fluoroquinolones and tetracyclines are generally adequate agents for empiric therapy based on susceptibilities of k. cryocrescens clinical isolates. |
hereditary non polyposis colorectal cancer (hnpcc ; mim 120435 and 120436) is an autosomal dominantly inherited disorder that predisposes males to an approximate colorectal cancer risk of 80% by 70 years of age and females to a 40% risk of bowel cancer and a 40% risk of endometrial cancer by the same age. the genetic basis of hnpcc has been linked to errors in dna mismatch repair [2 - 5 ], which leaves a characteristic tumour signature of dna microsatellite instability (msi) that can be used as a surrogate marker for this syndrome. at least four genes have been associated with dna mismatch repair and hnpcc and are hmsh2, hmlh1, hmsh6 and hpms2 ; for review see papadopoulos and lindblom. both hmsh2 and hmlh1 account for somewhere between 50% and 60% of all families that adhere to the amsterdam criteria (3 relatives with colorectal cancer (crc) ; one must be a first degree relative of the other two ; cross at least two generations ; crc must be diagnosed in one relative under the age of 50 years and familial adenomatous polyposis (fap) must be excluded). somewhere between 2% and 20% of hnpcc families defined by the bethesda criteria (excluding the amsterdam criteria families) are associated with mutations in these two genes [9 - 12 ]. the contribution of hpms2 and hmsh6 remains undefined, however, on current evidence both hpms2 and hmsh6 appear to account for approximately 5% of all hnpcc families. the remaining 30 - 40% of amsterdam criteria families that do not evidently harbour germline mutations in the genes described either contain exonic or whole gene deletions, cryptic alterations within their coding regions, harbour changes in promoter / enhancer regions or are due to other genes that await identification. the diagnosis of hnpcc families remains problematic for a variety of reasons despite knowledge about the genetic basis of the disease. the majority of mutations identified to date are determined by genetic amplification strategies using the polymerase chain reaction and thereafter, subsequent analyses of the amplified product. this strategy has been extremely valuable in identifying a large number of mutations that can be clearly associated with disease development. this approach, however, does not identify inframe deletions that potentially remove whole exons or the entire gene nor does it recognise duplications that are capable of either introducing premature stop codons or inserting transcribable coding sequence that culminates in the insertion of nonsense peptide which alters the functional properties of the protein. using a new approach, known as multiplex ligation - dependent probe amplification (mlpa), deletion changes can now readily be identified. in this report we have screened 118 probands who either came from families that adhered to the amsterdam or bethesda criteria for whole gene or exon deletions in hmlh1 and hmsh2. our results indicate that hmsh2 deletions are much more frequent in the australian population compared to hmlh1 deletions and that mlpa is a useful aid in identifying smaller deletions that occur in the proximity of the splice donor and acceptor sites within hmsh2 and hmlh1. all patients enrolled in this study have signed an informed consent document for genetic testing. the patients were from the state of new south wales or western australia, which has a total population of approximately 9 million inhabitants. the centre where the genetic testing was undertaken is a statewide service, which also includes the australian capitol territory. patients selected for mlpa testing had been previously diagnosed with cancer and had tested negative for point mutations in hmlh1 or hmsh2 by dgge or dhplc analysis. the majority of patients had obtained a positive result for immunohistochemistry staining (i.e. displayed absence of staining for either hmsh2 or hmlh1) and were considered to be prime candidates to harbour deletions within the respective gene. all families fulfilled the diagnostic criteria of hnpcc by adhering to either the amsterdam 1 or 2 criteria. dna was isolated from whole blood using the method described by miller (1988) without modification. a multiplex ligation - dependent probe amplification (mlpa) kit was obtained from mrc - holland, amsterdam, the netherlands. the mlpa method was developed by schouten and uses a probe mixture containing16 probe pairs for hmsh2 and 19 probe pairs for hmlh1 plus 7 control probe pairs. initially 250 ng of genomic dna in 5 l of te was heated to 95c for 5 minutes and allowed to cool to 25c before addition of the hmlh1 and hmsh2 probe mixture and buffer. the solution was reheated to 95c for 5 minutes followed by overnight hybridisation of the probe at 60c. the mixture was cooled to 54c before dna ligase and buffer were added, followed by ligation at 54c for 15 minutes and inactivation of the ligase at 98c for 5 minutes. the ligation products were then amplified with one unlabelled and one fam - labelled primer using the thermal cycling conditions advised by the manufacturer. following amplification, 0.75 l of the pcr reaction, 0.75 l of deionised water, 0.5 l of an internal standard (tamra-500) and 12 l of deionised formamide were mixed and incubated at 94c for 2 minutes. the samples were then analysed on an applied biosystems 310 capillary sequencer with a 47-cm capillary and pop4 genescan polymer. all peak areas were normalised by dividing each peak area by the combined peak areas of all peaks in that lane. the normalised peak area was then divided by the average normalised peak area of that probe amplification product of all samples. deletion of an exon for an allele is indicated by the reduction in a relative peak area for that probe amplification product of 30%, while duplication is indicated by an increase in the peak area by 30%. all single exon deletions were subject to dna sequencing to determine whether mutations occurred within the mlpa probe binding regions, thereby presenting as an apparent exon deletion. dna sequencing was performed on a semi - automated sequencing unit (model 310, perkin - elmer applied biosystems division, foster city, ca) according to the manufacturer 's instructions. all patients enrolled in this study have signed an informed consent document for genetic testing. the patients were from the state of new south wales or western australia, which has a total population of approximately 9 million inhabitants. the centre where the genetic testing was undertaken is a statewide service, which also includes the australian capitol territory. patients selected for mlpa testing had been previously diagnosed with cancer and had tested negative for point mutations in hmlh1 or hmsh2 by dgge or dhplc analysis. the majority of patients had obtained a positive result for immunohistochemistry staining (i.e. displayed absence of staining for either hmsh2 or hmlh1) and were considered to be prime candidates to harbour deletions within the respective gene. all families fulfilled the diagnostic criteria of hnpcc by adhering to either the amsterdam 1 or 2 criteria. dna was isolated from whole blood using the method described by miller (1988) without modification. a multiplex ligation - dependent probe amplification (mlpa) kit was obtained from mrc - holland, amsterdam, the netherlands. the mlpa method was developed by schouten and uses a probe mixture containing16 probe pairs for hmsh2 and 19 probe pairs for hmlh1 plus 7 control probe pairs. initially 250 ng of genomic dna in 5 l of te was heated to 95c for 5 minutes and allowed to cool to 25c before addition of the hmlh1 and hmsh2 probe mixture and buffer. the solution was reheated to 95c for 5 minutes followed by overnight hybridisation of the probe at 60c. the mixture was cooled to 54c before dna ligase and buffer were added, followed by ligation at 54c for 15 minutes and inactivation of the ligase at 98c for 5 minutes. the ligation products were then amplified with one unlabelled and one fam - labelled primer using the thermal cycling conditions advised by the manufacturer. following amplification, 0.75 l of the pcr reaction, 0.75 l of deionised water, 0.5 l of an internal standard (tamra-500) and 12 l of deionised formamide were mixed and incubated at 94c for 2 minutes. the samples were then analysed on an applied biosystems 310 capillary sequencer with a 47-cm capillary and pop4 genescan polymer. all peak areas were normalised by dividing each peak area by the combined peak areas of all peaks in that lane. the normalised peak area was then divided by the average normalised peak area of that probe amplification product of all samples. deletion of an exon for an allele is indicated by the reduction in a relative peak area for that probe amplification product of 30%, while duplication is indicated by an increase in the peak area by 30%. all single exon deletions were subject to dna sequencing to determine whether mutations occurred within the mlpa probe binding regions, thereby presenting as an apparent exon deletion. dna sequencing was performed on a semi - automated sequencing unit (model 310, perkin - elmer applied biosystems division, foster city, ca) according to the manufacturer 's instructions. the majority of patients enrolled in this study had had their tumours assessed for the presence or absence of hmlh1 or hmsh2 and this information was used as a guide to examine in detail which gene was most likely to harbour a deletion or duplication. in those instances where no knowledge was available about the offending dna mismatch repair gene 18 index cases were identified as having either a deletion or duplication in hmsh2 or hmlh1. by far the greatest proportion of confirmed exonic deletions (11) were identified in hmsh2 (~61%) with only 2 exonic deletions being identified in hmlh1 (~11%). two apparent exonic deletions were also identified by mlpa, which were due to a 2 base pair deletion (1706 - 1707delaa) in hmsh2 and a 5 base pair deletion (728 - 732delatggt) in hmlh1, respectively. these two changes occurred within the annealing sites of the respective probes used for the assay. a third potential deletion was observed in exon 16 of hmsh2 which could not be readily confirmed by virtue of there being no easily identifiable method of determining the 3 ' end of the deletion. similar to the other two apparent deletions a mutation was identified at position 2635, which was a substitution (c > g) that was within the annealing site of exon 16 mpla probe. two duplications were identified, one in hmsh2 encompassing exons 9 - 11 and one in hmlh1 involving exons 4 - 6. both of these changes were presumed to disrupt the coding sequence by the introduction of extraneous dna. the changes identified in hmsh2 include one whole gene deletion, two probands with exon 8 deleted, eight probands who harboured unique deletions (see table 1) and one patient that according to mlpa had an exon 15 deletion. this deletion could not be easily confirmed by an alternative method since it occurs in the most 3 ' exon of msh2 and the presumed breakpoint was not readily identifiable. disease characteristics of the 18 families tested by mlpa the two remaining exonic deletions in hmlh1 included a deletion of exons 1 and 2 in one patient and a deletion of exons 7 to 11 in a second unrelated patient. the majority of patients (83%) were diagnosed with colorectal cancer under the age of 50 years and there were three patients diagnosed with extra - colonic disease (two endometrial cancers and one ampulla of vater carcinoma). the family histories of disease were similar to that expected from hnpcc and the average age of confirmed colorectal cancer was 44.8 years, which was not significantly different to patients diagnosed with other types of mutation in either hmsh2 or hmlh1. there were 101 cases of cancer reported in the families of which 93 could be accurately identified. from the 93 reported cancer cases there were 65 cases of colorectal cancer, 11 cancers of the reproductive tract (both endometrial and ovarian cancers), 6 breast cancer cases, of which four occurred at an unusually early age and an assortment of unusual cancers that have been found to be over - represented in hnpcc families, including cancers of the central nervous system, bile duct, stomach and kidney. the identification of deletion mutations has traditionally been difficult as it entailed the use of southern blotting. since the introduction of the multiplex ligation - dependent probe amplification accurate estimates of the frequency of whole exon deletions or duplications in hnpcc can now be addressed and in our experience we believe that these types of mutation may account for up to 15% of all mutations identified in the australian hnpcc population. in the population used in this report exonic deletions or duplications of hmsh2 occurred with a significantly greater frequency compared to hmlh1 which is in agreement with the findings of gille, taylor, charbonnier and bunyan 2004 who identified primarily hmsh2 deletion / duplication mutations. the mlpa assay appears to be an extremely valuable screening tool prior to a more extensive mutation analysis as it not only identifies exonic deletions / duplications but is also capable of identifying point mutations that lie in the region of probe binding or ligation. when point mutations occur within these regions the resulting dosage appears as an apparent loss of the respective exon. in our experience, if the mlpa assay reveals a single exon deletion it should be confirmed by sequencing the entire segment of dna including both primer - binding sites. by undertaking this strategy we were able to identify small deletions in three patients, which were all deemed to be causative as they were predicted to interfere with exon splicing. when examining the family profiles of patients harbouring deletion or duplication mutations against families that harboured causative missense or nonsense mutations little difference between the disease characteristics could be observed. in total there were 104 cancers identified in 18 families and the average age of colorectal cancer diagnosis was 44.8 years compared to that observed in our previous study (45.77 years for hmsh2 mutation carriers and 47.16 for hmlh1 mutation carriers). all patients had a family history of disease indicating that these changes were not de novo. in addition to colorectal cancer, there was an over - representation of endometrial cancer (13 cases), followed by cns tumours (3 cases), renal tract cancers (3) and a variety of other cancers present in 2 or less patients. interestingly, breast cancer was also represented in this set of families with an average age of diagnosis being 50 years. within this group, however, there were two elderly patients ; one case was most likely to be a phenocopy (and could not be tested) as she was 82 years of age and the other one who was diagnosed with breast cancer at the age of 70 after having been diagnosed with colorectal cancer at the age of 63 years. all the other cases of breast cancer were diagnosed under the age of 50 years. there were two duplications identified in this study, one in hmsh2 and the other in hmlh1. there did not appear to be any difference between those families found to harbour duplications in either hmsh2 or hmlh1 compared to families that harboured deletions in either of these genes. the numbers of patients precluded any statistical analysis. in summary, there does not appear to be any overall difference in the disease profiles observed in families that harbour exonic deletions compared to those that have been shown to be associated with nonsense or causative missense changes. mlpa appears to be an extremely useful screening method that can identify up to 15% of the mutations that occur in hnpcc families. | hereditary non polyposis colorectal cancer (hnpcc) is characterized by the presence of early onset colorectal cancer and other epithelial malignancies. the genetic basis of hnpcc is a deficiency in dna mismatch repair, which manifests itself as dna microsatellite instability in tumours. there are four genes involved in dna mismatch repair that have been linked to hnpcc ; these include hmsh2, hmlh1, hmsh6 and hpms2. of these four genes hmlh1 and hmsh2 account for the majority of families diagnosed with the disease. notwithstanding, up to 40 percent of families do not appear to harbour a change in either hmsh2 or hmlh1 that can be detected using standard screening procedures such as direct dna sequencing or a variety of methods all based on a heteroduplex analysis.in this report we have screened a series of 118 probands that all have the clinical diagnosis of hnpcc for medium to large deletions by the multiplex ligation - dependent probe amplification assay (mlpa) to determine the frequency of this type of mutation. the results indicate that a significant proportion of australian hnpcc patients harbour deletion or duplication mutations primarily in hmsh2 but also in hmlh1. |
according to the census 2001, the population of the elderly (age 60 years and above) in india was 75.9 million, i.e. 7.4% of total population. it is projected to be 113 million, i.e. 8.9% of total population by the year 2016. the elderly, by themselves are a vulnerable group and non - communicable diseases (ncds) are clearly a major morbidity in this age group. in india, ncds were responsible for 53% of deaths and 44% of disability adjusted life years lost. developing countries, like india, are likely to face an enormous burden of ncds in future and of these diseases, hypertension is one of the most important treatable causes of mortality and morbidity in the elderly population. further, high blood pressure (bp) is a modifiable risk factor for cardiovascular disease (cvd). data from the framingham heart study showed increasing cardiovascular morbidity with increasing systolic or diastolic pressure in those aged 65 and over. one of the cornerstones of the primary prevention of cvds has been screening for high bp and anti - hypertensive drug treatment. the benefits of anti - hypertensive drug therapy for older persons have been clearly established. prior studies have shown that anti - hypertensive drug treatment for older hypertensive persons confers highly significant and clinically relevant reductions in cardiovascular morbidity and mortality rates. nevertheless, a considerable percentage of older persons with hypertension are not detected or are not adequately treated for hypertension. measures should be taken to diagnose hypertension and prevent or postpone its complications in this age group as the burden of hypertension is bound to increase due to increasing life expectancy rates. health seeking behavior of the elderly is influenced by their economic instability, reduced physical endurance, social isolation, reduced cognitive ability, dependency, and loneliness. this makes them more vulnerable to suffer or succumb to illnesses, which may be treatable, or whose disabling effects could be postponed. the current study was carried out to document the prevalence of hypertension, to understand the health seeking behavior and expenditure on treatment in the elder population of rural puducherry in south india. the study was reviewed and approved by the institute ethics committee of indira gandhi medical college and research institute. a community - based cross - sectional study was carried out in rural field practice area of the department of community medicine of indira gandhi medical college and research institute. one primary health center with five sub - centers provides health services in the field practice area. one of the sub - centers, serving a population of 7400, was selected randomly by lots for the study. the study area mainly comprised of rural population with agriculture and agriculture - related works as the main occupation. elderly persons (age 60 years and above) residing in the study area for at least 6 months were included in the study. elderly individuals who were critically ill and unable to comprehend questions were excluded. based on a prevalence of 55% in kerala, (a neighboring state in south india), among elderly from reviewed literature, assuming an absolute precision of 7% and error 5%, the required sample size came out to be about 202. a sample size of about 220 was decided in order to adjust for non - response. a pretested, structured interview schedule was used to collect data on demographic characteristics, health seeking behavior and expenditure on treatment for hypertension (in previously diagnosed cases). the list of houses where elderly individuals were available was taken from the census conducted by the field level health workers in december 2010. if more than one eligible was present in a selected house, one of them was selected randomly by lottery method. bp measurement was done as per the standard guidelines, i.e. using mercury sphygmomanometer in right arm in the sitting position with feet kept firmly on ground and arm kept at the level of the heart. bp was measured on two separate occasions with a minimum interval of at least 5 minutes between the two measurements. care was taken on the day of measurement that the participants did not smoke or take caffeine half an hour before the measurement of bp. bp measurements were not done for participants with any acute painful condition like dental pain or joint pain. a systolic bp of 140 mm hg and/or a diastolic bp of 90 mm hg measured on two separate occasions with a minimum interval of at least 5 minutes between the two measurements or a self - reported history of taking anti - hypertensive medications is defined as hypertension. expenditure on treatment for hypertension was self - reported and calculated based on the money spent on consultation with a private practitioner and that spent on purchasing anti - hypertensive medications outside the government health system. descriptive statistics like mean, median and proportions were calculated using statistical package for the social sciences (spss) version 13.0. descriptive statistics like mean, median and proportions were calculated using statistical package for the social sciences (spss) version 13.0. nine elderly were not included in the study as they were difficult to trace or houses were locked on two successive visits. mean age of study participants was 66 years (sd 6.9) [table 1 ]. one - fourth of subjects (25%) were economically dependent on other family members. nearly half (49.5%) of the elderly were not engaged in any occupation during the study period while one - third (33%) were engaged in agriculture and agriculture - related works. around half (51%) of participants current smokers (smoking at least for a month) and 33% were current smokeless tobacco users. thirteen percent (28 out of 211) of the participants reported that they were taking treatment for diabetes mellitus. breathing problems, joint pains, and sleeplessness were the other complaints among the study population. socio - demographic characteristics of study participants mean systolic bp and diastolic pressure in the study participants was 136.9 18.6 mm hg and 88.1 11.2 mm hg respectively. the overall prevalence of hypertension among study participants was 40.5% (95% ci : 33.7 - 47.4%). prevalence of hypertension among male subjects was 39.2% (95% ci : 25.8 - 53.8%) whereas it was 40.8% (95% ci : 33.1 - 48.9%) among female subjects. about 62% (53 out of 85 hypertensives) were already aware of their hypertensive status. of those under treatment (n=52), 86.5% were uncontrolled. among the previously diagnosed cases of hypertension, 54.7% (n=29) were diagnosed at government health facilities either at primary health centers or a government hospital whereas 45.3% (n=24) were diagnosed by private providers. about 65.9% were taking their anti - hypertensive medications from primary health centers and 32% were taking their medications from chemist shops. on an average, the elderly hypertensives were visiting the doctor once in a month (meansd : 27.1 19.2 days). seventy - five percent of the hypertensives had their bp checked once in 20 days on an average. forty - eight percent reported that they had missed at least one dose of anti - hypertensives in the last 3-month period. fifteen percent had reported that they skipped anti - hypertensives for a week and more. went to relative 's home and forgot to take medicine were the commonly told reasons for poor adherence to medication. about 33.3% reported that they had made changes in their diet pattern, like reduction in the consumption of oily foods and reduced salt intake after the diagnosis. when asked about the expected duration of treatment for hypertension, 92.6% of the elderly hypertensives reported that medications have to be continued for life time. expenditure on treatment of hypertension was calculated for the previously diagnosed cases that were on treatment. mean expenditure on consultation came out to be 77 (indian rupees) (sd 34.6) per visit and for anti - hypertensive medications was 290 per month (sd 183.7). nine elderly were not included in the study as they were difficult to trace or houses were locked on two successive visits. mean age of study participants was 66 years (sd 6.9) [table 1 ]. one - fourth of subjects (25%) were economically dependent on other family members. nearly half (49.5%) of the elderly were not engaged in any occupation during the study period while one - third (33%) were engaged in agriculture and agriculture - related works. around half (51%) of participants current smokers (smoking at least for a month) and 33% were current smokeless tobacco users. thirteen percent (28 out of 211) of the participants reported that they were taking treatment for diabetes mellitus. breathing problems, joint pains, and sleeplessness were the other complaints among the study population. mean systolic bp and diastolic pressure in the study participants was 136.9 18.6 mm hg and 88.1 11.2 mm hg respectively. the overall prevalence of hypertension among study participants was 40.5% (95% ci : 33.7 - 47.4%). prevalence of hypertension among male subjects was 39.2% (95% ci : 25.8 - 53.8%) whereas it was 40.8% (95% ci : 33.1 - 48.9%) among female subjects. about 62% (53 out of 85 hypertensives) were already aware of their hypertensive status. among the previously diagnosed cases of hypertension, 54.7% (n=29) were diagnosed at government health facilities either at primary health centers or a government hospital whereas 45.3% (n=24) were diagnosed by private providers. about 65.9% were taking their anti - hypertensive medications from primary health centers and 32% were taking their medications from chemist shops. on an average, the elderly hypertensives were visiting the doctor once in a month (meansd : 27.1 19.2 days). seventy - five percent of the hypertensives had their bp checked once in 20 days on an average. forty - eight percent reported that they had missed at least one dose of anti - hypertensives in the last 3-month period. fifteen percent had reported that they skipped anti - hypertensives for a week and more. went to relative 's home and forgot to take medicine were the commonly told reasons for poor adherence to medication. about 33.3% reported that they had made changes in their diet pattern, like reduction in the consumption of oily foods and reduced salt intake after the diagnosis. when asked about the expected duration of treatment for hypertension, 92.6% of the elderly hypertensives reported that medications have to be continued for life time. expenditure on treatment of hypertension was calculated for the previously diagnosed cases that were on treatment. mean expenditure on consultation came out to be 77 (indian rupees) (sd 34.6) per visit and for anti - hypertensive medications was 290 per month (sd 183.7). our findings provide an evidence of high burden of hypertension in the elderly age group in a rural area of puducherry. the prevalence of hypertension in our study was similar to a study done in rural areas of kerala. our study findings were also similar to a study done in the elderly in rural and urban areas of puducherry, south india where they reported a prevalence of 43.9% in rural elderly population. however, other studies done in assam, north - eastern india and a multi - centric study in bangladesh and india had reported higher prevalence of hypertension. however, a study in rural part of karnataka reported prevalence of hypertension among 60 - 69 years population to be about 30.5% and 32% in above 70 years population. moreover, a study done in kolkata, eastern india among the elderly in urban areas reported prevalence of 53.5%. the difference in prevalence levels may be due to different geographical factors and may be due to differences in dietary pattern. though 65% of the study participants were illiterate, 62% of all who were found to be hypertensive were already ware of their hypertensive status. this finding reflects the better health seeking behavior and well performing health system (both public and private) in this part of country. proximity of the study area to the primary health center (phc) and availability of 24 7 services in the phc might have contributed toward better health seeking behavior. in the study by kalavathy. in kerala, only 35% were aware of their hypertensive status. this finding shows the preference toward private practitioners and that the elderly were spending money from their pocket for diagnosis and as well as treatment of hypertension. awareness has to be created that diagnosis and treatment of hypertension are carried out routinely in the phcs. barriers for geriatric care at phcs have to be identified and rectified so that more elderly would seek care for their morbidity. about half of the hypertensives had reported missing at least one dose of anti - hypertensive drug. health workers during home visits should be able to recognize the hypertensive patients and advise them to be compliant to the treatment. the study was carried out in the community and sample was selected by a random method which adds to strengths of the study. findings of this study can not be generalized to state or national level since the study sample is confined to a limited geographical area but it offers an insight into the burden of the problem and puts forward the need to introduce mechanism for early diagnosis and management of hypertension in the elderly. information regarding caffeine intake, alcohol intake, non - steroidal anti - inflammatory medication use, body mass index, and dyslipidemia were not collected and would have given better insight. estimates of expenditure on treatment for hypertension and adherence to anti - hypertensive medication were based on smaller sample of individuals. as the elderly population is likely to increase in future, and there is definite shift in the disease pattern, i.e. from communicable to non - communicable, it is high time that the health care system gears itself to growing health needs of the elderly in an optimal and comprehensive manner. responding to the needs of the ever - increasing number of older people, the government of india (goi) announced the national policy on older persons (npop) in 1999 and national policy for senior citizens in 2011. the national programme for health care of the elderly (nphce) being implemented in india would be expanded to all states. under this programme, it is proposed that special screening of the 80 + population of villages and urban areas will be carried out recognizing the increase of ncds in the country. this community - based study has showed a high prevalence of hypertension among the elderly in rural south india. strategies should be identified to diagnose hypertension at an early stage and prevent or postpone its complications in this age group as burden of hypertension is bound to increase due to increasing life expectancy rates. | background : non - communicable diseases (ncds) are major contributors of morbidity and mortality in the elderly. estimating the prevalence of hypertension and studying the health seeking behavior is important.aim:this study was designed to estimate the prevalence of hypertension and understand the health seeking behavior among the elderly in rural puducherry, south india.materials and methods : a total of 211 elderly from a rural community were selected by systematic random sampling. blood pressure (bp) was measured. socio - demographic characteristics and health seeking behavior were assessed by interviews.results:prevalence of hypertension among study participants was 40.5%. prevalence of hypertension among elderly male subjects was 39.2% and in female subjects was 40.8%. about 62% (53 out of 85 hypertensives) were already aware of their hypertensive status. about 54.7% (29) were diagnosed at government health facilities either at primary health centers (phcs) or a government hospital.conclusion:burden of hypertension among the elderly is high in rural areas. strategies to detect and treat hypertension in the elderly have to be implemented early. |
yoghurt is a fermented milk product, made from standardized milk by streptococcus thermophilus and lactobacillus bulgaricus. these bacteria produce lactic acid during fermentation of lactose. in case of milk processed to yoghurt, the growth of lactic acid bacteria will cause ph to fall, favouring the growth of spoilage yeast. these preservatives are added to prolong the shelf life of foods. in dairy products, natamycin is used as an antimicrobial food preservative, produced during fermentation by the bacterium streptomyces natalensis. in addition, it is nearly insoluble in water so that it penetrates into the product, in hard cheeses < 1 mm and in rindless cheese. natamycin has also begun to be used as a preservative in yoghurt. according to the turkish food codex, the maximum natamycin concentration in cheese is 1 mg / dm and should not be detectable at 2 mm depth. although yoghurt is special fermented milk product, turkish food codex does not accept the use of any preservative in yoghurt [2, 5 ]. therefore, in the present study, simple, economical, rapid, accurate, reproducible, precise, and fully validated hplc method with good detection ranges for estimation of natamycin in yoghurt was developed. a number of analytical methods have been reported for the detection and quantification of natamycin including spectrophotometric, derivative spectrophotometric, and liquid chromatographic [814 ] methods. some of these studies are with respect to compounds in some food while others are with regard to compound in drugs or biological samples. methods of analysis in foods were based on organic solvents extraction followed by uv detection or further hplc separation with uv detection. although uv spectrum of natamycin shows three major absorption peaks in the range of 290320 nm, 304 nm is the wavelength commonly used to quantify the antifungal, because of spectral interference from other compounds. also, natamycin showed degradation in food and during the storage time ; it is uneven distribution in the solution ; for this reason, identification of natamycin peak was made by hplc - dad system in this study [15, 16 ]. a waters bondapak c8 (150 mm 4.6 mm 5 m) was used as column in agilent 1200 with setting of dad detector at 303 nm. the mobile phase consisted of methanol, water, and acetic acid (12 : 8 : 1 ; v / v / v) ; flow rate was 1.0 ml / min and temperature was 25c. injection volume was 20 l and detector wavelength was 303 nm. all chemicals and reagents were of analytical grade and water was distilled and filtered through a membrane filter (0.45 m natamycin powder was obtained from dr. methanol (hplc grade, merck, 106018, 99.8%) and acetonitrile (hplc grade, sigma, 27225, 99.8100.5%) acetic acid were used to prepare the dilute solution and mobile phase. all stock and working solutions were protected from light and stored in fridge at about 4c. stock solution of natamycin (0.5 mg / ml) was prepared by dissolution in methanol. 50 mg of pure natamycin (c33h47no13) was dissolved in methanol in a 100 ml one - mark volumetric flask, marked with water, mixed, and protected from light. stock solution of natamycin (5 mg / l) was prepared in mobile phase the concentration ranges of natamycin were 0.1 g / ml, 0.2 g / ml, 0.4 g / ml, 0.6 g / ml, and 0.8 g / ml, respectively. the calibration curve for hplc analysis was constructed by plotting the ratio of the peak area of the natamycin. an amount of 5 g homogenised yoghurt was weighed in conical flask and 50 ml of methanol was added. 25 ml of deionized water was added and placed in the conical flask in the freezer for about 60 min. cold extract was filtered through a folder filter paper (macherey - nagel 100 751/60/030) ; first filtrate was discarded. a potion of the filtrate was filtered through a membrane microfilter of pore size of 0.45 m (minisart rc25 17765) and then 0.20 m (minisart rc25). the minimum amount of test solution (filtrate) required is 20 l per injection for direct chromatographic measurement. plot peak area or peak height was obtained for each solution on the ordinate against the natamycin concentration, inmicrograms per millilitre, on the abscissa. precision. method precision (repeatability) was evaluated by assaying ten sets of test samples, all on the same day (intraday precision) and method precision was also determined by another person under the same experimental conditions. six solutions were prepared containing 0.01 mg / l, 0.1 mg / l, 0.2 mg / l, 0.4 mg / l, 0.6 mg / l, and 0.8 mg / l natamycin concentration, respectively. it is the lowest concentration of analyte in the sample that can be determined with the acceptable precision and accuracy under stated experimental condition. the solution was injected three times and the signal and the noise for each injection were recorded. the concentration of the solution was used to determine the detection limit if the average s / n ratio is between 3 and 10. also, loq value was calculated from the calibration curve using equation loq = 10 sd / b (where sd is standard deviation of intercepts of calibration and b is slope of corresponding calibration curve). the lod and loq values were found to be 0.499 mg / kg and 0.403 mg / kg for yoghurt samples, respectively. accuracy. accuracy was assessed by determination of the recovery of the method at two different concentrations (3 mg / kg and 6 mg / kg of the test solution concentration). 20 replicate injections of sample preparation were injected and number of theoretical plats, tailing factor, and relative standard deviation of peak area were determined. student 's t - test and anova f test were used to determine significant differences between dairy brands. although use of natamycin is restricted in yoghurt, some producers could add natamycin as preservative [5, 18 ]. however, it is accepted to use safe food additive in some countries. in this paper, an analytical hplc method for assay of natamycin in yoghurt products was developed and validated. the simultaneous determination of the chees samples was performed on a c8 column of (150 mm 4.6 mm) dimension and 5 m of particle size. a mixture of methanol, water, and acetic acid (12 : 8 : 1 ; v / v / v) was used as mobile phase with flow rate of 1 ml / min. according to iso 9223 - 2, 2007, preparation of test sample system suitability test can be defined as a test to ensure that the method can generate results of acceptable accuracy and precision. the requirements for the system suitability are usually designed after method development and validation have been completed. the components of the cheese samples did not show any interference at 304 nm and no detector signal was produced during the analysis. the calibration curves (dad) were constructed with five concentrations including the lower limit of quantification (loq) ranging from 0.01 to 0.8. the regression analysis was performed, showing the equation : y = 88.669x + 0.2894. retention time was 2.98 min ; the limit of detection (lod) and limit of quantification (loq) were determined as 0.320 mg / kg and 0.403 mg / kg, respectively, by taking 3 and 10 times the standard deviation using the slope of calibration curve of natamycin, respectively. recovery experiments were performed at three concentrations of 104% and the values of relative standard deviation (rsd) were 0.562%. both the standard natamycin mixture and the sample showed good linearity in the tested range. by applying linear regression analysis, figure 2(b) shows a typical chromatogram obtained for analysis of spiked natamycin in yoghurt. as shown in figure 2(b), the substances formed well shaped natamycin peak that was well separated from the mobile phase. the accuracy of the developed method was carried out by adding the known amount of natamycin pure drug to the preanalyzed yoghurt sample and subjected to the proposed method. the study was done at 3 mg / kg and 6 mg / kg of test concentration levels. all the results indicate that the method is highly accurate. a specimen hplc dad chromatogram and 3d spectrum of a sample solution precision of the assay was determined by intraday and intermediate assay of the developed method. intraday analysis refers to the use of the analytical procedure in a laboratory over a short period of time which was evaluated by assaying 10 sample solutions for each analyst, at the final concentration corresponding to 0.6 mg / kg of natamycin during the same day. the interday precision (repeatability) of the method was found as 1.874% and 3.442% rsd values for uv and dad detections. recovery studies were also carried out to determine accuracy and precision of the proposed method. the recovery procedure was carried out by spiking already analyzed samples of homogenized yoghurt with known concentrations of standard solution of natamycin. the results of the recovery analysis are shown in table 3. according to student 's t - test, the computed value is less than the table value and and accuracy parameter has been validated. the results obtained by two different analysts were compared with anova f test and they were not statistically different. in turkey, few companies use modern techniques in the production of yoghurt ; generally, yeast and moulds can occur in yoghurt after 1 week of storage ; also, use of basic technology could cause problem in the shelf life of yoghurts. according to turkish food codex, fermented dairy products should not contain any preservatives ; some producers use natamycin as a preservative to prevent the occurrence of yeast and moulds in yoghurt. a total of twenty - eight yoghurt samples purchased from the supermarkets were analyzed to evaluate the natamycin contents and results are listed in table 4. natamycin was detected in all yoghurt samples and it is not acceptable according to turkish codex. the rp - hplc method enables the determination of natamycin with good accuracy and precision, either in laboratory - made yoghurt samples or in the commercial yoghurt samples. high recovery shows that the method is free from the interference of the commonly used additives in the yoghurt product. | this study was aimed at developing rp - hplc method for determination of natamycin in turkish yoghurt. chromatographic separation was achieved on a c8 column (150 mm 4.6 mm 5 m) with a mobile phase of methanol : water : acetic acid (12 : 8 : 1 v / v / v), at 1 ml / min flow rate with a detection of 303 nm. natamycin was spiked into handmade yoghurt samples and used for validation. the method has been fully validated according to iso 9233 - 2, 2007 (idf 140 - 2, 2007). it was successfully applied to determination of 28 different turkish yoghurt products. findings dealing with the presence of natamycin in cheese samples are presented. |
gravitational waves, one of the more exotic predictions of einstein s general theory of relativity may, after decades of controversy over their existence, be detected within the next five years. sources such as interacting black holes, coalescing compact binary systems, stellar collapses and pulsars are all possible candidates for detection ; observing signals from them will significantly boost our understanding of the universe. new unexpected sources will almost certainly be found and time will tell what new information such discoveries will bring. gravitational waves are ripples in the curvature of space - time and manifest themselves as fluctuating tidal forces on masses in the path of the wave. the first gravitational - wave detectors were based on the effect of these forces on the fundamental resonant mode of aluminium bars at room temperature. initial instruments were constructed by joseph weber [310, 311 ] and subsequently developed by others. following the lack of confirmed detection of signals, aluminium bar systems operated at and below the temperature of liquid helium were developed [253, 261, 76, 170 ], although work in this area is now subsiding, with only two detectors, auriga and nautilus, continuing to operate. effort also continues to be pursued into cryogenic spherical bar detectors, which are designed to have a wider bandwidth than the cylindrical bars, with the two prototype detectors the dutch minigrail [233, 158 ] and brazilian mrio schenberg [159, 70 ]. however, the most promising design of gravitational - wave detectors, offering the possibility of very high sensitivities over a wide range of frequency, uses widely - separated test masses freely suspended as pendulums on earth or in a drag - free craft in space ; laser interferometry provides a means of sensing the motion of the masses produced as they interact with a gravitational wave. ground - based detectors of this type, based on the pioneering work of forward and colleagues (hughes aircraft), weiss and colleagues (mit), drever and colleagues (glasgow / caltech) [130, 129 ] and billing and colleagues (mpq garching), will be used to observe sources whose radiation is emitted at frequencies above a few hz, and space - borne detectors, as originally envisaged by peter bender and jim faller [126, 140 ] at jila, will be developed for implementation at lower frequencies. gravitational - wave detectors of long baseline have been built in a number of places around the world ; in the usa (ligo project led by a caltech / mit consortium) [45, 212 ], in italy (virgo project, a joint italian / french venture) [61, 304 ], in germany (geo600 project built by a collaboration centred on the university of glasgow, the university of hannover, the max planck institute for quantum optics, the max planck institute for gravitational physics (albert einstein institute), golm and cardiff university) [321, 151 ] and in japan (tama300 project) [78, 294 ]. a space - borne detector, called lisa [125, 217, 216 ], was until earlier this year (2011) under study as a joint esa / nasa mission as one l - class candidate within the esa cosmic visions program (a recent meeting detailing these missions can be found here). funding constraints within the us now mean that esa must examine the possibility of flying an l - class mission with european - only funding. the official esa statement on the next steps for lisa can be found here. when completed, this detector array would have the capability of detecting gravitational wave signals from violent astrophysical events in the universe, providing unique information on testing aspects of general relativity and opening up a new field of astronomy. it is also possible to observe the tidal effects of a passing gravitational wave by doppler tracking of separated objects. for example, doppler tracking of spacecraft allows the earth and an interplanetary spacecraft to be used as test masses, where their relative positions can be monitored by comparing the nearly monochromatic microwave signal sent from a ground station with the coherently returned signal sent from the spacecraft. by comparing these signals, a doppler frequency time series /0, where 0 is the central frequency from the ground station, can be generated. peculiar characteristics within the doppler time series, caused by the passing of gravitational waves, can be studied in the approximate frequency band of 10 to 0.1 hz. several attempts have been made in recent decades to collect such data (ulysses, mars observer, galileo, mars global surveyor, cassini) with broadband frequency sensitivities reaching 10 (see for a thorough review of gravitational - wave searches using doppler tracking). there are currently no plans for dedicated experiments using this technique ; however, incorporating doppler tracking into another planetary mission would provide a complimentary precursor mission before dedicated experiments such as lisa are launched. the technique of doppler tracking to search for gravitational - wave signals can also be performed using pulsar - timing experiments. millisecond pulsars are known to be very precise clocks, which allows the effects of a passing gravitational wave to be observed through the modulation in the time of arrival of pulses from the pulsar. many noise sources exist and, for this reason, it is necessary to monitor a large array of pulsars over a long observation time. further details on the techniques used and upper limits that have been set with pulsar timing experiments can be found from groups such as the european pulsar timing array, the north american nanohertz observatory for gravitational waves [190, 191 ], and the parkes pulsar timing array. all the above detection methods cover over 13 orders of magnitude in frequency (see figure 1) equivalent to covering from radio waves to x - rays in the electromagnetic spectrum. figure 1the sensitivity of various gravitational - wave detection techniques across 13 orders of magnitude in frequency. at the low frequency end the sensitivity curves for pulsar timing arrays (based on current observations and future observations with the square kilometre array) are extrapolated from figure 4 in. in the mid - range lisa, decigo and bbo are described in more detail in section 7, with the decigo and bbo sensitivity curves taken from models given in. at the high frequency the sensitivities are represented by three generations of laser interferometers : ligo, advanced ligo and the einstein telescope (see sections 6, 6.3.1 and 6.3.2). also included is a representative sensitivity for the auriga, allegro and nautilus bar detectors. the sensitivity of various gravitational - wave detection techniques across 13 orders of magnitude in frequency. at the low frequency end the sensitivity curves for pulsar timing arrays (based on current observations and future observations with the square kilometre array) are extrapolated from figure 4 in. in the mid - range lisa, decigo and bbo are described in more detail in section 7, with the decigo and bbo sensitivity curves taken from models given in. at the high frequency the sensitivities are represented by three generations of laser interferometers : ligo, advanced ligo and the einstein telescope (see sections 6, 6.3.1 and 6.3.2). also included is a representative sensitivity for the auriga, allegro and nautilus bar detectors. for a popular account of the development of the gravitational - wave field the reader should consult chapter 10 of black holes and time warps by kip s. thorne, or the more recent books, einstein s unfinished symphony, by marcia bartusiak and gravity from the ground up, by bernard schutz. a comprehensive review of developments toward laser interferometer detectors is found in fundamentals of interferometric gravitational wave detectors by peter saulson, and discussions relevant to the technology of both bar and interferometric detectors are found in the detection of gravitational waves edited by david blair. in addition to the wealth of articles that can be found on the home site of this journal, there are also various informative websites that can easily be found, including the homepages of the various international collaborative projects searching for gravitational waves, such as the ligo scientific collaboration. some early relativists were sceptical about the existence of gravitational waves ; however, the 1993 nobel prize in physics was awarded to hulse and taylor for their experimental observations and subsequent interpretations of the evolution of the orbit of the binary pulsar psr 1913 + 16 [185, 295 ], the decay of the binary orbit being consistent with angular momentum and energy being carried away from this system by gravitational waves. gravitational waves are produced when matter is accelerated in an asymmetrical way ; but due to the nature of the gravitational interaction, detectable levels of radiation are produced only when very large masses are accelerated in very strong gravitational fields. such a situation can not be found on earth but is found in a variety of astrophysical systems. gravitational wave signals are expected over a wide range of frequencies ; from 10 hz in the case of ripples in the cosmological background to 10 hz from the formation of neutron stars in supernova explosions. the most predictable sources are binary star systems. however, there are many sources of much - greater astrophysical interest associated with black - hole interactions and coalescences, neutron - star coalescences, neutron stars in low - mass x - ray binaries such as sco - x1, stellar collapses to neutron stars and black holes (supernova explosions), pulsars, and the physics of the early universe. for a full discussion of sources partly because observation of the velocity and polarisation states of the signals will allow a direct experimental check of the wave predictions of general relativity ; but, more importantly, because the detection of the signals should provide observers with new and unique information about astrophysical processes. it is interesting to note that the gravitational wave signal from a coalescing compact binary star system has a relatively simple form and the distance to the source can be obtained from a combination of its signal strength and its evolution in time. if the redshift at that distance is found, hubble s constant the value for which has been a source of lively debate for many years may then be determined with, potentially, a high degree of accuracy [282, 180 ]. only now are detectors being built with the technology required to achieve the sensitivity to observe such interesting sources. gravitational waves are most simply thought of as ripples in the curvature of space - time, their effect being to change the separation of adjacent masses on earth or in space ; this tidal effect is the basis of all present detectors. gravitational wave strengths are characterised by the gravitational - wave amplitude h, given by 1\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$h = { { 2\delta l } \over l},$$\end{document } where l is the change in separation of two masses a distance l apart ; for the strongest - allowed component of gravitational radiation, the value of h is proportional to the third time derivative of the quadrupole moment of the source of the radiation and inversely proportional to the distance to the source. the radiation field itself is quadrupole in nature and this shows up in the pattern of the interaction of the waves with matter. the problem for the experimental physicist is that the predicted magnitudes of the amplitudes or strains in space in the vicinity of the earth caused by gravitational waves even from the most violent astrophysical events are extremely small, of the order of 10 or lower [273, 272 ]. indeed, current theoretical models on the event rate and strength of such events suggest that in order to detect a few events per year from coalescing neutron - star binary systems, for example, an amplitude sensitivity close to 10 over timescales as short as a millisecond is required. if the fourier transform of a likely signal is considered it is found that the energy of the signal is distributed over a frequency range or bandwidth, which is approximately equal to 1/timescale. for timescales of a millisecond the bandwidth is approximately 1000 hz, and in this case the spectral density of the amplitude sensitivity is obtained by dividing 10 by the square root of 1000. thus, detector noise levels must have an amplitude spectral density lower than 10 hz over the frequency range of the signal. signal strengths at the earth, integrated over appropriate time intervals, for a number of sources are shown in figure 2. the weakness of the signal means that limiting noise sources like the thermal motion of molecules in the critical components of the detector (thermal noise), seismic or other mechanical disturbances, and noise associated with the detector readout, whether electronic or optical, must be reduced to an extremely low level. for signals above 10 hz ground based experiments are possible, but for lower frequencies where local fluctuating gravitational gradients and seismic noise on earth become a problem, it is best to consider developing detectors for operation in space. the earliest experiments in the field were ground based and were carried out by joseph weber of the university of maryland in the 1960s. with colleagues he began by looking for evidence of excitation of the normal modes of the earth by very low frequency gravitational waves. efforts to detect gravitational waves via the excitation of earth s normal modes was also pursued by weiss and block. weber then moved on to look for tidal strains in aluminium bars, which were at room temperature and were well isolated from ground vibrations and acoustic noise in the laboratory [310, 311 ]. the bars were resonant at 1600 hz, a frequency where the energy spectrum of the signals from collapsing stars was predicted to peak. despite the fact that weber observed coincident excitations of his detectors placed up to 1000 km apart, at a rate of approximately one event per day, his results were not substantiated by similar experiments carried out in several other laboratories in the usa, germany, britain and russia. it seems unlikely that weber was observing gravitational - wave signals because, although his detectors were very sensitive, being able to detect strains of the order of 10 over millisecond timescales, their sensitivity was far away from what was predicted to be required theoretically. development of weber bar type detectors continued with significant emphasis on cooling to reduce the noise levels, although work in this area is now subsiding with efforts continuing on auriga, nautilus, minigrail [233, 158 ] and mario schenberg [159, 70 ]. in around 2003, the sensitivity of km - scale interferometric gravitational - wave detectors began to surpass the peak sensitivity of these cryogenic bar detectors (10) and, for example, the ligo detectors reached their design sensitivities at almost all frequencies by 2005 (peak sensitivity 2 10 at 200 hz), see section 6.1 for more information on science runs of the recent generation of detectors. in addition to gaining better strain sensitivities, interferometric detectors have a marked advantage over resonant bars by being sensitive to a broader range of frequencies, whereas resonant bar are inherently sensitive only to signals that have significant spectral energy in a narrow band around their resonant frequency. the concept and design of gravitational - wave detectors based on laser interferometers will be introduced in the following section 3.2. an interferometric design of gravitational - wave detector offers the possibility of very high sensitivities over a wide range of frequency. it uses test masses, which are widely separated and freely suspended as pendulums to isolate against seismic noise and reduce the effects of thermal noise ; laser interferometry provides a means of sensing the motion of these masses produced as they interact with a gravitational wave (figure 3). this technique is based on the michelson interferometer and is particularly suited to the detection of gravitational waves as they have a quadrupole nature. waves propagating perpendicular to the plane of the interferometer will result in one arm of the interferometer being increased in length while the other arm is decreased and vice versa. the induced change in the length of the interferometer arms results in a small change in the intensity of the light observed at the interferometer output. as will be explained in detail in the next section 4, the sensitivity of an interferometric gravitational - wave detector is limited by noise from various sources. taking this frequency - dependent noise floor into account for example, the design sensitivity for initial ligo is show in figure 4 plotted alongside the achieved sensitivities of the three individual interferometers during the fifth science run (see section 6.1). such strain sensitivities are expected to allow a reasonable probability for detecting gravitational wave sources. however, in order to guarantee the observation of a full range of sources and to initiate gravitational - wave astronomy, a sensitivity or noise performance approximately ten times better in the mid - frequency range and several orders of magnitude better at 10 hz, is desired. therefore, initial detectors will be upgraded to an advanced configuration, such as advanced ligo, which will be ready for operation around 2015. figure 4measured sensitivity of the initial ligo interferometers during the s5 science run (see section 6.1.2). reproduced with permission from. measured sensitivity of the initial ligo interferometers during the s5 science run (see section 6.1.2). reproduced with permission from. for the initial interferometric detectors, a noise floor in strain close to 2 10 hz was achieved. detecting a strain in space at this level implies measuring a residual motion of each of the test masses of about 8 10 m / hz over part of the operating range of the detector, which may be from 10 hz to a few khz. this sets a formidable goal for the optical detection system at the output of the interferometer. in this section we discuss the main noise sources, which limit the sensitivity of interferometric gravitational - wave detectors. fundamentally it should be possible to build systems using laser interferometry to monitor strains in space, which are limited only by the heisenberg uncertainty principle ; however there are other practical issues, which must be taken into account. fluctuating gravitational gradients pose one limitation to the interferometer sensitivity achievable at low frequencies, and it is the level of noise from this source, which dictates that experiments to look for sub - hz gravitational - wave signals have to be carried out in space [291, 275, 93, 184 ]. in general, for ground - based detectors the most important limitations to sensitivity result from the effects of seismic and other ground - borne mechanical noise, thermal noise associated with the test masses and their suspensions, and quantum noise, which appears at high frequency as shot noise in the photocurrent from the photodiode, which detects the interference pattern and can appear at low frequency as radiation pressure noise due to momentum transfer to the test masses from the photons when using high laser powers. the significance of each of these sources will be briefly reviewed. seismic noise at a reasonably quiet site on the earth follows a spectrum in all three dimensions close to 10f m / hz (where here and elsewhere we measure f in hz) and thus if the disturbance to each test mass must be less than 3 10 m / hz at, for example, 30 hz, then the reduction of seismic noise required at that frequency in the horizontal direction is greater than 10. since there is liable to be some coupling of vertical noise through to the horizontal axis, along which the gravitational - wave - induced strains are to be sensed, a significant level of isolation has to be provided in the vertical direction also. isolation can be provided in a relatively simple way by making use of the fact that, for a simple pendulum system, the transfer function to the pendulum mass of the horizontal motion of the suspension point falls off as 1/(frequency) above the pendulum resonance. in a similar way isolation can be achieved in the vertical direction by suspending a mass on a spring. in the case of the virgo detector system the design allows operation to below 10 hz and here a seven - stage horizontal pendulum arrangement is adopted with six of the upper stages being suspended with cantilever springs to provide vertical isolation, with similar systems developed in australia and at caltech. for the geo600 detector, where operation down to 50 hz was planned, a triple pendulum system is used with the first two stages being hung from cantilever springs to provide the vertical isolation necessary to achieve the desired performance. this arrangement is then hung from a plate mounted on passive rubber isolation mounts and on an active (electro - mechanical) anti - vibration system [257, 298 ]. the upgraded seismic isolation for the total isolation will be provided by one external stage (hydraulics), two stages of in - vacuum active isolation, and being completed by the test mass suspensions [55, 166 ]. for clarity, the two stages of in - vacuum isolation are shown in figure 5, whereas the test - mass suspensions are shown separately in figure 6. figure 5internal stages of the large chamber seismic isolation system for advanced ligo (image is inverted for clarity). figure 6cad drawing of quad suspension system for advanced ligo, showing the mirror test mass at the bottom and where the uppermost section is attached to the third stage platform of the large chamber seismic isolation system shown in figure 5. internal stages of the large chamber seismic isolation system for advanced ligo (image is inverted for clarity). cad drawing of quad suspension system for advanced ligo, showing the mirror test mass at the bottom and where the uppermost section is attached to the third stage platform of the large chamber seismic isolation system shown in figure 5. in order to cut down motion at the pendulum frequencies, active damping of the pendulum modes has to be incorporated, and to reduce excess motion at low frequencies around the micro - seismic peak, low - frequency isolators have to be incorporated. these low - frequency isolators can take different forms tall inverted pendulums in the horizontal direction and cantilever springs whose stiffness is reduced by means of attractive forces between magnets for the vertical direction in the case of the virgo system, scott russell mechanical linkages in the horizontal and torsion bar arrangements in the vertical for an australian design, and a seismometer / actuator (active) system as shown here for advanced ligo and also used in geo600. such schemes can provide sufficiently - large reduction in the direct mechanical coupling of seismic noise through to the suspended mirror optic to allow operation down to 3 hz [98, 134 ]. however, it is also possible for this vibrational seismic noise to couple to the suspended optic through the gravitational field. gravity gradients, caused by direct gravitational coupling of mass density fluctuations to the suspended mirrors, were identified as a potential source of noise in ground - based gravitational - wave detectors in 1972. the noise associated with gravity gradients was first formulated by saulson and spero, with later developments by hughes and thorne and cella and cuoco. these studies suggest that the dominant source of gravity gradients arise from seismic surface waves, where density fluctuations of the earth s surface are produced near the location of the individual interferometer test masses, as shown in figure 7. figure 7time - lapsed schematic illustrating the fluctuating gravitational force on a suspended mass by the propagation of a surface wave through the ground. time - lapsed schematic illustrating the fluctuating gravitational force on a suspended mass by the propagation of a surface wave through the ground. the magnitude of the rms motion of the interferometer test masses, \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\tilde x(\omega)$\end{document }, can be shown to be 2\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\tilde x(\omega) = { { 4\pi g\rho } \over { { \omega ^2}}}\beta (\omega)\tilde w(\omega),$$\end{document } where is the earth s density near the test mass, g is newton s constant, is the angular frequency of the seismic spectrum, () ; is a dimensionless reduced transfer function that takes into account the correlated motion of the interferometer test masses in addition to the reduction due to the separation between the test mass and the earth s surface, and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\tilde w(\omega)$\end{document } is the displacement rms - averaged over 3-dimensional directions. in order to eliminate noise arising from gravity gradients, a detector would have to be operated far from these density fluctuations, that is, in space.. however, there are two proposed approaches for reducing the level of gravity - gradient noise in future ground - based detectors. a monitor and subtraction method can be used, where an array of seismometers can be distributed strategically around each test mass to monitor the relevant ground motion (and ground compression) that would be expected to couple through local gravity. a subtraction signal may be developed from knowing how the observed density fluctuations couple to the motion of each test mass, and can potentially allow a significant reduction in gravity - gradient noise. another approach is to choose a very quiet location, or better still, to also go underground, as is already going ahead for lcgt. since the dominant source of gravity - gradient noise is expected to arise from surface waves on the earth, the observed gravity - gradient noise will decrease with depth into the earth. current estimates suggest that gravity - gradient noise can be suppressed down to around 1 hz by careful site selection and going 150 m underground. the most promising approach (or likely only approach) to detecting gravitational waves whose frequency is below 1 hz is to build an interferometer in space. thermal noise associated with the mirror masses and the last stage of their suspensions is the most significant noise source at the low frequency end of the operating range of initial long baseline gravitational wave detectors. advanced detector configurations are also expected to be limited by thermal noise at their most sensitive frequency band [211, 238, 168, 121 ]. the thermal noise in the operating range comes from the tails of these resonant modes. for any simple harmonic oscillator such as a mass hung on a spring or hung as a pendulum, the spectral density of thermal motion of the mass can be expressed as 3\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${x^2}(\omega) = { { 4{k_{\rm{b}}}t\omega _ 0 ^ 2\phi (\omega) } \over { \omega m[{{(\omega _ 0 ^ 2 - { \omega ^2})}^2 } + \omega _ 0 ^ 4{\phi ^2}(\omega)]}},$$\end{document } where kb is boltzmann s constant, t is the temperature, m is the mass and () is the loss angle or loss factor of the oscillator of angular resonant frequency 0. this loss factor is the phase lag angle between the displacement of the mass and any force applied to the mass at a frequency well below 0. in the case of a mass on a spring, the loss factor is a measure of the mechanical loss associated with the material of the spring. for a pendulum, thus, the loss factor is lower than that of the material, which is used for the wires or fibres used to suspend the pendulum. indeed, following saulson it can be shown that for a pendulum of mass m, suspended on four wires or fibres of length l, the loss factor of the pendulum is related to the loss factor of the material by 4\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${\phi _ { { \rm{pend}}}}(\omega) = { \phi _ { { \rm{mat}}}}(\omega){{4\sqrt { tei } } \over { mgl}},$$\end{document } where i is the moment of the cross - section of each wire, and t is the tension in each wire, whose material has a young s modulus e. in general, for most materials, it appears that the intrinsic loss factor is essentially independent of frequency over the range of interest for gravitational - wave detectors (although care has to be taken with some materials in that a form of damping known as thermo - elastic damping can become important for wires of small cross - section and for some bulk crystalline materials). in order to estimate the internal thermal noise of a test mass, each resonant mode of the mass when the detector operating range is well below the resonances of the masses, following saulson, the effective spectral density of thermal displacement of the front face of each mass can be expressed as the summation of the motion of the various mechanical resonances of the mirror as also discussed by gillespie and raab. however, this intuitive approach to calculating the thermally - driven motion is only valid when the mechanical loss is distributed homogeneously and, therefore, not valid for real test - mass mirrors. the mechanical loss is known to be inhomogeneous due to, for example, the localisation of structural defects and stress within the bulk material, and the mechanical loss associated with the polished surfaces is higher than the levels typically associated with bulk effects. therefore, levin suggested using a direct application of the fluctuation - dissipation theorem to the optically - sensed position of the mirror substrate surface. this technique imposes a notional pressure (of the same spatial profile as the intensity of the sensing laser beam) to the front face of the substrate and calculates the resulting power dissipated in the substrate on its elastic deformation under the applied pressure. using such an approach we find that sx(f) can then be described by the relation 5\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${s_x}(f) = { { 2{k_{\rm{b}}}t } \over { { \pi ^2}{f^2}}}{{{w_{{\rm{diss } } } } } \over { f_0 ^ 2}},$$\end{document } where f0 is the peak amplitude of the notional oscillatory force and wdiss is the power dissipated in the mirror described as, 6\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${w_{{\rm{diss } } } } = \omega \int { \epsilon (r)\phi (r)\partial v }, $ $ \end{document } where (r) and (r) are the strain and mechanical loss located at specific positions within the volume v. this formalisation highlights the importance of where mechanical dissipation is located with respect to the sensing laser beam. in particular, the thermal noise associated with the multi - layer dielectric mirror coatings, required for high reflectivity, will in fact limit the sensitivity of second - generation gravitational - wave detectors at their most sensitive frequency band, despite these coatings typically being only 4.5 m in thickness. identifying coating materials with lower mechanical loss, and trying to understand the sources of mechanical loss in existing coating materials, is a major r&d effort targeted at enhancements to advanced detectors and for third generation instruments. in order to keep thermal noise as low as possible the mechanical loss factors of the masses and pendulum resonances further, the test masses must have a shape such that the frequencies of the internal resonances are kept as high as possible, must be large enough to accommodate the laser beam spot without excess diffraction losses, and must be massive enough to keep the fluctuations due to radiation pressure at an acceptable level. test masses currently range in mass from 6 kg for geo600 to 40 kg for advanced ligo. to approach the best levels of sensitivity discussed earlier the loss factors of the test masses must be 3 10 or lower, and the loss factor of the pendulum resonances should be smaller than 10. obtaining these values puts significant constraints on the choice of material for the test masses and their suspending fibres. geo600 utilises very - low - loss silica suspensions, a technology, which should allow detector sensitivities to approach the level desired for second generation instruments [105, 266, 268 ], since the intrinsic loss factors in samples of synthetic fused silica have been measured down to around 5 10. still, the use of other materials such as sapphire is being seriously considered for future detectors [104, 195, 266 ] such as in lcgt [234, 247 ]. the technique of hydroxy - catalysis bonding provides a method of jointing oxide materials in a suitably low - loss way to allow monolithic suspension systems to be constructed. a recent discussion on the level of mechanical loss and the associated thermal noise in advanced detectors resulting from hydroxy - catalysis bonds is given by cunningham.. images of the geo600 monolithic mirror suspension and of the prototype advanced ligo mirror suspension are shown in figure 8. figure 8monolithic silica suspension of (a) geo600 6 kg mirror test mass suspended from 4 fibres of thickness 250 m and (b) prototype monolithic suspension for advanced ligo at lasti (mirror mass of 40 kg, silica fibre thickness 400 m). monolithic silica suspension of (a) geo600 6 kg mirror test mass suspended from 4 fibres of thickness 250 m and (b) prototype monolithic suspension for advanced ligo at lasti (mirror mass of 40 kg, silica fibre thickness 400 m). for gravitational - wave signals to be detected, the output of the interferometer must be held at one of a number of possible points on an interference fringe. an obvious point to choose is halfway up a fringe since the change in photon number produced by a given differential change in arm length is greatest at this point (in practice this is not at all a sensible option and interferometers are operated at, or near, a dark fringe see sections 5.1 and 5.4). the interferometer may be stabilised to this point by sensing any changes in intensity at the interferometer output with a photodiode and feeding the resulting signal back, with suitable phase, to a transducer capable of changing the position of one of the interferometer mirrors. information about changes in the length of the interferometer arms can then be obtained by monitoring the signal fed back to the transducer. as mentioned earlier, it is very important that the system used for sensing the optical fringe movement on the output of the interferometer can resolve strains in space of 2 10 hz or lower, or differences in the lengths of the two arms of less than 10 m / hz, a minute displacement compared to the wavelength of light 10 m. a limitation to the sensitivity of the optical readout scheme is set by shot noise in the detected photocurrent. from consideration of the number of photoelectrons (assumed to obey poisson statistics) measured in a time it can be shown that the detectable strain sensitivity depends on the level of laser power p of wavelength used to illuminate the interferometer of arm length l, and on the time, such that : 7\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${\rm{detectable}}\;{\rm{strain}}\;{\rm{in}}\;{\rm{time}}\;\tau = { 1 \over l}{\left [{ { { \lambda hc } \over { 2{\pi ^2}p\tau } } } \right]^{1/2}},$$\end{document } or 8\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${\rm{detectable}}\;{\rm{strain}}\;{{\rm{(hz)}}^{- 1/2 } } = { 1 \over l}{\left [{ { { \lambda hc } \over { { \pi ^2}p } } } \right]^{1/2}},$$\end{document } where c is the velocity of light, h is planck s constant and we assume that the photodetectors have a quantum efficiency 1. thus, achievement of the required strain sensitivity level requires a laser, operating at a wavelength of 10 m, to provide 6 10 power at the input to a simple michelson interferometer. this is a formidable requirement ; however, there are a number of techniques which allow a large reduction in this power requirement and these will be discussed in section 5. as the effective laser power in the arms is increased, another phenomenon becomes increasingly important arising from the effect on the test masses of fluctuations in the radiation pressure. one interpretation on the origin of this radiation pressure noise may be attributed to the statistical uncertainty in how the beamsplitter divides up the photons of laser light. each photon is scattered independently and therefore produces an anti - correlated binomial distribution in the number of photons, n, in each arm, resulting in a \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$ \propto \sqrt n$\end{document } fluctuating force from the radiation pressure. this is more formally described as arising from the vacuum (zero - point) fluctuations in the amplitude component of the electromagnetic field. this additional light entering through the dark - port side of the beamsplitter, when being of suitable phase, will increase the intensity of laser light in one arm, while decreasing the intensity in the other arm, again resulting in anti - correlated variations in light intensity in each arm [109, 110 ]. the laser light is essentially in a noiseless coherent state as it splits at the beamsplitter and fluctuations arise entirely from the addition of these vacuum fluctuations entering the unused port of the beamsplitter. using this understanding of the coherent state of the laser, shot noise arises from the uncertainty in the phase component (quadrature) of the interferometer s laser field and is observed in the quantum fluctuations in the number of detected photons at the interferometer output. radiation pressure noise arises from uncertainty in the amplitude component (quadrature) of the interferometer s laser field. both result in an uncertainty in measured mirror positions. for the case of a simple michelson, shown in figure 3, the power spectral density of the fluctuating motion of each test mass m resulting from fluctuation in the radiation pressure at angular frequency is given by, 9\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\delta { x^2}(\omega) = \left({{{4ph } \over { { m^2}{\omega ^4}c\lambda } } } \right),$$\end{document } where h is planck s constant, c is the speed of light and is the wavelength of the laser light. in the case of an interferometer with fabry - prot cavities, where the typical number of reflections is 50, displacement noise x due to radiation pressure fluctuations scales linearly with the number of reflections, such that, 10\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\delta { x^2}(\omega) = { 50 ^ 2 } \times \left({{{4ph } \over { { m^2}{\omega ^4}c\lambda } } } \right).$$\end{document } radiation pressure may be a significant limitation at low frequency and is expected to be the dominant noise source in advanced ligo between around 10 and 50 hz. of course the effects of the radiation pressure fluctuations can be reduced by increasing the mass of the mirrors, or by decreasing the laser power at the expense of degrading sensitivity at higher frequencies. since the effect of photoelectron shot noise decreases when increasing the laser power as the radiation pressure noise increases, a fundamental limit to displacement sensitivity is set. for a particular frequency of operation, there will be an optimum laser power within the interferometer, which minimises the effect of these two sources of optical noise, which are assumed to be uncorrelated. this sensitivity limit is known as the standard quantum limit (sql) and corresponds to the heisenberg uncertainty principle, in its position and momentum formulation ; see [133, 109, 110, 221 ]. firstly, it is possible to reach the sql at a tuned range of frequencies, when dominated by either radiation - pressure noise or shot noise, by altering the noise distribution in the two quadratures of the vacuum field. there are a number of proposed designs for achieving this in future interferometric detectors, such as a squeezed - input interferometer [110, 300 ], a variational - output interferometer or a squeezed - variational interferometer using a combination of both techniques. this technique may be of importance in allowing an interferometer to reach the sql at a particular frequency, for example, when using lower levels of laser power and otherwise being dominated by shot noise. experiments are under way to incorporate squeezed - state injection as part of the upgrades to current gravitational - wave detectors, and where a squeezing injection bench has already been installed in the geo600 gravitational - wave detector, which expects to be able to achieve an up - to-6 db reduction in shot noise using the current interferometer configuration. similar experiments are also under way to demonstrate variational readout, where ponderomotive squeezing arises from the naturally - occurring correlation of radiation - pressure noise to shot noise upon reflection of light from a mirror [117, 271 ] secondly, if correlations exist between the radiation - pressure noise and the shot - noise displacement limits, then it is possible to bypass the sql, at least in principle. there are at least two ways by which such correlations may be introduced into an interferometer. one scheme is where an optical cavity is constructed, where there is a strong optical spring effect, coupling the optical field to the mechanical system. this is already the case for the geo600 detector, where the addition of a signal recycling cavity creates such correlation, where signal recycling is described in section 5.2. other schemes of optical springs have been studied, such as optical bars and optical levers [103, 101 ]. another method is to use suitable filtering at optical frequencies of the output signal, by means of long fabry - prot cavities, which effectively introduces correlation [201, 116 ]. as explained in section 4.4.1, high - power laser light is needed to overcome limitations of a detector s sensitivity due to photoelectron shot noise. the situation can be helped greatly if a multi - pass arrangement is used in the arms of the interferometer as this multiplies up the apparent movement by the number of bounces the light makes in the arms. the multiple beams can either be separate, as in an optical delay line [313, 95 ], or may lie on top of each other as in a fabry - prot resonant cavity, as shown in figure 9. figure 9michelson interferometers with (a) delay lines and (b) fabry - prot cavities in the arms of the interferometer. michelson interferometers with (a) delay lines and (b) fabry - prot cavities in the arms of the interferometer. optimally, the light should be stored for a time comparable to the characteristic timescale of the signal. thus, if signals of characteristic timescale 1 msec are to be searched for, the number of bounces should be approximately 50 for an arm length of 3 km. with 50 bounces the required laser power it can be shown that an optimum signal - to - noise ratio in a michelson interferometer can be obtained when the arm lengths are such that the output light is very close to a minimum (this is not intuitively obvious and is discussed more fully in). thus, rather than lock the interferometer to the side of a fringe as discussed above in section 4.4.1, it is usual to make use of a modulation technique to operate the interferometer close to a null in the interference pattern. an electro - optic phase modulator placed in front of the interferometer can be used to phase modulate the input laser light. if the arms of the interferometer are arranged to have a slight mismatch in length this results in a detected signal, which, when demodulated, is zero with the cavity exactly on a null fringe and changes sign on different sides of the null providing a bipolar error signal ; this can be fed back to the transducer controlling the interferometer mirror to hold the interferometer locked near to a null fringe (this is the rf readout scheme discussed in section 5.4). in this situation, if the mirrors are of very low optical loss, nearly all of the light supplied to the interferometer is reflected back towards the laser. in other words the impedance matching can be improved by placing another mirror of correctly chosen transmission a power recycling mirror between the laser and the interferometer so that a resonant cavity is formed between this mirror and the rest of the interferometer ; in the case of perfect impedance matching, no light is reflected back towards the laser [131, 278 ]. there is then a power build - up inside the interferometer as shown in figure 10. this can be high enough to create the required kilowatts of laser light at the beamsplitter, starting from an input laser light of only about 10 w. figure 10the implementation of power recycling on a michelson interferometer with fabry - prot cavities. the implementation of power recycling on a michelson interferometer with fabry - prot cavities. to be more precise, if the main optical power losses are those associated with the test mass mirrors taken to be a per reflection the intensity inside the whole system considered as one large cavity is increased by a factor given by (l)/(ca), where the number of bounces, or light storage time, is optimised for signals of timescale and the other symbols have their usual meaning. then : 11\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${\rm{detectable}}\;{\rm{strain}}\;{\rm{in}}\;{\rm{time}}\;\tau = { \left [{ { { \lambda ha } \over { 4\pi lp{\tau ^2 } } } } \right]^{1/2}}.$$\end{document } to enhance further the sensitivity of an interferometric detector and to allow some narrowing of the detection bandwidth, which may be valuable in searches for continuous wave sources of gravitational radiation, another technique known as signal recycling can be implemented [231, 292, 169 ]. this relies on the fact that sidebands created on the light by gravitational - wave signals interacting with the arms do not interfere destructively and so do appear at the output of the interferometer. if a mirror of suitably - chosen reflectivity is put at the output of the system as shown in figure 11, then the sidebands can be recycled back into the interferometer, where they resonate, and hence the signal size over a given bandwidth (set by the mirror reflectivity) is enhanced. figure 11the implementation of signal recycling on a michelson interferometer with fabry - prot cavities. the centre of this frequency band is set by the precise length of the cavity formed by the signal recycling mirror and the cavities in the interferometer arms. thus, control of the precise position of the signal recycling mirror allows tuning of the frequency at which the performance is peaked. often signal recycling will be used to provide a narrow bandwidth to search for continuous wave sources as mentioned above, however it may also be used with a relatively broad bandwidth, centred away from zero frequency, and this application is useful for matching the frequency response of the detector to expected spectral densities of certain broadband or chirping signals. using appropriate optical configurations that employ power and signal recycling as described in sections 5.1 and 5.2, the required laser power may thus be reduced to a level (in the range of 10 to 100 w) where laser sources are now available ; however stringent requirements on technical noise must be satisfied. power fluctuations as described above in section 5.1 gravitational - wave interferometers are typically designed to operate with the length of the interferometer arms set such that the output of the interferometer is close to a minimum in the output light. if the interferometer were operated exactly at the null point in the fringe pattern, then, in principle, it would be insensitive to power fluctuations in the input laser light. however in practice there will be small offsets from the null position causing some sensitivity to this noise source. in this case, it can be shown that the required power stability of the laser in the frequency range of interest for gravitational - wave detection may be estimated to be 12\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${{\delta p } \over p } \simeq h{(\delta l / l)^{- 1}},$$\end{document } where p / p are the relative power fluctuations of the laser, and l is the offset from the null fringe position for an interferometer of arm length l. from calculations of the effects of low - frequency seismic noise for the initial designs of long baseline detectors it can be estimated that the rms motion will be on the order of 10 m when the system is operating. taking strains of around 3 10 hz at 300 hz, as is the case for advanced ligo, requires power fluctuations of the laser not to exceed 13\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${{\delta p } \over p } \leq { 10^{- 7}}{\rm{h}}{{\rm{z}}^{- 1/2}}\;\;{\rm{at } } \simeq 300\;{\rm{hz}}.$$\end{document } to achieve this level of power fluctuation typically requires the use of active stabilisation techniques of the type developed for argon ion lasers. however, it should be noted that the most stringent constraint on laser - power fluctuations in future ground - based detectors, where circulating laser powers will approach 1 mw or even beyond, will ultimately arise from classical radiation pressure on the mirrors, as described in section 4.4.2. frequency fluctuations for a simple michelson interferometer it can be shown that a change x in the differential path length, x, of the interferometer arms causes a phase change in the light at the interferometer output given by = (2/c) (vx + xv) where is a change in the laser frequency and c is the speed of light. it follows that if the lengths of the interferometer arms are exactly equal (i.e., x = 0), the interferometer output is insensitive to fluctuations in the frequency of the input laser light, provided that, in the case of fabry - prot cavities in the arms, the fluctuations are not so great that the cavities can not remain on resonance. however, in practice, differences in the optical properties of the interferometer mirrors result in slightly different effective arm lengths, a difference of perhaps a few tens of metres. then the relationship between the limit to detectable gravitational - wave amplitude and the fluctuations d of the laser frequency is given by 14\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${{\delta \nu } \over \nu } \simeq h{(x / l)^{- 1}}.$$\end{document } hence, to achieve the target sensitivity used in the above calculation using a detector with arms of length 4 km, maximal fractional frequency fluctuations of, 15\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${{\delta \nu } \over \nu } \leq { 10^{- 21}}{\rm{h}}{{\rm{z}}^{- 1/2}},$$\end{document } are required. this level of frequency noise may be achieved by the use of appropriate laser - frequency stabilisation systems involving high finesse reference cavities.although the calculation here is for a simple michelson interferometer, similar arguments apply to the more sophisticated systems with arm cavities, power recycling and signal recycling discussed earlier and lead to the same conclusions. frequency stabilisation is also important in other applications such as in high resolution optical spectroscopy, optical frequency standards [223, 312 ] and fundamental quantum measurements. the best reference cavities, such as those developed by the ye and hnsch groups, have reported a frequency stability performance of around 10, which is broadly equivalent to that achieved for ground - based gravitational - wave detectors when scaling by cavity length. beam geometry fluctuations fluctuations in the lateral or angular position of the input laser beam, along with changes in its size and variations in its phase - front curvature may all couple into the output signal of an interferometer and reduce its sensitivity. these fluctuations may be due to intrinsic laser mechanical noise (from water cooling for example) or from seismic motion of the laser with respect to the isolated test masses. as an example of their importance, fluctuations in the lateral position of the beam may couple into interferometer measurements through a misalignment of the beamsplitter with respect to the interferometer mirrors. a lateral movement z of the beam incident on the beamsplitter, coupled with an angular misalignment of the beamsplitter of /2 results in a phase mismatch of the interfering beams, such that 16\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\delta \phi = (4\pi /\lambda)\alpha \delta z.$$\end{document } a typical beamsplitter misalignment of 10 radians means that to achieve sensitivities of the level described above using a detector with 3 or 4 km arms, and 50 bounces of the light in each arm, a level of beam geometry fluctuations at the beamsplitter of close to 10 m / hz at 300 hz is required.typically, and ignoring possible ameliorating effects of the power recycling cavity on beam geometry fluctuations, this will mean that the beam positional fluctuations of the laser need to be suppressed by several orders of magnitude. the two main methods of reducing beam geometry fluctuations are 1) passing the input beam through a single mode optical fibre and 2) using a resonant cavity as a mode cleaner [270, 288, 317, 83].passing the beam through a single mode optical fibre helps to eliminate beam geometry fluctuations, as deviations of the beam from a gaussian tem00 mode are equivalent to higher - order spatial modes, which are thus attenuated by the optical fibre. however, there are limitations to the use of optical fibres, mainly due to the limited power - handling capacity of the fibres ; care must also be taken to avoid introducing extra beam geometry fluctuations from movements of the fibre itself.a cavity may be used to reduce beam geometry fluctuations if it is adjusted to be resonant only for the tem00 mode of the input light. the use of a resonant cavity should allow the handling of higher laser powers and has the additional benefits of acting as a filter for fast fluctuations in laser frequency and power [288, 317 ]. this latter property is extremely useful for the conditioning of the light from some laser sources as will be discussed below. as described above in section 5.1 gravitational - wave interferometers are typically designed to operate with the length of the interferometer arms set such that the output of the interferometer is close to a minimum in the output light. if the interferometer were operated exactly at the null point in the fringe pattern, then, in principle, it would be insensitive to power fluctuations in the input laser light. however in practice there will be small offsets from the null position causing some sensitivity to this noise source. in this case, it can be shown that the required power stability of the laser in the frequency range of interest for gravitational - wave detection may be estimated to be 12\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${{\delta p } \over p } \simeq h{(\delta l / l)^{- 1}},$$\end{document } where p / p are the relative power fluctuations of the laser, and l is the offset from the null fringe position for an interferometer of arm length l. from calculations of the effects of low - frequency seismic noise for the initial designs of long baseline detectors it can be estimated that the rms motion will be on the order of 10 m when the system is operating. taking strains of around 3 10 hz at 300 hz, as is the case for advanced ligo, requires power fluctuations of the laser not to exceed 13\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${{\delta p } \over p } \leq { 10^{- 7}}{\rm{h}}{{\rm{z}}^{- 1/2}}\;\;{\rm{at } } \simeq 300\;{\rm{hz}}.$$\end{document } to achieve this level of power fluctuation typically requires the use of active stabilisation techniques of the type developed for argon ion lasers. however, it should be noted that the most stringent constraint on laser - power fluctuations in future ground - based detectors, where circulating laser powers will approach 1 mw or even beyond, will ultimately arise from classical radiation pressure on the mirrors, as described in section 4.4.2. frequency fluctuations for a simple michelson interferometer it can be shown that a change x in the differential path length, x, of the interferometer arms causes a phase change in the light at the interferometer output given by = (2/c) (vx + xv) where is a change in the laser frequency and c is the speed of light. it follows that if the lengths of the interferometer arms are exactly equal (i.e., x = 0), the interferometer output is insensitive to fluctuations in the frequency of the input laser light, provided that, in the case of fabry - prot cavities in the arms, the fluctuations are not so great that the cavities can not remain on resonance. however, in practice, differences in the optical properties of the interferometer mirrors result in slightly different effective arm lengths, a difference of perhaps a few tens of metres. then the relationship between the limit to detectable gravitational - wave amplitude and the fluctuations d of the laser frequency is given by 14\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${{\delta \nu } \over \nu } \simeq h{(x / l)^{- 1}}.$$\end{document } hence, to achieve the target sensitivity used in the above calculation using a detector with arms of length 4 km, maximal fractional frequency fluctuations of, 15\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${{\delta \nu } \over \nu } \leq { 10^{- 21}}{\rm{h}}{{\rm{z}}^{- 1/2}},$$\end{document } are required. this level of frequency noise may be achieved by the use of appropriate laser - frequency stabilisation systems involving high finesse reference cavities. although the calculation here is for a simple michelson interferometer, similar arguments apply to the more sophisticated systems with arm cavities, power recycling and signal recycling discussed earlier and lead to the same conclusions. frequency stabilisation is also important in other applications such as in high resolution optical spectroscopy, optical frequency standards [223, 312 ] and fundamental quantum measurements. the best reference cavities, such as those developed by the ye and hnsch groups, have reported a frequency stability performance of around 10, which is broadly equivalent to that achieved for ground - based gravitational - wave detectors when scaling by cavity length. beam geometry fluctuations fluctuations in the lateral or angular position of the input laser beam, along with changes in its size and variations in its phase - front curvature may all couple into the output signal of an interferometer and reduce its sensitivity. these fluctuations may be due to intrinsic laser mechanical noise (from water cooling for example) or from seismic motion of the laser with respect to the isolated test masses. as an example of their importance, fluctuations in the lateral position of the beam may couple into interferometer measurements through a misalignment of the beamsplitter with respect to the interferometer mirrors. a lateral movement z of the beam incident on the beamsplitter, coupled with an angular misalignment of the beamsplitter of /2 results in a phase mismatch of the interfering beams, such that 16\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\delta \phi = (4\pi /\lambda)\alpha \delta z.$$\end{document } a typical beamsplitter misalignment of 10 radians means that to achieve sensitivities of the level described above using a detector with 3 or 4 km arms, and 50 bounces of the light in each arm, a level of beam geometry fluctuations at the beamsplitter of close to 10 m / hz at 300 hz is required. typically, and ignoring possible ameliorating effects of the power recycling cavity on beam geometry fluctuations, this will mean that the beam positional fluctuations of the laser need to be suppressed by several orders of magnitude. the two main methods of reducing beam geometry fluctuations are 1) passing the input beam through a single mode optical fibre and 2) using a resonant cavity as a mode cleaner [270, 288, 317, 83 ]. passing the beam through a single mode optical fibre helps to eliminate beam geometry fluctuations, as deviations of the beam from a gaussian tem00 mode are equivalent to higher - order spatial modes, which are thus attenuated by the optical fibre. however, there are limitations to the use of optical fibres, mainly due to the limited power - handling capacity of the fibres ; care must also be taken to avoid introducing extra beam geometry fluctuations from movements of the fibre itself. a cavity may be used to reduce beam geometry fluctuations if it is adjusted to be resonant only for the tem00 mode of the input light. the use of a resonant cavity should allow the handling of higher laser powers and has the additional benefits of acting as a filter for fast fluctuations in laser frequency and power [288, 317 ]. this latter property is extremely useful for the conditioning of the light from some laser sources as will be discussed below. from equation (7) it can be seen that the photon - noise limited sensitivity of an interferometer is proportional to \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\sqrt p$\end{document } where p is the laser power incident on the interferometer, and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\sqrt \lambda$\end{document } where is the wavelength of the laser light. thus, single frequency lasers of high output power and short wavelength are desirable. with these constraints in mind, argon - ion lasers emitting light at 514 nm were used to illuminate several interferometric gravitational - wave detector prototypes, see, for example, [287, 264 ]. however, their efficiency, reliability, controllability and noise performance has ruled them out as suitable laser sources for current and future gravitational wave detectors. nd : yag lasers, emitting at 1064 nm or frequency doubled to 532 nm, present an alternative. the longer (infrared) wavelength may initially appear less desirable than the 514 nm of the argon - laser and the frequency doubled 532 nm, as more laser power is needed to obtain the same sensitivity. in addition, the resulting increase in beam diameter leads to a need for larger optical components. for example in an optical cavity the diameter of the beam at any point is proportional to the square root of the wavelength and to keep diffractive losses at each test mass below 1 10, it can be shown that the diameter of each test mass must be greater than 2.6 times the beam diameter at the test mass. thus, the test masses for gravitational wave detectors have to be 1.4 times larger in diameter for infrared than for green light. however, nd : yag sources at 1064 nm have demonstrated some compelling advantages, in particular the demonstration of scaling the power up to levels suitable for second generation interferometers (200 w) combined with their superior efficiency [286, 306, 200 ]. frequency - doubled nd : yvo lasers at 532 nm have currently only been demonstrated to powers approaching 20 w and have not been actively stabilised to the levels needed for gravitational - wave detectors. an additional problem associated with shorter wavelength operation is the potential for increased absorption, possibly leading to photochemistry (damage) in the coating materials, in addition to increased scatter. for this reason, all the initial long - baseline interferometer projects, along with their respective upgrades, have chosen some form of nd : yag light source at 1064 nm. as an example, the laser power is being upgraded from 10 w in initial ligo to 180 w for advanced ligo to improve the snr of the shot - noise - limited regime. this power will be delivered by a three stage injection - locked oscillator scheme [120, 237, 157, 144 ]. the first stage uses a monolithic non - planar ring oscillator (npro) to initially produce 2 w of output power. this output is subsequently amplified by a four - head nd : yvo laser amplifier to a power of 35 w, which is in turn delivered into an injection locked nd : yag oscillator to produce 200 w of output power. other laser developments are being pursued, such as high - power - fibre lasers, which are currently being investigated by the aei in germany and prototyped for advanced virgo by grverie. in france. fibre amplifiers show great potential for extrapolation to higher laser powers in addition to lower production costs. third - generation interferometric gravitational - wave detectors, such as the einstein telescope, require input laser powers of around 500 w at 1064 nm in order to achieve their high - frequency shot - noise - limited sensitivities. low - frequency sensitivity is expected to be achieved through the use of separate low - power interferometers with silicon optics operating at cryogenic temperatures [269, 262 ]. longer wavelengths are proposed here due to excessive absorption in silicon at 1064 nm and the expected low absorption (less than 0.1 ppm / cm) at around 1550 nm. worldwide laser developments may provide new baseline light sources that can provide different wavelength and power options for future detectors. however, the stringent requirements on the temporal and spatial stability for gravitational - wave detectors are beyond that sought in other laser applications. therefore, a dedicated laser - development program will be required to continue beyond the second - generation interferometers in order to design and build a laser system that meets third - generation requirements, as discussed in more detail in. another key area of laser development, targeted at improving the sensitivity of future gravitational - wave detectors, is in the use of special optical modes to reduce thermal noise. it can be shown that the amplitude of thermal noise associated with the mirror coatings is inversely proportional to the beam radius. the configuration within current interferometers is designed to inject and circulate tem00 optical modes, which have a gaussian beam profile. to keep diffraction losses suitably low for this case (< 1 ppm), a beam radius of a maximum size 35% of the radius of the test mass mirror can be used. the thermal noise could be further reduced if optical modes are circulated that have a larger effective area, yet not increasing the level of diffraction losses. this would be possible through the use of higher - order laguerre - gauss beams, and other a more in - depth discussion of how these optical schemes can be implemented and the potential increase in detector sensitivity attainable can be found in. despite the very - low levels of optical absorption in fused silica at 1064 nm, thermal loading due to high - levels of circulating laser powers within advanced gravitational - wave detectors will cause significant thermal loading. in the case of advanced ligo, thermal lensing will be most significant in the input test masses of the fabry - prot cavities, where the beam must transmit through the substrate in addition to the high - power within the cavity being incident on the coating surfaces. thermal distortion in the optics will be sensed by hartmann sensors and coupled to two schemes of thermal compensation. firstly, ring resistance heaters will be installed around the barrel of the input mass in order to compensate for the beam heating the central region of the optics, as demonstrated for radius of curvature tuning in geo600. secondly, a flexible co2 laser based system will be used to deposit heat onto the reaction mass (otherwise called the compensation plate) for the input test mass, as demonstrated in initial ligo [209, 308 ]. the laser beam shape and intensity can be easily modified from outside with the vacuum system and can therefore adapt to non - uniformities in the absorption and other changes in the interferometer s thermal state. it should also be noted that energy can couple from the optical modes resonating in the interferometer fabry - prot cavities and the acoustic modes of the test masses. when there is sufficient coupling between these optical and mechanical modes, and the mechanical modes have a suitably high - quality factor, then mechanical resonances can be rung - up by the large circulating laser power to the point where the interferometer is no longer stable, a phenomenon called parametric instabilities. mechanical dampers that are tuned to damp at high - frequency yet not significantly increasing thermal noise at low - frequency are being considered in possible upgrades to advanced detectors, in addition to other schemes, such as active feedback to damp problematic modes provided through the electrostatic actuators. there are various schemes that can be applied to readout the gravitational - wave signal from an interferometer. if the interferometer laser has a frequency of f1 then a passing gravitational wave, with frequency fgw, will introduce sidebands onto the laser with a frequency of fs = f1 fgw. a readout scheme must be able to decouple the gravitational - wave component, with frequencies of order 100 hz, from the far higher laser frequency at hundreds of tera - hz. to do this it needs to be able to compare the sideband frequency with a known stable optical local oscillator. ideally this oscillator would be the laser light itself (a homodyne scheme), but the initial generation of gravitational - wave detectors are operated at a dark fringe (i.e., the interferometer is held, so as the light from the arms completely destructively interferes at the beam splitter), so no light at the laser frequency exits (a gravitational wave will alter the arms lengths and constructively interfere, causing light to exit, but only at the sideband frequency). the standard scheme used by the initial interferometers is a radio frequency (rf) heterodyne readout. in this case the laser light is modulated at an rf (called schnupp modulation) prior to entering the interferometer arms, giving rise to sidebands offset from the laser frequency at the rf. the interferometer is set up to allow these rf sidebands to exit at the output port. this can be used as a local optical oscillator with which to demodulate the gravitational wave sidebands. however, the demodulation will introduce a beat between the rf and the gravitational wave frequency, which must be removed by a second (hence heterodyne) demodulation at the rf. the preferred method for future detectors is a dc scheme (see [147, 309, 177 ] for motivations and advantages of using such a scheme). in this instead the interferometer is held just off the dark fringe, so some light at the laser frequency reaches the output to serve as the local oscillator. prior to the start of the 21st century there existed several prototype laser interferometric detectors, constructed by various research groups around the world at the max - planck - institt fr quantenoptik in garching, at the university of glasgow, at the california institute of technology, at the massachusetts institute of technology, at the institute of space and astronautical science in tokyo and at the astronomical observatory in tokyo. these detectors had arm lengths varying from 10 m to 100 m and had either multibeam delay lines or resonant fabry - prot cavities in their arms. the 10 m detector that used to exist at glasgow figure 12the 10 m prototype gravitational wave detector at glasgow. the 10 m prototype gravitational wave detector at glasgow. the sensitivities of some of these detectors reached a level better than 10 for millisecond bursts such that the technology could be considered sufficiently mature to propose the construction of detectors of much longer baseline that would be capable of reaching the performance required to have a real possibility of detecting gravitational waves. an international network of such long baseline gravitational wave detectors has now been constructed and commissioned, and science - quality data from these has been produced and analysed since 2002 (see section 6.1 and section 6.2 for a review of recent science data runs and results). the american ligo project comprises two detector systems with arms of 4 km length, one in hanford, washington, and one in livingston, louisiana (also known as the ligo hanford observatory 4k [lho 4k ] and ligo livingston observatory 4k [llo 4k ], or h1 and l1, respectively). one half length, 2 km, interferometer was also contained inside the same evacuated enclosure at hanford (also known as the lho 2k, or h2). the design goal of the 4 km interferometers was to have a peak strain sensitivity between 100200 hz of 3 10 hz (see figure 15), which was achieved during the fifth science run (section 6.1). a birds - eye view of the hanford site showing the central building and the directions of the two arms is shown in figure 13. in october 2010 the ligo detectors shut down and decommissioning began in preparation for the installation of a more sensitive instrument known as advanced ligo (see section 6.3.1). figure 13a bird s eye view of the ligo detector, sited in hanford, washington. a bird s eye view of the ligo detector, sited in hanford, washington. the french / italian virgo project comprises a single 3 km arm - length detector at cascina near pisa. as mentioned earlier, it is designed to have better performance than the other detectors, down to 10 hz. the tama300 detector, which has arms of length 300 m, at the tokyo astronomical observatory was the first of the beyond - prototype detectors to become operational. this detector is built mainly underground and partly has the aim of adding to the gravitational - wave detector network for sensitivity to events within the local group of galaxies, but is primarily a test bed for developing techniques for future larger - scale detectors. initial operation of the interferometer was achieved in 1999 and power recycling was implemented for data taking in 2003. all the systems mentioned above are designed to use resonant cavities in the arms of the detectors and use standard wire - sling techniques for suspending the test masses. the german / british detector, geo600, built near hannover, germany, is somewhat different. it makes use of a four - pass delay - line system with advanced optical signal - enhancement techniques, utilises very - low loss - fused silica suspensions for the test masses, and, despite its smaller size, was designed to have a sensitivity at frequencies above a few hundred hz comparable to the first phases of virgo and ligo during their initial operation. it uses both power recycling (section 5.1) and tunable signal recycling (section 5.2), often referred to together as dual recycling. to gain the most out of the detectors as a true network, the ligo and geo600 teams signed a memorandum of understanding (mou), under the auspices of the ligo scientific collaboration (lsc), allowing complete data sharing between the two groups. part of this agreement has been to ensure that both ligo and geo600 have taken data in coincidence (see below). coincident data taking, and joint analysis, has also occurred between the tama300 project and the lsc detectors. the virgo collaboration also signed an mou with the lsc, which has allowed data sharing since may 2006. the operation and commissioning of these detectors is a continually - evolving process, and the current state of this review only covers developments until late-2010. for the most up - to - date information on detectors readers are advised to consult the proceedings of the amaldi meetings, gwdaw / gwpaw (gravitational wave data analysis workshops), and gwadw (gravitational wave advanced detectors workshops) see for a list of past conferences. for the first and second generations of detector, much effort has gone into estimating the expected number of sources that might be observable given their design sensitivities. in particular, for what are thought to be the strongest sources : the coalescence of neutron - star binaries or black holes (see section 6.2.2 for current rates as constrained by observations). these estimates, based on observation and simulation, are summarised in table 5 of and the realistic rates suggest initial detectors would expect to see 0.02, 0.004 and 0.007 events per year for neutron - star - binary, black - hole - neutron - star, and black - hole - binary systems, respectively (it should be noted that there is a range of possible rates consistent with current observations and models). second generation detectors (see section 6.3.1), which can observe approximately 1000 time more volume than the initial detectors might, expect to see 40, 10, and 20 per year for the same sources. with such rates a great deal of astrophysics could be possible the commissioning and improvement of the various gravitational - wave detectors has been suspended at various stages to take data for astrophysical analysis. these have been times when it was considered that the detectors were sensitive and stable enough (or had made sufficient improvements over earlier states) to make astrophysical searches worthwhile. within the lsc these have been called the science (s) runs, for virgo they have been the virgo science runs (vsr), and for tama300 they have been the data taking (dt) periods. a time - line of science runs for the various interferometric detectors, can be seen in figure 14. figure 14a time - line of the science runs of the first generation interferometric gravitational - wave detectors, from their first lock to mid-2011. a time - line of the science runs of the first generation interferometric gravitational - wave detectors, from their first lock to mid-2011. a figure of merit for the sensitivity of a detector is to calculate its horizon distance. this is the maximum range out to which it could see the coalescence of two 1.4 m neutron stars that are optimally oriented and located (i.e., with the orbital plane perpendicular to the line - of - sight, and with this plane parallel to the detector plane, so that the antenna response is at its maximum) at a signal - to - noise ratio of 8. the horizon distance can be converted to a range that is an average over all sky locations and source orientations (i.e. not the best case scenario) by dividing it by 2.26) we shall use this angle averaged range throughout the rest of this review. the first interferometric detector to start regular data taking with sufficient sensitivity and stability to enable it to potentially detect gravitational waves from the the galactic centre was tama300. over the period between august 1999 to january 2004 tama had nine data - taking periods (denominated dt1 - 9) over which time its typical strain noise sensitivity, in its most sensitive frequency band improved from 3 10 hz to 1.5 10 hz. tama300 operated in coincidence with the ligo and geo600 detectors for two of the science data - taking periods. more recently focus has shifted to the cryogenic laser interferometer observatory (clio) prototype detector [324, 164 ], designed to test technologies for a future second - generation japanese detector called the large - scale cryogenic gravitational - wave telescope (lcgt) (see section 6.3.1). the first ligo detector to achieve lock (meaning having the interferometer stably held on a dark fringe of the interference pattern, with light resonating throughout the cavity) all three detectors had achieved lock and have since undergone many periods of commissioning and science data taking. over the period from mid-2001 to mid-2002 the commissioning process improved the detectors peak sensitivities by several orders of magnitude, with l1 going from 10 10 hz at 150 hz. in summer 2002 it was decided that the detectors were at a sensitivity, and had a good enough lock stability, to allow a science data - taking run. from 23 august to 9 september 2002 the three ligo detectors, along with geo600 (and, for some time, tama300), undertook their first coincident science run, denoted s1 (see for the state of the ligo and geo600 detectors at the time of s1). at this time the most sensitive detector was l1 with a peak sensitivity at around 300 hz of 23 10 hz. the best strain amplitude sensitivity curve for s1 (and the subsequent ligo science runs) can be seen in figure 15. the amount of time over the run that the detectors were said to be in science mode, i.e., stable and with the interferometer locked, called their duty cycle, or duty factor, was 42% for l1, 58% for h1 and 73% for h2. for the most sensitive detector, l1, the inspiral range was typically 0.08 mpc. the s6 curve is preliminary and based on h(t) data that has not been completely reviewed and may be subject to change. the s6 curve is preliminary and based on h(t) data that has not been completely reviewed and may be subject to change. for the second science run (s2), from 14 february to 14 april 2003, the noise floor was considerably improved over s1 by several upgrades including : improving and stabilising the optical levers used to measure the mirror orientation to reduce the low frequency (50 hz) noise ; replacing the coil drivers that are used as actuators to control the position and orientation of suspended mirrors, to improve the mid - frequency (50200 hz) noise floor ; and increasing the laser power in the interferometer to reduce shot noise and improve the high frequency (200 hz) sensitivity (see section iia of for a more thorough description of the detector improvements made for s2). these changes improved the sensitivities by about an order of magnitude across the frequency band with a best strain, for l1, of 3 10 hz between 200300 hz. the duty factor during s2 was 74% for h1, 58% for h2 and 38% for l1, with a triple coincidence time when all three detectors were in lock of 22% of the run. the average inspiral ranges during the run were approximately 0.9, 0.4 and 0.3 mpc for l1, h1 and h2 respectively. for the the third science run (s3), from 31 october 2003 to 9 january 2004, the detectors were again improved, with the majority of sensitivity increase in the mid - frequency range. the best sensitivity, which was for h1, was 5 10 hz between 100200 hz. the duty factors were 69% for h1, 63% for h2 and only 22% for l1, with a 16% triple coincidence time. l1 s poor duty factor was due to large levels of anthropogenic seismic noise near the site during the day. the fourth science run (s4), from 22 february to 23 march 2005, saw less - drastic improvements in detector sensitivity across a wide frequency band, but did make large improvements for frequencies 70 hz. between s3 and s4 a better seismic isolation system, which actively measured and countered for ground motion, was installed in l1, greatly reducing the amount of time it was thrown out of lock. for h1 the laser power was able to be increased to its full design power of 10 w. the duty factors were 80% for h1, 81% for h2 and 74% for l1, with a 56% triple coincidence time. by mid - to - late 2005 the detectors had equaled their design sensitivities over most of the frequency band and were also maintaining good stability and high duty factors. it was decided to perform a long science run with the aim of collecting one year s worth of triple coincident data, with an angle - averaged inspiral range of equal to, or greater than, 10 mpc for l1 and h1, and 5 mpc or better for h2. november 2005 (l1 started slightly later on 14 november) until 1 october 2007, and the performance of the detectors during it is summarised in. one year of triple coincidence was achieved on 21 september 2007, with a total triple coincidence duty factor of 52.5% for the whole run. the average insprial range over s5 was 15 mpc for h1 and l1, and 8 mpc for h2. after the end of s5 the ligo h2 detector and geo600 were kept operational while possible in an evening and weekend mode called astrowatch. this observing mode continued until early 2009, after which h2 was retired. during this time commissioning of some upgrades to the 4 km ligo detectors took place for the sixth and final initial ligo science run (s6) the aim of these upgrades, called enhanced ligo, was to try and increase sensitivity by a factor of two. enhanced ligo involved the direct implementation of technologies and techniques designed for the later upgrade to advanced ligo (see section 6.3.1) such as, most notably, higher - powered lasers, a dc readout scheme (see section 5.4), the addition of output mode cleaners and the movement of some hardware into the vacuum system. the lasers, supplied by the albert einstein institute and manufactured by laser zentrum hannover, give a maximum power of 30 w, which is around 3 times the initial ligo power. the upgrade to higher power required that several of the optical components needed to be replaced. these upgrades were only carried out on the 4 km h1 and l1 detectors due to the h2 detector being left in astrowatch mode during the commissioning period. the upgrades were able to produce 1.52 times sensitivity increases at frequencies above 200 hz, but generally at lower frequencies various sources of noise meant sensitivity increases were not possible. s6 took place from july 2009 until 20 october 2010, at which point decommissioning started for the full upgrade to advanced ligo. typically the detectors ran with laser power at 10 w during the day (at higher power the detector was less stable and the higher level of anthropogenic noise during the day meant that achieving and maintaining lock required lower power) and 20 w at night, leading to inspiral ranges from 1020 mpc. geo600 achieved first lock as a power - recycled michelson (with no signal recycling) in late 2001. commissioning over the following year, detailed in, included increases in the laser power, installation of monolithic suspensions for the end test masses (although not for the beam splitter and inboard mirrors), rearrangement of the optical bench to reduce scattered light and implementation of an automatic alignment system. for the s1 run, carried out in coincidence with ligo (and, in part, tama300), the detector was kept in this configuration (see for the status of the detector during s1). it had a very high duty factor of 98%, although its strain sensitivity was 2 orders of magnitude lower than the ligo instruments. the auto - alignment system in geo600 has since meant that it has been able to operate for long periods without manual intervention to regain lock, as has been the case for initial ligo. following s1 the signal recycling mirror was installed and in late 2003 the first lock of the fully dual - recycled system was achieved (see [289, 318, 163 ] for information on the commissioning of geo600 as a dual - recycled detector). other upgrades included the installation of the final mirrors, suspended as triple pendulums, and with monolithic final stages. once installed it was found that there was a radius of curvature mismatch with one of the mirrors, which had to be compensated for by carefully heating the mirror. due to this commissioning effort geo600 did not participate in the s2 run. very soon after the implementation of dual - recycling geo600 took part in the s3 run. this occurred over two time intervals from 511 november 2003, dubbed s3i, and from 30 december 2003 to 13 january 2004, dubbed s3ii. during s3i geo600 operated with the signal - recycling cavity tuned to 1.3 khz, and had a 95% duty factor, but was then taken off - line for more commissioning work. in the period between s3i and ii various sources of noise and lock loss were diagnosed and mitigated, including noise from a servo in the signal recycling cavity and electronic noise on a photo - diode. this lead to improved sensitivity by up to an order of magnitude at some frequencies (see figure 16). for s3ii the signal recycling cavity was tuned to 1 khz and, due to the upgrades, had an increased duty factor of 99%. geo600 operated during the whole of s4 (22 february to 24 march 2004), in coincidence with ligo, with a 97% duty factor. it used the same optical configuration as s3, but had sensitivity improvements from a few times to up to an order of magnitude over the s3 values. also shown is the theoretical noise budget for the detector when tuned to 550 hz the operating position for the s5 run. the typical strain sensitivities from the geo600 science runs s1 through s5. also shown is the theoretical noise budget for the detector when tuned to 550 hz the operating position for the s5 run. the main changes to the detector after s4 were to shift the resonance condition of the signal recycling cavity to a lower frequency, 350 hz, allowing better sensitivity in the few hundred hz regime, and increasing the circulating laser power, with an input power of 10 w. the pre - s5 peak sensitivity was 4 10 hz at around 400 hz, with an inspiral range of 0.6 mpc. geo600 did not join s5 at the start of the ligo run, but from 21 january 2006 was in a night - and - weekend data - taking mode whilst noise hunting studies and commissioning were conducted. it went into full - time data taking from 1 may to 16 october 2006, with an instrumental duty factor of 94%. the average peak sensitivity during s5 was better than 3 10 hz (see for a summary of geo600 during s5). after this it was deemed more valuable for geo600 to continue more noise hunting and commissioning work, to give as good a sensitivity as possible for when the ligo detectors went offline for upgrading. geo600 continued operating in astrowatch mode between november 2007 and july 2009 after which upgrades began. the plans for the geo600 detector are to continue to use it as a test - bed for more novel interferometric techniques whilst focusing on increasing in sensitivity at higher frequencies (greater than a few hundred hz). the main upgrades started during 2009 were to change the read - out scheme from an rf read - out to a dc read - out system (also see section 5.4), install an output mode cleaner, place the read - out system in vacuum, injecting squeezed light [302, 114 ] into the output port, and finally increasing the input laser power to 35 w. running the interferometer with squeezed light will be the first demonstration of a full - scale gravitational - wave detector operating beyond the standard quantum limit. geo - hf participated in s6 in an overnight and weekend mode, alongside a commissioning schedule, and is continuing in this mode following the end of s6. in summer 2002 virgo completed the commissioning of the central area interferometer, consisting of a power - recycled michelson interferometer, but without the 3 km fabry - prot arm cavities. over the next couple of years various steps were made towards commissioning the full - size interferometer. in early 2004 first lock with the 3 km arms was achieved, but without power - recycling, and by the end of 2004 lock with power recycling was achieved. during summer 2005 the commissioning runs provided order - of - magnitude sensitivity improvements, with a peak sensitivity of 6 10 hz at 300 hz, and an inspiral range of over 1 mpc. in late 2005 see [58, 59, 60, 61 ] for more detailed information on the commissioning of the detector. virgo joined coincident observations with the ligo and geo600 s5 run with 10 weekend science runs (wsrs) starting in late 2006 until march 2007. over this time improvements were made mainly in the mid - to - low frequency regime (300 hz). full - time data taking, under the title of virgo science run 1 (vsr1), began on 18 may 2007 and ended with the end of s5 on 1 october 2007. during vsr1, the science - mode duty factor was 81% and by the end of the run maximum neutron - star - binary inspiral range was frequently up to about 4.5 mpc. the best sensitivity curves for wsr1, wsr10 and vsr1 can be seen in figure 17. figure 17the best strain sensitivities from the virgo weekend and full time science runs wsr1, wsr10, vsr1 and vsr2 [305, 57 ]. the best strain sensitivities from the virgo weekend and full time science runs wsr1, wsr10, vsr1 and vsr2 [305, 57 ]. at the same time as commissioning for enhanced ligo was taking place the main upgrade was to the lasers to increase their power from 10 to 25 w at the input mode cleaner, with upgrades also to the thermal compensation system on the mirrors, the control electronics, mode cleaners and injection optics [64, 141 ]. virgo+ started taking data with enhanced ligo for virgo science run 2 (vsr2) and sensitivities of virgo+ close to the initial virgo design sensitivity were reached. this was followed by vsr3, which began on 11 august 2010 and ran until 20 october 2010. prior to the advent of the large scale interferometric detectors there had been some limited effort to produce astrophysical results with the prototype interferometers. the caltech 40 m detector was used to search for, and set an upper limit on, the gravitational wave emission from pulsar psr j1939 + 2134, and on the rate of neutron star binary inspirals in our galaxy, using coincident observations with the university of glasgow prototype and, more recently, on its own. coincident observations using the prototype detectors at the university of glasgow and max planck institute for quantum optics, in garching, germany, were used to set an upper limit on the strain of gravitational wave bursts. the garching detector was used to search for periodic signals from pulsars, and in particular set a limit on a potential source in sn 1987a. however since the start of science data taking for the large scale detectors there has been a rapid rise in the number, and scope, of science result papers being published. with the vastly improved sensitivities pushing upper limits on source populations and strengths towards astrophysically interesting areas. the recent analysis efforts have generally been split into four broad areas depending on the expected signal type : unmodelled transients or bursts, e.g., supernovae ; modelled transients, e.g., inspirals and ring - downs (or more specifically compact binary coalescences, cbc) ; continuous sources ; stochastic sources. within each area a variety of different sources could exist and a variety of analysis techniques have been developed to search for them. some electromagnetic sources, such as radio pulsars and -ray bursts, are also used to enhance searches. a good review of the data analysis methods used in current searches, and the astrophysical consequences of some of the results described below, can be found in. here we will mainly focus on those produced by the ligo scientific collaboration detectors ligo and geo600 from s1 to the s5 run. at the time of writing not all of the data from the s6 run had been fully analysed, with more results expected over the next year. reviews of some early s5, and prior science run, results can also be found in [254, 139 ]. in none of the searches searches for unmodelled bursts, e.g., from supernova core - collapse, are based on looking for short duration periods of excess power in the detectors. transients are common features in the data, so to veto these events from true gravitational - wave signals they must be coincident in time, and to some extent amplitude and waveform, between multiple detectors. various methods to assess instrumental excess power, and inter - detector correlations, are used, some examples of which can be found here [202, 77, 284, 228, 107, 112, 113 ]. these algorithms will produce triggers, which are periods of excess power that cross a predetermined signal - to - noise ratio threshold (determined by tuning the algorithms on a section of playground data, so that the output produces a desired false alarm rate). real signals can not be turned off, and detectors can not be shielded from them, so the background rate has to be approximated by time shifting one detector s data stream with respect the the others. time shifts should only leave triggers due to random coincidences in detector noise and there should be no contribution from real signals. once a background is calculated, the statistical significance of the foreground rate can be compared to it. to assess the sensitivity of these searches, hardware (the interferometer mirrors are physically moved via the control system) and software signals are injected into the data stream at various strengths and the efficiency of the algorithms at detecting them data from the ligo s1 run was searched for gravitational - wave bursts of between 4 to 100 ms, and within the frequency band 150 to 3000 hz. no plausible candidate event was found, but a 90% confidence upper limit on the event rate of 1.6 events per day was set. the search was typically sensitive, at a 50% detection efficiency, to bursts with amplitudes of hrss 10 10 hz (defined in terms of \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${h_{{\rm{rss } } } } \equiv \sqrt { \int { |h{|^2}{\rm{d}}t}}$\end{document }, which is the root - sum - squared strain amplitude spectral density). due, in part, to its lower sensitivity geo600 data was not used in this analysis. the s2 data s improved sensitivity, and advances in the analysis techniques, allowed a sensitivity to signals (in the frequency range 1001100 hz) in the amplitude range hrss 10 10 hz. interpreting the best sensitivities astrophysically gave order of magnitude estimates on the visible range of 100 pc for a class of theoretical supernova waveforms, and 1 mpc for the merger of 50 m black holes. again no signal was seen, but a 90% upper limit of 0.26 events per day was set for strong bursts. in the frequency range 7002000 hz tama300 data was also used in the search giving amplitude sensitivities of hrss 13 10 hz and decreasing the rate upper limit to 0.12 events per day. one used the 8 days of triple coincidence data from the three ligo detectors to search for sub - second bursts in the frequency range 1001100 hz. the search was sensitive to signals with amplitudes over hrss 1 10 hz, but did not include an astrophysical interpretation of the limit or event rate upper limit. this run included coincident operation with the italian auriga bar detector and this data has been analysed. this search looked for short bursts of less than 20 ms within the 850950 hz band (around the bar s sensitive resonant frequency). this had comparable sensitivity to the ligo - only s2 search and produced a 90% confidence rate upper limit of 0.52 events / day. for s4 15.5 days of ligo data this was sensitive to signals with hrss 10 and set a 90% confidence rate upper limit of 0.15 per day. these show that there would be a 50% detection efficiency to signals of sine - gaussian nature (at the most sensitive frequency of 153 hz and quality factor q = 8.9) at a distance of 10 kpc for an energy of 10 mc, and would be sensitive to signals out to the virgo cluster (16 mpc) for an energy release of 0.25 mc. there was also a burst search combining s4 geo600 and ligo data for the first time. this searched data between 7682048 hz where the sensitivities were most comparable and used 257 hours of quadruple coincidence between the detectors and saw no gravitational wave events. for the analysis on the first year of s5 data the frequency range for the all - sky burst search was split a low frequency search covered the most sensitive region between 602000 hz, and a high frequency search covering 16 khz (this being the first time an untriggered burst search looked at frequencies above 3 khz). the high frequency search set a 90% upper limit on the rate of 5.4 events per year for strong events. the low frequency search analysed more data than the high frequency and set an event rate limit of 3.6 events per year. the second year of s5 ligo data was analysed with geo600 and virgo vsr1 data to search for bursts over the whole 506000 hz band. combining this with the earlier s5 searches gave hrss upper limits for a variety of simulated waveforms of 6 10 hz to 2 10 hz, and a 90% confidence event rate for signals between 642048 hz of less than two per year. modeled bursts generally mean the inspiral and coalescence stage of binaries consisting of compact objects, e.g., neutron stars and black holes. the signals are generally well approximated by post - newtonian expansions of the einstein equation, which give the amplitude and phase evolution of the orbit. more recently signal models have started to include numerical relativity simulations of the merger stage. as mentioned in section 6 the best estimate of the number of signals observable with initial ligo at design sensitivity (i.e. during s5) would be 0.02 per year (based on an event rate of 1 10 per year per mweg). the majority of inspiral searches make use of matched filtering in which a template bank of signal models is built [250, 252 ], with a maximum mismatch between templates that is generally of order 10%. these templates are then cross - correlated with the data and statistically significant triggers (i.e. times when the template and data are highly correlated) from this are looked for. triggers must be coincident between detectors and the significance of any trigger is judged against a background calculated in the same way as described in section 6.2.1. see for a good description of the search method. the first search for an inspiral signal with data from the ligo s1 run looked for compact object coalescences with component masses between 13 m and was sensitive to such sources within the milky way and magellanic clouds. it gave a 90% confidence level upper limit on the rate of 170 per year per mweg. for the s2 ligo analysis the search was split into 3 areas covering neutron - star binaries, black - hole binaries and primordial black - hole binaries in the galactic halo. the neutron - starbinary search used 15 days of data with coincidence between either h1 and l1 or h2 and l1. it had a range of 1.5 mpc, which spanned the local group of galaxies, and gave a 90% event rate upper limit on systems with component masses of 13 m of 47 per year per mweg. the black - hole - binary search looked for systems with component masses in the 320 m range using the same data set as the neutron - star - binary search. this search had a 90% detection efficiency for sources out to 1 mpc and set a 90% rate upper limit of 38 per year per mweg. the third search looked for low mass (0.21 m) primordial black - hole binaries in a 50 kpc radius halo surrounding the milky way. this placed a 90% confidence - rate upper limit of 63 events per year per milky way halo. the s2 search was performed in coincidence with the tama300 dt8 period and an inspiral search for neutron - star binaries was performed on data when tama300 and at least one of the ligo sites was operational. this gave a total of 584 hours of data for the analysis, which set a 90% rate upper limit of 49 per year per mweg, although this search was only sensitive to sources within the majority of the milky way. the search for neutron - star - black - hole binaries in s3 ligo data used techniques designed specifically for systems with spinning components. it searched for systems with component masses in the range 120 m and analysed 167 hours of triple coincident data and 548 hours of h1h2 data to set the upper limits. for a typical system with neutron - star and black - hole mass distributions centred on 1.35 m and 5 m (from the population statistics discussed in) this search produced a 90% confidence - rate upper limit of 15.9 per year per l10. the search for a wide range of binary systems with components consisting of primordial black holes, neutron stars, and black holes with masses in the ranges given above 788 hours of s3 data and 576 hours of s4 data were used and no plausible gravitational - wave candidate was found. the highest mass range for the black - holebinary search was set at 40 m for s3 and 80 m for s4. at peak in the mass distribution of these sources 90% confidence - rate upper limits were set at 4.9 per year per l10 for primordial black holes, 1.2 per year per l10 for neutron - star binaries, and 0.5 per year per l10 for black - hole - binaries. s4 data has also been used to search for ring - downs from perturbed black holes, for example following black - hole - binary coalescence. the search was sensitive to ring - downs from 10500 m black holes out to a maximum range of 300 mpc, and produced a best 90% confidence upper limit on the rate of ring - downs to be 1.6 10 per year per l10 for the mass range 85390 m. one other kind of modeled burst search is that looking for gravitational waves produced by cusps in cosmic (super)strings. just over two weeks of ligo s4 data were used to search for such signals. this was used to constrain the rate and parameter space (string tension, reconnection probability, and loop sizes), but was not able to beat limits set by big bang nucleosynthesis. data from the first and second year of s5 (prior to virgo joining with vsr1) have been searched for low - mass binary coalescences with total masses in the range 235 m. the second year search results have produced the more stringent upper limits with 90% confidence rates for neutron - star - binaries, black - hole - binaries and neutron - star - black - hole systems respectively of 1.4 10, 7.3 10 and 3.6 10 per year per l10. five months of overlapping s5 and vsr1 data were also searched for the same range of signals giving 90% confidence upper rates of 8.7 10 per year per l10, 2.2 10 per year per l10, and 4.4 10 per year per l10. the whole 2 years of ligo s5 data were also used to search for higher mass binary coalescences with component mass between 199 m and total masses of 25100 m. no signal was seen, but a 90% confidence upper limit rate on mergers of black - hole - binary systems with component masses between 19 and 28 m, and with negligible spin, was set at 2.0 mpc myr. many gravitational wave burst sources will be associated with electromagnetic (or neutrino) counterparts, for example short -ray bursts (grbs) are potentially caused by black - hole and neutronstar coalescences. joint observation of a source as both a gravitational wave and electromagnetic event also greatly increases the confidence in a detection. therefore many searches have been performed to look for bursts coincident (temporally and spatially) with external electromagnetic triggers, such as grbs observed by swift for example. these searches have used both excess power and modeled matched - filter methods to look for signals. during s2 a particularly bright -ray burst event (grb 030329) occurred and was specifically targeted using data from h1 and h2. the search looked for signals with duration less than 150 ms and in the frequency range 802048 hz. this produced a best strain upper limit for an unpolarised signal around the most sensitive region at 250 hz of hrss = 6 10 hz. the first target was the hyperflare from the soft -ray repeater sgr 180620 (sgrs are thought to be magnetars, neutron stars with extremely large magnetic fields of order 10 gauss) on 27 december 2004 (this actually occurred before s4 in a period when only the h1 detector was operating). the search looked for signals at frequencies corresponding to short duration quasi - periodic oscillations (qpos) observed in the x - ray light curve following the flare. the most sensitive 90% upper limit was for the 92.5 hz qpo at hrss = 4.5 10 hz, which corresponds to an energy emission limit of 4.3 10 mc (of the same order as the total electromagnetic emission assuming isotropy). the other search used ligo data from s2, s3 and s4 to look for signals associated with 39 short duration -ray bursts (grbs) that occurred in coincidence with these runs. the grb triggers were provided by ipn, konus - wind, hete-2, integral and swift as distributed by the grb coordinate network. the search looked in a 180-second window around the burst peak time (120 seconds before and 60 seconds after) and for each burst there were at least two detectors contributing data. no signal coincident with a grb was observed and the sensitivities were not enough to give any meaningful astrophysical constraints, although simulations suggest that for s4, as in the general burst search, it would have been sensitive to sine - gaussian signals out to tens of mpc for an energy release of order a solar mass. the first search of virgo data in coincidence with a grb was performed on data from a commissioning run in september 2005. the long duration grb 050915a was observed by swift on 15 september 2005 and virgo data was used to search for an unmodelled burst in a window of 180 seconds around (120 s before and 60 s after) the grb peak time. the search produced a strain upper limit of order 10 in the frequency range 2001500 hz, but was mainly used as a test - bed for setting up the methodology for future searches, including coincidence analysis with ligo. data from the s5 run has been used to search for signals associated with even more -ray bursts. one search looked specifically for emissions from grb 070201 [156, 155 ], which showed evidence of originating in the nearby andromeda galaxy (m31). the data around the time of this burst was used to look for an unmodelled burst and an inspiral signal as might be expected from a short grb. the analysis saw no gravitational - wave event associated with the grb, but ruled out the event being a neutron - star - binary inspiral located in m31 with a 99% confidence. again, assuming a neutron - star - binary inspiral, but located outside m31, the analysis set a 90% confidence limit that the source must be at a distance greater than 3.5 mpc. assuming a signal again located in m31, the unmodelled burst search set an upper limit on the energy emitted via gravitational waves of 4.4 10 mc, which was well within the allowable range for this being an sgr hyper - flare in m31. searches for 137 grbs (both short and long grbs) that were observed, mainly with the swift satellite, during s5 and vsr1 have been performed again using unmodelled burst methods and for (22 short bursts) inspiral signals. the unmodeled burst observations were used to set lower limits on the distance to each grb, with typical limits, assuming isotropic emission, at \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$d \sim 15{\mkern 1mu } { \text{mpc}}{(e_{{\text{gw}}}^{{\text{iso}}}/0.01{\mkern 1mu } { m _ \odot } { c^2})^{1/2}}$\end{document}. the inspiral search, which was sensitive to cbcs with total system masses between 2 m and 40 m, was able to exclude with 90% confidence any bursts being neutron - star - black - hole mergers within 6.7 mpc, although the peak distance distribution of grbs is well beyond this. another search has been to look for gravitational waves associated with flares from known sgrs and anomalous x - ray pulsars (axps), both of which are thought to be magnetars. during the first year of s5 there were 191 (including the december 2004 sgr 180620 event) observed flares from sgrs 180620 and 1900 + 14 for which at least one ligo detector was online, and 1279 flare events if extending that to six known galactic magnetars and including all s5 and post - s5 astrowatch data including virgo and geo600. the data around each event was searched for ring - down signals in the frequency range 13 khz and with decay times 100400 ms as might be expected from f - mode oscillations in a neutron star. no gravitational bursts were seen from any of the events. for the earlier search the lowest 90% upper limit on the gravitational - wave energy from the ring - down search was \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$e_{{\rm{gw}}}^{90\% } = 2.4 \times { 10^{48}}$\end{document } erg for an sgr 180620 burst on 24 august 2006. the lowest 90% upper limit on the unmodeled search was \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$e_{{\rm{gw}}}^{90\% } = 2.9 \times { 10^{45}}$\end{document } erg for an sgr 180620 burst on 21 july 2006. the smallest limits on the ratio of energy emitted via gravitational waves to that emitted in the electromagnetic spectrum were of order 10100, which are into a theoretically - allowed range. the latter search gave the lowest gravitational - wave emission - energy upper limits for white noise bursts in the detector - sensitive band, and for f - mode ring - downs (at 1090 hz), of 3.0 10 erg and 1.4 10 erg respectively, assuming a distance of 1 kpc. the f - mode energy limits approach the range seen emitted electromagnetically during giant flares. one of these flares, on 29 march 2006, was actually a storm of many flares from sgr 1900 + 14. for this event a more sensitive search has been performed by stacking data around the time of each flare. waveform dependent upper limits of the gravitational - wave energy emitted were set between 2 10 erg and 6 10 erg, which are an order of magnitude lower than the previous upper limit for this storm (included in the search of) and overlap with the range of electromagnetic energies emitted in sgr giant flares. another possible source of gravitational waves associated with electromagnetically - observed phenomenon are pulsar glitches. during these it is possible that various gravitational - wave - emitting vibrational modes of the pulsar may be excited. a search has been performed for fundamental modes (f - modes) in s5 data following a glitch observed in the timing of the vela pulsar in august 2006. over the search frequency range of 13 khz this provided upper limits on the peak strain of 0.61.4 10 depending on the spherical harmonic that was excited. already efforts are under way to invert this process of searching gravitational - wave data for external triggers, and instead supplying gravitational - wave burst triggers for electromagnetic follow - up. this is being investigated across the range of the electromagnetic spectrum from radio, through optical (e.g., [198, 119 ]) and x - ray/-ray, and even looking for coincidence with neutrino detectors [86, 258, 111 ]. having multi - messenger observations can have a large impact on the amount of astrophysical information that can be learnt about an event. there are fully targeted searches, which look for gravitational waves from known radio pulsars in which the position and spin evolution of the objects are precisely known. there are semi - targeted searches, which look at potential sources in which some, but not all, the source signal parameters are known, for example neutron stars in x - ray binary systems, or sources in supernova remnants where no pulses are seen, which have known position, but unknown frequency. finally, there are all - sky broadband searches in which none of the signal parameters are known. the targeted searches tend to be most sensitive as they are able to perform coherent integration over long stretches of data with relatively low computational overheads, and have a much smaller parameter space leading to fewer statistical outliers. due to various neutron - star population statistics, creation rates and energetics arguments, there is an estimate that the amplitude of the strongest gravitational - wave pulsar observed at earth will be h0 4 10 (a more thorough discussion of this argument can be found in), although this does not rule out stronger sources. the various search techniques used to produce these results all look for statistically - significant excess power in narrow frequency bins that have been doppler demodulated to take into account the signal s shifting frequency caused by the earth s orbital motion with respect to the source (or also including the modulations to the signal caused by the source s own motion relative to the earth, such as for a pulsar in a binary system). the statistical significance of a measured level of excess power is compared to what would be expected from data that consisted of gaussian noise alone. a selection of the searches are summarised in, but for more detailed descriptions of the various methods see [100, 206, 188, 33, 29, 132 ]. in s1 a fully - coherent targeted search for gravitational waves from the then - fastest millisecond pulsar j1939 + 2134 was performed. this analysis and the subsequent lsc known - pulsar searches assume that the star is triaxial and emitting gravitational waves at exactly twice its rotation frequency. all the data from ligo and geo600 was analysed and no evidence of a signal was seen. a 95% degree - of - belief upper limit on the gravitational - wave strain amplitude was set using data from the most sensitive detector, l1, giving a value of 1.4 10. this result was also interpreted as an ellipticity of the star given a canonical moment of inertia of 10 kg m at = 2.9 10. however, this was still of order 100 000 times higher than the limit that can be set by equating the star s rate of loss of rotational kinetic energy with that emitted via gravitational radiation the number of known pulsar sources searched for with ligo data increased from 1 to 28, although all of these were isolated pulsars (i.e. not in binary systems, although potentially still associated with supernova remnants or globular clusters). this search used pulsar timing data supplied by lyne and kramer from jodrell bank observatory to precisely reconstruct the phase of the gravitational - wave signal over the period of the run. the lowest 95% upper limit on gravitational - waves amplitude was 1.7 10 for psr j19105959d, and the smallest upper limit on ellipticity (again assuming the canonical moment of inertia) was 4.5 10 for the relatively - close pulsar psr j21243358, at a distance of 0.25 kpc. the pulsar closest to its inferred spin - down limit was the crab pulsar (psr j0534 + 2200) with an upper limit 30 times greater than that from spin - down. s2 also saw the use of two different all - sky - wide frequency band searches that focused on isolated sources, but also including a search for gravitational waves from the low mass x - ray binary scorpius x1 (sco - x1). the first search used a semi - coherent technique to search 60 days of s2 data in the frequency band between 200400 hz and with signal spin - downs between 1.1 10 and 0 hz s. this gave a lowest gravitational - wave strain 95% upper limit of 4.4 10 for the l1 detector at around 200 hz. the other all - sky search was fully coherent and as such was computationally limited to only use a few hours of the most sensitive s2 data. it searched frequencies between 160728.8 hz and spin - downs less than 4 10 hz s for isolated sources and gave a 95% upper limit across this band from 6.6 10 to 1 10. the search for gravitational waves from sco - x1 used the same period of data. it did not have to search over sky position as this is well known, but did have to search over two binary orbital parameters the projected semi - major axis and the orbital phase reference time. the frequency ranges of this search relied on estimates of the spin - frequency from quasi - periodic oscillations in the x - rays from the source and covered two 20 hz bands from 464484 hz and 604624 hz (it should be noted that it is now thought that these estimates of the spin - frequency are unreliable). in these two ranges one search that was carried out purely on ligo s3 data was the coherent all - sky wide - band isolated pulsar search using the distributed computing project einstein@home. the project is built upon the berkeley open infrastructure for network computing and allows the computational workload to be distributed among many computers generally contributed by the general public who sign up to the project. this used the most sensitive 600 hours of data from h1 and cut it into 60 ten hour stretches on each of which a coherent search could be performed. the data was farmed out to computers owned by participants in the project and ran as a background process or screen saver. the search saw no plausible gravitational - wave candidates and the result is described at, but it was not used to produce an upper limit. in the known pulsar search the number of sources searched for using the combined ligo data from s3 and s4 was increased to 78. for many of the pulsars that overlapped with the previous s2 analysis results were improved by about an order of magnitude. the lowest 95% upper limit on gravitational - waves amplitude was 2.6 10 for psr j16037202, and the smallest ellipticity was again for psr j21243358 at just less than 10. the upper limit for the crab pulsar was found to be only 2.2 times above that from the spin - down limit. three different, but related, semi - coherent all - sky continuous wave searches were performed on s4 ligo data, looking for isolated neutron stars in the frequency range from 501000 hz and the spin - down range from 1 10 to 0 hz s. the best 95% upper limit based on an isotropically - distributed, randomly - oriented for one of the searches, which combined data from the different detectors, an isolated pulsar emitting at near 100 hz, and with an extreme ellipticity of 10 could have been seen at a distance of 1 kpc, although for a more realistic ellipticity of 10 only a distance of less than 1 pc would be visible over the entire ligo band. the einstein@home project was also used to search the most sensitive data from s4, which consisted of 300 hours of h1 data and 210 hours of l1 data. the search performed a coherent analysis on 30-hour stretches of this data and covered the frequency range of 501500 hz. the range of spin - downs was chosen by using a minimum spin - down age and having f/ < < 0.1 f/ (small spin - ups are allowed as some pulsars in globular clusters exhibit this due to their doppler motions within the clusters), with = 1000 years for signals below 300 hz and = 10 000 years above 300 hz. approximately 6000 years of computational time spread over about 100 000 computers were required to perform the analysis. no plausible gravitational - wave candidates were found, although the results suggest that 90% of sources with strain amplitudes greater than 10 would have been detected by the search. a search designed to produce a sky map of the stochastic background was also used to search for gravitational waves from sco - x1 using a method of cross - correlating h1 and l1 data. this produced a 90% root - mean - squared upper limit on gravitational wave strain of h = 3.4 10(f/200 hz) for frequencies greater than 200 hz. the first 8 months of s5 have been used to perform an all - sky search for periodic gravitational waves. this search used a semi - coherent method to look in the frequency range 501100 hz and spin - down range 5 10 0 hz s and used data from the h1 and l1 detectors. it obtained 95% strain upper limits of less than 10 over a frequency band of 200 hz. the search would have been sensitive to a neutron star with equatorial ellipticity greater than 10 within around 500 pc. einstein@home has been used to search for periodic waves of 501500 hz in 860 hours of data from a total span of 66 days of s5 data. it would have been sensitive to 90% of sources in the 125225 hz band with amplitudes greater than 3 10. the first approximately 9 months of s5 data was used for a coherent search for gravitational waves from the crab pulsar. in this search two methods were used : the first followed the method of the targeted search and assumed that the gravitational waves are phase locked to the electromagnetic pulses ; the second allowed for some mechanism, which would cause a small mismatch between the two phases. two 95% upper limits were set, one using astrophysical constraints on the pulsar orientation angle and polarisation angle and the other applying no such constraints. with the first method these 95% upper limits were 3.4 10 and 2.7 10 respectively, which correspond to ellipticities of 1.8 10 and 1.4 10 (assuming the canonical moment of inertia). these beat the crab pulsar s spin - down limit by 4 to 5 times and can be translated into the amount of the available spin - down power that is emitted via gravitational waves, with the lower of these limits showing that less than 4% of power is going into gravitational waves. for the second search the uniform and restricted prior analyses gave upper limits of 1.7 10 and 1.2 10 respectively. the whole of s5 was used to search for emissions from 116 known pulsars. during this search the crab limit was further brought down to be less than a factor of 7 below the spin - down limit, and the spin - down limit is reached for one other pulsar psr j05376910. of the other pulsars, the best (lowest) upper limit on gravitational - wave amplitude was 2.3 10 for psr j16037202 and our best (lowest) limit on the inferred pulsar ellipticity is 7.0 10 for psr j21243358. a semi - targeted search was performed with 12 days of s5 data, although this time searching for a source with a known position in the cassiopeia a (cas a) supernova remnant, but for which there is no known frequency. the search looked in the frequency band between 100300 hz and covered a wide range of first and second frequency derivatives and no signal was seen, but it gave 95% amplitude and ellipticity upper limits over the band of (0.71.2) 10 and (0.44) 10 respectively. these results beat indirect limits on the emission based on energy - conservation arguments (similar, but not the same as the spin - down limits) and were also the first results to be cast as limits on the r - mode amplitude. the vela pulsar has a spin frequency of 11 hz and was not accessible with current ligo data. however, virgo vsr2 data had sensitivity in the low frequency band that made a search for it worthwhile. using 150 days of virgo data, no signal was seen, but a 95% upper limit on the amplitude of 2 10 was set, which beat the spin - down limit by 1.6 times. other than the crab pulsar, this is currently the only other object for which the spin - down limit has been beaten. searches are conducted for a cosmological, or astrophysical, background of gravitational waves that would show up as a coherent stochastic noise source between detectors. this is done by performing a cross - correlation of data from two detectors as described in. in s1 the most sensitive detector pair for this correlation was h2-l1 (the h1h2 pair are significantly hampered by local environmental correlations) and they gave a 90% confidence upper limit of gw < 44 9 within the 40314 hz band, where the upper limit is in units of closure density of the universe and for a hubble constant in units of 72 km s mpc. this limit was several times better than previous direct - detector limits, but still well above the concordance acdm cosmology value of the total energy density of the universe of 0 1 (see, e.g.,). no published stochastic background search was performed on s2 data, but s3 data was searched and gave an upper limit that improved on the s1 result by a factor of 10. the most sensitive detector pair for this search was h1-l1 for which 218 hours of data were used. upper limits were set for three different power - law spectra of the gravitational - wave background. for a flat spectra, as predicted by some inflationary and cosmic string models, a 90% confidence upper limit of gw(f) = 8.4 10 in the 69156 hz range was set (again for a hubble constant of 72 km s mpc). this is still about 60 times greater than a conservative bound on primordial gravitational waves set by big - bang nucleosynthesis (bbn). for a quadratic power law, as predicted for a superposition of rotating neutron - star signals, an upper limit of gw(f) = 9.4 10(f/100 hz) was set in the range 73244 hz, and for a cubic power law, from some pre - big - bang cosmology models, an upper limit of gw(f) = 8.1 10(f/100 hz) in the range 76329 hz was produced. for s4 354 hours of h1-l1 data and 333 hours of h2-l1 data were used to set a 90% upper limit of gw(f) < 6.5 10 on the stochastic background between 51150 hz, for a flat spectrum and hubble constant of 72 km s mpc. this result is still several times higher than bbn limits. about 20 days of h1 and l1 s4 data was also used to produce an upper limit map on the gravitational wave background across the sky as would be appropriate if there was an anisotropic background dominated by distinct sources. this search covered a frequency range between 501800 hz and had spectral power limits (which come from the square of the amplitude h) ranging from 1.2 10 hz (100 hz / f) and 1.2 10 hz(100 hz / f) for an f source power spectrum, and limits of 8.5 10 hz and 6.1 10 hz for a flat spectrum. data from s4 was also used to perform the first cross - correlation between an interferometric and bar detector to search for stochastic backgrounds. l1 data and data from the nearby allegro bar detector were used to search in the frequency range 850950 hz, several times higher than the ligo only searches. a 90% upper limit on the closure density of gw(f) 1.02 (for the above hubble constant) was set, which beat previous limits in that frequency range by two orders of magnitude. this limit beats what would be achievable with llo - lho cross correlation of s4 data in this frequency range by a factor of several tens, due to the physical proximity of llo and allegro. the entire two years of s5 data from the ligo detectors has been used to set a limit on the stochastic background around 100 hz to be gw(f) < 6.9 10 at 95% confidence (for a flat gravitational - wave spectrum). all the current detectors have upgrades planned over the next several years. these upgrades will give rise to the second generation of gravitational - wave detectors, which should start to open up gravitational - wave astronomy as a real observational tool. there are also currently plans being made for third generation detectors, which could provide the premier gravitational - wave observatories for the first half of the century. a brief summary of the planned upgrades to current and future detectors is given below. some of the technologies for these upgrades are discussed earlier in this review (e.g. section 5). advanced ligo (aligo) [166, 214, 66 ] and advanced virgo (advvirgo) [62, 67 ] are the second generation detectors. they are planned to have a sensitivity increase over the levels of the initial detectors by a factor of 1015 times. these increased sensitivity levels would expand the volume of space observed by the detectors by 1000 times meaning that there is a realistic detection rate of neutron - star - binary coalescences of around 40 yr [3, 205 ]. the technological issues required to reach these sensitivities, such as choice of test mass and mirror coating materials, suspension design, interferometric layout, control and readout, would need a separate review article to themselves, but we shall very briefly summarise them here. advanced ligo will consist of three 4 km detectors in the current ligo vacuum system ; two at the hanford site and one at livingston. it will apply some of the technologies from the geo600 interferometer, such as the use of a signal recycling mirror at the output port and monolithic silica suspensions for the test masses, rather than the current steel wire slings. larger test masses will be used with an increase from 11 to 40 kg, although the masses will still be made from fused silica. the mirror coating is likely to consist of multiple alternating layers of silica and tantala, with the tantala layers doped with titania to reduce the coating thermal noise. the seismic isolation systems will be replaced with improved versions offering a seismic cut - off frequency of 10 hz as opposed to the current cut - off of 40 hz. as stated for enhanced ligo (in section 6.1.2), the laser power will be greater than for initial ligo and a dc readout scheme will be used. initial / enhanced ligo was shut down to begin the installation of these upgrades on 20 october 2010. the design strain amplitude sensitivity curve for aligo (and advvirgo and lcgt) is shown in figure 18. figure 18design sensitivity curves for the advanced ligo, advanced virgo and lcgt second - generation detectors. the advanced ligo curve comes from, the advanced virgo curve comes from, and the lcgt curve comes from. these curves are based on specific configurations of the detectors and are therefore subject to change. design sensitivity curves for the advanced ligo, advanced virgo and lcgt second - generation detectors. the advanced ligo curve comes from, the advanced virgo curve comes from, and the lcgt curve comes from. these curves are based on specific configurations of the detectors and are therefore subject to change. advvirgo will apply similar upgrades to those for aligo and over a similar timescale (for details see and). plans are to add a signal recycling mirror, monolithic suspensions, increased laser power to 200 w, improved coatings, and to potentially use non - gaussian beams (see, e.g.,), although this option is unlikely. the seismic isolation system will not be changed. the large - scale cryogenic gravitational - wave telescope (lcgt) [234, 247, 207 ] is a planned japanese detector to be sited underground in the kamioka mine. the lgct will consist of a detector with 3 km arms, using sapphire mirrors and sapphire suspensions. initially it will operate at room temperature, but will later be cooled to cryogenic temperatures. this detector is planned to have similar sensitivities to aligo and advvirgo, with a reach for binary coalescences of about 200 mpc with snr of 10. there currently exists a technology demonstrator called the cryogenic laser interferometer observatory (clio) [324, 164 ], which has a 100 m baseline and is also sited in the kamioka mine. this is to demonstrate the very stable conditions (i.e., low levels of seismic noise) existing in the mine and also the cryogenically - cooled sapphire mirrors suspended from aluminium wires. in experiments with clio at room temperature (i.e. 300 k), using a metallic glass called bolfur for its wire suspensions, it has already been used to produce an astrophysics result by looking for gravitational waves from the vela pulsar, giving a 99.4% confidence upper limit of h = 5.3 10. tests with the cryogenic system activated and using aluminium suspensions allowed two mirrors to be cooled to 14 k. having a network of comparably - sensitive detectors spread widely across the globe is vital to gain the fullest astrophysical insight into transient sources. position reconstruction for sources relies on triangulating the location based on time - of - flight delays observed between detectors. therefore, having long baselines, and different planes between as many detectors as possible, gives the best positional reconstruction in it is shown that for the 2 us aligo sites sky localisation will be on the order of 1000 square degrees, whereas this can be brought down to a few square degrees with the inclusion of more sites and detectors. observation with multiple detectors also provides the best way to give confidence that a signal is a real gravitational wave rather than the accidental coincidence of background noise. finally, multiple, differently - oriented, detectors will increase the ability to reconstruct a transient sources waveform and polarisation. currently design studies are under way for a third - generation gravitational - wave observatory called the einstein telescope (et). this is a european commission funded study with working groups looking into various aspects of the design including the site location and characteristics (e.g. underground), suspensions technologies ; detector topology and geometry (e.g. an equilateral triangle configuration) ; and astrophysical aims. the preliminary plan is to aim for an observatory, which improves upon the second - generation detectors by an order of magnitude over a broad band. there are many technological challenges to be faced in attempting to make this a reality and research is currently under way into a variety of these issues. investigations into the interferometric configuration have already been studied (see [145, 174, 178 ]), with suggestions including a triple interferometer system made up from an equilateral triangle, an underground location, and potentially a xylophone configuration (two independent detectors covering different frequency ranges, i.e., ultimately giving six detectors in total, although constructed over a period of years). three potential sensitivity curves are plotted in figure 19 for different configurations of detectors. figure 19potential sensitivities of the einstein telescope for 3 different design concepts : et - b, et - c and et - d. the curves are available from potential sensitivities of the einstein telescope for 3 different design concepts : et - b, et - c and et - d. some of the most interesting gravitational - wave signals, resulting from the mergers of supermassive black holes in the range 10 to 10 m and cosmological stochastic backgrounds, will lie in the frequency region below that of ground - based detectors. the most promising way of looking for such signals is to fly a laser interferometer in space, i.e. to launch a number of drag - free spacecraft into orbit and to compare the distances between test masses in these craft using laser interferometry. until early 2011, the laser interferometer space antenna (lisa) see, for example, [125, 272, 217, 216 ] was under consideration as a joint esa / nasa mission as one l - class candidate within the esa cosmic visions program. funding constraints within the us now mean that esa must examine the possibility of flying an l - class mission with european - only funding. accordingly all three l - class candidates are undergoing a rapid redesign phase with the goal of meeting the new european - only cost cap. financial, programmatic and scientific issues will be reassessed following the redesigns and it is currently expected that the selection of the first l - class mission will take place in 2014. however, for the rest of this article we will discuss the plans for lisa prior to these developments. lisa would consist of an array of three drag - free spacecraft at the vertices of an equilateral triangle of length of side 5 10 km, with the cluster placed in an earth - like orbit at a distance of 1 au from the sun, 20 behind the earth and inclined at 60 to the ecliptic. a current review of lisa technologies, with expanded discussion of, and references for, topics touched upon below, can be found in. here we will focus upon a couple of topics regarding the interferometry needed to give the required sensitivity. proof masses inside the spacecraft (two in each spacecraft) form the end points of three separate, but not independent, interferometers. each single two - arm michelson - type interferometer is formed from a vertex (actually consisting of the proof masses in a central spacecraft), and the masses in two remote spacecraft as indicated in figure 21. the three - interferometer configuration provides redundancy against component failure, gives better detection probability, and allows the determination of the polarisation of the incoming radiation. the spacecraft, which house the optical benches, are essentially there as a way to shield each pair of proof masses from external disturbances (e.g., solar radiation pressure). drag - free control servos enable the spacecraft to follow the proof masses to a high level of precision, the drag compensation being effected using proportional electric thrusters. illumination of the interferometers is by highly - stabilised laser light from nd : yag lasers at a wavelength of 1.064 microns, laser powers of 2 w being available from monolithic, non - planar ring oscillators, which are diode pumped. for lisa to achieve its design performance strain sensitivity of around 10 hz, adjacent arm lengths have to be sensed to an accuracy of about 10 pm(hz). because of the long distances involved and the spatial extent of the laser beams (the diffraction - limited laser spot size, after travelling 5 10 km, is approximately 50 km in diameter), the low photon fluxes make it impossible to use standard mirrors for reflection ; thus, active mirrors with phase locked laser transponders on the spacecraft will be implemented. telescope mirrors will be used to reduce diffraction losses on transmission of the beam and to increase the collecting area for reception of the beam. with the given laser power, and using arguments similar to those already discussed for ground - based detectors with regard to photoelectron shot noise considerations, means that for the required sensitivity the transmitting and receiving telescope mirrors on the spacecraft will have diameters of 40 cm. figure 20a design sensitivity amplitude spectral density curve for lisa created using the standard parameters in the online generator at. the curve assumes equal length arms, sensitivity averaged over the whole sky and all polarisations, and an snr of 1. also included is a curve showing the expected background noise from galactic white - dwarf - binary systems, which will dominate over the instrumental noise in the range from 0.11 mhz. a design sensitivity amplitude spectral density curve for lisa created using the standard parameters in the online generator at. the curve assumes equal length arms, sensitivity averaged over the whole sky and all polarisations, and an snr of 1. also included is a curve showing the expected background noise from galactic white - dwarf - binary systems, which will dominate over the instrumental noise in the range from 0.11 mhz. further, just as in the case of the ground - based detectors, the presence of laser frequency noise is a limiting factor. it leads to an error in the measurement of each arm length. if the arms are equal, these errors cancel out, but if they are unequal, the comparison of lengths used to search for gravitational waves may be dominated by frequency noise. for the 5 10 m long arms of lisa, then, for a relative arm length measurement of 2 10 m hz (the error budget level allowed in the lisa design for this noise source), equation (14) suggests that a laser stability of 6 10 hz hz is required, a level much better than can be achieved from the laser on its own. the first method of stabilisation is to lock the frequency of one laser in the system on to a local frequency reference, e.g., a fabry - prot cavity mounted on one of the craft (see, for example,), and then to effectively transfer this stability to other lasers in the system by phase locking techniques. with the temperature fluctuations inside each craft limited in the region of 3 mhz to approximately 10 k hz by three stages of thermal insulation, a cavity formed of material of low expansion coefficient such as ule allows a stability level of approximately 30 hz hz (again at 3 mhz). this level of laser frequency noise is clearly much worse than the required 1.2 10 hz hz (at 3 mhz) and a further correction scheme is needed. a second possible stage of frequency stabilisation is arm - locking, which relies on the fact that, by design, the fractional stability of the lisa arms is of order /l 10 hz to derive an error signal from the phase difference between the local laser and the received light. as the received light is phase locked with the local laser from the craft that sent it, it caries a replica of the frequency noise of the local laser noise delayed by one round trip time = 33 s. using this fact, this noise can be suppressed at frequencies smaller than the round trip frequency f = 1/ = 30 mhz. this scheme requires no additional hardware and can be completely implemented in software, but it will still leave frequency noise that is several orders of magnitude above required levels. a third stage frequency stabilisation scheme, which is a post - processing step, is time - delay interferometry (tdi). this makes use of the fact that, because the beams coming down each arm are not combined, the phase of each beam can be measured and recorded. therefore, correlations in the frequency noise can be calculated and subtracted by algebraically combining phase measurements from different craft delayed by the multiples of the time delay between the spacecraft. the accuracy of this is set by the phase measurement accuracy, which allows frequency noise subtraction to below the required level. a simple tdi scheme, for a much simplified constellation, was first based in the frequency domain, but due to complexities in taking into account changing arm lengths and a more complex interferometric scheme, subsequent implementations have been in the time domain. a mathematical overview of the tdi scheme, along with moving spacecraft and unequal arm lengths, can be found in. one of the major components of lisa is the disturbance reduction system (drs), which is responsible for making sure the test masses follow, as far as possible, purely gravitational orbits. this consists of the gravitational reference sensor (grs) and the control and propulsion systems used to keep the spacecraft centred on the test mass. the test masses for lisa are 1.96 kg cubes, with sides of 46 mm and made of an alloy of 75% gold and 25% platinum, chosen because of its very small magnetic susceptibility. the masses are housed in a cube of electrodes designed to capacitively sense their position and to have measurement noise levels of 1.8 nm hz. the masses need to be tightly held in place during launch and then released, so a caging mechanism has been designed consisting of 8 hydraulic fingers (one for each corner of the mass) pushing with 1200 n of force. there will be adhesion between the fingers and the masses, which will require about 10 n of force per finger to break. to provide this force two plungers will push on the the top and bottom surfaces of the masses releasing them from the fingers, followed by pushing smaller release tips in each plunger, and quickly retracting them, to overcome their adhesion to the masses. charged particles produced by cosmic radiation interacting with the surrounding spacecraft can cause the test masses to become charged at a rate of about 50 electrons per second. current plans are to use uv light from mercury lamps (or potentially uv leds) to discharge the masses. another key technology for the drs are the micro - newton thrusters, which provide the fine control needed for drag - free flight. these will mainly be used to counteract solar radiation pressure on the spacecraft, which requires about 10 n per relevant thruster. thrust noise as a function of frequency is required to be smaller than \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$0.1\mu { \rm{n}}\;{\rm{h}}{{\rm{z}}^{{\rm{- 1/2 } } } } \times \sqrt { 1 + { { \left({{{10\;{\rm{mhz } } } \over f } } \right)}^4}.}$$\end{document } two types of system, both of which meet the requirements, will be tested on lisa pathfinder : the us colloid micro - newton thruster (cmnt) ; and the european field emission electric propulsion system (feep). the cmnt uses small drops of a colloid, which it ionises through field emission, accelerates and ejects from the thruster. two designs of feep currently exist, one using indium and the other caesium and with different geometries, which, instead of ionising a droplet of colloid, just use single ions. many of the systems above are being tested in the lisa pathfinder mission (see below) and use the nominal lisa designs. there are many other issues associated with laser interferometry, and other aspects of the mission mentioned above, for lisa, which are not dealt with here and the interested reader should refer to [183, 192, 193 ] for a discussion of some of these. for lisa an industrial contract was awarded to astrium gmbh for the lisa mission formulation study. within the current esa science programme lisa is in the cosmic vision 20152025 programme, and launch after 2020 seems likely. in 2007 the national research council report on the nasa beyond einstein program (soon to become the physics of the cosmos program) gave lisa the highest scientific ranking, and it has been rated very highly in the astro2010 decadal survey. however, as stated above, as of earlier this year (2011) esa is considering the feasibility of lisa as a single agency mission. several of the key technologies for lisa are being testing on the lisa pathfinder mission (formerly smart-2). lisa pathfinder will fly the lisa technology package (ltp), which essentially consists of a downscaled version of one lisa arm compressed from 5 million km to 38 cm. the ltp arm contains two test masses (an emitter and a receiver) with a doppler link between them. the three main things it will measure are : the acceleration phase noise caused by the relative motion of the emitter and receiver from non - gravitational forces ; the readout noise ; and noise caused by the departure of the doppler link from the ideal scheme, due to the fact that we are not truly measuring the relative accelerations of two point particles, but instead a more complex system of multiple doppler links and extended masses. it is designed to test the accuracy of these to within an order of magnitude of that required by the full lisa. other aspects of the mission that will be tested are the discharging of the test masses, the caging and release of the masses following launch and the micro - newton thrusters. as much as possible lisa pathfinder is currently scheduled for launch in mid 2013, after which it will orbit the l1 point, with a 180-day mission plan. lisa is the most advanced space - based project, but there exist concepts for at least two more detectors. decigo (deci - hertz interferometer gravitational wave observatory) [274, 199 ] is a japanese project designed to fill the gap in frequency between ground - based detectors and lisa, i.e. the 0.110 hz band. it would have a similar configuration to lisa with three drag - free spacecraft, but have far shorter arm lengths at 1000 km. although still early in its design there are plans for two precursor technology demonstration missions (decigo pathfinder and pre - decigo), with a the main mission having a launch date in the mid-2020s. a similar mission, in terms of the frequency band it seeks to cover, is the us big bang observer (bbo) (see [122, 124, 167 ] for overviews of the proposal). one of its main aims will be to detect the stochastic background from the early universe, but it can also be used for high precision cosmology. the current configuration would consist of three lisa - like constellations of three spacecraft each, with 50 000 km arm lengths, and separated in their orbit by 120. the launch of this mission would be after decigo, but is designed to be 23 times as sensitive. at lower frequencies than lisa, 0.1 hz1 mhz, there are chinese proposals for superastrod (super astrodynamical space test of relativity using optical devices). significant effort worldwide has been invested, and is continuing to be invested, in the development of both ground and spaced based gravitational - wave detectors. over the coming years, ground - based detectors will reach sensitivities that will enable the direct observation of gravitational waves as predicted by einstein s general theory of relativity and open the exciting new field of gravitational - wave astronomy. studying the gravitational - wave sky could radically change our understanding of the universe, expanding our knowledge of fundamental physics, cosmology and relativistic astrophysics. gravitational - wave observations will allow us to gaze into the heart of the most violent events in the universe, and will help answer some of the biggest questions, particularly in cosmology when combined with other astronomical observations. | significant progress has been made in recent years on the development of gravitational - wave detectors. sources such as coalescing compact binary systems, neutron stars in low - mass x - ray binaries, stellar collapses and pulsars are all possible candidates for detection. the most promising design of gravitational - wave detector uses test masses a long distance apart and freely suspended as pendulums on earth or in drag - free spacecraft. the main theme of this review is a discussion of the mechanical and optical principles used in the various long baseline systems in operation around the world ligo (usa), virgo (italy / france), tama300 and lcgt (japan), and geo600 (germany / u.k.) and in lisa, a proposed space - borne interferometer. a review of recent science runs from the current generation of ground - based detectors will be discussed, in addition to highlighting the astrophysical results gained thus far. looking to the future, the major upgrades to ligo (advanced ligo), virgo (advanced virgo), lcgt and geo600 (geo - hf) will be completed over the coming years, which will create a network of detectors with the significantly improved sensitivity required to detect gravitational waves. beyond this, the concept and design of possible future third generation gravitational - wave detectors, such as the einstein telescope (et), will be discussed. |
in the past decade, written communications about biliary - enteric drainage (bed) have significantly decreased throughout the world. in fact, the indications for this surgical modality of common bile duct stone (cbds) have been limited with the advances and increasing experience in laparoscopic and endoscopic procedures. the advantages offered by this procedure, as well as its risks and disadvantages, also have been well noted in most of the already quoted bibliographic references. however, with three exceptions, the long - term results of choledochoduodenostomy (cds), particularly in reference to the incidence of complications such as cholangitis, sump syndrome and risk of late development of cholangiocarcinoma have not been documented clearly. this retrospective study reviews the results of 51 consecutive cases of open biliary - enteric bypass carried out for cbds over a period of 5 years. our objective was to review the indications and outcome of this procedure in the endoscopic and laparoscopic era. we conducted a descriptive retrospective chart review of all the patients undergoing open bed for cbds in general surgery department at farhat hached hospital of sousse in tunisia between january 2005 and december 2009. data were collected from patient 's medical folders with particular attention to perioperative course, symptoms, complications, hospital length of stay, and mortality. short - term outcomes were assessed through clinical reports at outpatient follow - up and hospital readmissions. the main surgical technique used was a side to side cds after washing systematically the cbd without verifying its clearance by a choledocoscope or a cholangiogram. the anastomosis was constructed with 4 - 0 vicryl suture with small atraumatic needles in a continuous running (anterior and posterior rows) or interrupted fashion : it was left to the surgeon 's discretion to select the type of suturing. ninety - three patients were treated for cbds with several procedures during the 5 years period. only 51 (40 women [78% ] and 11 men) underwent open bed. the age range was 36 - 87 years with mean age 72 years (standard deviation [sd ] 9,7). they presented with acute cholangitis (28%), abdominal pain (86%), jaundice (21%), acute pancreatitis (6%), and acute cholecystitis with asymptomatic cbd stone (6%). demographic and clinical details of 51 patients the main indications for biliary enteric bypass were elderly patients (90% were over 65 years old), multiple (atleast 5) stones (54.9%) and unextractable calculi (15.7%). the remaining patients had different clinical and intra - operative presentation : congenital biliary cyst (1 case), accidental intra - operative transection of cbd with transversal choldochotomy (1 case), cholcysto - choledochal fistulas (1 case), suspicious chronic pancreatitis associated with cbd calculi (1 young woman) and a recurrent lithiasis in an old woman with permanent biliary stenting. we performed side to side cds in 49 patients (96%) and end to side type in one patient who had iatrogenic transection of cbd. the cbd diameter was larger than 16 mm in 37.3% of the patients and 10 mm in 98% of the patients. post - operative complications [table 2 ] occurred in 5 (10%) patients, including one case of intra - abdominal abscess (2%) successfully managed by transparietal drainage, wound infection (4%), two cases of heart failure (3.9%) and one patient had post - operative peritonitis subordinate to ruptured liver abscess previously unnoticed, he was re - operated and had successful peritoneal toilet. post - operative deaths : two patients (3.9%) died in the hospital during the same hospitalization for their cds. the first was an 80-year - old woman with cardiac history admitted for acute cholecystitis associated to asymptomatic cbds ; she died of post - operative heart failure. the second was a 76-year - old woman admitted for severe acute cholangitis with hemodynamic failure. post - operative complications the mean hospital stay was 10.9 days (sd 6.5) and the mean post - operative stay was 7 days (sd 3.8). eight patients left the hospital in good condition and were lost to follow - up, so it is presumed that their intervention was initially successful. they never come to outpatient control. during the follow - up (1 - 6 years) of the remaining patients, two had further intervention for right subcostal incisional hernia and one presented non - resectable head pancreatic cancer with multiple liver metastases 1 year after cds. our data corroborating others, as illustrated in table 3, indicate that side to side cds is a very safe procedure with low morbidity and mortality. no long - term complications, as cholangitis, sump syndrome, or cholangiocarcinoma were observed. comparison of outcomes of different studies it should be noticed that we opted for bed because of not only its benefits mentioned further in the text but also the lack of interventional endoscopist and laparoscopic instruments particularly the helical basket in our hospital. indeed cds provides a safe and effective biliary drainage when carried out for carefully chosen indications. the procedure may still be required in selected cases, even with all the endoscopic and laparoscopic advances. we performed the side to side type of cds for 49 patients (96%) including two young women (36- and 38-years - old) as it is technically easier and faster than its end to side variety and hjs. it also permits to avoid the particular problem of residual stone. these criteria are capital when choosing among bed modalities particularly for high risk and elderly patients. furthermore, the latter with their limited life expectancy should not develop long - term complications. reports on laparoscopic cds have been published, but these procedures need considerable experience and expensive technologies. the two most notable objections to cds are the possibility of cholangitis and the presence of the symptoms produced by the so called sump / blind sac syndrome. thus many authors suggested a cbd diameter larger than 16 mm precaution to allow a cds with an opening no smaller than 14 mm in diameter.[17 ] our study, with no such complications, disproves this condition (only 37% of patients had cbd diameter larger than 16 mm). moreover, de almeida ensured that a wide anastomosis can be constructed even on a duct 10 - 12 mm wide, and this concur with our results (98% of our patients had cbd 10 mm). such well performed cds prevents bile stasis, avoids the buildup of excessive intra - ductal pressure and permits a free flow of common duct contents including duodenal reflux as well as eventual retained / recurrent lithiasis back in the duodenum. the prevalence of blind sac syndrome is reported between 2.5% and 9.6% in several studies. it presumably derives from stasis and refluxed duodenal contents (food, stones, or debris) in to the terminal common duct, with bacterial overgrowth enhancing bile salt deconjugation. so factors leading to sump syndrome are a wide distal choledoque, papillary stenosis, retained stones, and anastomosis stricture. this phenomenon leads to diarrhea, as observed in the blind - loop syndrome arising anywhere else in the gut, can cause secondary liver abscess and facilitates deposition and reformation of calcium bilirubinate stones which could not be documented in spite of an exhaustive search. a very wide anastomosis could be the explanation.[810 ] in order to avoid this so feared syndrome, lasnier recommended end to side cds for young patient or in case of incomplete clearance of cbd and/or oddi dysfunction. otherwise they opted for side to side cds specially for elderly patients, in case of altered anatomy after billroth ii surgery, and when he faced up to technical difficulties due to inflammation, sclerosis and portal hypertension. however, mavrogiannis reported a considerably high recurrence rate 19% (6 patients/31) of the syndrome (restenosis of the sphincterotomy opening) after initially successful such management. it occurred 31 - 72 months (median : 58.5 months) after the initial treatment and new endoscopic treatment with a papillotomy was effective. in case of stoma stricture the best treatment is the endoscopic balloon cholangioplasty.[413 ] the blind sac syndrome associated to side to side hjs is extremely rare. indeed stricture is the main complication of hjs and rates, in most series, varies from 0% to 12% (with most being around 8%) and from 2.4 to 4.8% in case of biliary lithiasis. technique requirements for a good anastomosis are good submucosal apposition, single - layer closure, and mucosal inversion. in case of hepatolithiasis, bed results in high frequency of reflux cholangitis and thus should be abandoned. in recent years the number of reports of cholangiocarcinoma occurring in patients who previously had bed many years ago, has been increasing, so it has been suggested as a possible long - term complication of these surgical procedures. its incidence ranges from 2.8 - 7.4% at interval average of 10 years after choldechoenterostomy.[1720 ] the direct connection between the intestinal tract and biliary tree and reflux of activated pancreatic juice and intestinal bacteria into the biliary tract are considered as the factors causing chronic relapsing cholangitis, the latter has been suspected as a predisposing factor for the late development of cholangiocarcinoma. bed permits free communication between gastro - intestinal and biliary tracts, depriving the anti - reflux function of the sphincter of oddi. histologic examination of resected specimens, including cholangiocarcinoma of the biliary tract arising from long - standing cholangitis secondary to a bilioduodenal drainage, has shown, at sites distant from the malignancy, various degrees of hyperplastic lesions occurring in the area of cholangitis. this strengthens the hyperplasia - dysplasia - carcinoma progression hypothesis of a biliary carcinogenesis mechanism. the roux - en - y technique is the one expected to produce lower rates of cholangitis because it is immune to pancreatic reflux and less prone to intestinal bacterial backflow because of the protective action exerted by the interposed jejunal loop. tocchi reported a retrospective study of 1003 patients who underwent biliary enteric anastomosis (transduodenal sphincteroplasty, cds, and hjs) for benign disease with 129.6 years mean follow - up : univariate and multivariate analysis showed a significantly higher incidence of cholangiocarcinoma in the cds group (p = 0.02), and cholangitis was confirmed to be the only independent factor affecting its incidence (p = 0.001, odds ratio 35.7). thus hjs should be considered straightaway for young patients in spite of cds benefits. they also noted that patients with such anastomosis had higher incidence of gastric lesions when compared to those with t - tube drainage or endoscopic sphincterotomy. the fact that one of our patients presented non - resectable and metastatic head pancreatic cancer 1 year after the cds, suggests that the tumor was present at the time of bd but was not detected. large retrospective studies and prospective randomized trials are needed to establish the definitive risk for late biliary carcinogenesis after bilio - intestinal drainage. thus we suggest that any patient treated with these procedures and experiencing relapsing cholangitis should be monitored for the late development of bile duct cancer. in case of asymptomatic cbs, we readily opt for minimally invasive techniques (laparoscopy and/or endoscopy) for young patients or fine cbd. indeed considering cds inconvenients, it was performed for only old patients with dilated cbd provided that surgery is nt considered inappropriate. open biliary drainage procedures may still indicated in select patients where the facility or expertise for minimally invasive biliary procedures is not available. side - to - side cds is a safe and highly effective therapeutic measure, even when performed on ducts less than 15 mm wide, provided a few technical requirements are respected. it should only be considered as obsolete after extensive, long - term, prospective, randomized assessments of laparoscopic or combined laparo - endoscopic approaches have been shown to be as effective as or superior to cds even in young population. patients experiencing relapsing cholangitis after bed should be closely monitored for the late development of biliary tract malignancies. the procedure should be regarded as an essential in the general surgical knowledge and training. | background : nowadays, biliary - enteric drainage (bed) is regarded as a last resort or obsolete therapeutic method for common bile duct stone (cbds) not only because of advances in minimally invasive therapeutic modalities but also due to fears of higher morbidity, cholangitis, and sump syndrome.aim:the present study aimed at evaluating the outcome of this procedure for choledocholithiasis.materials and methods : it is a retrospective review of 51 patients who underwent open choledochoenterostomy for cbds between january 2005 and december 2009.results:about 40 women (78%) and 11 men underwent open bed (mean age 72 years). indications were elderly patients (90%), multiple stones (54.9%) and unextractable calculi (15.4%). we performed 49 (96%) side to side choledochoduodenostomies, one end to side choledochoduodenostomy (cds) and one end to side hepaticojejunostomy. the mortality rate was 3.9%. overall morbidity was 12% with no biliary leakage. with a decline of 1 - 6 years, neither sump syndrome nor cholangiocarcinoma occurred.conclusions:side-to-side cds is a safe and highly effective therapeutic measure, even when performed on ducts less than 15 mm wide, provided a few technical requirements are respected. patients experiencing relapsing cholangitis after bed should be closely monitored for the late development of biliary tract malignancies. |
the scientific path taken to understand cancer is paved with thousands of theories and scientific findings, from the time of hippocrates to genomics today. chemical carcinogenesis was brought to light with the observations of paracelsus on cancer onset and environmental exposure to chemicals undertaken over six centuries ago 1 - 3. the relationship between cancer and chemicals captured the interest of scientists since the 18 century, and this having had a greater impact in the last century. this knowledge, previously unproved, is a reality in today 's research and plays a vital role in the understanding of cancer. exactly a century ago, in the book, zur frage der entstehung maligner tumoren, boveri presented his theory on cancer that was based on chromosomal abnormality 4 - 6. boveri had no knowledge on the chromosome structure ; however, his theory today is considered a pillar of modern molecular approaches to cancer. the discovery of the dna structure and the achievements of molecular biology have reaffirmed boveri 's theory and given technological support to developments in this field. without doubt, the first human transforming gene, and more broadly, oncogenes and anti - oncogenes, had been previously theorized by theodor boveri. nowadays, even though scientific research moves ahead rapidly, cancer still needs solid and reliable solutions. nevertheless, science should look back to historical achievements in advancing new theories and data. considering the past, and reviewing the milestones of every scientific finding could help in molding future experimental approaches in cancer research. cancer has been in existence on earth even before the appearance of man, as evidenced by paleontological findings of tumors in animals 1, 2. the first description of human cancer can be found in the edwin smith papyrus dated 3000 bc that illustrated a case of breast cancer. other documented proof includes the ebers papyrus dating from 1500 bc that describes several types of tumors concerning skin, uterus, stomach, and rectum. these old egyptian documents recorded cancer as a grave incurable disease and associated it to ' the curse of the gods ' 1. interestingly, this belief continued to be accepted right up to hippocrates (460 - 370 bc), who postulated the earliest scientific theory about cancer. he believed that cancers, and more broadly any disease, developed whenever the balance in the four body humours (blood, phlegm, yellow bile, and black bile) were lost. whenever black bile became the dominant humours in some part of the body, this developed cancer. hippocrates theory established the foundations of experimental approaches in understanding and confronting cancer, thereafter practiced by all scientists. claudius galen (130 - 200), a greek physician who practiced medicine in rome, implemented the hippocrates theory. he proposed that black bile caused incurable cancer, whereas yellow bile caused curable cancer. additionally, the terms and words used to name this disease, and whatever it was associated with, were created by these early ' scientists '. hippocrates used the word carcinoma, comparing the disease to a crab () that adheres to its surroundings with his claws 1, 2. the physician celsus (25 bc-50), later translated this word into cancer, the latin word for crab. galen (130 - 200) described tumors using the greek term for swelling, oncos. in the middle ages, religious beliefs inhibited the advancement of knowledge, and people began to believe that cancer was an infectious disease. eventually, medical science gained a foothold during the renaissance when scientists began performing autopsies, combining the study of the human body and that of cancer, leading to important achievements. in the 16 century, the anatomist andreas vesalius and others were able to demonstrate the non - existence of black bile. nevertheless, hippocrates ' theory still continued to have great number of supporters, and it took some time before it was superseded by a new hypotheses. in the same century, paracelsus studied tumors of mine workers, and suggested there were deposits of sulfur and arsenic salts in blood of these workers, causing their cancers 1 - 3. at this time it was not known how the elements of the environment caused disease ; however, this observation pre - empted the future of chemical carcinogenesis research, being the first association between the work environment and cancer. a century later, boerhaave suggested, ' that cancer was most likely induced by viruses present in water or in soil. once acquired, the cancer viruses remained in the body, and they could be transferred by contagious infections or by heredity '. this theory was by no means new, as people during middle ages understood that cancer was a contagious disease. moreover, at that time the term virus indicated some kind of toxic substance, therefore in this respect, boerhaave 's hypothesis was similar to that of paracelsus. however, boerhaave first introduced the new concept that cancer could be hereditary 1, 2. in the 18 century morgagni, founded scientific oncology by performing autopsies after death to identify the patient 's disease. consequently, he reported that cancer was the result of an ' organ lesion '. other scientists and surgeons suggested cancer was a destructive growth of the organ, caused by their internal structural transformation, or derived from coagulated lymph 1, 2. during the 18 century, different physicians expanded the hypothesis about the association between cancers and chemical exposure. their observations had recalled the paracelsus hypothesis, and this established the new and modern approach of cancer epidemiology. interestingly, in 1775 percivall pott noticed that chimney sweepers frequently developed cancer of the scrotum in later life, and suggested that soot was the cause of this type of cancer. in the last decades of the 19 century, ludwig rehn, observed an association between exposure to the aniline dye and bladder cancer in industrial workers 2, 3. the use of the microscope represented an opportunity for researchers to investigate tumors in great detail. rudolf virchow stated that ' cancer is a disease of cells ', and david hansemann proposed, ' the cell of the malignant tumor is a cell with a certain abnormal chromatin content ' 2, 4, 6. at the beginning of the 20 century, several researchers experimentally confirmed that specific chemicals in the workplace and environment were correlated with the carcinogenic process, demonstrating their multistage and multifactor nature. katsusaburo yamagiwa and koichi ichikawa, in 1915, were able to induce cancer in rabbits by applying coal tar to the skin of these laboratory animals 3. it is worthwhile to mention what boveri wrote, ' tumor growth is based on.. a particular, incorrect chromosome combination which is the cause of the abnormal growth characteristics passed on to daughter cells ' 4 - 6. a major milestone for modern era cancer was the discovery of the first tumor viruses 7, 8 in chickens by ellerman and bang (1908) and peyton rous (1911). these and other important achievements on cancer research, from prehistoric times up to present day are summarized by date in table 1. of important note, in the beginning of 1900 external factors such chemical ; radiation and viruses were accepted as etiological agents of cancer 3 - 8. amazingly, both chemical and viral carcinogenesis produced the same results 9, since, independently, they provided evidence that tumors are correlated to mutated genes. ellerman and bang in 1908, induced erythro - myeloblastic leukemia in healthy chickens by an acellular filtrate collected from diseased chickens 7, 8, which later proved to be an rna virus. in 1910, peyton rous isolated a filterable agent from the sarcoma in the breast muscle of a plymouth rock hen. this agent was later named the rous sarcoma virus (rsv) and was regarded as the first rna tumor virus, as in the early 1900 's leukemia was not considered a cancer. however, the scientific world at this time was skeptical and did not note the importance of these developments. for instance in 1936, r. bittner demonstrated that the mammary carcinoma of c3h mice was induced by viruses and this observation was fundamental in rna tumor virus research 7, 8. later, on 1953, l. gross isolated the first virus inducing leukemia in mice 7, 8. it is worthwhile to mention that in 1907 the italian researcher, g. ciuffo, demonstrated the transmission of human warts by inoculating himself with a cellular filtrate 7, 8. the significance of this finding was understood only many years later, when the association between cancer and some types of these viruses was demonstrated, currently known as papillomaviruses. dna tumor virus research and more broadly of other human tumor viruses, will not be discussed in this review ; however, in table 1 the principal achievements in these fields are listed. cell culture techniques implementation and the definition of dna structure have been crucial in determining infections and transformation mechanisms of these tumor viruses, permitting the isolation and characterizations of mutants 10 - 12. table 2 shows the achievements on molecular biology and biochemistry from the end of 1800, up to the advent of the human genome. if table 1 and table 2 are compared, it becomes quite evident that the deeper knowledge of cellular molecular processes and the implementation of new experimental approaches were decisive in understanding present day tumor genetics. figure 2 summarises the major achievements obtained in molecular cancer research and molecular biology. in 1961, rsv was shown to contain an rna genome 13, and it became the prototype, ' rna tumor virus ' 14. moreover, experiments performed by temin with actinomycin and amethopterin, inhibitors of nucleic acid, suggested that the virus replicated by transferring the genetic information of the infecting virus, from rna to dna, and back again to the rna of the progeny virus 15. these results have to be considered as a milestone in tumor virology, although the rna genome of the virus represented a dilemma hard to resolve. in 1964, temin proposed the theory of provirus, '.... virus enters a cell and directs formation of a dna containing the genetic information of the virus. this new dna, the provirus, then acts as a template for the formation of new nucleic rna, for the virion.. ' 16. the provirus theory was based on the revolutionary hypothesis that rsv replicates through a synthetized dna intermediate using as a template the rna genome 17. up to this time, no evidence existed on the possibility of transfering genetic information from rna to dna and back to rna. hence, this innovative model of viral replication did not receive much attention from the scientific community. however, researchers had the urgency to resolve the inexplicable results on rna tumor virus replication obtained with inhibitors of nucleic acid synthesis. in 1969, this theory postulated that dna of most, or all of, the eukaryotic cells carried vertically transmitted rna tumor virus information, this being known as virogene 18. these viral sequences contain the oncogene, which may transform normal cells into tumor cells. these rna tumor viruses are present in a repressed form in the cells and they may be ' switched on ' by various environmental factors, and also are dependent on the host genotype. the ' switching on ' of these sequences can produce an infectious virus and/or induce the cellular transformation. the virogene hypothesis differs from the provirus hypothesis, since the latter implies that an exogenous virus infects the cell. instead, the virogene / oncogene is part of the genetic background of all vertebrate cells. in 1970, temin and baltimore individually isolated the reverse transcriptase, which was able to mediate the rna - dependent dna synthesis 19, 20. this enzyme represented the key element of the provirus hypothesis, and after this event the rna tumor viruses were renamed retroviruses. reverse transcriptase had definitively changed the approach in studying rna tumor viruses and permitted new experimental methods in investigating their neoplastic mechanisms. in 1976, dominique stehelin, harold varmus, michael bishop, and peter vogt characterized the sequences of rsv responsible for its neoplastic potential, the src gene 21. moreover, they demonstrated the cellular origin of this transforming gene, since its normal counterpart is present in the normal eukaryotic cells. following this, many more transforming genes of other tumorigenic retroviruses were characterized, and their cellular origin confirmed. these achievements recalled the theory of virogene / oncogene by huebner and todaro, and these genes were actually named oncogenes, and their cellular counterpart proto - oncogene 22. around this time, bruce ames demonstrated that carcinogens could mutate bacterial genes 23. the experiments proved that carcinogens could damage the genes of target cells, confirming the theory that cancer cells carried mutant cellular genes, which may confer a growth advantage. the achievements on the transformation mechanisms of retroviruses and the findings on the mutagen activity of carcinogens demonstrated that tumors had gene alteration as a common denominator, independent of their etiology 6, 22, 23. researchers had felt the urge to go further, and formulated experimental protocols in defining possible relationships between viral oncogenes and genes mutated by physical and chemical carcinogens. early in the 1970s, graham and van der eb developed a procedure for gene transfer in mammalian cells 24. the researchers used this method to introduce dna of virally transformed cells into nih 3t3 recipient cells, obtaining foci of transformation in their recipient cells several weeks later 25. hence, this approach appeared to be a suitable method to analyse the genetic background of cancer cells, and to eventually reveal the genes involved in the neoplastic process. dna transfection was then used to analyze the presence of transforming active sequences in dna extracted from several chemically transformed 3-methylcholanthrene (3-mc) mouse - cell lines. this experiment yielded a large number of foci, and non - transforming or activated endogenous viruses being detected in these mouse cells. it was probable that the transforming activity was derived from a cellular gene mutated by the 3-mc into a transforming active allele 25. later, researchers used the gene transfer protocol to investigate the presence of active transforming genes in human tumor cell lines and primary tumors. remarkably, three different research groups demonstrated that dna extracted from human tumors cells lines could transform nih/3t3 cells. furthermore, the characterization of the transforming sequences detected in the cell line t24/ej, established from a human bladder carcinoma, evidenced their homology to ras, the transforming gene of harvey, and the balb sarcoma viruses (ha - ras) 26 - 28. molecular comparisons between the human ras transforming gene and its normal allele revealed that the malignant potential was activated by a single point mutation. also, the ras gene can acquire a transforming activity from mechanisms other than capture by retroviral sequences 9. investigators continued with the transfection approach analyzing dna extracted from human solid and hematopoietic tumors 29, 30. surprisingly, most of the activated transforming sequences detected in these tumors were homologous to viral ras genes (ha - ras and ki- ras), although these later experiments also revealed activated transforming sequences that did not have viral counterparts 30. moreover, ras transforming sequences were detected also in animal tumors induced by chemical carcinogens 9, 31. these results rolled back again to the boveri 's concept of neoplastic process in consequence of genetic alterations. in the mid-1980s, dna - transfection experiments confirmed the old theories on cancer onset and development, and fitted in well with tumor virology and chemical carcinogenesis. despite, these extraordinary achievements, investigators soon underlined the limits of viral transforming genes had been used to investigate at molecular level tumors and their cells. these molecular approaches highlighted the fact that human proto - oncogenes can acquire transforming activity and drive cells to the neoplastic phenotype if rearranged, translocated or amplified 22. one of the most relevant examples is the translocated philadelphia chromosome associated with chronic myelogenous leukemia, a hematopoietic tumor. this translocation involves the abl gene on chromosome 9 and the bcr (breakpoint cluster region) gene on chromosome 22 32. also, bukitt 's lymphoma cells were shown to present three types of translocations, all involving a fragment from the end of the long arm of chromosome 8, which can be translocated to the long arm of chromosomes 14, or eventually, to chromosomes 2 or 22. this myc sequence can be translocated to chromosome 14, near to the immunoglobulin heavy chain locus, or to chromosomes 2 or 22, near to the two immunoglobulin light chain genes 33, 34. myc gene amplification was found in the human promyelocytic leukaemia line hl60, and other cancer lines 35. moreover, it was well known from cytogenetic analysis that tumor cells may show loss of genetic information. researchers developed sophisticated protocols based on cell hybrids to investigate at molecular - level these genetic abnormalities. by cell hybridization of normal fibroblasts with the human cancer cells, hela non - tumorigenic clones were obtained, some of which reverted to neoplastic phenotype 36. molecular cytogenetic analysis of these clones had evidenced that the transformed phenotype correlated with the loss of the fibroblast chromosomes 11 and 14. together, these findings proved that neoplastic phenotype and loss of genetic sequences are correlated. furthermore, the molecular characterization of retinoblastoma and wilms ' tumor evidenced deletions on both pairs of chromosomes 13 and 11, respectively 37, 38. additionally, transfection of a normal allele of chromosomes 11 into tumorigenic wilms ' tumor cells can revert to their phenotype. the chromosomal locus / genes correlated with these two neoplasia had been cloned and characterized, following elaborate cloning strategies. the genes, associated with retinoblastoma and wilms ' tumor development were named rb1 and wt1, respectively. their characterization demonstrated that germ - line mutations are present in the heritable form 22, 37, 38. however, this inherited mutation is not sufficient for tumor formation ; a second mutational event is required. similarly, for the development of non - heritable tumors, two somatic events must occur and inactivate both genes. these results proved the two - hit hypothesis of knudson according to ' a complete inactivation of both alleles of a tumor - suppressor gene is required for cancer onset and development... '. hence, these sentinel genes acting against tumors have been named anti - oncogenes or tumor suppressor genes 39, 40. characterization of the protein products of proto - oncogenes and tumor suppressor genes revealed their role in regulating intra- and extracellular signaling related to cell growth and division. usually proto - oncogene products are correlated with cellular functions that stimulate cell growth and division. tumor suppressor genes are normal genes, mainly devoted to the cellular processes correlated with cell growth inhibition and programmed cell death (apoptosis) 6, 22, 33. mutation, amplification, deletion or translocation of these genes disrupt their function, leading to an uncontrollable growth and cancer 6, 22. these changes alter the dna and associated proteins without changing the dna sequence, modulating the chromatin structure and dna accessibility. through these mechanisms, epigenetic changes may alter the transcriptional status of cancer genes, or regions of chromosomes that control their expression, inducing the neoplastic process 41. interestingly, anti - oncogenes are involved in tumor development induced by some dna tumor viruses. these viruses code proteins that are capable of inhibiting tumor suppressor proteins. an example, is the binding between p53 protein and viral transforming proteins such e6 of oncogenic hpv 40, 42. boveri had predicted all these findings, when he wrote, ' another possibility is that there is a specific inhibitory mechanism in every normal cell that only permits cell division to take place when this mechanism is overcome by some special stimulus... a tumor cell that proliferated without restraint would be generated if these ' inhibitory chromosomes ' were eliminated ' 4, 5. in 1914, boveri wrote, ' it is conceivable that for any one cell type there is one particular abnormal combination of chromosomes that endows the cell with the properties of malignancy ', predicting cancer to be a complex multifaceted process. today, it is understood that more than one gene / chromosomal alteration contributes to tumor development, and these must be known and characterized to fully comprehend the molecular process of tumor development. in 1985, dulbecco stated in support of these factors, ' a major gap in our understanding of cancer is how the activity of an oncogene is related to the events of progression. but, the first task is to ascertain whether the dna of an advanced cancer is as heterogeneous as the phenotype of its cells. if we wish to learn more about cancer, we must now concentrate on the cellular genome.. ', and in consequence, he advocated the human genome project (hgp) 43. as dulbecco had foreseen, the sequencing of the human genome represented the corner stone for the construction of physical and genetic maps and the identification of genes involved in human cancer. integrating the knowledge on human genomes with the new powerful technologies for dna sequencing and inspection of protein expressions (mass - spectrometry), has permitted the collection of large amounts of data on genetic alterations and aberrant gene expression correlated to onset and development of different cancers 44, 45. the cancer genome atlas (tcga, http://cancergenome.nih.gov/) and the international cancer genome consortium (icgc, https://icgc.org/) provide clinical data and the genomic profiles of thousands of normal and tumor samples for a variety of cancer types. the catalogue - that includes oncogenes, anti - oncogenes, epigenetic regulators, cell cycle - associated genes, dna damage response / repair genes, metabolic regulators, and micrornas - has not yet been completed 46 - 49. cellular and genomic data from a variety of tumors have confirmed and highlighted their extraordinary heterogeneity. this heterogeneity has been evidenced among tumors from patients with the same cancer type, inter - patients heterogeneity, and within cancer cells of an individual patient 's tumor, intra - tumor heterogeneity. heterogeneity represents, indeed, a thorny challenge to overcome in order to provide clinics with a suitable cancer genomic data. the inter - patient heterogeneity is most likely tied to the different genetic backgrounds and lifestyles, and they should be considered and evaluated. therefore, a molecular investigation at single - patient level may be required for a correct diagnosis and optimal therapy 48, 49. moreover, intra - tumor heterogeneity should also be taken into account, and the mechanisms producing it. to explain the intra - tumor heterogeneity, one of the proposed models is the ' clonal evolution model '. it assumes that different mutant clones acquire a survival advantage, because of the natural competition with other clones. stochastic genetic or epigenetic changes are claimed as the most probable mechanisms conferring growth advantage to cancer cells. likely, macro and micro - environments play an important role in the development of the subpopulations of cancer cells, enabling them to initiate and/or propagate the disease, or even to permit its remission 50, 51. recently, cancer stem cells (cscs) have gained the interest of scientists as a good candidate and ' model ' to investigate tumor heterogeneity. much experimental evidence has highlighted the presence of cscs in a broad spectrum of malignancies. it has been suggested that some tumors may arise from small populations of cscs that give rise to phenotypically diverse cancer cells, playing important roles in tumor heterogeneity, favoring growth, dormancy or recurrence, and metastasis 52. the cscs imply that tumors are organized as an adult tissue, and their genetic heterogeneity of cancer propagating cells may derived from ' clonal evolution ' within the stem cell pool by the darwin 's premise of ' natural selection '. these models of cancer initiation and spreading refers back to the ' seed and soil ' and ' embryonal ' theories proposed by paget and cohenhein, respectively, more than hundred years ago (table 1). moreover, they emphasize that a tumor is composed of complex mixtures of cells with various genetic alterations and functions. they then suggest a single - cell analysis as feasible approach to overcome the dubious evaluation caused by multiple mixtures of mutations within tumor cells. nowadays, single - cell genomic sequencing and single - cell transcriptomic sequencing have been explored as potential techniques to investigate cancer heterogeneity. single - cell genomic sequencing mainly focuses on the general landscape of mutations, such as single nucleotide variations, insertions, and deletions in a single cell 53. single - cell transcriptomic sequencing can be useful for analyzing genetic network regulation, including differentiation, reprogramming and transdifferentiation. single - cell transcriptomic sequencing can highlight transcriptomic alterations (mrnas, micrornas, retained introns, alternative splicings, long - noncoding rnas and fusion genes) 50 - 53. data coming from such analysis could be relevant in understanding the role and the fate of single cells in a tumor, mainly cscs. these techniques, along with appropriate ' in vitro ' and ' in vivo ' cscs models to mimic tumor growth, could give an insight into genetic alteration / modification. it could also provide an understanding of the gene regulation networks responsible for physiological functions, behaviors, and phenotypes in response to signals and microenvironmental changes. the incoming data could be useful for revising and making available the huge amount of genomics information for the world research community. moreover, this information, when cross - referenced and integrated, could be crucial to define better personalized therapeutic strategies 48 - 53, as well as to design a broad spectrum of preventive and therapeutic strategies that are broadly relevant. the analysis of expressed biomarkers in the cancer is one of the most used approaches for the disease 's treatment, and recently, particular attention has been given to cscs, as a main factor in fighting cancer. different therapeutic strategies have been developed and are being designed to attack the cscs and to destroy their surrounding environment ; some of these strategies are under preclinical and clinical evaluation. some strategies rely on using monoclonal antibodies against surface markers that are different from normal stem cells. monoclonal antibodies against these markers and conjugated with cytotoxic agents permit the targeting of the cscs cells and eventually kill them. cscs cells play also an important role in chemo - resistance acquisition. to overcome this problem, the mechanism responsible for the chemo - resistance must be identified and characterized. one of the most recognized mechanisms is the efflux of cellular cytotoxic drugs by the atp - binding cassette (abc) transporter proteins. the trans - membrane proteins using the energy from the atp hydrolysis expel the drugs from the cells. moreover, cscs could be attacked modifying the specific pathways involved in apoptosis and resulting in suppression of cellular characteristics. particular attention has to be given to the tumor micro - environment, since it plays a relevant role in cscs growth and selection, as well as in protecting them from drugs. for instance, experimental evidence shows a protective role of bone stromal cells in b cell malignancies. finally, several therapeutic approaches are focusing on the angiogenesis processes as a mechanism to control cscs growth and differentiation. a different strategy is to target genes that are affected in a high percentage of tumor types, such as myc or ras, and to which cancer cells appear to be ' addicted ' 54, 55. approximately 30% of human tumors harbor a mutant ras gene, with an altered gtpase activity. targeting therefore, many efforts have been directed at alternative strategies such as the synthetic lethal approach, which aims at targeting proteins that are crucial for the survival of tumor cells with an activated ras protein. theoretically, it may be possible to kill tumorigenic cells expressing a mutated ras gene without affecting normal cells 55. these are just few examples of the recent efforts to relocate the modern achievements in cancer research into the diagnosis and therapy fields. however, much more is still to be done. more genomics data, more animal - models that are suitable, more implemented personalized medicine trials, and new expertise such as onco - bio - informatics, will be crucial to implement onco - knowledge into clinical evaluation. moreover, scientists should always consider the ethical, legal, and social implications that clinical - genomics raise. for instance, the high costs of such therapeutic and diagnostic approaches that can put a financial strain on people selecting treatments that they can or can not afford. this, in the end, could determinate who may or may not undergo the most appropriate cure. the purpose of this review is to underline past developments that may help to achieve important new goals in cancer research. interpreting and construing the past giants theories relating to the modern achievements have been a quite exciting, but easy task. however, predicting the future of cancer research, which is based on current and past knowledge, is difficult and should take in account two considerations. the second is the complexity : the modern technologies generate thousands of results, and each one is a small element looking for its place in an extraordinary puzzle. therefore, these peculiarities of current cancer research suggest that future investigations should be based on worldwide cooperation, the best ' technology ' for reaching the daring goals to understand cancer and ' eventually ' cure it. cancer research evolved down a long path, since hippocrates proposed his theory of black bile. boveri 's vision of cancer as a genetic disease a century ago, cloning of the first human oncogene almost thirty years ago, and decoding of the human genome almost ten years ago, represent major steps in the understanding of cancer. nowadays, dna sequencing and gene expression profiles may have a relevant prognostic value in a wide variety of malignancies. for instance, dna copy number variations and gene expression signatures are employed to define tumor subtypes and as prognostic indicators in chronic myeloid leukaemia, breast cancers and other tumors 56, 57. bioinformatics technologies and cancer databases are used to combine molecular, clinical, and population data for diagnostic purposes and for formulating therapeutic strategies. by analyzing individual genetic profiles, it may be possible to foresee risks of cancer development and devise personalized therapies 58, 59. we are now in the era of cancer ' omics ', which investigate genomes, epigenomes, methylomes, transcriptomes, mirnomes, metabolomes, proteomes and so on, easily accumulating ponderous amounts of information, often causing intractable problems 46. there are still huge gaps among these large datasets and in the understanding of cancer biology. how the cancer genomes and gene expression programs interact to dictate cancer cell behaviour, the role of cancer stem cells, the mutual influence of the cancer cells with the cells of the microenvironment, are all aspects that have to be confronted. still cancer is an unresolved problem, and researchers must work harder to get to the root of all these open ended questions. past scientific achievements could represent the milestones for building future bridges along the path of cancer research understanding, ' we should not forget the past '. | cancer has been in existence longer than human beings, and man has been facing the illness ever since he made his appearance on earth. amazingly, the first human cancer gene was cloned only thirty years ago. this, and other extraordinary scientific goals achieved by molecular cancer research in the last 30 years, seems to suggest that definitive answers and solutions to this severe disease have been finally found. this was not the case, as cancer still remains to be defeated. to do so, cancer must be first understood.this review highlights how cancer onset and progression has been tackled from ancient times to present day. old theories and achievements have provided the pillars of cancer understanding, in laying the basis of ' modern era ' cancer research, are discussed. the review highlights the discovery of oncogenes and suppressor tumor genes, underlining the crucial role of these achievements in cancer diagnosis and therapies. finally, an overview of how the modern technologies have given impetuous to expedite these goals is also considered. |
previous studies have reported the incidence of motor weakness following traumatic brain injury as 956%. transtentorial herniation is one of the causes of motor weakness in traumatic brain injury, along with diffuse axonal injury, deep cerebral hemorrhage, focal cortical contusion, and hypoxic - ischemic injury. transtentorial herniation can bring about corticospinal tract injury by compression of the cerebral peduncle, which was caused by downward displacement of medial brain structures out of the cranium through the tentorial notch following head trauma. conventional brain mri can not visualize or estimate a neural tract at the cerebral peduncle ; therefore, it is difficult to demonstrate the recovery of the corticospinal tract injured by transtentorial herniation. by contrast, diffusion tensor imaging allows for visualization and estimation of the corticospinal tract in three dimensions. some recent diffusion tensor imaging studies have reported on the usefulness of diffusion tensor imaging in diagnosis of transtentorial herniation in patients with traumatic brain injury ; however, little is known about recovery of the corticospinal tract injured by transtentorial herniation. in this study, we present with a patient who showed recovery of a corticospinal tract injured by transtentorial herniation following head trauma, using diffusion tensor imaging. a 39-year - old, right - handed male patient underwent decompressive craniectomy due to traumatic intracerebral hemorrhage, which occurred after falling from a height of 3 meters. brain ct images, which were taken after surgery, showed intracerebral hemorrhage in the left fronto - temporal lobe and left transtentorial herniation (figure 1a). brain mri (1 and 27 months after intracerebral hemorrhage onset) revealed shrinkage in the left cerebral peduncle (figure 1a). brain ct, mri images and diffusion tensor tractography results of the corticospinal tract in the included patient. (a) brain ct images after surgery show intracerebral hemorrhage in the left fronto - temporal lobes and left transtentorial herniation (arrow). brain mri images (1 and 27 months after onset) reveal shrinkage of the left cerebral peduncle (arrow). the first (1 month after head trauma) and second (27 months after onset) diffusion tensor tractography for the corticospinal tracts (yellow) in the right hemispheres showed that fiber tracts passed along the known corticospinal tract pathway. on the first diffusion tensor tractography of the affected (left) hemisphere, the corticospinal tract (red) was disrupted below the cerebral peduncle (blue arrow) and connected to the right hemisphere via transpontine fibers. the transpontine connection fibers (red) in the right hemisphere may be related to compensatory mechanism after motor weakness or corticospinal tract injury. however, the left corticospinal tract (red) originated from the left primary motor cortex and descended through the left cerebral peduncle (blue arrow) on the second diffusion tensor tractography. the standardized motricity index and medical research council scale were used for determination of motor function at the time of intracerebral hemorrhage onset and 1, 6 and 27 months after onset. the motricity index is a measure of the integrity of extremity motor function, with a maximum score of 100. morticity index has two different evaluation scores between hand and other joints (shoulder, elbow, hip, knee and ankle) ; hand prehension : 0 (non - movement), 33 (beginning of prehension), 56 (grips cube without gravity), 65 (holds cube against gravity), 77 (grips against pull, but weaker than other side), and 100 (normal) ; other joints : 0 (non - movement), 28 (palpable contraction), 42 (movement without gravity), 56 (movement against gravity), 74 (movement against resistance, but weaker than normal), and 100 (normal). in addition, six categories are included in the medical research council scale : 0, no contraction ; 1, palpable contraction, but no visible movement ; 2, movement without gravity ; 3, movement against gravity ; 4, movement against a resistance lower than the resistance overcome by the healthy side ; 5, movement against a resistance equal to the maximum resistance overcome by the healthy side. reliability and validity of the motricity index are well - established. the patient presented with severe paralysis of the right extremities at the time of intracerebral hemorrhage onset (motricity index, onset : 0 point, 1 month after onset (first diffusion tensor imaging) ; 24 points), but slowly recovered some functions, to the point of being able to move the affected extremities against some resistance at about 6 months after onset (motricity index : 75 points) (table 1). at 27 months after onset (second diffusion tensor imaging), the motor function of the right extremities in the patient had recovered to a nearly normal state (motricity index : 96 points). changes of motor function in the included patient diffusion tensor imagings were obtained twice (1 month and 27 months after onset) using a multi - channel head coil on a 1.5-t philips gyroscan intera (philips, best, the netherlands) with single - shot echo - planar imaging. imaging was performed using a 6-channel head coil. for each of the 32 non - collinear diffusion - sensitizing gradients, we acquired 67 contiguous slices parallel to the anterior commissure - posterior commissure line. imaging parameters were as follows : acquisition matrix = 96 96, reconstruction matrix = 128 128 matrix, field of view = 221 221 mm, repetition time / echo time = 10 726/76 ms, parallel imaging reduction factor (sense factor) = 2, echo planar imaging factor = 49 and b = 1 000 s / mm, number of excitations = 1, and a slice thickness of 2.3 mm (acquired isotropic voxel size 2.3 2.3 2.3 mm). eddy current - induced image distortions and motion artifacts were removed using affine multi - scale two - dimensional registration. preprocessing of diffusion tensor imaging datasets was performed using the oxford centre for functional magnetic resonance imaging of brain software library. we evaluated the corticospinal tract using diffusion tensor imaging -studio software (cmrm, johns hopkins medical institute, usa). for analysis of the corticospinal tract, the region of interest was placed on the corticospinal tract portion of the pons (anterior blue portion on the color map). fiber tracking was performed with an fa threshold of > 0.2 and direction threshold 0.2 and direction threshold 0.2 and direction threshold < 60. diffusion tensor tractography for the corticospinal tracts in the right hemispheres showed that fiber tracts originated from the primary motor cortex, and passed along the known corticospinal tract pathway (figure 1b). through the diffusion tensor tractography, the corticospinal tracts on the affected (left) hemisphere were disrupted below the cerebral peduncle and crossed to the right hemisphere via the transpontine fibers at 1 month after onset, and they originated from the left primary motor cortex and descended through the left cerebral peduncle at 27 months after onset. diffusion tensor imaging parameters of regions of interest on the corticospinal tract pathway are summarized in table 2. through the diffusion tensor imaging, the fractional anisotropy value (0.18) of the corticospinal tract area in the left cerebral peduncle was lower than that (0.71) of the right hemisphere at 1 month after onset and it increased to 0.63 at 27 months after onset. by contrast, the apparent diffusion coefficient value of that area increased to 1.39 at 1 month after onset and it decreased to 0.94 at 27 months after onset. in this study, we demonstrated recovery of a corticospinal tract injured by transtentorial herniation in a patient with traumatic brain injury by diffusion tensor tractography and diffusion tensor imaging findings as well as clinical symptoms. through the diffusion tensor tractography, the left corticospinal tract was disrupted below the cerebral peduncle and connected to the right hemisphere via transpontine fibers at 1 month after onset and the disrupted left corticospinal tract was restored at 27 months after onset. these diffusion tensor tractography findings are consistent with diffusion tensor imaging findings : fractional anisotropy and apparent diffusion coefficient values of the corticospinal tract area in the cerebral peduncle were decreased and increased, respectively, compared with those of the unaffected side. however, fractional anisotropy value increased and apparent diffusion coefficient value decreased at 27 months after onset. fractional anisotropy value represents the degree of directionality of microstructures (e.g., axons, myelin, and microtubules). apparent diffusion coefficient value indicates the magnitude of water diffusion in tissue, which can increase with some forms of pathology, particularly vasogenic edema or accumulation of cellular debris from neuronal damage. therefore, the decrease of fractional anisotropy value with the increase of apparent diffusion coefficient value on 1-month diffusion tensor imaging suggests neural injury ; in contrast, the increase of fractional anisotropy value with the decrease of apparent diffusion coefficient value indicates recovery of neural injury. the patient presented with complete weakness of the right extremities at the onset of traumatic brain injury and showed slow motor recovery, to the point of being able to move the affected extremities against some resistance at 6 months after onset, which demonstrates the nearly normal motor function at 27 months after onset. this recovery course suggests that recovery of the motor function of the affected side progresses through brain plasticity. several diffusion tensor imaging studies on a corticospinal tract injured by transtentorial herniation in patients with stroke or traumatic brain injury have been reported. however, to the best of our knowledge, only one study on recovery of an injured corticospinal tract by transtentorial herniation has been reported. using diffusion tensor imaging and transcranial magnetic stimulation, kwon reported on a patient with spontaneous intracerebral hemorrhage who showed recovery of the corticospinal tract after injury by brain ct and mri - confirmed transtentorial herniation. their results showed that through the diffusion tensor tractography, the left corticospinal tract was disrupted below the cerebral peduncle at 3 weeks after onset, however, the disruption had recovered, with motor recovery, at 1 year after onset ; through the transcranial magnetic stimulation, there was no motor - evoked potential for the affected hemisphere at 3 weeks after onset, and motor - evoked potentials compatible with a regenerated corticospinal tract were obtained from the muscle of the affected hand at 6 months after onset. in conclusion, we demonstrated recovery of a corticospinal tract injured by transtentorial herniation in a patient with head trauma. this result has important implications for brain rehabilitation in terms of recovery of injured neural tracts following traumatic transtentorial herniation. with regard to the motor recovery mechanism in patients with traumatic brain injury, therefore, this is the first diffusion tensor imaging study to demonstrate recovery of a corticospinal tract after injury by traumatic transtentorial herniation. | transtentorial herniation is one of the causes of motor weakness in traumatic brain injury. in this study, we report on a patient who underwent decompressive craniectomy due to traumatic intracerebral hemorrhage. brain ct images taken after surgery showed intracerebral hemorrhage in the left fronto - temporal lobe and left transtentorial herniation. the patient presented with severe paralysis of the right extremities at the time of intracerebral hemorrhage onset, but the limb motor function recovered partially at 6 months after onset and to nearly normal level at 27 months. through diffusion tensor tractography, the left corticospinal tract was disrupted below the cerebral peduncle at 1 month after onset and the disrupted left corticospinal tract was reconstructed at 27 months. these findings suggest that recovery of limb motor function in a patient with traumatic transtentorial herniation can come to be true by recovery of corticospinal tract. |
most of our decisions are made under uncertainty, and many of our actions are geared toward reducing this uncertainty. information seeking actions take many shapes and forms that span the gamut of cognitive function. at one end of the range are simple orienting acts whereby we use our sensory receptors to sample task - relevant information such as looking at a relevant stimulus or listening for a relevant sound. at the other end are elaborate behaviors such as scientific research, which systematically search for information over extended time scales. and at an intermediate level there are exploration / exploitation tradeoffs, whereby we may temporarily forego a valuable action in order to learn about more uncertain but potentially more lucrative paths. understanding how the brain regulates its information seeking behaviors is significant for understanding core cognitive functions. it is key for understanding the control of attention, which is our main information selection mechanism and is implicated in a range of psychiatric disorders. it is critical for understanding the active control of learning and memory how a neural system determines which ones of our experiences will leave a lasting trace. it is critical for understanding development and the ways in which infants and children actively choose which task they wish to learn or investigate. last but not least, it is critical for understanding curiosity, the drive to know. one question is how subjects build explanatory models of their environment, and how these models further constrain the sampling of additional information. a related question is how the brain generates the intrinsic motivation to seek information when physical rewards are absent or unknown, and how this impacts cognitive development in the long term. at the level of neuronal systems, a key question concerns the relationship between neural processes encoding uncertainty and risk, and cognitive mechanisms of learning, memory and attention. the goal of this collection is to provide a home for papers on these and related topics. we accept studies using a range of methodologies including computations, behavioral, cognitive and neural systems investigations, and a broad range of format including full research articles, brief communications and review / opinion or correspondence articles. | the importance of exploratory behaviors by which agents actively sample information has been long appreciated in a range of disciplines. however, because of their complexity and cognitive nature, these behaviors have been difficult to characterize. in recent years, a resurgence of interest in this question has been based on a confluence of ideas from multiple fields, including machine learning, development, perceptual learning and attention and decision making. this collection of articles in f1000research aims to provide a home for a broad range of studies addressing this topic. we welcome full length research articles, brief communications, single figure studies, and review / opinion articles, and studies using computational, behavioral or neural approaches. here, we provide an introduction to the collection which we hope will grow and become a valuable resource for the researchers exploring this topic. |
abstractepigenetic machinery have become a major focus for new targeted cancer therapies. our previous report described the discovery and biological activity of a potent, selective, orally bioavailable, irreversible inhibitor of lysine demethylase 1 (lsd1), gsk2879552. a proliferation screen of cell lines representing a number of tumor types indicated that small cell lung carcinoma (sclc) was sensitive to lsd1 inhibition. the sclc lines that undergo growth inhibition in response to gsk2879552 exhibit dna hypomethylation of a signature set of probes suggesting this may be used as a predictive biomarker of activity. this targeted mechanism coupled with a novel predictive biomarker make lsd1 inhibition an exciting potential therapy for sclc. |
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bacterial vaginosis (bv), the most common bacterial genital infection in women of reproductive age, has been linked to considerable gynecologic and obstetric morbidity. bv may be a cofactor in male - to - female and female - to - male human immunodeficiency virus type (hiv) transmission and other sexually transmitted infections (stis) including herpes simplex virus type-2 (hsv-2) [14 ]. the highest prevalence of bv has been found in sub - saharan africa where hiv infection is also common [3, 5 ]. it has been postulated that bv may increase susceptibility to hiv infection through a variety of mechanisms including altering the host 's defense system, absence of the normal h2o2-producing vaginal lactobacilli (which maintain the natural acidic ph of the vagina), loss of the protective myeloperoxidase halide - hydrogen peroxide system, and by production of metabolic by - products that may increase hiv replication [7, 8 ] or activate target cells for hiv infection. if any of these hypotheses are correct, reduction of bv prevalence could significantly reduce female hiv acquisition. furthermore, the high prevalence of bv in sub - saharan africa remains unexplained. though considerable evidence supports sexual transmission of bv, factors like clothing, climate, and hygiene may also influence vaginal flora. of the various risk factors associated with bv, the contribution of each to causing bv may vary depending on hygienic, sexual, and other practices. bv treatment with metronidazole although effective in the initial clearing of symptoms and signs of bv has since proven disappointing even in industrialized countries with recurrence rates reaching 40% within 3 to 6 months after treatment with oral or intravaginal preparations [1113 ]. in uganda, administration of metronidazole, azithromycin, and ciprofloxacin to large populations of adults of reproductive age every ten months did not reduce the prevalence of bv below 50%. barrier contraceptive devices, such as the diaphragm, lubricants, and microbicides have been associated with changes in vaginal flora [1417 ]. specifically, contraceptive use of the diaphragm with nonoxynol-9 (n-9-) based spermicide has been associated with altered vaginal bacterial microflora including decreased rates of lactobacillus colonization and urinary tract infections (utis) [15, 16 ]. however, most have regarded alteration of vaginal flora and increased risk of uti as a direct result of the n-9 spermicide and not related to the diaphragm alone. importantly, buffergel a candidate microbicidal gel with the ability to maintain a low vaginal ph was associated with profound reductions in bv after a short period of use among participants in a phase 1 safety trial. in several small trials, miphil, a gel with acid - buffering properties, demonstrated effectiveness in treating women with bv. here, we studied the effect of the diaphragm used with replens, a commercially available vaginal lubricant with weak ph buffering capacity, on the presence of bv for up to 24 months of followup among women enrolled in a large randomized controlled hiv prevention trial, the methods for improving reproductive health in africa (mira) study. the mira trial recruited sexually active (an average of at least four sex acts per month), hiv - seronegative, women aged 1849 years who were free of chlamydia and gonorrhea between september, 2003 and september, 2005. women were recruited from family planning, well - baby, and general health clinics, and from community - based organizations, through printed media and radio. two thousand four hundred and ninety - nine women were enrolled at the zimbabwe site, where the mira - bv ancillary study was conducted (the main mira trial was also conducted in johannesburg and durban, south africa). participants who met protocol inclusion and exclusion criteria were then randomized into one of two groups : the intervention group, who received a clinician - fitted diaphragm (all - flex arcing spring diaphragm ; ortho - mcneil pharmaceutical, raritan, nj, usa), a supply of lubricant gel (replens, lil ' drug store products, cedar rapids, ia, usa), and male condoms and the control group, who received male condoms only. all participants received a comprehensive hiv prevention package consisting of hiv / sti pretest and posttest counseling, treatment of curable, laboratory - diagnosed stis, and intensive risk reduction counseling, which emphasized condom negotiation and was tailored to each participant 's individual circumstances. the follow - up period for the study was designed to be staggered, with the first enrolled participants were followed up for 24 months, the last enrolled were followed up for 12 months, and an overall expected average of 18 woman - months of followup per participant. the study protocol was reviewed and approved by the university of california san francisco institutional review board committee on human research and by the medical research council of zimbabwe ethical review committee, the medicines control authority of zimbabwe and western institutional review board. an independent external audit, sponsored by ibis reproductive health, was done by the quintiles corporation in november 2005, after study accrual was completed. the study was conducted as an ancillary study of the mira trial in two clinics within 30 km of harare : chitungwiza, a periurban municipality and epworth, a slightly poorer and less developed suburb. bv study procedures followed almost identically procedures for the main mira trial, which have been published in detail. briefly, at screening, we obtained verbal consent to assess initial eligibility, followed by written informed consent for screening procedures, including diagnostic hiv and sti testing and answering an interviewer - administered questionnaire on demographics and sexual behavior. the enrolment visit was scheduled within 2 weeks and not more than 30 days after screening, for participants who met the initial eligibility criteria. by design, approximately 500 participants from the two zimbabwe clinics were enrolled serially into the bv - ancillary study. at their enrolment visit, participants provided written informed consent for the main trial and for the bv - ancillary study, women had to give consent to have an additional vaginal swab obtained at each visit. women were reevaluated for study eligibility and had a pelvic examination that included collection of a vaginal swab for gram stain evaluation of bv using nugent criteria. after randomization in to the main mira trial women in the intervention group were counseled to insert the diaphragm into their vagina at any time that was convenient to them before coitus and to leave it in place for at least 6 hours after sex. they were given detailed instructions on maintenance, cleaning, and storage of their diaphragm. women were asked to empty an applicator of gel (about 2.5 g) into the dome of the diaphragm at the time of insertion, to spread gel onto the rim to facilitate insertion, and to insert another applicator of gel into the vagina before each act of vaginal sex. at each visit, women received a 3-month supply of gel and were counseled that the effectiveness of the diaphragm and lubricant gel for the prevention of hiv infection was not known. to prevent hiv, they were asked to use condoms regardless of whether or not they used the diaphragm and gel. participants were also told that they should not use the diaphragm and gel as a method of contraception. women were advised to use other contraceptive methods and were provided with hormonal contraceptives through the clinic. they were encouraged to return to the clinic if they experienced any problems or needed more study products. at all visits, participants in both study groups received counseling on risk reduction and as many male condoms as desired. counselors emphasized that condoms are the only known method to prevent hiv and stis and that condoms should be used for every act of sex. participants returned 2 weeks after enrolment for resupply of products, for counseling, and to have any problems assessed. thereafter, follow - up visits were scheduled quarterly to assess hiv and sti status and to obtain a vaginal swab for gram stain evaluation of bv. recent medical history, use of study products, and sexual behavior were ascertained through a face - to - face clinician - administered questionnaire and audio computer - assisted self - interview (acasi). (e.g., soap, commercial douche, and natural products) during the past two - weeks was assessed through a face - to - face - administered questionnaire. women were counted as having attended their quarterly visit if they visited during the period from 14 days before, to 73 days after, the scheduled date. clinicians addressed any medical problems ; a pelvic examination and urinalysis or wet mount were done when clinically indicated, and treatment was provided when appropriate. data were managed at the center for international data evaluation and analysis at the university of california san francisco. the primary outcome was bv detection by nugent 's criteria during followup. to minimize recall bias, per - protocol analyses were based on a priori product use at last sex as one of our two measures of diaphragm and condom use in all analyses. we also assessed a measure of cumulative use of the methods since last study visit (always, sometimes, or never). for calculation of sample size we assumed that bv detection during followup would be compared between arms in an intent - to - treat (itt) analysis using generalized estimating equation (gee) logistic regression model controlling for a within - participant correlation between repeated outcomes of 0.2, with an annual loss to followup of 5%. based on these assumptions, 250 women per arm insured 80% power to detect a 25% difference in prevalence between the two study arms as significant at the 5% level (based on a two - sided test). this corresponded to a relative risk (odds ratio) of 0.75 (0.70). the itt analysis compared serial bv prevalence during followup between groups using a logistic regression model, including a binary indicator of group assignment as the primary predictor variable and accounting for possible within - participant correlation between repeated outcomes using generalized estimating equations (gees) methods. results of the primary analysis were summarized by the estimated odds ratios comparing bv serial prevalence during followup in women in the intervention group to that in the control arm, with associated 95% confidence intervals (cis). we used the same criteria for conducting the per - protocol analyses as were performed for the determination of the effect of the diaphragm on hiv acquisition. in the per - protocol analyses the between - group comparison of the primary outcome, excluding follow - up periods where participants in the intervention group (diaphragm, replens, and condom promotion) (1) did not report diaphragm use at last sex and (2) did not report use of the diaphragm consistently since the last follow - up visit, generally three months. person time in the control group was included even if no condom use was reported but was excluded if diaphragm use was reported. the sponsor, the bill & melinda gates foundation maintained oversight of the trial through regular progress reports and meetings with investigators, and its program officer had input into key scientific decisions as a member of the study technical advisory committee. the sponsor had no other role in the data collection, data analysis, data interpretation, or writing of the report. the authors had access to all the data and shared final responsibility for the decision to submit for publication. five hundred and forty - three women were enrolled in the bv ancillary study between february and october 2004 ; 9 subjects did not have baseline and 6 subjects did not have follow - up bv results leaving 528 (97%) evaluable subjects (figure 1). baseline sociodemographic characteristics, sexual behavior, and stis were similar between the intervention and control arms (table 1). at enrolment, 102 (39%) in the intervention arm and 111 (42%) in the control arm had bv. since the prevalence of bv during followup differed predictably between women with and without bv at enrollment, we analyzed both groups separately. over the course of the study, participants randomized to the intervention arm reported consistent (always) diaphragm use during the previous three months at 904 (60.1%) out of 1505 visits. women also reported always using gel at 908 (60.3%) visits indicating that in most instances when the diaphragm was used, gel was used as well. at enrollment, 29% of women reported always use a condom during the previous three months while 74% of women reported condom use at last sex with no significant differences found between study arms (table 1). during followup a greater proportion of women enrolled in the control arm reported condom use at last sex (range per visit : 78%88%) than in the intervention arm (range per visit : 45%60%). for participants with bv at baseline, the odds of prevalent bv decreased an average of 12% per visit with a 60% overall decline from baseline during followup (or = 0.40, 95% ci 0.250.65) ; this decline was similar between arms (p = 0.99 ; figure 2). for those without bv at enrollment, the odds of prevalent bv increased nonsignificantly during the study in comparison to baseline an average of 6% per visit with a 50% overall increase for participants in the intervention arm (or = 1.5, 95% ci 0.992.25) in comparison to a 3% per visit and a 24% overall increase from baseline for women in the control arm (or = 1.24, 95% ci 0.771.99) ; this increase was not significantly different between arms (p = 0.55 ; figure 2). only 2.1% of participants were treated for symptomatic bv, and antibiotic use during the last four weeks for any indication was reported infrequently during the course of the study (range per visit : 0.2%0.7%). use of water to clean the vagina in the past two weeks was common during followup and did not significantly differ by study arm ; intervention arm range per visit : 67%77% ; control arm range per visit : 64%76%. use of other products to clean the vagina in the past two weeks was less commonly reported overall and did not differ by study arm during the course of the study either ; intervention arm range per visit : 6%12% ; control arm range per visit : 5%12%. in the itt analysis, bv prevalence over time did not differ between the intervention and control groups for women who had bv at enrollment (or = 1.01, 95% ci 0.521.94, p = 1.0) and did not have bv at enrollment (or = 1.21, 95% ci 0.652.27, p = 0.5) (figure 2, table 2). in the per - protocol analysis, limited to women reporting diaphragm use at last sex, women in the intervention arm with and without bv at enrollment did not have an altered odds of bv (or = 0.90, 95% ci 0.461.76 ; and or = 1.34, 95% ci 0.682.62, resp.) in comparison to the control arm (table 2). we performed a second per - protocol analysis limited to women reporting consistent diaphragm use since the last visit in the intervention arm ; women without bv at enrollment had a nonsignificant increased odds of bv (or = 1.83, 95% ci 0.903.71) compared to those in the control arm ; the intervention was not associated with an altered odds of bv in women with bv at enrollment (or = 1.17, 95% ci 0.562.45) (table 2). first, we compared the prevalence of bv between groups at followup, restricted to women with normal vaginal flora (nugent 's score 03) at enrollment. participants in the intervention group with normal vaginal flora at enrollment did not have an altered odds of bv during followup in comparison to the control group in itt (or = 1.37, 95% ci 0.623.07) and in per - protocol analysis limited to women who reported diaphragm use at last sex (or = 1.64, 95% ci 0.713.82). however, women with normal vaginal flora at enrollment reporting consistent diaphragm use since their last visit had an increased odds of bv during followup (or = 2.52, 95% ci 1.026.22) in comparison to the control arm (table 2). next, we compared the prevalence of bv among participants with normal lactobacillus (score : 0 - 1) found on gram stain at enrollment. among women with normal lactobacillus on gram stain at enrollment, being in the intervention arm was not associated with an altered odds of bv during followup in itt or in the two per - protocol analyses (table 2) in comparison to the control arm. our study demonstrated no difference in the risk of bv (as measured by nugent score) for women provided with the diaphragm plus replens gel in addition to male condoms compared to those provided male condoms alone. however, in the analysis limited to women with normal vaginal flora at enrollment, participants who reported consistent diaphragm use over the preceding three months had a significantly increased odds of bv during followup compared to the control arm. this result did not seem to stem from differential vaginal cleansing practices or antibiotic use including treatment for symptomatic bv between study arms. it is worth noting the statistical power was reduced following stratification of the results by presence versus absence of bv at enrollment. thus, we can not rule out smaller differences between arms in regards to bv prevalence at followup as was our original intention. furthermore, while we believe that our data on use of the diaphragm and gel are valid, we could not confirm their use in this trial. as has previously been reported, the study attained a high rate of condom use that was maintained over time. however, as in the multisite mira hiv prevention trial, women enrolled in this ancillary study and randomized to the control arm reported higher condom use than those in the intervention arm, suggesting that diaphragm use may have been compensatory, that is, that women provided with the diaphragm were less likely to negotiate condom use with their partners. sexual intercourse without a condom has been associated with prevalent and recurrent bv [23, 24 ], providing further evidence that a sexually transmitted factor plays a role in bv pathogenesis. thus, we hypothesize that increased condom use in both arms led to the significant decline in bv over time among those with bv at enrollment and that differential condom use between arms and/or diaphragm use in the intervention arm may have contributed at least partially to the lower prevalence of bv among those with normal vaginal flora at enrollment randomized to the control arm in our per - protocol analysis. alternatively, we can not rule out that the device itself could predispose to abnormal vaginal flora. studied the safety of the diaphragm used with ky personal lubricant and cellulose sulfate (cs) gel and cs gel alone over a six - month period in a similar population. although they did not report any statistically significant difference between treatment groups, bv appeared to be most common among women in the diaphragm and cs arm (34.2% by amsel 's and 39.5% by nugent 's score) and least common among those in the cs - alone group (15.0% by amsel 's and 22.5% by nugent 's score ; p = 0.07 and p = 0.14, resp.). it is possible that even intermittent use of the diaphragm could physically affect the vaginal distribution of immune factors such as immunoglobulin (ig) a, secretory leukocyte protease inhibitor (slpi), cytokines, and chemokines originating from the endocervix and endometrium ; evidence suggests that these factors inhibit growth of abnormal vaginal flora while promoting colonization of the vagina by lactobacilli [26, 27 ]. a prior investigation of young women including university students in the usa demonstrated an increased risk of bv in women using the diaphragm for contraception, an association that had previously been thought to be due to the use of n-9 [15, 16 ]. among women initiating new contraceptive methods, the diaphragm used with n-9 was associated with increased vaginal colonization by escherichia coli, enterococcus species, anaerobic gram - negative rods, and bv. while data related to diaphragm use without a spermicide is not available, recent studies have investigated the effects of replens on vaginal microflora. replens contains carbopol and polycarbophil, negatively charged polymers that maintain the vaginal ph in the physiologic range, that may have microbicidal properties and have some activity against bv - associated bacteria [18, 28 ]. although we did not directly assess use of replens, we believe there was very little diaphragm use reported without gel. thus, when used with a diaphragm, replens gel did not appear to protect against bv. following the hiv results of the mira trial, wider - scale use of the diaphragm for hiv prevention is unlikely at this time. however, women who choose to use the diaphragm for pregnancy prevention should be counseled about the potential increased risk of bv. with the development of multipurpose technologies to prevent hiv and pregnancy underway, this investigation provides additional insight regarding bv pathogenesis with important implications for microbicide, cervical barrier, and combination product research. | background. bacterial vaginosis (bv) has been linked to female hiv acquisition and transmission. we investigated the effect of providing a latex diaphragm with replens and condoms compared to condom only on bv prevalence among participants enrolled in an hiv prevention trial. methods. we enrolled hiv - seronegative women and obtained a vaginal swab for diagnosis of bv using nugent 's criteria ; women with bv (score 710) were compared to those with intermediate (score 46) and normal flora (score 03). during quarterly follow - up visits over 1224 months a vaginal gram stain was obtained. the primary outcome was serial point prevalence of bv during followup. results. 528 participants were enrolled ; 213 (40%) had bv at enrollment. overall, bv prevalence declined after enrollment in women with bv at baseline (or = 0.4, 95% ci 0.29.56) but did not differ by intervention group. in the intention - to - treat analysis bv prevalence did not differ between the intervention and control groups for women who had bv (or = 1.01, 95% ci 0.521.94) or for those who did not have bv (or = 1.21, 95% ci 0.652.27) at enrollment. only 2.1% of participants were treated for symptomatic bv and few women (516%) were reported using anything else but water to cleanse the vagina over the course of the trial. conclusions. provision of the diaphragm, replens, and condoms did not change the risk of bv in comparison to the provision of condoms alone. |
frailty affects a significant proportion of the elderly population and requires a unique approach to caregiving.1 the multidimensional nature of frailty as a medical syndrome of age - associated decline in physiologic reserve and function across multiple organ systems, resulting in diminished strength and endurance, increased vulnerability to stressors, risk of falls, disability, hospitalization, and mortality, has been broadly accepted.25 however, there is no consensus regarding a single definition of frailty for clinical use.3,4 frailty can be physical or psychological or a combination of both.2,4,6 popular definitions of physical frailty include a specific phenotype model consisting of five items2,7 and a frailty index defined as the proportion of accumulated deficits.810 although a number of other definitions have been developed, the frailty criteria worked out by fried still constitute a reference frame for many studies in community - dwelling populations,5,7,1113 as opposed to geriatric unit inpatient populations.2 a consensus exists that one of the primary purposes of diagnosing frailty is to identify nonrobust and nondisabled older patients at risk of adverse health outcomes in the near future.3 however, frailty can coexist with disability and comorbidity;2,14,15 thus, the diagnosis of frailty can be even more useful in managing older people with chronic diseases and disability.3 fried excluded patients with a history of parkinson s disease, stroke, and considerable cognitive impairment (mini - mental scores 10 lbs (4.5 kg) or 5% of body mass in the last year (obtained from patient, caregiver, or medical records);weakness (assessment based on the handgrip strength measurement ; interpretation of results takes into account sex and body mass index [bmi ]). a kern digital dynamometer was used for grip strength measurement;exhaustion (audited information based on two questions from center for epidemiological studies depression (ces - d) scale;33 a score from 1 [fatigue or exhaustion felt rarely or not at all ] to 4 [fatigue or exhaustion felt most of the time ], 3 or 4 points means that the test is positive for decreased physical activity);slow gait (walking time over a distance of 15 ft [4.57 m ] ; interpretation of results takes into account sex and height);low physical activity (energy expenditure weekly rate calculated on the basis of the modified questionnaire minnesota leisure time activity questionnaire).34,35 unintentional weight loss of > 10 lbs (4.5 kg) or 5% of body mass in the last year (obtained from patient, caregiver, or medical records) ; weakness (assessment based on the handgrip strength measurement ; interpretation of results takes into account sex and body mass index [bmi ]). a kern digital dynamometer was used for grip strength measurement ; exhaustion (audited information based on two questions from center for epidemiological studies depression (ces - d) scale;33 a score from 1 [fatigue or exhaustion felt rarely or not at all ] to 4 [fatigue or exhaustion felt most of the time ], 3 or 4 points means that the test is positive for decreased physical activity) ; slow gait (walking time over a distance of 15 ft [4.57 m ] ; interpretation of results takes into account sex and height) ; low physical activity (energy expenditure weekly rate calculated on the basis of the modified questionnaire minnesota leisure time activity questionnaire).34,35 patients who fulfilled none of the criteria were considered nonfrail, patients who fulfilled 1 and 2 criteria were classified as prefrail, and patients who fulfilled 3 criteria were classified as frail.2 we had expected that some components of frailty criteria, for example, gait speed assessment, would be impossible to perform or assess in the part of our study population. for this reason, we decided to distinguish patients for whom we could obtain all criteria (diagnostic group or d group) and patients for whom one or more criteria could not be obtained (nondiagnostic group or nd group). the obtained data were analyzed using the statistica software version 10 (statsoft, inc., usa). in the analysis of differences between groups, we used test and the mann whitney u statistic. the study protocol was registered with the bioethical committee of the medical university of silesia in katowice. in a statement, the committee determined that the study is characterized by record review and in the context of law is not a medical experiment and does not require assessment by the bioethical committee (letter knw/0022/kb/207/13). based on this decision, written informed consent was not required of our study nor was separate patient consent required for our statistical analysis or research since patient data is not disclosed outside internal hospital ward staff. the study comprised 500 consecutive patients aged 79.08.4 years (xstandard deviation[sd ]), among them 67% women and 33% men were admitted to the subacute geriatric ward department of geriatrics at the large multiprofile university hospital no 7, upper silesian medical center in katowice, poland, between october 2013 and may 2014. cga included a structured interview, physical examination, geriatric functional assessment, blood sampling, ecg, abdominal ultrasound, and chest x - ray. the mini - mental state examination (mmse)28 was used to assess global cognitive performance and geriatric depression scale (gds)-short form to identify depression.29 the barthel index of activities of daily living (barthel index)30 and lawton s instrumental activities of daily living (iadl) scale31 were used to determine the functional status. the mmse scores range from 0 to 30, the barthel index scores range from 0 to 100, and iadl scores range from 9 to 27 ; higher scores indicate better functional state. geriatric depression scale - short form scores range from 0 to 15 with higher scores indicating higher depression probability. modified get - up and go test,32 scored from 0 to 10 with lower values indicating higher balance disorders, was used to assess the risk of falls. frailty was diagnosed according to the fried2 criteria. a polish language version of the protocol frailty assessment components : standardized protocols was used. these criteria include five components : unintentional weight loss of > 10 lbs (4.5 kg) or 5% of body mass in the last year (obtained from patient, caregiver, or medical records);weakness (assessment based on the handgrip strength measurement ; interpretation of results takes into account sex and body mass index [bmi ]). a kern digital dynamometer was used for grip strength measurement;exhaustion (audited information based on two questions from center for epidemiological studies depression (ces - d) scale;33 a score from 1 [fatigue or exhaustion felt rarely or not at all ] to 4 [fatigue or exhaustion felt most of the time ], 3 or 4 points means that the test is positive for decreased physical activity);slow gait (walking time over a distance of 15 ft [4.57 m ] ; interpretation of results takes into account sex and height);low physical activity (energy expenditure weekly rate calculated on the basis of the modified questionnaire minnesota leisure time activity questionnaire).34,35 unintentional weight loss of > 10 lbs (4.5 kg) or 5% of body mass in the last year (obtained from patient, caregiver, or medical records) ; weakness (assessment based on the handgrip strength measurement ; interpretation of results takes into account sex and body mass index [bmi ]). a kern digital dynamometer was used for grip strength measurement ; exhaustion (audited information based on two questions from center for epidemiological studies depression (ces - d) scale;33 a score from 1 [fatigue or exhaustion felt rarely or not at all ] to 4 [fatigue or exhaustion felt most of the time ], 3 or 4 points means that the test is positive for decreased physical activity) ; slow gait (walking time over a distance of 15 ft [4.57 m ] ; interpretation of results takes into account sex and height) ; low physical activity (energy expenditure weekly rate calculated on the basis of the modified questionnaire minnesota leisure time activity questionnaire).34,35 patients who fulfilled none of the criteria were considered nonfrail, patients who fulfilled 1 and 2 criteria were classified as prefrail, and patients who fulfilled 3 criteria were classified as frail.2 we had expected that some components of frailty criteria, for example, gait speed assessment, would be impossible to perform or assess in the part of our study population. for this reason, we decided to distinguish patients for whom we could obtain all criteria (diagnostic group or d group) and patients for whom one or more criteria could not be obtained (nondiagnostic group or nd group). the obtained data were analyzed using the statistica software version 10 (statsoft, inc., usa). in the analysis of differences between groups, we used test and the mann whitney u statistic. the study protocol was registered with the bioethical committee of the medical university of silesia in katowice. in a statement, the committee determined that the study is characterized by record review and in the context of law is not a medical experiment and does not require assessment by the bioethical committee (letter knw/0022/kb/207/13). based on this decision, written informed consent was not required of our study nor was separate patient consent required for our statistical analysis or research since patient data is not disclosed outside internal hospital ward staff. our study group consisted of patients who represent a wide range of clinical conditions and functional states (barthel index 72.228.2 scores in the range from 0 to 100 and mmse 23.67.1 scores in the range from 0 to 30). a common feature was multimorbidity (mean number of diseases 6.02.8 in the range from 1 to 15), with hypertension, osteoarthritis, coronary artery disease, chronic heart failure, diabetes, and dementia as the primary conditions leading to morbidity (table 1). all five frailty criteria as defined by fried were possible to assess in 65% of our study group (325 patients : 213 women and 112 men ; 95% ci = 60.869.2), while assessment of all criteria was not possible in 35% of patients (175 patients in the entire group : 123 women and 52 men ; 95% ci = 30.839.2). nd group, as compared to d group, presented similar comorbidity but represented increased age, higher prevalence of dementia, lower prevalence of hypertension and osteoarthritis, lower systolic blood pressure, bmi, mmse, barthel index, and iadl scores, as well as lower modified get - up and go test scores (table 1). assessing weight loss from 1 year ago proved to be the most difficult criterion to assess (in 137 patients), because data on body weight from a year ago were seldom available. specific reasons were inability to obtain any information because of cognitive disorders in 87 patients (17% of the entire study group) or the lack of body weight measurement in the past (50 patients or 10% of all). cognitive impairment was also an essential cause of diagnostic failure in other frailty criteria (table 2). ninety patients (51.4%) in the nd group had three or four positive frailty criteria, while 181 patients (55.7%) had three or more positive criteria (p=0.361) in the d group (table 3). in the entire study group, 271 patients (54.2%) had 3 positive components and, thus, fulfilled the diagnosis of frailty as defined by fried frailty criteria. apart from the body weight component, all tests for frailty gave worse results in nd group patients both in women and men (table 4). analysis of our study group revealed frailty in 31.7% of patients aged 6069 years and in 67.6% of patients aged 90 years or older, with an average of 54.2% rate of frailty for the entire study group (table 5). there is increasing evidence of the prognostic significance of frailty in elderly patients for a variety of different medical conditions. these conditions include diabetes,36 heart failure,37 acute coronary syndrome,38 femoral fracture,23 cancer,39 alzheimer s disease,40 major surgery,41 and hospitalization at the internal medicine ward.25 thus, frailty assessment in elderly populations may be significant for patient management personalization, allowing reduction in both excessive complications and costs of undertreatment and overtreatment.40,4244 however, the lack of consensus for a universal definition of frailty for clinical use limits the application of the diagnosis of frailty syndrome in clinical practice. an impediment for a universal definition of frailty is the extreme heterogeneity of elderly populations with regard to health and functional status. among different conceptual approaches, fried physical phenotype model2 remains a reference standard for many studies.1113 however, phenotype diagnostic components require a patient to be sufficiently fit to complete the questionnaires, to perform the handgrip tests, and to walk a 15 ft length twice. these challenges may limit the applicability of the fried2 frailty assessment method in geriatric patients, but a range of these restrictions has not previously been extensively studied. to assess both the usefulness and limitations of frailty components in the elderly population with worsened health, we performed an observational study on geriatric ward inpatients. completion of all five fried frailty assessment criteria was possible in 65% of studied patients (d group), which suggests that effective application of the assessment is possible in 60.8%69.2% (95% confidence interval [ci ]) of individuals in similar inpatient groups. as might be expected, cognitive impairment was an important cause of diagnostic failure in all five fried frailty assessment criteria (table 2). surprisingly, the lack of previous weight control, which is one of the simplest health measures, appeared also to be an important diagnostic problem in at least 10% of patients. paradoxically, this finding of the study may be the most important message for the elderly population and medical professionals. the study revealed a very high prevalence of frailty in the studied population, indicating need of urgent introduction of frailty assessment in hospitalized geriatric patients. there is sufficient evidence that management personalization of elderly patients without assessing this syndrome is unfeasible. despite the limitations experienced in assessing 35% of our study group, we found that the prevalence of positive fried frailty assessment in the nd group was not less than in the d group. a possible method for addressing patients who can not be assessed by fried frailty components is to apply a combination of frailty assessment methods. if we consider that a positive fried frailty assessment needs no further evaluation, then perhaps complementary frailty assessment methods may be useful for cases where fried frailty assessment is incomplete. frailty definitions to consider may include the tilburg frailty indicator45 or the clinical frailty scale.26 the most auspicious proposal to solve some limitations of the fried frailty phenotype method in relation to geriatric inpatients seems operationalized components proposed by rockwood.10 a limitation of this study was the lack of validation of frailty assessment methods for our specific population. different population characteristics may necessitate the adjustment of frailty assessment values.7 this issue requires further study. in summary, we found that fried frailty criteria are useful in geriatric inpatients, despite diagnostic limitations in a considerable proportion of this specific population. frailty prevalence exceeded 50%, increasing with age from 31.7% in sexagenarians to 67.6% in nonagenarians, which was substantially higher than in other described elderly populations.2,7,12,15 very high prevalence of frailty in this group indicates the need of routine frailty appraisal as a part of a cga. high prevalence of frailty among geriatric inpatients suggests that evaluation for frailty should be considered a part of the cga. | backgroundmanagement of geriatric patients would be simplified if a universally accepted definition of frailty for clinical use was defined. among definitions of frailty, fried frailty phenotype criteria constitute a common reference frame for many geriatric studies. however, this reference frame has been tested primarily in elderly patients presenting with relatively good health status.objectivethe aim of this article was to assess the usefulness and limitations of fried frailty phenotype criteria in geriatric inpatients, characterized by comorbidity and functional impairments, and to estimate the frailty phenotype prevalence in this group.patients and methods : five hundred consecutive patients of the university hospital subacute geriatric ward, aged 79.08.4 years (67% women and 33% men), participated in this cross - sectional study. comprehensive geriatric assessment and fried frailty phenotype component evaluation were performed in all patients.resultsmultimorbidity (6.02.8 diseases) characterized our study group, with a wide range of clinical conditions and functional states (barthel index of activities of daily living 72.228.2 and mini - mental state examination 23.67.1 scores). all five fried frailty components were assessed in 65% of patients (95% confidence interval [ci ] = 60.869.2) (diagnostic group). one or more components were not feasible to be assessed in 35% of the remaining patients (nondiagnostic group) because of lack of past patient s body mass control and/or cognitive or physical impairment. patients from the nondiagnostic group, as compared to patients from the diagnostic group, presented with more advanced age, higher prevalence of dementia, lower prevalence of hypertension, lower systolic and diastolic blood pressure, body mass index, mini - mental state examination and barthel index of activities of daily living. despite diagnostic limitations, we found 3 positive criteria (thus, frailty diagnosis) in 54.2% of the study group (95% ci = 49.858.6), with prevalence from 31.7% in sexagenarians to 67.6% in nonagenarians.conclusionfried frailty phenotype criteria seem useful for geriatric inpatient assessment, despite diagnostic limitations. high prevalence of frailty among geriatric inpatients suggests that evaluation for frailty should be considered a part of the comprehensive geriatric assessment. |
cerebral palsy (cp) is defined as a permanent neurological disorder caused by a non - progressive brain injury or damage to the brain1. cp primarily affects muscle tone, coordination, control movement, and balance1, 2. many children with cp have neuromuscular deficits, including the lack of motor control, abnormal muscle tone, impaired coordination, sensory problems, and impaired balance control. balance control is imperative to all movements, and a major factor restricting functional ability is poor balance control2. in addition, this helps to demonstrate that an improved balance ability results from the interventions performed in the clinical setting3, 4. several clinical balance assessments have been developed and used ; however, few of balance tools for children exist3. the pediatric balance scale (pbs), modified by based on berg balance scale (bbs) has been used in several studies to assess balance ability in children, especially those with balance problem4, 5. however, it is suitable for assessing balance ability in a group of lower - functioning children ages 6 because of its propensity to display ceiling effects6. to assess balance function in a group of higher - functioning individuals, the fullerton advanced balance (fab) although the fab is more suitable for higher - functioning individuals compared to the pbs, the study does not report on the item difficulty between the pbs and the fab in children with cp. the purpose of this study is to compare the level of difficulty of the pbs with that of the fab scale in children with cp using rasch analysis. forty children with cp (male=17, female=23), who received physical therapy as outpatients at a hospital with a level 1 or 2 based on the gross motor function classification system (gmfcs), and who had sufficient cognition to participate in the study by the mini - mental state examination - korean (mmse - k) score of 23 or higher, were included. the exclusion criteria were as follows : (1) history of any orthopedic injuries, and other disease or conditions that could influence standing and gait performance within 3 months, (2) any medical intervention affecting balance function before study. prior to initiation of the study, the principal investigator explained all procedures and safety in detail to the participants. the participants agreed to the publishing of their study data and signed an informed consent form. all participants completed a general characteristics questionnaire with the following information : gender, age, and medical history. thereafter, the balance function of each subject was assessed using the pbs and fab. the assessment was administered by trained one examiner who had experience completing these tests in patients with cp. the subjects were asked to rest to minimize the effect of the previous measurement. the pbs and fab scale data obtained were analyzed using rasch analysis with winstep version 3.71.0 (linacare, chicago, il, usa). rasch analysis is based on the probability that the response patterns of individuals to the total sample to measure person ability and item difficulty8. it converts ordinal - scale values to interval - scale values, which are calibrated on a single linear measurement continuum divided into equal intervals, or logits, for each item. therefore, rasch analysis has an advantage that it s focus on the hierarchy of items, i.e., from easy to difficulty to perform in each domain9. based on these results, item difficulty was compared and analyzed after combining the two assessment tools using logit values. the general characteristics of the participants are presented in table 1table 1.demographic characteristics of the participants (mean sd)parametersmale (n=17)female (n=23)total (n=40)age (yrs)12.4 2.710.6 3.111.4 3.1hemiplegia (%) 9 (52.9)12 (52.2)21 (52.5)diplegia (%) 8 (47.1)11 (47.8)19 (47.5)pbs score45.5 6.444.9 9.345.2 8.1fab score23.9 7.622.4 11.423.00 9.9pbs : pediatric balance scale ; fab : fullerton advanced balance scale. among the 24 items of combined pbs and fab, the most difficult item was walk with head turns (fab item 6) ; the easiest item was sitting with back unsupported and feet supported on the floor table 2table 2.item difficulty of the combined pbs and fabnoitemlogiterrorfab 9walk with head turns2.820.2fab 6stand on one leg2.390.18fab 5tandem walk2.190.18pbs 9standing on one foot1.930.18pbs 8standing with one foot in front1.870.18fab 7stand on foam, eyes closed1.080.18fab 2reaching forward to an object0.880.18fab 4step up and over0.880.18fab 10reactive postural control0.880.18pbs 13placing alternate foot on step stool while standing unsupported0.740.19pbs 14reaching forward with outstretched arm while standing0.520.19fab 8tow - footed jump0.330.2pbs 10turning 360 degrees0.210.2fab 3turn in full circle0.210.2pbs 11turning to look behind left and right shoulders while standing still0.090.21fab 1standing with feet together and eyes closed0.330.23pbs 7standing unsupported with feet together0.550.24pbs 12pick up object from the floor from a standing position1.370.3pbs 1sitting to standing1.90.36pbs 3transfers1.90.36pbs 6standing unsupported with eyes closed1.90.36pbs 4standing unsupported2.040.38pbs 2standing to sitting2.890.57pbs 5sitting with back unsupported and feet supported on the floor3.971mean00.28 shows the items of the combined pbs and fab, arranged by order of difficulty. the purpose of this study is to compare the item difficulty between the pbs and fab scales using rasch analysis, which has recently been widely used for the evaluation of the construction and validation of functional assessment tools in various fields. item difficulty is expressed by logit value in rasch analysis, with a higher value indicating a high item difficulty level8, 9. the pbs and fab scales were combined to compare the relative item difficulty. the results show that among the 24 items of the combined pbs and fab scale, the most difficult item was walk with head turns, and the easiest item was sitting with back unsupported and feet supported on the floor. item difficulty estimates are presented in logits, where a logit value of 0 is the average of the item difficulty measures10. in this study, the turning to look behind left and right shoulders while standing still the items with higher positive logit values are most difficult than those with lower positive or negative logit values. of the 14 pbs items, five items (item 8, 9, 10, 13, and 14) presented positive logit values, and nine items (item 1, 2, 3, 4, 5, 6, 7, 11, and 12) negative logit values. these findings are consistent with previous research and similar to developmental skill acquisitions in children3. in addition, of the 10 fab scale items, only one (item 1) showed a negative logit value, and the rest showed positive logit values. therefore, pbs has relatively easier items than the fba scale. the lack of items assessing higher function is the primary weakness of the pbs ; thus, the assessment of higher - functioning individuals is limited3. furthermore, the pbs is not enough to distinguish the difference of function in children with cerebral palsy11. in conclusion, the fab scale is a more appropriate tool for assessing balance ability in a group of higher - functioning individuals than the pbs. first, the participants were not selected from a representative population, and data were obtained only one hospital ; thus, the generalization of our results is limited. second, other psychometric properties, such as, age, cognition, fear of falling, and cp classification that may affect balance function in children with cp were not considered. further study is required for incorporate data of children with cp in various regions, and other psychometric properties affecting balance function should be considered. | [purpose ] the purpose of this study was to compare the item difficulty degree between the pediatric balance scale and fullerton advanced balance scale for children with cerebral palsy. [subjects and methods ] forty children with cerebral palsy (male=17, female=23) voluntarily participated in the study. item difficulty was expressed in the rasch analysis using a logit value, with a higher value indicative of increasing item difficulty. [results ] among the 24 items of the combined pediatric balance scale and fullerton advanced balance scale, the most difficult item was walk with head turns, whereas, the easiest item was sitting with back unsupported and feet supported on the floor. among the 14 items of the pediatric balance scale, 9 items (item 1, 2, 3, 4, 5, 6, 7, 11, and 12) had negative logit values, whereas for the fullerton advanced balance scale, only 1 item (item 1) had a negative logit value. [conclusion ] the fullerton advanced balance scale is a more appropriate tool to assess balance ability than the pediatric balance scale in in a group of higher functioning children with cerebral palsy. |
honey bees (apis mellifera) pollinate numerous agricultural crops, including almonds, apples, blueberries, and oil seed crops, valued at a total of $ 14.6 billion annually (1). they are also responsible for pollination of plant species that augment the biodiversity of agricultural and nonagricultural landscapes. increased annual losses of honey bee colonies in the united states, canada, and europe have alarmed agricultural specialists, beekeepers, growers, scientists, and the general public. these losses are in part due to a phenomenon known as colony collapse disorder (ccd), which occurs predominantly over the fall / winter months. the cause(s) of increased colony losses remain unknown, but the current prevailing theory is that multiple factors (i.e., pathogens, poor bee nutrition, and agrochemical exposure) acting in concert can reduce colony longevity (2, 3). while no specific pathogen(s) has been shown to be consistently associated with ccd and/or overwinter losses, pathogen incidence and abundance correlate with colony loss (2, 4). therefore, continued efforts to monitor and discover bee pathogens are extremely important to agriculture and global food production. furthermore, these research efforts lead to extremely interesting biological findings, including those of li. discovered that a plant virus, tobacco ring spot virus (trsv), infects and replicates in honey bees. this is a fascinating example of an rna virus that can infect both insect and plant hosts. furthermore, li. determined that weak honey bee colonies, defined by low colony population size and collapse, had a higher prevalence of trsv than healthy colonies (5). these findings expand the realm of pathogens that may play important roles in honey bee health. the majority of honey bee viruses are positive - sense, single - stranded rna (ssrna) viruses in the order picornavirales (reviewed in reference 6). taxonomic classification, as inferred from phylogenetic analyses of virus - encoded rna - dependent rna polymerases (rdrp), further delineates picorna - like viruses into the picornaviridae, dicistroviridae, and iflaviridae families (7). common honey bee viruses in the dicistroviridae family include acute bee paralysis virus (abpv), black queen cell virus (bqcv), israeli acute paralysis virus (iapv), and kashmir bee virus (kbv). iflaviruses include sacbrood virus (sbv), deformed wing virus (dwv), and slow bee paralysis virus (sbpv). additional viruses, including chronic bee paralysis virus (cbpv), kakugo virus, and the lake sinai viruses (lsvs), remain unclassified (810). virus infections in honey bees can be asymptomatic, cause deformities and/or paralysis, or result in death. advances in molecular techniques, including genome sequencing, pcr - mediated detection, microarray - based detection and discovery, and ultra - high - throughput (or next - generation) sequencing have increased our understanding of virus prevalence, phylogenetic relatedness, and association with colony health (4, 6, 1014). recently, next - generation sequencing of honey bee - associated viruses determined the prevalence of israeli acute paralysis virus in samples from ccd - affected bees (12), identified a new honey bee - associated strain of aphid lethal paralysis virus (alpv ; strain brookings) (10, 15), and resulted in the discovery of a new group of honey bee - infecting viruses, the lsvs (10), which have been associated with ccd in the united states (4) and overwinter losses in belgium (16)., underscore the importance of research aimed at discovering, detecting, and characterizing the pathogenesis, phylogenetic relatedness, and geographic distribution of honey bee viruses, as well as the investigation of these pathogens in the context of the entire honey bee microbiome (1719). like the majority of honey bee viruses, trsv was first observed in infected tobacco and is known to infect numerous plant species, including crops, weeds, and ornamentals. trsv is vectored by nematodes and insects (e.g., aphids, thrips, grasshoppers, and the tobacco flea beetle). the work by li. significantly expands the known host range of trsv to include honey bees. the phylogenetic relationship between common honey bee viruses and trsv inferred from the rdrp sequence (7), which includes honey bee viruses on two branches and trsv on the third branch of a large virus clade composed of chromalveolate-, plant-, and arthropod - infecting viruses. interestingly, the majority of viruses transmitted by insects to animal hosts (i.e., arboviruses) replicate within insect vectors, whereas the majority of insect - transmitted plant viruses do not replicate in insect vectors (20). furthermore, the distribution of trsv in honey bees differs from that seen in insects that serve as vectors primarily for plant viruses. specifically, trsv was detected throughout the honey bee body, whereas plant viruses that replicate in aphid and thrip vectors are found primarily in the gut and salivary gland. li. demonstrated that the varroa destructor mite, an ectoparasite of honey bees that transmits some honey bee viruses, also harbors trsv. the distribution pattern of trsv in the varroa destructor mite, as demonstrated by in situ hybridization, is similar to that of arthropod viral vectors. future studies that determine whether trsv replicates in varroa, and whether it indeed transmits tsrv to honey bees, are important for understanding the transmission dynamics of this newly described honey bee pathogen. the phylogenetic relationship among the honey bee, varroa mite, and pollen - associated trsv isolates described by li., as well as plant trsv isolates in the ncbi database, was inferred from a 731-nucleotide, capsid protein - encoding region of the genome. the resulting phylogenetic tree indicated that the bee, mite, and pollen isolates are indistinguishable from each other but distinct from the plant isolates. full - length genome sequences of trsv isolates are needed to address several questions, including the following. are there host - specific trsv mutations, particularly in viral proteins that interact with the host (e.g., capsid proteins) ? do trsvs isolated from plants have increased conservation in the genome region encoding the movement protein (mp), which facilitates virus passage through plant plasmodesmata ? will the tsrv vpg (virus protein, genome - linked) genome region in plant and bee isolates diverge over time ? does this virus regularly shuttle between plant and insect hosts, and if so, will host - specific mutations be difficult to confirm ? comparative analyses of trsv isolates from historic samples and currently circulating isolates from diverse geographic regions may be used to begin to address some of these exciting, biologically relevant questions. likewise, additional studies aimed at assessing the host range of trsv in other insects (e.g., bees, wasps, and ants) will further our understanding of the interspecies transmission. clearly the findings by li. will excite scientists from all fields, since the discovery that a plant virus (trsv) infects and replicates in honey bees is a unique finding with broad implications. the observation that trsv infection correlates with poor colony health and colony collapse warrants further investigation and may result in a better understanding of increased honey bee losses. this work is particularly relevant for researchers examining correlations between individual bee and colony health with pathogen- and host - associated microbial profiles. poor colony health and colony losses are associated with increased pathogen incidence and abundance. since the majority of honey bee pathogens are rna viruses, it is likely that rna interference (rnai)-mediated antiviral strategies are employed by honey bees to control viral invasion (2123), much like fruit flies and mosquitos (reviewed in references 24 and 25). however, basic questions regarding the mechanism(s) of honey bee antiviral immunity remain unanswered. for example, our studies demonstrate that double - stranded rna (dsrna) of any specificity decreases virus load in honey bees, which indicates the potential of additional dsrna - triggered immune mechanisms (26) ; likewise, other studies demonstrate unexpected off - target effects of dsrna (27). together, these studies suggest that antiviral defense in honey bees involves rnai and additional dsrna - triggered antiviral immune pathways. future studies are needed to determine the molecular mechanisms and relative importance of these responses in specific bee - virus interactions. as li we anticipate that future experiments in this field will lead to the discovery of additional bee pathogens and reveal the mechanisms of honey bee antiviral immunity. these studies will result in a greater understanding of the impact of viruses on honey bee health. such discoveries may redefine our understanding of honey bee health, immunity, and disease transmission and are paramount to understanding the current crisis facing honey bees. | abstracthoney bees are eusocial insects that are commercially managed to provide pollination services for agricultural crops. recent increased losses of honey bee colonies (averaging 32% annually since 2006) are associated with the incidence and abundance of pathogens. in their study in mbio, j. l. li. [mbio 5(1):e00898 - 13, 2014, doi:10.1128/mbio.00898 - 13 ] share their discovery that a plant virus, tobacco ring spot virus (trsv), replicates in honey bees and that the prevalence of this virus was high in weak colonies. their findings increase our understanding of the role of viruses in honey bee colony losses and underscore the importance of surveying for new and/or emerging viruses in honey bees. furthermore, their findings will pique the interest of virologists and biologists across all disciplines. the discovery that a plant virus (trsv) replicates, spreads, and negatively affects the health of an insect host will lead to additional studies on the mechanisms of host - specific adaptation and the role of cross - kingdom infections in the transmission of this virus. |
inherited platelet function disorders (pfds) are a group of rare bleeding disorders characterized by qualitative defects in platelet adhesion, aggregation, secretion, or hemostatic activity.1,2 bleeding associated with pfds varies in frequency and intensity, although typical manifestations include predominantly mucocutaneous symptoms, such as epistaxis, gingival hemorrhage, menorrhagia, and easy bruising. bleeding in response to trauma can also be severe, and therefore, surgical procedures often require careful hemostatic management. glanzmann s thrombasthenia (gt) is one of the well - known rare pfds, occurring in an estimated 1 per 1 million individuals and is associated with abnormalities in glycoprotein iib / iiia (gpiib / iiia), the integrin glycoprotein complex responsible for platelet aggregation through binding to fibrinogen. because of the autosomal recessive nature of gt, its prevalence is highest among ethnic groups in which consanguinity is common.3 unlike other rare pfds, literature on gt extends beyond case reports to include an international survey and a large patient registry, and therefore gt often serves as a prototype for broader discussions of pfd diagnosis and management. coagulation disorders may be considered in cases of unexplained bleeding or in patients with an individual or family history of severe bleeding or easy bruising. diagnosing gt can be challenging because of the similarity in bleeding patterns across a variety of platelet disorders and the absence of diagnostic indicators revealed through initial laboratory assessments.4 unlike quantitative platelet disorders (thrombocytopenias), patients with gt have normal platelet counts and a normal prothrombin time (pt) and partial thromboplastin time (ptt). in most institutions, assessment of closure time (a screening test performed via platelet function analyzer [pfa-100 ]) can be used to detect abnormalities in platelet function ; however, this tool lacks disease specificity and may be most effectively used as a screening tool or to exclude severe platelet defects.5 advanced hematologic assessments necessary to diagnose gt require consultation with a hematologist and include platelet aggregometry (the gold standard diagnostic tool) or flow cytometry for gpiib / iiia. the standard treatment for severe bleeding and perioperative management in patients with gt is platelet transfusion,1,2,6 although less severe episodes may be controlled with local measures such as fibrin sealants and topical thrombin, or with antifibrinolytics or desmopressin.7,8 an important limitation in the use of platelet transfusions is the potential for platelet refractoriness after repeated transfusions, which often involves development of (apa) targeting human leukocyte antigens (hlas) or deficient glycoproteins (gpiib / iiia in patients with gt).2,7,9,10 interestingly, apa have been reported in patients who have not received platelet transfusions, suggesting that alternative mechanisms for sensitization may occur, such as molecular mimicry, partial gp expression, or exposure to platelets / platelet microparticles via red blood cell transfusions.11 because of the rarity of gt, consistent epidemiologic data regarding the incidence of refractoriness or typical number of exposures associated with the development of apa or refractoriness are lacking ; however, the risk of developing refractoriness is thought to increase with repeated exposures.7,8 the potential for apa development may be reduced by administering platelets obtained from hla - matched donors1,8 or by using leukocyte - depleted blood components;12 however, platelet availability may be limited because of short shelf life and variable blood bank capacity,13 leading to difficulties in obtaining hla - matched platelets in a timely manner when faced with severe bleeding, including that occurring in the perioperative setting. an additional challenge in patients with gt is monitoring hemostatic response to platelet transfusions ; unlike thrombocytopenias, which can be followed with platelet counts, response in patients with gt must be assessed functionally, either through clinical improvements in bleeding or through laboratory testing of platelet function.14 however, these assessments may be difficult to perform, may take several hours, and are not available at all hospitals. an important alternative therapeutic option is recombinant activated coagulation factor vii (rfviia, novoseven rt ; novo nordisk a / s, bagsvaerd, denmark).7,8 rfviia was approved by the european medicines agency (ema) in 2004 for the treatment of bleeding episodes and prevention of bleeding during surgery in patients with gt who are refractory to platelets and have apa15 and was approved by the food and drug administration (fda) in 2014 for the treatment of bleeding episodes and perioperative management in adults and children with gt with refractoriness to platelet transfusions, with or without apa.16 here we review data regarding the clinical experience with rfviia in patients with gt and platelet refractoriness and discuss the importance of rapidly recognizing refractoriness and initiating alternative treatments. following early investigations into the use of rfviia in von willebrand s disease and thrombocytopenia, the first reported use of rfviia for gt was in 1995 for refractory epistaxis in a 2-year - old child.17 subsequent reports include case reports and series,18 an international survey regarding the use of rfviia in patients with gt,19 an open - label iranian study including clinical responses to rfviia,20 and a multinational gt registry (gtr).21,22 the largest source of data regarding rfviia in patients with gt and apa or refractoriness is the gtr, a multinational registry study initiated as an ema postmarketing surveillance commitment, which evaluated use of systemic hemostatic treatments in gt.21,22 at the request of the fda, apa status, refractoriness, and rfviia efficacy data from the gtr were independently evaluated by a hematology expert panel, which reviewed data on a case - by - case basis using all available patient information. of 266 rfviia - treated bleeding episodes and 160 rfviia - treated surgical procedures evaluated by this panel, most bleeding episodes were in children aged 16 years or younger (65%), and most surgical procedures were in adults more than 16 years of age (86%).16 the most common types of bleeding episodes were epistaxis (44%), gum bleeding (18%), menorrhagia (14%), tooth or dental extraction related (11%), and gastrointestinal (9%), and the most common types of surgical procedures were dental (66%), endoscopic (8%), nasal (5%), excision related (4%), gastrointestinal (4%), and orthopedic (4%). overall, rfviia was considered effective in 251 (94.4%) bleeding episodes and 159 (99.4%) surgical procedures (table 1). adjudicated rfviia efficacy was also high across treatment categories (rfviia platelets other hemostatic agents) and across apa or refractory groups. among patients with refractoriness, the recommended dosing for severe bleeding episodes is 90 g / kg every 26 hours and for surgery 90 g / kg every 2 hours.16 additional clinical information was provided by an international survey including physician reports from 37 hospitals in 14 countries across europe, north america, australia, and asia.19 use of rfviia was reported in 142 events (108 bleeding episodes and 34 invasive procedures) which occurred in 59 patients ; most of these patients were female (59%) and median age was 22 years (range, 172 years). reasons for using rfviia included prevention of antiplatelet immunization (43%), history of apa (42%), prevention of blood - borne pathogen transmission (37%), and platelet inefficacy for previous bleeds (35%) or for the current bleed (14%). among 103 evaluable bleeding episodes, rfviia was reported as effective (defined as the cessation of bleeding and a lack of rebleeding within 48 hours) in 69 episodes (67%) ; furthermore, investigators reported comparable efficacy between patients with and without apa or refractoriness. among 31 evaluable surgical procedures, rfviia was considered effective in 29 procedures (94%) ; however, the number of surgeries in patients with apa and/or refractoriness was not reported. two serious adverse events possibly related to rfviia were reported by the investigators (deep venous thrombosis with pulmonary embolism and clotting in one ureter). multiple case reports have also described rfviia use in patients with gt and were supportive of the fda approval of rfviia in gt.18 of 49 available reports of patients with gt who received rfviia, 36 bleeding episodes and 29 invasive, surgical, or dental procedures were documented. of reported bleeding events, rfviia was considered effective in 25 bleeding episodes (69%) and 28 procedures (97%). this rate of efficacy is comparable to that reported among the subset of patients with reported apa and/or refractoriness (n=15) ; within this population, an effective response was reported for 4 of 8 bleeding episodes (50%) and 9 of 10 procedures (90%). overall, one serious adverse event (jugular vein thrombosis) and two nonserious adverse events (nausea and vomiting, experienced by a single patient) were reported as possibly or probably related to rfviia administration. defining platelet refractoriness in patients with pfds is a significant challenge, as standard assessments for effectiveness of platelet transfusions have been established only in the context of thrombocytopenia.23 developing a standard measurement of platelet transfusion effectiveness independent of platelet count is therefore a critical unmet need. for hospital - based physicians, persistent external (visible) or internal bleeding (ie, a continuous drop in blood hemoglobin concentration or continuous drainage from surgical drain) after platelet transfusions should be viewed as a potential indicator of platelet refractoriness (figure 1). hospital physicians should be aware of alternative treatments such as rfviia for patients experiencing an inadequate response to platelet transfusions for severe bleeding episodes or for surgical management after failing to respond to local or topical (eg, gelatin sponges, packing) and antifibrinolytic (oral or intravenous) therapies. bleeding in these patients should be managed preferably in consultation with a hematologist with expertise in coagulation disorders, and those with suspected platelet refractoriness should be referred to an experienced hematologist to ensure optimal long - term management. because of the risk of alloimmunization with repeated platelet transfusions, rfviia may be administered as a preemptive alternative to platelet transfusions, even in patients without evidence of refractoriness, especially when early treatment is important. interesting data regarding real - world use of rfviia were obtained from the gtr, which showed that despite the labeled indication in europe of gt with refractoriness and apa, 177 of 266 rfviia - treated bleeds (67%) and 66 of 160 rfviia - treated surgeries (41%) in this registry occurred in patients with neither refractoriness nor apa (or who had unknown status for both). because platelet transfusions are standard treatment for severe bleeding episodes and perioperative management in patients with gt, the potential development of refractoriness is an important clinical concern. uncontrolled bleeding can be life threatening, and therefore hospital - based physicians should be on alert to identify patients with ongoing bleeding suggestive of platelet refractoriness and should be aware of the importance of adapting treatment regimens for the effective management of bleeding. | patients with rare qualitative platelet disorders or platelet function disorders (pfds) may present to the hospital physician with severe bleeding episodes or excessive surgical bleeding. although standard treatment consists of platelet transfusions, repeated transfusions may result in the development of antiplatelet antibodies (apa) or clinical refractoriness, rendering further platelet therapy ineffective. in such settings, an approved treatment option for patients with glanzmann s thrombasthenia (gt), one of the well - known rare pfds, is recombinant activated coagulation factor vii (rfviia). data regarding the efficacy of rfviia in patients with gt and platelet refractoriness are available from a large patient registry, an international survey, and multiple case reports and demonstrate efficacy in patients with and without refractoriness or apa. this article reviews the rfviia clinical data in patients with gt and platelet refractoriness and discusses clinical implications relevant to the hospital - based physician. because uncontrolled bleeding can be life - threatening, hospital physicians should be alert to the signs of platelet refractoriness, be able to recognize continued internal or external bleeding, and know how to adapt treatment regimens for the effective management of bleeding. the management of patients who receive rfviia should occur in consultation with a hematologist with experience in pfds, and patients with suspected platelet refractoriness should be referred to such a hematologist as early as possible. a critical unmet need is the development of a definition of an adequate response to platelet transfusion, which would facilitate early recognition of platelet refractoriness in patients with pfds who exhibit a normal platelet count. |
triple a or allgrove syndrome is characterized by three main conditions : achalasia, alacrima (defective tear formation) and addison disease1). it is an uncommon idiopathic primary neuromascular disorder involving the smooth muscle layer of the esophagus and the lower esophageal sphincter. this sphincter is the functional barrier between the stomach and esophagus which is impaired in achalasia syndrome. it is characterized by incomplete relaxation of the lower esophageal sphincter and decreased peristalsis of the smooth muscle esophageal3). achalasia can be the first manifestation of triple - a syndrome. adrenal insufficiency and alacrima are asymptomatic in some cases ; therefore, closer examination should be performed to detect these disorders. some affected individuals show slowly progressive neurological symptom such as dysautonomia and bulbar involvement4). allgrove syndrome is very rare and one such case was presented to us as an edentulous child. a 15-year - old boy complaining of missing teeth referred to oral medicine department of dental faculty of hamadan university of medical sciences (fig. 1). he could not eat properly and suffered from his appearance. in the oral examination, he had a very poor hygiene condition and wore a very inappropriate partial denture. in the past he had complained about pain associated with swallowing difficulties, vomiting and weight loss. in his medical history, x - ray examination after barium ingestion by the patient, showed that the contrast passed through different parts of the esophageal. 3). sever constriction in the distal esophagus in cardia area with achalasia view was evident. during esophageal dilation, parietal irregularities caused by spasms could be seen locally. fairly uniform opacity based on the upper half of the right lung and pre hillary was evident, causing the disappearance of pericardium and mediastinum. uniform opacity could be seen in other parts of the right lung and left middle lung. on further investigation, it was found that he suffered of adrenal insufficiency (addison disease) and alacrima. prednisolone, vitamin d and calcium supplements were prescribed for him. at this time, height was 150 cm, weight 41 kg and body mass index was 18.2 kg / m. the patient was reported to have some symptoms such as fatigue, anxiety, tendency to eating sweet and salty foods, moodiness and poor concentration few years ago. laboratory tests revealed decrease of cortisole hormone (4.3 mcg / dl ; references range, 523 mcg / dl) and increase of plasma adrenocorticotropic hormone (acth) concentration (77 pg / ml ; references range, 1060 pg / ml). the symptoms being improved, he was treated with hydrocortisone and was currently using maintenance dose. due to the gastric reflux, repeated vomiting, chemical erosion, tendency to eating sweet foods and poor oral hygiene, the teeth have decayed rapidly. also, due to inadequate root length remaining after the removal of decay, all of them were extracted. implant treatment was delayed because of the lower age of the patient and economic problem. maxillary and mandibular temporary complete dentures were provided for the patient in prosthetic dental department of university (fig. triple - a syndrome, also known as allgrove syndrome, is a rare autosomal recessive disorder. this multisystemic disease appears during early childhood and is characterized by prominent features of esophageal achalasia, alacrima, and atypical primary adrenal insufficiency. while acth insensitivity could barely have manifestation such as asthenia, it might be wrongly diagnosed as muscle fatigue. the hormone values must be checked - up yearly because acute adrenal crisis could lead to sudden death4). it is characterized by degeneration of enteric nervous system that innervates the esophageal muscles and lower esophageal sphincter. since then, almost 200 patients with this disorder have been reported around the world4). papageorgiou.8) reported the genetic basis of triple - a syndrome in a greek family. all family members (parents and her sibling) had mutation in aaa gene and the parents were affected. nakamura.9) focused on the neurological disorder in a 60-year - old japanese man with allgrove syndrome, and his parents and siblings were unaffected. gebril10) reported an egyptian family with two affected siblings. unlike our case, they had neurological symptoms. they were diagnosed in childhood as well as our patients. while these patients usually consult with physicians because of vomiting, dysphagia and weight which loss, our case suffered from similar symptoms 3). yassaee.11) investigated 5 unrelated families clinically diagnosed with allgrove syndrome for aaa gene mutation. accordingly, while some cases do not have adrenal insufficiency, our case was affected11). genetic examination indicated mutations in both alleles of aaa gene coding for a special 546-amino - acid protein denominated aladin. although scientists have discovered that the mutations are related to chromosome 12q13, they have not identified the role of aladin protein, yet4). therefore, the genetic cause of the syndrome is unknown in this child patient. to date, 90 families with affected members have been reported worldwide8). in our report, although the parents were nonconsanguineous and healthy, the patient 's sister was affected. our case was diagnosed in childhood and did not have dark pigmentation lesions on skin and neurologic disorders. the parents of our case were healthy and he did not have mutation in genetic test. autonomic dysfunction and peripheral neurologic manifestation is detected in some adult cases that may progress slowly during the time4). since achalasia is a rare condition in children, affected child with adrenal insufficiency should undergo more medical examinations. in conclusion, we report an edentulous boy with achalasia, adrenal insufficiency, and alacrima, which are main conditions of triple - a syndrome. | triple - a syndrome, also known as allgrove syndrome, is a rare autosomal recessive disorder. the 3 features of this syndrome are achalasia, adrenal insufficiency, and alacrima. achalasia could be the first manifestation of the triple - a syndrome ; however, its etiology is unclear. alacrima is generally asymptomatic but can be detected by obtaining patient history. although adrenal insufficiency could have manifestations such as asthenia, it might be wrongly diagnosed as muscle fatigue. vitamin d and calcium supplements are usually prescribed for the prevention of osteoporosis. neurologic manifestations could be present in adults. in some individuals with this disorder, genetic examination indicates mutations in both alleles of the aaas gene, which encodes a special 546-amino - acid protein designated aladin, and in chromosome 12q13. the genetic cause of the triple a syndrome in some patients who do not have an identified mutation is unknown. while very few such cases have been reported till date, one such case was presented to us as an edentulous child. |
implementation of a fair, systematic, and reliable evaluation system of the faculty members activities is a challenging issue. this evaluation is mostly performed in the areas of teaching, research and innovation, professional practice, commitment, and citizenship and should be able to show the interpersonal differences in the above - mentioned areas. it would help universities to make decisions regarding promotion, appointment, faculty compensation, recruiting, granting tenure, and rewarding excellence based on objective criteria. on the other hand, expectations and the evaluation criteria should be clear and announced to the faculty members. in spite of the global interest for these kinds of evaluation systems, few studies have assessed systems that can cover all the activities of the faculty members. this system should be able to differentiate among faculty members, observe the mission of the university, and be applicable to all faculty members. some medical schools started to evaluate the activities of their faculty members based on mission based management (mbm) presented by association of american medical colleges and computer sciences corporation in the late 1990s. mbm emerged as a way to understand the costs and revenues associated with the multiple missions of the medical school ; align faculty members activities with the school 's mission ; provide transparent data ; and make decisions based on those data. evaluation of the activities and productivity of the faculty members, especially in education, is very complicated ; however, several solutions have been proposed including the experiences of the universities of dalhousie, texas health science center, and wisconsin. moreover, several papers have been published in this regard since 1995. medical school of tehran university of medical sciences (tums) implemented an objective metrics system to evaluate the faculty members performance quantitatively in the areas of education, research, and service provision in 2009. this system is called shoa, a persian abbreviation for a phrase meaning academic performance metrics and valuing and is designed based on mbm concepts. in this system, the activities of faculty members are arranged in five categories including education (theoretical teaching, teaching in laboratories and practice, clinical and field training, educational workshops, consultation and supervision, evaluation, educational products, research in education, and self - promotion), research (research projects, articles and research products, research workshops and consultation and supervision), clinical services (patient care and service providing with and without presence of students), administrative affairs (management positions and participation in official meetings), and out of university academic activities. data are collected through a web - based software in which each faculty member has a password protected home page. faculty members self - report their activities along with their details during each evaluation period (1-month) and at the end send them to a verifier who is either department 's or clinical ward 's dean. the verifier can return some data to the faculty member for more explanation or correction. each activity has a relative value scale based on the time required for preparation and presentation, group or individual nature of the activity and its importance. a relative value unit (rvu) after 5 years, the coverage of the shoa system is 79% and 75% in clinical and basic sciences departments respectively. some applications of the system include determining the expectancies from faculty members, directing the activities of the faculty members and educational departments, promotion, and assessment of the effective physical presence. there is no evidence - based structured model for faculty members metrics system in other universities as well. therefore, we decided to meta - evaluate the shoa system to devise a model for evaluation of the activities of the faculty members. for meta - evaluation, the personal evaluation standards presented by joint committee on standards for educational evaluation were used. these standards are provided in four areas of propriety standards (7 standards), utility standards (6 standards), feasibility standards (3 standards), and accuracy standards (11 standards). this mix method study with explanatory design and qual - quan approach was performed in tums medical school in 2014. the school has 10 basic sciences and 25 clinical departments with 99 (10%) and 884 (90%) faculty members, respectively. then we conducted semi - structured interviews with the faculty members regarding joint committee meta - evaluation standards. then, through content analysis of the interviews, we designed a questionnaire which was completed online by faculty members. in the final stage, using questionnaire 's factor analysis, the components of the model of the evaluation of the faculty members activities were determined. the validity of the system had been evaluated previously during developing shoa by reviewing the activities list by all department deans (35 deans) to ensure complete coverage of all faculty members activities. to evaluate the reliability of the system, we test - retested self - reported data for september 2013. forty faculty members were randomly selected proportionate to size from basic sciences and clinical departments. four of them who had not entered their data in september 2013 and two who had refused to participate were excluded. 1-month after the evaluation period, we saved and removed the entered information off the system and asked the participants to re - enter their activities. the reliability of the system was calculated through assessing the correlation of the rvus of the activities in the two stages. to evaluate the stance of regarding joint committee meta - evaluation standards in the shoa system, we conducted semi - structured interviews with faculty members who had at least 2 years of experience with the shoa. a same interviewer conducted all interviews and asked the faculty members to discuss issues such as how to provide a list of academic activities and their values, how to increase the validity and reliability of the system, the process of evaluation and data verification, and how obtained information could be utilized. two experts performed content analysis and the results were compared to achieve trustworthiness. in the next stage, according to the codes and themes extracted from the interviews, we designed a questionnaire. the questionnaire included 13 demographic and 33 likert - type main questions ranging from 0 (very little) to 4 (very much). evaluation of the reliability with retest after 2 weeks in a sample of 25 faculty members showed a correlation coefficient (r) of 0.81. finally, three sources were used to design the model : the results of the factor analysis of the questionnaire, the results of interviews content analysis, anda focus group with the participation of medical education experts for finalizing the model. the results of the factor analysis of the questionnaire, the results of interviews content analysis, and a focus group with the participation of medical education experts for finalizing the model. as the study is a mix method one, we first present the results of quantitative and qualitative parts separately and then introduce the draft and final model. comparison of the rvus obtained from test and retest using paired t - test showed no significant difference. reliability of faculty members activities metrics system in medical school we asked interview participants questions relating to eight domains of the academic activities list, activities values, validity, reliability, workflow, data verification, application of information, and effects of system in faculties performance. using qualitative content analysis, we coded the transcribed text and extracted 96 subthemes from which 65 items remained after omitting phrases with overlapping statements. then we reviewed these 65 subthemes to put relevant concepts in a same theme with an appropriate name. finally, we reached 10 main themes which were used as the factors of the final model. main themes with some of their relevant subthemes resulted from qualitative content analysis of interviews also, interviews showed that the status of meta - evaluation standards was acceptable except for the standard of functional reporting in the area of utility standards which was not met. functional reporting meant that reports should be clear, timely, accurate, and germane so that they were of practical value to the evaluates and other appropriate audiences. a total number of 488 faculty members completed the questionnaire (response rate = 49.6%). since, the faculty members were informed for participation in the survey by their academic email addresses and many of them used other email services, it seems that the corrected response rate could be higher. table 3 shows the characteristics of the participants in the survey whose mean age was 45 (7.9) years. demographic characteristics of the faculty members activities metrics system survey participants in medical school we used exploratory factor analysis to extract main factors. kaiser - mayer - olkin index was 0.914, and bartlett 's test of sphericity was 5238.162 (p < 0.001) that showed the high adequacy of the items and its suitability for factor analysis. to extract factors, we used varimax rotation method with four, five, and six factors solutions. finally, it seemed that the six factors solution was the most suitable one. in this solution, eigenvalues were greater than one and could explain 66.20% of the variability. eight questions which were not loaded in these six factors and their removal had no significant effect on the variance explanation were removed. table 5 shows the factors labels with their cronbach 's alpha, mean and standard deviation. since, the average of all factors was above 2 (the midpoint of the likert scale), they were all used in designing the model. labels and related questions of extracted factors of faculty members activities metrics system survey characteristics of extracted factors of faculty members activities metrics system survey the model draft had six dimensions and nine factors. the dimensions of the model should be considered in the whole evaluation system of the faculty members activities and were mainly derived from factor analysis of the questionnaire. factors included the executive components of the system and were mainly obtained via qualitative content analysis of the interviews with faculty members. the dimensions of the model draft included : mission alignment the evaluation system of the faculty members activities should be effective in promoting their performance. the performance should cover the school 's missions including education, research, clinical services, and administrative and managerial affairs.. the system should be able to help the faculty members and managers to identify performance weaknesses and strengths. moreover, it should be able to identify the excellent performance.accuracy the information derived from the system should be accurate as much as possible. the list of the activities should cover all academic affairs, and their value should be close to their real ones.explicit faculty members should receive adequate information on how to complete the required data, evaluation results usages, and guidelines, and bylaws. they should also be informed on the clear definition of the activities listed in the system.satisfaction faculty members should be satisfied with the way their questions and ambiguities regarding the system are answered, and the way and time of verifying their self - reported data.appropriateness the minimum expectancies from faculty members in the areas of education, research, and clinical services should be appropriate.constructiveness the evaluation system should encourage faculty members to perform activities on the list that they did not do and, on the other hand, keep from activities that they previously did but are not included in the list because they are not in line with the missions. the system should also encourage faculty members to modify their activities according to the system. mission alignment the evaluation system of the faculty members activities should be effective in promoting their performance. the performance should cover the school 's missions including education, research, clinical services, and administrative and managerial affairs. the system should be able to help the faculty members and managers to identify performance weaknesses and strengths. accuracy the information derived from the system should be accurate as much as possible. the list of the activities should cover all academic affairs, and their value should be close to their real ones. explicit faculty members should receive adequate information on how to complete the required data, evaluation results usages, and guidelines, and bylaws. they should also be informed on the clear definition of the activities listed in the system. satisfaction faculty members should be satisfied with the way their questions and ambiguities regarding the system are answered, and the way and time of verifying their self - reported data. appropriateness the minimum expectancies from faculty members in the areas of education, research, and clinical services should be appropriate. constructiveness the evaluation system should encourage faculty members to perform activities on the list that they did not do and, on the other hand, keep from activities that they previously did but are not included in the list because they are not in line with the missions. the system should also encourage faculty members to modify their activities according to the system. the executive factors of the model draft included : consensus the list of the activities and their values should be prepared upon the consensus of all the faculty members. the activities should be revised independently in each department and customized accordingly.self-reporting the activities should be self - reported and then verified by an informed and acceptable person.web-based system data entry of the activities, their verification and correction, data analysis, and notifications should be done through a web - based software.evaluation period faculty members activities data should be entered in the system in the predefined evaluation period, preferably 1-month.minimum expectancies the expected rvus from faculty members in the areas of education and research should be clear and announced formally.analysis intervals although the evaluation period was 1-month, the mean rvus for each faculty members should be calculated in 1-year intervals. in this way, different amounts of activities in different months did not cause any problems in the evaluation.verifiers each verifier should be in charge of verifying the data of a maximum of 15 faculty members to increase the accuracy and speed of the process.flexibility all the components of the evaluation system including the list of activities, their values, minimum expectancies, and workflow should be flexible and revised every 1 or 2 years.decision making the scores (rvus) obtained from the system should not replace the school administrators decisions and are only used to help them in the process of decision making.validity the validity of the system should be constantly evaluated. for this reason, the documents and evidences of the activities of a number of randomly selected faculty members would objectively be assessed. consensus the list of the activities and their values should be prepared upon the consensus of all the faculty members. self - reporting the activities should be self - reported and then verified by an informed and acceptable person. web - based system data entry of the activities, their verification and correction, data analysis, and notifications should be done through a web - based software. evaluation period faculty members activities data should be entered in the system in the predefined evaluation period, preferably 1-month. minimum expectancies the expected rvus from faculty members in the areas of education and research should be clear and announced formally. analysis intervals although the evaluation period was 1-month, the mean rvus for each faculty members should be calculated in 1-year intervals. in this way, different amounts of activities in different months did not cause any problems in the evaluation. verifiers each verifier should be in charge of verifying the data of a maximum of 15 faculty members to increase the accuracy and speed of the process. flexibility all the components of the evaluation system including the list of activities, their values, minimum expectancies, and workflow should be flexible and revised every 1 or 2 years. decision making the scores (rvus) obtained from the system should not replace the school administrators decisions and are only used to help them in the process of decision making. for this reason, the documents and evidences of the activities of a number of randomly selected faculty members would objectively be assessed. a focus group was held with the participation of the medical school dean, three medical education experts, and four faculty members who were experienced in the area of evaluation to finalize the above - mentioned model. in this session which took 4 h, all the factors of the model were discussed and reviewed. since, the components of the model were directly extracted from the views of the faculty members, caution was exercised not to change or modify the items only based on taste. the only modification was removing validity from factors since the members of the focus group believed that it was part of accuracy and did not require a separate entity. comparison of the rvus obtained from test and retest using paired t - test showed no significant difference. we asked interview participants questions relating to eight domains of the academic activities list, activities values, validity, reliability, workflow, data verification, application of information, and effects of system in faculties performance. using qualitative content analysis, we coded the transcribed text and extracted 96 subthemes from which 65 items remained after omitting phrases with overlapping statements. then we reviewed these 65 subthemes to put relevant concepts in a same theme with an appropriate name. finally, we reached 10 main themes which were used as the factors of the final model. main themes with some of their relevant subthemes resulted from qualitative content analysis of interviews also, interviews showed that the status of meta - evaluation standards was acceptable except for the standard of functional reporting in the area of utility standards which was not met. functional reporting meant that reports should be clear, timely, accurate, and germane so that they were of practical value to the evaluates and other appropriate audiences. a total number of 488 faculty members completed the questionnaire (response rate = 49.6%). since, the faculty members were informed for participation in the survey by their academic email addresses and many of them used other email services, it seems that the corrected response rate could be higher. table 3 shows the characteristics of the participants in the survey whose mean age was 45 (7.9) years. demographic characteristics of the faculty members activities metrics system survey participants in medical school we used exploratory factor analysis to extract main factors. kaiser - mayer - olkin index was 0.914, and bartlett 's test of sphericity was 5238.162 (p < 0.001) that showed the high adequacy of the items and its suitability for factor analysis. to extract factors, we used varimax rotation method with four, five, and six factors solutions. finally, it seemed that the six factors solution was the most suitable one. in this solution, eigenvalues were greater than one and could explain 66.20% of the variability. eight questions which were not loaded in these six factors and their removal had no significant effect on the variance explanation were removed. table 5 shows the factors labels with their cronbach 's alpha, mean and standard deviation. since, the average of all factors was above 2 (the midpoint of the likert scale), they were all used in designing the model. labels and related questions of extracted factors of faculty members activities metrics system survey characteristics of extracted factors of faculty members activities metrics system survey the dimensions of the model should be considered in the whole evaluation system of the faculty members activities and were mainly derived from factor analysis of the questionnaire. factors included the executive components of the system and were mainly obtained via qualitative content analysis of the interviews with faculty members. the dimensions of the model draft included : mission alignment the evaluation system of the faculty members activities should be effective in promoting their performance. the performance should cover the school 's missions including education, research, clinical services, and administrative and managerial affairs. the system should be able to help the faculty members and managers to identify performance weaknesses and strengths. moreover, it should be able to identify the excellent performance.accuracy the information derived from the system should be accurate as much as possible. the list of the activities should cover all academic affairs, and their value should be close to their real ones.explicit faculty members should receive adequate information on how to complete the required data, evaluation results usages, and guidelines, and bylaws. they should also be informed on the clear definition of the activities listed in the system.satisfaction faculty members should be satisfied with the way their questions and ambiguities regarding the system are answered, and the way and time of verifying their self - reported data.appropriateness the minimum expectancies from faculty members in the areas of education, research, and clinical services should be appropriate.constructiveness the evaluation system should encourage faculty members to perform activities on the list that they did not do and, on the other hand, keep from activities that they previously did but are not included in the list because they are not in line with the missions. the system should also encourage faculty members to modify their activities according to the system. mission alignment the evaluation system of the faculty members activities should be effective in promoting their performance. the performance should cover the school 's missions including education, research, clinical services, and administrative and managerial affairs. the system should be able to help the faculty members and managers to identify performance weaknesses and strengths. accuracy the information derived from the system should be accurate as much as possible. the list of the activities should cover all academic affairs, and their value should be close to their real ones. explicit faculty members should receive adequate information on how to complete the required data, evaluation results usages, and guidelines, and bylaws. they should also be informed on the clear definition of the activities listed in the system. satisfaction faculty members should be satisfied with the way their questions and ambiguities regarding the system are answered, and the way and time of verifying their self - reported data. appropriateness the minimum expectancies from faculty members in the areas of education, research, and clinical services should be appropriate. constructiveness the evaluation system should encourage faculty members to perform activities on the list that they did not do and, on the other hand, keep from activities that they previously did but are not included in the list because they are not in line with the missions. the system should also encourage faculty members to modify their activities according to the system. the executive factors of the model draft included : consensus the list of the activities and their values should be prepared upon the consensus of all the faculty members. the activities should be revised independently in each department and customized accordingly.self-reporting the activities should be self - reported and then verified by an informed and acceptable person.web-based system data entry of the activities, their verification and correction, data analysis, and notifications should be done through a web - based software.evaluation period faculty members activities data should be entered in the system in the predefined evaluation period, preferably 1-month.minimum expectancies the expected rvus from faculty members in the areas of education and research should be clear and announced formally.analysis intervals although the evaluation period was 1-month, the mean rvus for each faculty members should be calculated in 1-year intervals. in this way, different amounts of activities in different months did not cause any problems in the evaluation.verifiers each verifier should be in charge of verifying the data of a maximum of 15 faculty members to increase the accuracy and speed of the process.flexibility all the components of the evaluation system including the list of activities, their values, minimum expectancies, and workflow should be flexible and revised every 1 or 2 years.decision making the scores (rvus) obtained from the system should not replace the school administrators decisions and are only used to help them in the process of decision making.validity the validity of the system should be constantly evaluated. for this reason, the documents and evidences of the activities of a number of randomly selected faculty members would objectively be assessed. consensus the list of the activities and their values should be prepared upon the consensus of all the faculty members. self - reporting the activities should be self - reported and then verified by an informed and acceptable person. web - based system data entry of the activities, their verification and correction, data analysis, and notifications should be done through a web - based software. evaluation period faculty members activities data should be entered in the system in the predefined evaluation period, preferably 1-month. minimum expectancies the expected rvus from faculty members in the areas of education and research should be clear and announced formally. analysis intervals although the evaluation period was 1-month, the mean rvus for each faculty members should be calculated in 1-year intervals. in this way, different amounts of activities in different months did not cause any problems in the evaluation. verifiers each verifier should be in charge of verifying the data of a maximum of 15 faculty members to increase the accuracy and speed of the process. flexibility all the components of the evaluation system including the list of activities, their values, minimum expectancies, and workflow should be flexible and revised every 1 or 2 years. decision making the scores (rvus) obtained from the system should not replace the school administrators decisions and are only used to help them in the process of decision making. validity the validity of the system should be constantly evaluated. for this reason, the documents and evidences of the activities of a number of randomly selected faculty members would objectively be assessed. a focus group was held with the participation of the medical school dean, three medical education experts, and four faculty members who were experienced in the area of evaluation to finalize the above - mentioned model. in this session which took 4 h, all the factors of the model were discussed and reviewed. since, the components of the model were directly extracted from the views of the faculty members, caution was exercised not to change or modify the items only based on taste. since the members of the focus group believed that it was part of accuracy and did not require a separate entity. conceptual models are employed to better identify and understand the phenomena that are conceptualized in the brain and can be used for structuration of a managerial problem in order to summarize the views of the experts on what is right. in this study, we evaluated a system that was operational for some years to present a model from the viewpoint of its main users, that is, faculty members. therefore, we determined its validity and reliability. then we investigated the views of the faculty members through qualitative interviews, and a questionnaire was designed based on the results of the interviews. joint committee personnel evaluation standards were observed in all the stages. in the end, the model was presented considering the results of the content analysis of the interviews, factor analysis of the questionnaire, and experts opinion through a focus group. one of the important points affecting this study is how the shoa system has been installed and operated in tums. the little resistance to its installation, when the system was designed, has faded over time since the system is flexible and revised periodically. the shoa represents the views of the faculty members who have used it in the past years. for this reason, it was not surprising that the views of the faculty members were close to the running system, and in qualitative interviews, data were saturated after about the 10 interview, though we continued to 18 interviews to cover different educational groups. joint committee personnel evaluation standards were acceptable, especially the validity and reliability of the system which were higher than expectations. only the standard functional reporting in the area of utility standards was not met. therefore, after the project, a module was added to the system that enabled the faculty members to evaluate their activities in a period of time of their choice and calculate their own rvus based on different categories. the majority of the reports in universities with similar systems concerns and explains the installation of the system, determining the list of activities, and their weights ; and mainly focuses on multiple source decision making and analysis of the obtained data. in this study, we tried to present a model based on the viewpoints of the faculty members considering the 5-year experience of the shoa system. the main objective of mbm is to align the activities of the faculty members to the school missions. from the standpoint of faculty members, the evaluation system should differentiate between clinical and basic sciences faculty members fairly. unlike basic sciences faculty members, clinical faculty members obtain a great proportion of their rvus through clinical activities. therefore, evaluation of activities should be based on the spectrum of mission - aligned activities in each department ; otherwise, the results always falsely show that some departments are less active than others. this concern mostly exists for educational activities and is resolved by the factor of minimum expectancies in the model ; in other words, calculation of minimum rvus expected from faculty members for educational activities is performed independently in each department. in the shoa, educational expectancy calculation is norm - referenced for each department, and the mean minus one standard deviation is announced as the mean expected rvus for that department. on the other hand, research activities, the faculty members insisted that managerial activities should not be included in the minimum expectancies and the rvus obtained from administrative affairs should only be included in the total score. as a result, for the promotion of faculty members, three domains of education, research, and total scores should be independently evaluated. due to the insistence of the faculty members in this regard, appropriateness the dimensions of accuracy, explicit, and satisfaction are very close to personnel evaluation standards and were specially emphasized by faculty members. one of the considerations in the evaluation of faculty members is its effect on their performance or being constructive. the faculty members also focused on the importance of the issue which was discussed under constructiveness. the faculty members in this study put emphasis on cooperation in different stages of designing the evaluation system, especially preparing the list of activities and their values, which was added as the factor of consensus to the model. moreover, constant correction and modification of the system proportionate to changes in programs and job descriptions of departments were another demand of the faculty members which were addressed as flexibility in the model factors. self - reporting and the role of verifiers were other discussed issues. the faculty members believed that they should be trusted although they insisted on verification of their self - reported activities with consideration of their respected position. having a web - based evaluation system to make the process easier was one of the discussed issues. although data entry intervals were 1-month, calculation of obtained scores for decision making was based on the monthly average of their activities through a year. since activities vary in different months, their monthly average provides a better estimate of the activities of the faculty members in 1-year. finally, the faculty members expected the school managers to evaluate their activities intelligently and to use the shoa results as an assistant in the process of decision making rather than making decision only based on obtained rvus. in this study, the questionnaire was mailed only to participants academic email address that could be considered as a limitation since some of them may use other email services. the model in this study was only presented based on the viewpoints of the faculty members as evaluates ; therefore, other stakeholders may have other dimensions and factors in mind which should be investigated in future studies. moreover, we did not investigate how the list and weight of the activities were determined, what were the problems of collecting the data of the faculty members activities especially educational ones, and how the quality of the activities and its relationship with their quantity should be assessed, which require further studies. in this study, we evaluated a faculty members performance metrics system and investigated faculty members views through qualitative interviews and a quantitative survey. self - reported faculty members activities had acceptable reliability and validity and would be used for decision making. the proposed model for faculty members activities evaluation consisted of six dimensions : mission alignment, accuracy, explicit, satisfaction, appropriateness, and constructiveness and nine executive factors : consensus, self - reporting, web - based system, evaluation period, minimum expectancies, analysis intervals, verifiers, flexibility, and decision making and could be used for designing and implementing such systems in medical schools. am contributed in the conception and design of the work and acquisition, analysis, and interpretation of data, conducting the study, drafting and revising the manuscript, approval of the final version of the manuscript, and agreed for all aspects of the work. ksa contributed in the design of the work, revising the draft, approval of the final version of the manuscript, and agreed for all aspects of the work. rm contributed in the design of the work, revising the draft, approval of the final version of the manuscript, and agreed for all aspects of the work. mj contributed in the design of the work, revising the draft, approval of the final version of the manuscript, and agreed for all aspects of the work. hkv contributed in the design of the work, revising the draft, approval of the final version of the manuscript, and agreed for all aspects of the work. | background : there is a global interest for deploying faculty members activities evaluation systems, however implementing a fair and reliable system is a challenging issue. in this study, the authors devised a model for evaluation of faculty members activities with regard to their viewpoints and meta - evaluation standards.materials and methods : the reliability of the current faculty members activities metrics system was investigated in medical school of tehran university of medical sciences in 2014. then authors conducted semi - structured interviews regarding meta - evaluation standards and designed a questionnaire based on interviews results which were delivered to faculty members. finally, they extracted the components of the model regarding interviews content analysis and questionnaire 's factor analysis and finalized them in a focus group session with experts.results:reliability of the current system was 0.99 (p < 0.05). the final model had six dimensions (mission alignment, accuracy, explicit, satisfaction, appropriateness, and constructiveness) derived from factor analysis of the questionnaire and nine factors (consensus, self - reporting, web - based system, evaluation period, minimum expectancies, analysis intervals, verifiers, flexibility, and decision making) obtained via qualitative content analysis of the interviews.conclusion:in this study, the authors presented a model for faculty members activities evaluation based on meta - evaluation of the existing system. the model covered conceptual and executive aspects. faculty members viewpoints were the core component of this model, so it would be acceptable in a medical school to use the model for evaluating their activities. |
the demand for health care services continues to grow as the population in most developed countries ages. to make health care more affordable, policy makers and health organizations try to align financial incentives with the implementation of care processes based on best practices and the achievement of better patient outcomes. the length of stay (los) in hospitals is often used as an indicator of efficiency of care and hospital performance. it is generally recognized that a shorter stay indicates less resource consumption per discharge and cost - saving while postdischarge care is shifted to less expensive venues. it motivates the endeavor to develop a diagnosis - related group (drg) for patient classification based on the type of hospital treatments in relation to the costs incurred by the hospital. this quality assurance scheme was then linked to the prospective payment system (pps) and adopted by the federal government in the united states for the medicare program in 1983. this payment system was found to moderate hospital cost inflation due to a significant decline in the average length of stay (alos), which refers to the average number of days that patients spend in hospital. under the assumption that patients sharing common diagnostic and demographic characteristics require similar resource intensity, the aim of drg is to quantify and standardize hospital resource utilization for patients. other than diagnostic attributes, most research focuses on two types of factors to explain the variation in los : patient characteristics and hospital characteristics. in examining data for the national health service (nhs) in the united kingdom, the variation in los for those over age 65 was consistently larger across all regions. it was observed that the variation in los between hospitals was larger compared to that between doctors in the same hospital. it was found that psychiatrists were able to predict los with significant accuracy, but only for patients they treated. moreover, the prediction by a hospital coordinator involved in all patient treatments was significantly more correlated to the true los than psychiatrists ' predictions. a comparison of data from 24 hospitals in japan showed that inpatient capacity and the ratio of involuntary admissions correlated positively to longer los. a higher level of caregiver interaction among nurses and physicians, such as communication, coordination, and conflict management, was significantly associated with lower los. the ability to predict los as an initial assessment of patients ' risk is critical for better resource planning and allocation, especially when the resources are limited, as in icus [10, 11 ]. considered timing for los prediction in three clinical stages for burn patients : admission, acute, and posttreatment. using three different regression models, the best mean absolute error (mae) in the los predictions was around 9 days in both the admission and the acute stage and 6 days in the posttreatment stage. with three more treatment - related variables, the results showed that the prediction accuracy was significantly improved in the posttreatment stage. an accurate prediction of los can also facilitate management with higher flexibility in hospital bed use and better assessment in the cost - effectiveness treatment [12, 13 ]. this prediction can even stratify patients according to their risk for prolonged stays [14, 15 ]. spratt. used a multivariate logistic regression method to identify factors associated with prolonged stays (> 30 days) for patients with acute ischemic stroke. in addition to advanced age (> 65), diabetes and in - hospital infection were significantly associated with prolonged los. analyzed los data on childhood gastroenteritis in australia and, using either the robust gamma mixed regression or linear mixed regression method, found that both gastrointestinal sugar intolerance and failure to thrive significantly affected prolonged los. schmelzer. used the multiple logistic regression method and found that both the american society of anesthesiologists (asa) scores and postoperative complications were significant in the prediction of prolonged los after a colectomy. studied the los variation for medicare patients after coronary artery bypass graft surgery (cabg) in 28 hospitals. other than age and gender, the most powerful predictors were history of mitral valve disease or cerebrovascular disease and preoperative placement of an intra - aortic balloon pump. different hospitals varied significantly in their los, and the readmission rate was linearly related to longer los. constructed a logistic regression model to predict the probability for patients requiring 3 or more days in icu after cabg. only 60% of the patients predicted to be high risks had a prolonged icu stay. chang. identified that, among preoperative factors, age of more than 75 years and having chronic obstructive pulmonary disease (copd) were associated with increased los for patients who underwent elective infrarenal aortic surgery. even though diagnosis had been considered the primary factor affecting hospital stays, patients ' clinical conditions, such as the number of diagnoses and the intensity of nursing services required, might be as critical in determining los variations within some drgs. one study showed that only 12% of the variation could be explained by patient characteristics and general hospital characteristics for patients with a primary diagnosis of acute myocardial infarction (ami). for heart failure patients, whellan. patients with longer los had a higher disease severity and more comorbidities, such as hypertension, cardiac dysrhythmias, diabetes mellitus, copd, and chronic renal insufficiency or failure. however, the overall model based on characteristics at the time of admission explained only a modest amount of los variation. the purpose of this study is to develop artificial neural network (ann) models to predict los for inpatients with one of the three primary diagnoses : coronary atherosclerosis (cas), heart failure (hf), and acute myocardial infarction (ami) in a cardiovascular unit in a christian hospital in taipei, taiwan. a better recognition in critical factors before admission that determine los, or a capacity to predict an individual patient 's los, could promote the development of efficient admission policy and optimize resource management in hospitals. this study aims to use ann to predict los for patients with three primary diagnoses : coronary atherosclerosis (cas), heart failure (hf), and acute myocardial infarction (ami) in a cardiovascular unit. moreover, two stages in los prediction are presented : one uses all clinical factors, designated as the predischarge stage, and the other uses only factors available before admission, designated as the preadmission stage. the prediction results obtained at the predischarge stage are then used to evaluate the relative effectiveness in predicting los at the preadmission stage. the remainder of this paper is organized as follows. in section 2, the method including steps in data collection and processing and prediction model construction is introduced. then, the prediction results of various artificial neural network (ann) models are presented in section 3. the discussion of the results and the conclusion of the research finding is given in section 4, with the limitations and future research directions. this study was approved by the mackay memorial hospital institutional review board (irb) for protection of human subjects in research. clinical and administrative data were obtained for cardiology patients discharged between october 1, 2010, and december 31, 2011, in a christian hospital with two locations in the metropolitan area of taipei, taiwan : taipei branch and tamshui branch. a total of 2,424 admission cases were collected for patients with one of three primary diagnoses : cas, hf, and ami. then 47 admissions were identified as outliers, with more than three standard deviations from the mean, when fitting for both forward addition regression and backward elimination regression models. for the remaining 2,377 cases, 933 were coronary atherosclerosis (cas) patients, 872 heart failure (hf) patients, and 572 acute myocardial infarction (ami) patients, as summarized in table 1. the los of any patient in this cardiology unit was defined as from the time of admission to the time of discharge with range from 1 to 35 days, with an average of 5.73, a standard deviation of 5.44, and a median of 4 days. the age ranges from 21 to 99 years, with an average of 67.07, a standard deviation of 14.35, and a median of 68 ; 35% were 75 years or older. an admission case might have zero to multiple comorbidities and similar medical histories were aggregated into comorbidity factors. for example, the history of hypertensive disease includes four types of diseases as identified by icd-9 codes 401 (essential hypertension) to 404 (hypertensive heart and chronic kidney disease). each case might have zero to multiple interventions during the admission. out of a total of 46 types of intervention or diagnostic ancillary services found in the dataset, only the top 6 interventions with more than 5% occurrence in the entire dataset were adopted in this study. the last characteristic, tw - drg pay, was regarding whether the admission case had been reimbursed by the pay - per - case (i.e., tw - drg) system implemented by the national health insurance administration (nhia). the nhia in taiwan provides a universal health insurance system and covers approximately 99% of the population. except for using fee - for - service payment system, the nhia started introducing the first phase of tw - drg with 164 groups from 2010. since cases in the same drg are reimbursed with the same amount, it is to encourage hospitals to improve their financial performance by better utilizing medical resources. among the data collected, 25% were reimbursed through tw - drg payment by the nhia. pearson 's correlation coefficients were used to study the relationships between los and each inpatient 's characteristics. as summarized in table 2, it was observed that all characteristics were significantly correlated with los except for the comorbidity of chronic airway obstruction (icd 496). as for the risk factors, the top three significant positive correlated variables for longer los were patients with heart failure (icd 428) as main diagnosis, who were older and female. it was consistent with the findings about factors related to prolonged los from literature : female, increasing age, and comorbidities such as cerebrovascular disease and diabetes mellitus [18, 19, 22 ]. the top three significant negative correlated variables for longer los were patients with coronary atherosclerosis (icd 414) as main diagnosis, who went through either percutaneous transluminal coronary angioplasty (ptca) or percutaneous coronary intervention (pci). as shown in figure 1, the distribution in los data was skewed with few cases staying longer than 14 days. the average and standard deviation of los for cas patients were 2.63 days and 2.25 days, respectively. for ami and hf patients, the average and standard deviations of los were 7.74 days and 5.93 days, respectively. the distribution of los for cas patients was significantly different than that for patients with either ami or hf (with p value < 0.0001), which suggested different prediction models should be built for cas patients and for non - cas patients or referred to as ami and hf patients. with the profound growth in clinical knowledge and technology, the development of more sophisticated information systems to support clinical decision making is essential to enhance quality and improve efficiency. artificial neural networks (anns) are useful in modeling complex systems and have been applied in various areas, from accounting to school admission. walczak and cerpa proposed four design criteria in artificial neural network (ann) modeling : the appropriate input variables, the best learning methods, the number of hidden layers, and the quantity of neural nodes per hidden layer. the learning method of ann can be either supervised or unsupervised, depending on whether the output values should be known before or should be learned directly from the input values. for supervised learning, backpropagation is one of the most commonly used methods due to its robustness and ease of implementation. the clinical benefits of using ann had been notable in specific areas, such as cervical cytology and early detection of acute myocardial infarction (ami). compared with logistic regression, anns were found useful in predicting medical outcomes due to their nature of nonlinear statistical principles and inference. dybowski. adopted an ann to predict the survival results for patients with systematic inflammatory response syndrome and hemodynamic shock. after improving the performance of ann iteratively, utilized an ann model to predict the icu survival outcome and the los for traumatic patients. the results showed that the mean predicted los using ann was not significantly different than the mean of actual los. developed ann models to predict the prolonged los (13 days and above) for elder emergency patients (age 80 and over). based on the biomedical literature from pubmed, dreiseitl and ohno - machado showed that the discriminatory performance of ann models was better or not worse in 93% of the surveyed papers compared to the logistic regression method. grossi. found ann models outperformed traditional statistic methods in accuracy in various diagnostic and prognostic problems in gastroenterology. li. found that the ann model using all input variables yielded a slightly higher predictive accuracy than the one using a subset of variables filtered by correlation analysis. hence, we decided to consider all inpatient characteristics, including gender, age, location, main diagnosis, eight types of comorbidity, six types of intervention, and whether the case met the criteria for tw - drg reimbursement. these input variables were then categorized into two stages : preadmission stage and predischarge stage, as shown in table 3. variables in the preadmission stage included information available prior to hospitalization, such as gender, age, hospital branch (location) to be admitted to, main diagnosis, and comorbidities. in the predischarge stage, additional to variables in the preadmission stage, it includes interventions and whether the case was reimbursed by tw - drg payment. a case is to be reimbursed by tw - drg payment, not default pay - per - service, depending on the actual discharge condition such as surgical procedure, treatment, and discharge status according to the nhia guideline. separate anns were built to predict los : one for coronary atherosclerosis (cas) patients and the other for heart failure or acute myocardial infarction ami and hf patients. the output layer has only one neuron and it generates a number ranged from 0 to 35 to represent the predicted los., the prediction model using input variables in the predischarge stage is referred to as the predischarge model. likewise, the model using variables in the preadmission stage is referred to as the preadmission model. for a predischarge model, the input layer in an ann model has 18 neurons (n0 = 18) for cas patients. for ami and hf patients, the value of n0 is 19 with one additional neuron with boolean value to represent whether the major diagnosis is hf in a predischarge model. in preadmission models, the value of n0 is 11 and 12 for cas patients and for ami and hf patients, respectively. as for the hidden layer, more neurons were found to enable a better closeness - of - fit with lower training errors. however, large ann size also required more training efforts and could result in overfitting. some research suggested the number of neural nodes in hidden layers to be between 2/3 to 2 times of the size of the input layer [26, 39, 40 ]. in this section, the los prediction in predischarge model and preadmission model using anns is benchmarked with the results using linear regression (lr) models. all prediction models are implemented using ibm spss v.21 and ibm spss neural networks 21. similar to the preliminary trial run, the original data was separated into training dataset and test dataset. the training dataset included 744 admissions for cas patients and 1,155 admissions for ami and hf patients, and the test dataset consisted of 189 admissions for cas patients and 289 admissions for ami and hf patients. during training any ann model, 70% of the training dataset were randomly assigned to the training set and the remaining 30% to the validation set. the training stops when the number of training epochs reaches 2,000 or there is no improvement in validation error for 600 epochs consecutively. for lr models, since the los predictions obtained by ann or lr models are continuous numbers, we further define that a prediction of los is considered accurate if the difference is within 1 day from the actual los for cas patients. moreover, the effectiveness of predictability was measured according to the mean absolute error (mae) and the mean relative error (mre), defined as follows:(1)mae=i=1ny~iyin, mre=i=1ny~iyi / yin, where y~i and yi are the predicted los and actual los for the ith test data, i = 1,2,, n, and n is the number of testing instances. to incorporate the randomness in data selected for training ann, the results showed in table 4 are the 95% confidence intervals (95% ci) for accuracy, mae, and mre based on 30 runs. all models were quite effective in predicting los, with the accuracy rate ranging from 88.07% to 91.53%, the mae from 1 to 1.11 days, and the mre from 0.44 to 0.47. figure 3 shows a detailed look at the distribution in the accurate los prediction in the test dataset. it is observed that lr model performed better than ann model for patients with los of 2 days, which was about 60% of the test dataset. however, both lr and ann models were unable to predict correctly for los more than 5 days, which accounted for 3.7% of the test dataset. same performance indices are used to evaluate the effectiveness of prediction models for ami and hf patients. results summarized in table 5 show that these models are not as effective in predicting los as for cas patients, with the accuracy rate ranging from 32.99% to 36.33%. the mae of all models has been quite stable, ranging from 3.76 to 3.97 days and the mre from 0.69 to 0.77. further, considering the high degree in the variation of los distribution, the definition of accuracy has been extended to include two more scenarios : a tolerance of 1 day is allowed (the difference of los prediction to the actual los is less than 2 days) or a tolerance of 2 days is allowed (the difference of los prediction to the actual los is less than 3 days). however, the accuracy rate of these models is increased from 63.69% to 67.47% only even with 2 days of deviation in prediction being allowed as in table 5. figure 4 shows the breakdown in the accurate los prediction with no tolerance in the test dataset. it is observed that both lr and ann models performed better in predicting los between 8 and 11 days. in the predischarge model, ann performs better than lr model for patients with los of 3, 5, 6, or 7 days, which is about 60% of the test dataset. moreover, as shown in the resized charts in figure 4, ann models were able to predict correctly for cases with los greater than 11 days, which accounts for 14.5% of the test dataset. however, both lr and ann models were unable to predict correctly for los greater than 18 days, which accounts for 5.9% of the test dataset. to determine a proper structure for ann used in this study, a preliminary trial run was first conducted to identify a proper structure for ann models while assuming that the neuron activation function used for each neuron in the hidden layer was log - sigmoidal function with outputs between 0 and 1. the training dataset included the first 12-month data, from october 1, 2010, to september 30, 2011, with 744 admissions for cas patients and 1,155 admissions for ami and hf patients. the test dataset consisted of the data in the last 3 months, with 189 admissions for cas patients and 289 admissions for ami and hf patients. to avoid overfitting, the training dataset was further separated into two sets : a training set, to update the weights and biases, and a validation set, to stop training when the ann might be overfitting. in this study, the size of training set and validation set in training all ann models was assumed to be 70% and 30% of the training dataset. the weights in an ann were modified using a variable learning rate gradient decent algorithm with momentum. the training stopped when the number of training epochs reached 2,000 or there is no improvement in the validation error for consecutively 600 epochs. after an ann was trained, the model was then used to obtain the predicted los in the test dataset. furthermore, to avoid the effect of randomness when comparing the results, a fixed training set and validation set were used when training the backpropagation anns. figure 5 shows the root mean squared error (rmse) for the training set, validation set, and test dataset of trained ann models with different numbers of neurons in the hidden layer, ranging from 10 to 30. the training errors were found to be slightly decreasing as more neurons were included in the hidden layer. however, no overfitting was observed and the test errors had been quite stable for both models. to balance between the required training effort and the test errors improvement, the number of neurons in the hidden layer, or the value of n1 in figure 2, was set to be 13 for all ann models. figure 6 shows the weight distribution between input neurons and hidden neurons, as each dot indicates the weight of one of the input neurons to some hidden neuron, and each input neuron has a total of thirteen dots (weights) linked to the hidden layer. it further validates the size of ann used in this study since the weights had been scattered evenly from 1.5 to 1.5 with only a few dots (weights) close to zero. this study proposed the use of the neural network techniques to predict los for patients in a cardiovascular unit with one of three primary diagnoses : coronary atherosclerosis (cas), heart failure (hf), and acute myocardial infarction (ami). the major observation based on the results was that the preadmission models were as effective in predicting los as the predischarge models. it was even found that some preadmission models performed slightly better than predischarge models as shown in tables 4 and 5. this observation indicates that whether a patient might be reimbursed by tw - drg did not provide additional predictive ability in los, and the assumption that a shorter los would be preferred in the sake of hospitals ' financial performance when drg was implemented was not applicable in our case hospital. the benefit of using ann models was more significant when predicting prolonged los for hf and ami patients. when predicting prolonged los, most literature formulated the prediction models to determine whether an admission might belong to a prolonged stay [14, 15, 17 ] or whether the los might be within a fixed range of los days [16, 22 ]. the study by mobley. predicted the exact los days for patients in a postcoronary care unit. with 629 and 127 admissions in the training and test file, a total of 74 input variables were used to predict 1 to 20 los days in anns. the mean los was 3.84 days and 3.49 days in the training file and the test file. they showed no significant difference in the distribution from the predicted los and from the actual los in the test file. however, ann with two or three hidden layers made no prediction of los beyond 5 days. in this study, the mean los was 2.65 days and 2.53 days in the training dataset and the test dataset for cas patients. with only 18 input variables, our models were able to predict correctly for patients with los up to 5 days as shown in figure 3. for ami and hf patients, the mean los was 7.86 days and 7.23 days in the training dataset and the test dataset. compared with lr method, the ann model was able to predict patient stays longer than 11 days, as shown in figure 4. in general, it is observed that the lr model performed slightly better than ann models in terms of accuracy as in tables 4 and 5. it might be due to the reason that each ann model was built by only 70% of the training dataset, which consisted of the first 12-month data, and the test dataset, which was the remaining 3-month data, had been highly consistent with the previous 12 months. this phenomenon implies that the clinical pathways were well - established in our case hospital. limitation of this research is that the major diagnosis and comorbidities for patients are assumed to be well - known in the preadmission stage. further study is suggested to fully assess the use of ann models in los prediction, especially for patients who might require longer los. instead of predicting the actual los, it might be practical to first categorize los into risk groups. more patient characteristics, such as vital signs or lab readings at the time of admission, can be included to improve the performance of los predictability. as the bed supply is limited, the utilization of hospital beds is considered economically critical for most hospitals and any policy related to improving bed utilization has profound impacts on the perception of quality in the provided care and satisfaction of patients and physicians. currently, hospitalists rely on only aggregated data, such as occupancy rates and average los, to access the performance and competitiveness among clinics in the hospital. a reliable los prediction in the preadmission stage could further assist in identifying abnormality or potential medical risks to trigger additional attentions for individual cases. it might even allow bed managers to foresee any bottlenecks in bed availability when admitting patients to avoid unnecessary bed transfer between wards. | for hospitals ' admission management, the ability to predict length of stay (los) as early as in the preadmission stage might be helpful to monitor the quality of inpatient care. this study is to develop artificial neural network (ann) models to predict los for inpatients with one of the three primary diagnoses : coronary atherosclerosis (cas), heart failure (hf), and acute myocardial infarction (ami) in a cardiovascular unit in a christian hospital in taipei, taiwan. a total of 2,377 cardiology patients discharged between october 1, 2010, and december 31, 2011, were analyzed. using ann or linear regression model was able to predict correctly for 88.07% to 89.95% cas patients at the predischarge stage and for 88.31% to 91.53% at the preadmission stage. for ami or hf patients, the accuracy ranged from 64.12% to 66.78% at the predischarge stage and 63.69% to 67.47% at the preadmission stage when a tolerance of 2 days was allowed. |
the total number of diabetics is estimated to rise from 171 million in 2000 to 366 million in 2030. diabetic foot ulcers (dfus) are one of the most serious complications of diabetes. the lifetime risk of developing foot ulceration in persons with diabetes is as high as 25%. over 1424% of these patients in fact, complications of dfus are the number one cause of nontraumatic lower extremity amputations, which is also associated with a high rate of morbidity and mortality with a 5-year survival rate as low as 31% for major limb amputees. wound healing is a complex process, which includes four overlapping phases : coagulation, inflammation, migration - proliferation, and remodeling. peripheral vascular disease, trauma, infection, and neuropathy complicate the treatment of these wounds and thus necessitate a multidisciplinary approach. appropriate wound management varies mainly according to the cause of the wound, such as aggressive debridement, adequate pressure offloading, treatment of infection, hyperbaric oxygen therapy, bypass surgery for revascularization, and local dressings. however, those concomitant or sequential therapeutic approaches are highly resistant and indolent in some cases, such as antimicrobial therapy aiming to cure the infection, not to heal the wound, while surgery to cure ulcers may result in secondary ulceration and other complications. therefore, there has been increased interest in novel therapies for dfus that have been refractory to standard treatments. stem cells have been shown to mobilize and find home for ischemic and wounded tissues where they secrete chemokines and growth factors to promote angiogenesis and extracellular matrix remodeling [11, 12 ]. several types of stem cells, such as bm - mscs, have been reported to promote wound healing in dfus [1315 ]. these pluripotent stem cells are capable of differentiation into numerous cells types, including fibroblasts, osteoblasts, chondrocytes, adipocytes, myocardial cells, vascular endothelial cells, neurones, hepatocytes, epithelial cells, and other tissue cells [16, 17 ]. many clinical trials also demonstrated that autologous bm - mscs transplantation could improve wound healing in patients with dfus [14, 18, 19 ]. however, the biological mechanisms for this improvement have not yet been identified. in the present study, we established a delayed wound healing model in diabetic rats and evaluated the impact of allogeneic bm - mscs transplantation on delayed wound healing and the possible underlying mechanisms of bm - mscs in accelerating wound healing. we also determined which transplantation method is more effective in the improvement of wound healing. all animals were treated humanely according to the guidelines for the care and use of laboratory animals published by the chinese ministry of public health. diabetes was induced in four - month - old male wistar rats of spf grade (experimental animal center of traditional chinese medicine of gansu province, china). briefly, rats were starved for at least 12 h before a single intraperitoneal injection of streptozotocin (stz ; sigma, usa) dissolved in sodium citrate buffer (0.1 mm, ph 4.4) at a dose of 60 mg / kg body weight. seven days following stz injection, blood samples were obtained from the angular vein, and the blood glucose levels were measured by glucometer. stz - treated rats with blood glucose levels above 16.7 mmol / l were considered diabetic and were used in this study. the animal model was established on 36 diabetic rats and 12 age - matched nondiabetic rats by using previously described methods [21, 22 ]. briefly, rats were anesthetized with an intraperitoneal injection of 10% chloral hydrate at 3 ml / kg body weight. the skin was disinfected with 75% ethanol, a rectangle was marked on the hind dorsal surface of the feet (both left and right) in each rat, and then a layer of skin in full thickness (standard area was 3 mm 7 mm) was removed. the wound was then bandaged by sterilized dressing until bm - mscs therapy was initiated. after surgery, the rats were returned to their cages in the animal holding room after they had regained consciousness and were housed separately. briefly, 2 male wistar rats (4-week - old) of spf grade were sacrificed by c - spine luxation and soaked in 75% alcohol for 15 minutes. a needle was inserted into the bone, and cells were aspirated followed by several flushes through the bone using a 1 ml syringe filled with culture medium, until all the bone marrow was flushed out of the bone. the marrow was scattered into single cell suspension by repeated flushes using 1 ml pipette filled with culture medium, and the residual marrow was filtered. the suspended cells were cultured by plastic adherence in dulbecco 's modified eagle medium (sigma, usa) with low glucose, 10% fetal bovine serum (fbs, hangzhou sijiqing biological engineering materials co., ltd, china), and antibiotics. after 24 h culture at 37c in a 5% co2 atmosphere, the nonadherent cells were removed, and the media were changed every 3 days thereafter. when the cells were 80% confluent, adherent cells were harvested with 0.25% trypsin solution (sigma, usa) for passage. experiments in this study were carried out using cells from the third passage. for in vivo tracing of bm - mscs, the third - passage bm - mscs were labeled with dapi. cells were incubated in the dapi (20 g / ml) containing medium for 2 h in the dark, following by thoroughly rinsing with pbs 5 times prior to trypsinization. subsequently, dapi - labeled allogeneic bm - mscs (8.0 10 cells / ml, according to our previous studies, data not shown) suspension was harvested for the following experiments. before experimental use, the third - passage bm - mscs were confirmed with their specific markers by flow cytometry. briefly, a single cell suspension of 1.0 10 cells / ml was placed in 100 l of pbs and was incubated in the dark for 30 min on ice with saturating concentrations of fluorochromes - conjugated mouse monoclonal antibodies against rat cd44-pe (santa cruz, ca, usa), cd90-pe, cd34pe, and cd45fitc (becton dickinson, ca, usa). and then cells were washed with pbs (1% fbs) 3 times, centrifuged at 1000 rpm for 5 min, and resuspended at a density of 1.0 10 cells / ml. cell fluorescence was evaluated immediately in a facscalibur flow cytometer (becton dickinson, ca, usa), and the data was analyzed using cell quest software (becton dickinson, ca, usa). forty - eight male wistar rats were randomly divided into four groups (n = 12 per group). in group i (non - diabetic controls) and group ii (diabetic controls), rats were subcutaneously injected with pbs into their left hind legs and intramuscularly injected with pbs into their right hind legs. in group iii, diabetic experimental rats were subcutaneously injected with dapi - labeled bm - mscs into their two hind legs. in group iv, diabetic experimental rats were intramuscularly injected with dapi - labeled bm - mscs into their two hind legs. on day 0, rats were treated with subcutaneous injections (6 sites, 5 mm away from the wound edge, 0.05 ml cells suspension or pbs per site) or by intramuscular injections (6 sites, 0.5 cm 0.5 cm distance, 1 cm deep, 0.05 ml cells suspension or pbs per site). three animals were sacrificed on each time point (days 2, 5, 8, and 11) after transplantation to evaluate the wound healing. the size of wounded area was recorded by digital photographs taken from each rat 's foot at each time point after injecting corresponding preparations (pbs for control group and bm - mscs for experimental groups). pictures were taken using a digital camera (dmc - lx5gk, panasonic, japan). the size of the wounded area was analyzed by image - pro plus 4.5 software. the rate of wound contraction was calculated by the following formula : rate of wound contraction (%) = (original wound area detected wound area)/original wound area 100%. at each time point, one part was cut in frozen sections, and the second part was fixed in 4% phosphate - buffered paraformaldehyde, embedded in paraffin, and sectioned for he and immunohistochemical staining. the third part was preserved in liquid nitrogen for later elisa and rt - pcr use. for histological examinations the frozen sections were immediately used to observe the trace of dapi - labeled bm - mscs and the intensity and distribution of blue fluorescence in wound tissues by a fluorescence microscope. the he staining was used to detect granulation tissue formation. to study the angiogenesis during wound healing, the expression of cd31, a marker of endothelial cells, was detected using a polyclonal rabbit cd31 antibody (santa cruz, ca, usa). cellular proliferation was assessed by the expression of ki-67 using a monoclonal rabbit antibody (abcam, uk). for the immunohistochemical staining, the paraffin sections (5 m) were deparaffinized, endogenous peroxidase was inactivated for 10 min with 3% h2o2, and the antigen was retrieved using sodium citrate buffer. after blocking with 5% bsa for 30 min, the slides were exposed to primary antibody at 37c for 1 h. after washing with pbs, the slides were then incubated with biotinylated goat anti - rabbit secondary antibodies (wuhan boster bioengineering limited company, china) at 37c for 30 min, further developed with 3, 3-diaminobenzidine tetrahydrochloride solution, and finally counterstained with hematoxylin. positive staining was indicated by dark brown color. for immunostaining quantification, ten random digital images from each selected area the number of cd31-positive small blood vessels and ki-67-positive cells per high power field (hpf) were counted. at every time point, wound tissue samples in each group were collected, and the level of vegf in the wounded tissues was determined by using previously described methods. briefly, tissue samples were homogenized in 1 pbs containing complete protease inhibitors (wuhan boster bioengineering limited company, china). homogenates were centrifuged (2500 rpm for 20 min) to remove debris, and the pellet was resuspended and filtered through a 1.2 m pore syringe filter. the level of vegf was determined using a rat vegf specific elisa kit (wuhan boster bioengineering limited company, china) according to manufacturer 's protocol. total rnas of the wound tissues were extracted using rnaiso plus (takara, japan) according to the manufacturer 's instructions. 1 g of total rna was used for reverse transcription into complementary dna by primescript rt reagent kit (takara, japan). gene primers for rat vegf and glyceraldehydes-3-phosphate dehydrogenase (gapdh, internal control) were summarized in table 1. real - time pcr was performed using sybr premix ex taq ii (takara, japan). the samples were subject to the following conditions in 7300 real - time pcr system (applied biosystems, usa) : after initial denaturation at 95c for 30 s, pcr amplification was performed for 40 cycles at 95c for 5 s and 60c for 31 s. we selected the data of group ii on day 2 as a calibrator. the equal efficiencies of amplification of the target and reference mrna allowed for the comparative ct (2) method to be used to determine the relative level of gene expressions. statistical analysis was performed by one - way anova when more than two groups were present and lsd - t for two groups (when there were differences between all groups). after the injection of stz, an increase of blood glucose levels was observed over time, and this was accompanied by a reduction of body weight, polydipsia, polyuria, and increased diet. blood glucose levels in diabetic rats used in the present study were consistently higher than 16.7 mmol / l, and these levels were not changed during the whole experimental process (data not shown). diabetic rats did not gain weight after stz injection and after bm - mscs transplantation. compared to their body weight pre - stz injection, diabetic rats lost average 36 5 g, 39 7 g, 41 10 g, and 43 12 g body weight at 2, 5, 8, and 11 days after bm - mscs transplantation, respectively, while the nondiabetic controls gained 20 5 g, 22 5 g, 26 7 g, and 29 8 g, respectively. after three passages in culture, the isolated cell population became homogeneous, showing a monolayer consisting of adherent cells displaying further traits of bm - mscs, demonstrating a typical fibroblast - like morphology (figure 1(a)). adhesion to the bottom of the culture flask also served as a criterion to distinguish bm - mscs from other free - floating cells. after being incubated in dapi - containing medium for 2 h, we have also found that some cells have two micronuclei which were extremely bright and were probably just before cell divisions (figure 1(b)). isolated bm - mscs were confirmed by flow cytometry analysis using bm - mscs specific markers. at passage 3, most of the bm - mscs showed high levels of cd44 and cd90 expressions (72.07% and 89.53%, resp.) but were virtually negative for cd34 and cd45 expressions (1.55% and 2.16%, resp.), suggesting that the major population of the adherent cells were bm - mscs (figure 2). to compare the wound contraction rate in different groups, macroscopic gross differences of the wound surface were observed (figure 3). on day 2, all the wounds were covered with a little purulent fluid. size of wound was significantly reduced with formation of black scabs and alleviation of the inflammatory responses in all groups. on days 8 and 11, some scabs become detached, and part of new epidermis appeared in groups i, iii, and iv. table 2 summarizes the average rate of wound contractions in different groups. at every time point, the rate of wound contraction was higher in rats of group i as compared with other groups (p < 0.05). furthermore, the wound area was smaller in group iv compared to that in groups ii and iii on day 11 (p < 0.05). however, we did not found statistical significance between groups ii and iii during the whole experimental process. these results suggested that intramuscular transplantation of bm - mscs accelerated delayed wound healing in diabetic rats. in order to trace the dapi - labeled bm - mscs in wound tissues, we made frozen sections on days 2, 5, 8, and 11, and the tissue was immediately observed under fluorescence microscope (figure 4). there was no blue fluorescence at any time point in groups i and ii (data not shown). while the intensity and distribution of blue fluorescence were obviously detected in groups iii and iv, with the highest intensity observed on day 5, suggesting that the dapi - labeled bm - mscs migrated to the wound area to participate in the wound healing process, interestingly, the intensity and distribution of blue fluorescence in group iv were found much higher compared to that in group iii on days 2 and 5. as shown in figure 5, the granulation tissue deposition showed no differences among these groups on day 2 (data not shown). however, the granulation tissue was thicker in groups i and iv compared to that in groups ii and iii on day 5. on days 8 and 11, reepithelialization around the wound edge was the predominant process during the late stage of wound healing. these findings suggested that more bm - mscs may participate in and promote the formation of granulation tissue by intramuscular transplantation. small blood vessels of the wounds were characterized by cd31 immunohistochemical staining (figure 6(a)). the average vessel density is quantified and summarized in table 3. we found that the number of blood vessels increased along with the wound healing until day 8 in all groups. the angiogenesis in rats of group ii (diabetic controls) was the weakest among those groups. on days 5 and 8, the vessel density in group iv was higher when compared with groups ii and iii (p < 0.05). and the increased number of small blood vessels in group iii was detected compared to that in group ii on days 8 and 11 (p < 0.05). next, we evaluated the cellular proliferation in the wound tissues using ki67 staining (figures 6(b) and 6(c)). the ki67-positive cells were detected in vascular endothelial cells, fibroblasts, hair follicle epithelial cells, and basal cells. the average number of ki67-positive cells in groups iii and iv at every time point was significantly higher compared to group ii (p < 0.01) ; no statistical significance was detected among group i, group iii, and group iv (table 4). although those groups demonstrated angiogenesis and cellular proliferation in wound tissues and wound edge, intramuscular transplantation of bm - mscs may result in the best efficacy of angiogenesis and cellular proliferation during wound healing. to investigate the possible mechanism involved in the accelerated wound healing, we examined the expression level of angiogenic factor, vegf in the wound tissues by elisa. it is clear that the expression of vegf augmented during wound healing process in groups i, iii, and iv when compared with group ii on days 5 and 8 (p < 0.01). on day 11, the vegf level in group iv was significantly higher compared to that in the other three groups (p < 0.001). to further confirm the results from elisa, relative rt - pcr was employed to quantify the in vivo expression of vegf mrna. relative values for vegf expression were highest on day 8 in groups i, ii, and iii. however, the expression level of group ii was always the lowest at all - time points. a clear trend of increase in vegf expression was observed in group iv, and its expression level was significantly higher than that of other groups on day 11 (p < 0.001). these results confirmed that transplantation of bm - mscs promoted the expression of vegf in wound tissues, especially intramuscular transplantation of bm - mscs at the later stage of wound healing. normal wound healing is a dynamic and complex process involving a series of coordinated events, including bleeding and coagulation, acute inflammation, cell migration, proliferation, differentiation, angiogenesis, re - epithelialization, and synthesis and remodeling of the extracellular matrix. many factors can impair wound healing in dfus, including infection, tissue hypoxia, necrosis, exudates, and excess levels of inflammatory cytokines, which prolong one or more phases of inflammation, proliferation, or remodeling. the ideal modality of wound healing in dfus should have synergistic effects of reducing inflammation, forming vital granulation tissue, and accelerating cellular proliferation and angiogenesis. functional characteristics of bm - mscs that may benefit wound healing include their ability to migrate to the site of injury or inflammation, participate in regeneration of damaged tissues, stimulate proliferation and differentiation of resident progenitor cells, promote recovery of injured cells through growth factor secretion and matrix remodeling, and exert immunomodulatory and anti - inflammation effects [2931 ]. in this study, transplantation of bm - mscs demonstrated a positive effect towards enhancing delayed wound healing in diabetic rats. the major findings of this study are the following : (1) transplanted bm - mscs highly migrated or found home for the wound tissues and contributed to tissue cells ; (2) implantation of bm - mscs promoted granulation tissue formation and re - epithelialization ; (3) transplanted bm - mscs significantly enhanced angiogenesis and cellular proliferation ; and (4) a high expression of vegf in wound tissue, especially in rats with intramuscular transplantation of bm - mscs, was detected. the wounds in our experimental rats demonstrated inflammatory characteristics, such as purulent fluid, redness, and swelling. intramuscularly injected bm - mscs can migrate from muscles to sites of injury in response to chemotactic signals to modulate inflammation and contribute to tissue remodeling. many studies found that engrafted bm - mscs can differentiate into keratinocytes, epithelial cells, and endothelial cells in the skin [12, 32, 33 ]. differentiation and paracrine signaling have both been reported to implicate as mechanisms by which bm - mscs improve tissue repair. bm - mscs differentiation contributes to regeneration of damaged tissues, whereas bm - mscs paracrine signaling is likely the primary mechanism for reducing inflammation, promoting angiogenesis, and inducing migration and proliferation. granulation tissue is essential to wound healing, as it is formed on the surface of wounds to protect and provide nutrition to wounds, consisting of fibroblasts, new capillaries, and infiltrated inflammatory cells. on day 5, the blue fluorescence was observed and peaked in granulation tissue and surrounding area on rats of groups iii and iv. the granulation tissue was thicker than that in diabetic controls, indicating that transplanted bm - mscs accelerated thicker granulation tissue formation and accelerated wound closure. senescent fibroblasts produce elevated levels of the proteolytic enzymes, such as collagenase, elastase, stromelysin, and decreased levels of metalloproteinase inhibitors timp-1 and timp-3. in our study, cellular proliferation and regeneration were examined in terms of ki-67 expression in wound tissues and wound margin area. our results showed that not only fibroblasts but also endothelial vascular cells, basal cells, and hair follicle epithelial cells demonstrated increased proliferation during the healing process, suggesting that bm - mscs transplantation promoted wound healing by increasing cell proliferation, which in turn facilitated the wound healing process and led to enhanced tissue regeneration. another important component of granulation tissue is blood vessels which are necessary to support the formed granulation tissue. in this study, we demonstrated that bm - mscs - treated wounds had enhanced capillary density, suggesting that bm - mscs promote angiogenesis. indeed, we also found increased vegf levels in wounds of bm - mscs transplanted rats compared to diabetic control rats. vegf is one of the most important proangiogenesis factors that promote angiogenesis, stimulate cell migration and proliferation, delay senility, inhibit apoptosis, and promote cell survival [3840 ]. chronic wounds in diabetes are mainly due to the lack of angiogenesis. our results suggested that bm - mscs transplantation in the wound accelerated release of vegf in wound tissues, which may be partially responsible for bm - mscs - mediated enhanced angiogenesis and wound healing. however, there is not yet an ideal therapy that is widely acceptable at present. becaplermin is the only growth factor that has demonstrated therapeutic efficacy in randomized controlled trials of significant numbers of patients. however, the cost of this treatment is high, and its half - time is short, which greatly limits its application. stem cells are a particularly promising therapy for the treatment of chronic nonhealing wounds ; the mechanisms should be revealed clearly to develop more efficient treatment strategies. long - term systemic effects of stem cell therapy have yet to be established, and the security of stem cell therapy should also be of concern., our study provides evidence that bm - mscs are beneficial in the treatment of delayed wound healing in rats. and intramuscular transplantation of exogenous isogeneic stem cells may be suitable for clinical application in the treatment of dfus, although the safety of this therapy should be considered. however, future studies are necessary to overcome the limitations of our experimental design, including clarification of the characteristics and the phenotype changes of these transplanted stem cells during the wound healing process and establishing a more suitable animal model for mimicking patients who suffered from dfus. | in this paper, we established a delayed wound healing model on diabetic rat to mimic the pathophysiology of clinical patients who suffered from diabetic foot ulcers. we also evaluated if transplantation of allogeneic bone marrow - derived mesenchymal stem cells could promote the delayed wound healing and investigated the possible underlying biological mechanisms and stem cell behavior involved in this process. the results showed that bone marrow - derived mesenchymal stem cells had a positive effect on delayed wound healing in diabetic rats. intramuscular transplantation demonstrated the best efficacy. this effect is associated with granulation tissue formation, angiogenesis, cellular proliferation, and high vascular endothelial growth factor expression in wound tissues. in addition, bone marrow - derived mesenchymal stem cells have been shown to mobilize and find home for ischemic and wounded tissues to participate in the process of wound healing. intramuscular transplantation of exogenous isogeneic stem cells may be suitable for clinical application in the treatment of diabetic foot ulcers although the safety of this therapy should be considered. |
gap formation at cavity wall - restorative material interface is always of great importance, because of its significance to microleakage, postoperative sensitivity and secondary caries formation. bond strength and polymerization shrinkage of restorative materials are directly related to their adaptation to cavity walls through their flow, modulus of elasticity and wettability. cavity factors such as number of walls, access to the walls and wall quality, along with operator factors such as material type, material handling and method of insertion are also very important to wall adaptation. if these factors are not properly handled, a gap of variable size will be formed between cavity walls and filling material, during or following the hardening of the material. light, scanning electron, laser and confocal microscopy, dyes, tracers, air - pressure, electrical conductance and optical coherence tomography have all been used in the investigation of this gap. most of the above methods measure representative two - dimensional sections of cavities, interpolating the results for three - dimensional estimations. although, efforts in measuring the three - dimensional space in small cavities (2 mm in diameter) were made, in minimally invasive interventions for caries, the question of how well a material can be adapted to the walls of cavities with smaller diameters (1.0 - 1.5 mm), still remains. opdam. found that voids and wall gaps were greater for thicker in consistency materials than medium or thin materials. in another study, olmez. found that internal voids correlated with marginal microleakage in class ii composite restorations. since material voids are equally important to gap space, and both are depended on the manipulation of the material, it is important to see if voids are also affected by the size of the cavity, during clinicians effort to insert the material to the bottom of the cavity. in the study of lioumi, it was noted that the width of material - cavity interface was not uniformly distributed over the entire cavity walls but it was different in different parts of the cavity, due to certain parameters. the widest gap formed at the internal line angles of the cavity when margins were beveled, etched and bonded, while a significant amount of voids appeared within the material close to or in contact with the interface. investigating the effect of configuration factor on gap formation in composites found that high c - factor values generate large gap formation. x - ray computed micro - tomography (micro xct) is a method that uses x - rays to create two - dimensional density images of the specimen 's cross sections and then reconstruct the specimen in a three - dimensional model. for this reason, it is very useful in many areas in dentistry including the investigation of material adaptation to cavity wall and margins, with relative accuracy, visualizing the different in density entities of the specimens. until now, a number of studies have been published on the adaptation of several different composite materials on cavity walls. however, most of these studies were focused on polymerization shrinkage in cavities of normal dimensions and the attention was given to a rather laboratory technique in order to facilitate measurements. for these reasons, a method that could permit the investigation of gaps in a more clinically relevant setting of the entire cavity preparation, and in dimensions closer to a very conservative cavity would be of significant importance to the study of gap formation and location in such restorations. the purpose of this study was to validate a microtomographic procedure for the comparison of voids (densities) in different ultraconservative cavities and to test the hypotheses that : the materials used for direct restoration of the cavities present no differences in the amount of internal voids or at the material - wall interface, voids and gaps are not related to cavity dimension, andlocation of voids is not dependent to a specific restorative material. the materials used for direct restoration of the cavities present no differences in the amount of internal voids or at the material - wall interface, voids and gaps are not related to cavity dimension, and location of voids is not dependent to a specific restorative material. for the purpose of this study, 12 human molar teeth were used for the preparation of 48 cylindrical cavities and their restoration with four different materials. the required sample size at a = 0.05, a power of 0.85 and an effect size of 0.6, was a priori computed using g power v.3.1.5 (franz ful, universitat kiel, germany). visually intact teeth were carefully selected from recently extracted teeth, without the presence of hypomineralized or hypolplastic areas, caries or fracture lines on their buccal or lingual surfaces. teeth were kept continually in tap water until cavity preparations. in order to prepare cavities, as much as possible on the same horizontal tooth plane, the roots of all teeth, 2 - 3 mm below their cemento - enamel junction, were cut and removed. two buccal ans two linguall cavities of 2.5 mm in depth were prepared in each tooth under air - water spray, with a diamond bur rotating in a high - speed hand piece. two of the cavities were opened with a 0.8 mm in diameter bur (fgd0, strauss and co) and two with a 1.0 mm diameter (fgd1, strauss and co). cavity depth was established with a length mark on each bur and cutting direction was facilitated by guiding the tooth against a stabilized hand piece with a horizontally rotating bur [figure 1 ]. no special attention was given for opening the cavities with an exact width or depth, in order to have a range of cavities with differences in width or depth and facilitate their correlation with restorations gaps and voids. only four cavities were allowed to be opened by the same bur and cavities of different diameter were distributed equally to labial and lingual surfaces. after preparation of the cavities, all teeth were stored in water of 37c until their first microtomographic examination by skyscan 1072 microtomographer (micro xct, model 1072, skyscan aartselaar, belgium). the cavities were examined before insertion of the filling material, to calculate the precise cavity dimensions and assist the estimation of gaps and voids in the restored cavities at the second microtomographic examination [figure 2 ]. operating parameters used for skyscan 1072 microtomographer upper left : microtomographic section of a tooth showing the prepared cavities. lower : a screen image showing the binarization settings with tview software, before calculation starts. the restorative materials used to fill the experimental cavities are shown in table 2, and the procedure followed for each material is described below. the assignment of restorative material and bur diameter on tooth surfaces was based on a latin square design, in order to have teeth, with all the designed treatment variables, in a systematic rather than a random fashion. all cavities were acid etched with 37% ortho - phosphoric acid for 30 s, rinsed with water for 2 - 3 s and dried with air for 4 - 5 s and 1 - 2 absorbent paper points. cavities assigned to receive composite resins were lined carefully with adhesive according to manufacturer instructions (prime and bond nt, dentsply int inc, milford, usa) and polymerized for 20 s, using a curing unit with a light intensity of 550 - 600 mw / cm (demetron lc / kerr gmbh, rastatt, germany). it must be mentioned that adhesive material was used with caution since excess of adhesive remain undetected by x - rays and even a small excess could be measured as gap. materials used in the study aeliteflo / bisco inc, chicago, usa (af) was placed in the cavity with a small diameter tip on material syringes. the tip was initially placed at the bottom of the cavity and was gradually withdrawn in an outward direction as the material was injected into the cavity. the material was polymerized for 30 s, and the excess, was finished and polished using medium, fine and superfine sof - lex discs (3 m espe, dental products, st. permaflo / ultradent products inc, stlouis / usa(pf) was placed in the cavity exactly as af. aelite aesthetic / bisco inc, chicago, usa (aa) was placed in the cavity in small parts and condensed with the use of a dentin excavator, modified to a small diameter condenser (0.8 mm), in order to enter the cavities. after condensation and polymerization, the same finishing and polishing procedure as in af was followed. fuji ix / gc corporation, tokyo, japan (fu) was inserted in the cavity after activation of the capsule according to manufacturer 's instructions, by placing the capsule tip at the entrance of the cavity and condensing the material with the use of the same condenser, as before. immediately after finishing and polishing of restorations, teeth were placed in distilled water of 37c and retrieved after 24 h to receive 500 thermal cycles (5 - 55c, 3 min each). the second x - ray microtomographic examination was made under the same operating parameters with the first examination, immediately after thermocycling procedure. the volume amount of empty spaces within the material or between material and cavity walls was calculated on this second series of digital sections, using the tview v.1.3 software (skyscan n.v. the software calculates on binary images of sample 's tomographic sections, the different in density areas, within a predefined region of interest (roi). running all relative sections, the total volume (vt) and the volume fraction percent (vf %) of the interested densities (called active pixels) in the roi can be calculated. a specific process had to be followed, to ensure that all calculations were based exactly on the same roi. aartelaar, belgium, the number of horizontal sections covering the width of the cavity was determined. the sections showing all four cavities at their largest width were located and copied on a photoshop cs file. a circular area over the cavity was drawn on a transparent layer with the marquee tool. then, the circular marquee line was copied in new transparent layers, moved and applied to all four cavities for an exact positioning and all four layers were combined and saved as a single.bmp file. this process was repeated for all tooth cavities [figure 2 ] and the saved.bmp files were placed as the last tomographic image, in order to remain available, but outside the range of the selected sections. the inserted image in each set of tooth microtomographs was located and the roi was drawn over the selected areas. the upper and lower sections of each cavity were inserted, and the color palette was set to binary colors (247 in level-1 and 255 in level-2), for best discrimination of empty spaces from tooth / material substance [figure 2 ]. the three - dimensional analysis was used, in order to collect information from the set of predefined horizontal sections on the variables described below. this three - dimensional analysis was repeated three times for each cavity and for all teeth. data collected concerned the following morphometric values : vt, the total selected volume throughout all the cross sections, vf %, the volume percent of the selected item (empty space) in an area, st, the total surface area of the item. the interest was centered on vf % and its relation to the initial volume of cavity dimensions and restorative material type. vt, the total selected volume throughout all the cross sections, vf %, the volume percent of the selected item (empty space) in an area, st, the total surface area of the item. the interest was centered on vf % and its relation to the initial volume of cavity dimensions and restorative material type. the location of voids and gaps was evaluated using the tview software and its no1 color palette. all relative to each cavity.bmp images (1024 1024 pixels) were viewed by two examiners for the location of voids if present, on a 19 computer screen. for their classification three locations were recorded : the material itself, the side walls of the cavity andthe bottom of the cavity. voids at the walls or bottom of the cavity were actually the gaps at the material / cavity interface. for each cavity, one, two or all three locations were recorded, always based on the agreement of both examiners. voids at the walls or bottom of the cavity were actually the gaps at the material / cavity interface. for each cavity, one, two or all three locations were recorded, always based on the agreement of both examiners. kolmogorov - smirnov tests for normality and bartlett 's test for the equality of variances indicated the use of kruskal - wallis nonparametric test to analyze the data for differences between materials, in respect to the morphometric parameters. mann - whitney test was also used to search for possible differences between low and high viscosity materials, for each morphometric parameter. the degree of correlation of cavity dimensions with all morphometric parameters was analyzed by spearman 's test. all statistical tests performed with medcalc v.10.0 (medcalc software, mariakerke, belgium) at a 0.05 level of significance. for the purpose of this study, 12 human molar teeth were used for the preparation of 48 cylindrical cavities and their restoration with four different materials. the required sample size at a = 0.05, a power of 0.85 and an effect size of 0.6, was a priori computed using g power v.3.1.5 (franz ful, universitat kiel, germany). visually intact teeth were carefully selected from recently extracted teeth, without the presence of hypomineralized or hypolplastic areas, caries or fracture lines on their buccal or lingual surfaces. in order to prepare cavities, as much as possible on the same horizontal tooth plane, the roots of all teeth, 2 - 3 mm below their cemento - enamel junction, were cut and removed. two buccal ans two linguall cavities of 2.5 mm in depth were prepared in each tooth under air - water spray, with a diamond bur rotating in a high - speed hand piece. two of the cavities were opened with a 0.8 mm in diameter bur (fgd0, strauss and co) and two with a 1.0 mm diameter (fgd1, strauss and co). cavity depth was established with a length mark on each bur and cutting direction was facilitated by guiding the tooth against a stabilized hand piece with a horizontally rotating bur [figure 1 ]. no special attention was given for opening the cavities with an exact width or depth, in order to have a range of cavities with differences in width or depth and facilitate their correlation with restorations gaps and voids. only four cavities were allowed to be opened by the same bur and cavities of different diameter were distributed equally to labial and lingual surfaces. after preparation of the cavities, all teeth were stored in water of 37c until their first microtomographic examination by skyscan 1072 microtomographer (micro xct, model 1072, skyscan aartselaar, belgium). the cavities were examined before insertion of the filling material, to calculate the precise cavity dimensions and assist the estimation of gaps and voids in the restored cavities at the second microtomographic examination [figure 2 ]. operating parameters used for skyscan 1072 microtomographer upper left : microtomographic section of a tooth showing the prepared cavities. lower : a screen image showing the binarization settings with tview software, before calculation starts. the restorative materials used to fill the experimental cavities are shown in table 2, and the procedure followed for each material is described below. the assignment of restorative material and bur diameter on tooth surfaces was based on a latin square design, in order to have teeth, with all the designed treatment variables, in a systematic rather than a random fashion. all cavities were acid etched with 37% ortho - phosphoric acid for 30 s, rinsed with water for 2 - 3 s and dried with air for 4 - 5 s and 1 - 2 absorbent paper points. cavities assigned to receive composite resins were lined carefully with adhesive according to manufacturer instructions (prime and bond nt, dentsply int inc, milford, usa) and polymerized for 20 s, using a curing unit with a light intensity of 550 - 600 mw / cm (demetron lc / kerr gmbh, rastatt, germany). it must be mentioned that adhesive material was used with caution since excess of adhesive remain undetected by x - rays and even a small excess could be measured as gap. materials used in the study aeliteflo / bisco inc, chicago, usa (af) was placed in the cavity with a small diameter tip on material syringes. the tip was initially placed at the bottom of the cavity and was gradually withdrawn in an outward direction as the material was injected into the cavity. the material was polymerized for 30 s, and the excess, was finished and polished using medium, fine and superfine sof - lex discs (3 m espe, dental products, st. permaflo / ultradent products inc, stlouis / usa(pf) was placed in the cavity exactly as af. aelite aesthetic / bisco inc, chicago, usa (aa) was placed in the cavity in small parts and condensed with the use of a dentin excavator, modified to a small diameter condenser (0.8 mm), in order to enter the cavities. after condensation and polymerization, the same finishing and polishing procedure as in af was followed. fuji ix / gc corporation, tokyo, japan (fu) was inserted in the cavity after activation of the capsule according to manufacturer 's instructions, by placing the capsule tip at the entrance of the cavity and condensing the material with the use of the same condenser, as before. immediately after finishing and polishing of restorations, teeth were placed in distilled water of 37c and retrieved after 24 h to receive 500 thermal cycles (5 - 55c, 3 min each). the second x - ray microtomographic examination was made under the same operating parameters with the first examination, immediately after thermocycling procedure. the volume amount of empty spaces within the material or between material and cavity walls was calculated on this second series of digital sections, using the tview v.1.3 software (skyscan n.v. the software calculates on binary images of sample 's tomographic sections, the different in density areas, within a predefined region of interest (roi). running all relative sections, the total volume (vt) and the volume fraction percent (vf %) of the interested densities (called active pixels) in the roi can be calculated. a specific process had to be followed, to ensure that all calculations were based exactly on the same roi. aartelaar, belgium, the number of horizontal sections covering the width of the cavity was determined. the sections showing all four cavities at their largest width were located and copied on a photoshop cs file. a circular area over the cavity was drawn on a transparent layer with the marquee tool. then, the circular marquee line was copied in new transparent layers, moved and applied to all four cavities for an exact positioning and all four layers were combined and saved as a single.bmp file. this process was repeated for all tooth cavities [figure 2 ] and the saved.bmp files were placed as the last tomographic image, in order to remain available, but outside the range of the selected sections. the inserted image in each set of tooth microtomographs was located and the roi was drawn over the selected areas. the upper and lower sections of each cavity were inserted, and the color palette was set to binary colors (247 in level-1 and 255 in level-2), for best discrimination of empty spaces from tooth / material substance [figure 2 ]. the three - dimensional analysis was used, in order to collect information from the set of predefined horizontal sections on the variables described below. this three - dimensional analysis was repeated three times for each cavity and for all teeth. data collected concerned the following morphometric values : vt, the total selected volume throughout all the cross sections, vf %, the volume percent of the selected item (empty space) in an area, st, the total surface area of the item. the interest was centered on vf % and its relation to the initial volume of cavity dimensions and restorative material type. vt, the total selected volume throughout all the cross sections, vf %, the volume percent of the selected item (empty space) in an area, st, the total surface area of the item. the interest was centered on vf % and its relation to the initial volume of cavity dimensions and restorative material type. aartelaar, belgium, the number of horizontal sections covering the width of the cavity was determined. the sections showing all four cavities at their largest width were located and copied on a photoshop cs file. a circular area over the cavity was drawn on a transparent layer with the marquee tool. then, the circular marquee line was copied in new transparent layers, moved and applied to all four cavities for an exact positioning and all four layers were combined and saved as a single.bmp file. this process was repeated for all tooth cavities [figure 2 ] and the saved.bmp files were placed as the last tomographic image, in order to remain available, but outside the range of the selected sections. the inserted image in each set of tooth microtomographs was located and the roi was drawn over the selected areas. the upper and lower sections of each cavity were inserted, and the color palette was set to binary colors (247 in level-1 and 255 in level-2), for best discrimination of empty spaces from tooth / material substance [figure 2 ]. the three - dimensional analysis was used, in order to collect information from the set of predefined horizontal sections on the variables described below. this three - dimensional analysis was repeated three times for each cavity and for all teeth. data collected concerned the following morphometric values : vt, the total selected volume throughout all the cross sections, vf %, the volume percent of the selected item (empty space) in an area, st, the total surface area of the item. the interest was centered on vf % and its relation to the initial volume of cavity dimensions and restorative material type. vt, the total selected volume throughout all the cross sections, vf %, the volume percent of the selected item (empty space) in an area, st, the total surface area of the item. the interest was centered on vf % and its relation to the initial volume of cavity dimensions and restorative material type. the location of voids and gaps was evaluated using the tview software and its no1 color palette. all relative to each cavity.bmp images (1024 1024 pixels) were viewed by two examiners for the location of voids if present, on a 19 computer screen. for their classification three locations were recorded : the material itself, the side walls of the cavity andthe bottom of the cavity. voids at the walls or bottom of the cavity were actually the gaps at the material / cavity interface. for each cavity, one, two or all three locations were recorded, always based on the agreement of both examiners. voids at the walls or bottom of the cavity were actually the gaps at the material / cavity interface. for each cavity, one, two or all three locations were recorded, always based on the agreement of both examiners. kolmogorov - smirnov tests for normality and bartlett 's test for the equality of variances indicated the use of kruskal - wallis nonparametric test to analyze the data for differences between materials, in respect to the morphometric parameters. mann - whitney test was also used to search for possible differences between low and high viscosity materials, for each morphometric parameter. the degree of correlation of cavity dimensions with all morphometric parameters was analyzed by spearman 's test. all statistical tests performed with medcalc v.10.0 (medcalc software, mariakerke, belgium) at a 0.05 level of significance. unfilled cavity dimensions were measured and found to have a mean length of 2.6 0.4 mm and a mean width 1.30 0.19 mm. the mean values of all morphometric elements (vt, vf and st), estimated by three - dimensional analysis for the cavities filled with four restorative materials are shown in table 3. morphometric parameters of voids and gaps upper left : a tooth section showing two of the filled cavities with voids within fuji ix (fu) material. lower right : voids at the bottom of an fu and in the middle of an aa restoration. statistical analysis by kruskal - wallis nonparametric analysis of variance indicated significant differences among filled cavity groups, for all three parameters (kwvt = 38.0, kwvf = 38.29, kwst = 29.28, p < 0.0001). post - hoc analysis based on pairwise comparisons showed significantly higher values of vf % in cavities filled with fu than those filled with other materials (p < 0.05) [table 3 ]. restorations with af and pf materials showed the lowest vf % values of all (p < 0.05). mann - whitney test also indicated a significant difference between cavities filled with high and low viscosity materials (u = 565.5, p < 0.0001). cavity length, width and volume of high and low viscosity materials as well as in most individual materials correlated poorly and not significantly with vf %. however, in cavities filled with aa, a significant correlation was found between vf % and depth of the cavity. spearman 's rho coefficients of vf % with cavity dimensions the proportion of cavities with voids at different locations (within the material, bottom and side walls of the cavity) is shown in table 5. in 50 - 75% of the restorations, voids were present within the material, without difference in their proportion among the different materials. in 90 - 100% of the cavities filled with aa and fu, gaps were present at the bottom of the cavity, and significantly higher than those filled with af or pf. all cavities filled with aa showed also gaps at the side walls of the cavity, significantly higher than all other materials. lower and upper limits of the 95% ci for the proportion of cavities with voids within the material, and gaps at the bottom or side walls of the cavity the results showed that the procedure followed to compare voids fraction among different cavities on the same sample, is capable of revealing significant differences. the study rejected all three hypotheses : for no differences between materials in vf % of voids (and gaps),for no association of cavity dimensions with vf % of voids (and gaps), andfor no association of voids location with a specific restorative material. for no differences between materials in vf % of voids (and gaps), for no association of cavity dimensions with vf % of voids (and gaps), and for no association of voids location with a specific restorative material. the procedure introduced in this study for the selection of exactly the same roi on all cavities was not a simple one, but since differences between repeated measurements ranged only from 0.002 to 0.005 vf %, this indicates that the method was able to reveal small differences in the volume of voids in very conservative cavities. however, the need to add extra digital sections to a number of cavities smaller in depth than others, or the inclusion in the experiment of materials with high variability in the volume of voids, like fu material, may introduce an error to the measurements. in our study, the low viscous materials were the best among different materials in filling the ultraconservative cavities without gap and this is in agreement with the results of other studies for normal cavities. found that thinner composites had fewer problems with voids and wall adaptation than thicker materials. peutzfeldt and asmussen, who also studied gap formation, found a positive correlation of high viscosity and polymerization shrinkage with gap formation. moreira da silva. found a correlation of gap and voids formation with high viscous flow and low flexural modulus of composite materials. these results can be explained by the high thixotropic effect of low viscous composite materials, and their low polymerization shrinkage due to their low modulus of elasticity. opdam. showed that injection technique, like the one we used with low viscous composites, leads to reduced voids formation within the material and a smaller interfacial gaps. hence, in cavities of very small diameter (1.0 - 1.5 mm) where the insertion of the restorative material and its adaptation to the cavity walls is difficult, low viscous material should be syringed deep into the cavity instead of using higher viscosity composites. thicker composite materials even if they are managed to be inserted in such cavities can potentially form internal voids between layers, and for this reason their use should be done with caution. glass - ionomer material was inserted with difficulty in small cavities, due to its adherence to the instruments and the cavity walls during insertion. as a result of this this type of material should not be used in such small cavities unless perhaps a special small in diameter tip designed to allow proper insertion of the material deep into the cavity before its hardening. our study also showed a significant correlation of initial cavity volume with vf of spaces around and within aa filling material only. this positive correlation of deeper or wider cavities with the presence of voids is probably a result of higher polymerization contraction in larger restorations, as he. their study showed that in larger cavities with a high c - factor (class i and v), the bonding was riskier than in small cavities. therefore, larger cavities filled with high viscous composite material may present a higher amount of voids and gaps. our study showed that the voids within the restorative material were not associated with a specific type of material. however, gaps at the bottom of the cavity were more frequently associated with the more viscous aa and fu materials, as other studies indicated. they presented a higher polymerization contraction than af and pf materials, and need a higher force and more attention to make a closer contact with the bottom of the cavity. the reason is probably that polymerization contraction, which is evident with all cavity walls in a nonbonded restoration, creates a space at the sides of the cavity, more evidently than in other materials. fu, for instance, has the ability of creating a bond with the side walls of the cavity, but also seems to create larger gaps at the bottom of the cavity, which helps to accommodate a significant part of its contraction during hardening. it must be noticed that pixel size in microtomographic images was 14 in this study, although system 's detection limit was 1.8. for this reason, smaller in size voids could not be recorded or located and voids of such dimensions may exist in all types of materials and locations. this is probably the limitation of the suggested method and the reason why low viscous materials show a low proportion of voids or gaps. however, newer ct technology (x - ray computed nanotomography) will be able to estimate gaps and voids more accurately. voids are present in the same frequency within the mass of all restorative materials, but gaps are present more frequently in restorative materials with high viscosity, at the bottom and the side cavity walls. for these reasons, more research is needed to refine the available techniques for placing correctly and precisely a restorative material within a very conservative cavity, the way of using adhesives in cavities of a very small opening, the type of material that can actually minimize the hardening shrinkage of the material and of course the measurement of voids of very small dimensions. there were differences between restorative materials in the vf % of voids and gaps remaining after condensation of the materials in the cavities.low viscous composite resins were the best in filling cavities without voids or gaps.high viscous composite and glass - ionomer materials produced the highest amount of internal voids and gaps.cavity depth, width and volume do correlate with the amount of voids and gap spaces, but only for the high viscous composite material.voids are located in the same frequency within all materials, but gaps are more frequently located within high viscous composites, both at the bottom and the side cavity walls. there were differences between restorative materials in the vf % of voids and gaps remaining after condensation of the materials in the cavities. high viscous composite and glass - ionomer materials produced the highest amount of internal voids and gaps. cavity depth, width and volume do correlate with the amount of voids and gap spaces, but only for the high viscous composite material. voids are located in the same frequency within all materials, but gaps are more frequently located within high viscous composites, both at the bottom and the side cavity walls. the authors of this manuscript declare that they have no conflicts of interest, real or perceived, financial or non - financial in this article. the authors of this manuscript declare that they have no conflicts of interest, real or perceived, financial or non - financial in this article. | background : volume fraction (vf) and location of internal voids and gaps in relation to material type and cavity dimensions in ultraconservative restorations were investigated in this study.materials and methods : forty - eight round cavities of 1.3 mm mean diameter and 2.6 mm mean depth were made on buccal and lingual surfaces of recently extracted human teeth. these were filled and thermocycled with two low viscosity composites (aeliteflo lv [af ], permaflo [pf ]), one high viscosity composite (aelite aesthetic enamel [aa ]) and one glass - ionomer (gcfuji ix gp). x - ray microtomography, following a specific procedure, was applied to all cavities before and after their restoration, using skyscan-1072 microtomographer. vf percent (vf%) and location of voids and gaps were recorded and analysed statistically at a = 0.05. kruskal - wallis nonparametric analysis of variance, post - hoc analysis, mann - whitney test, spearman 's correlation analysis were used to analyze data.results:cavities filled with af and pf showed significantly lower vf % of voids and gaps than all other restorations (p 0.05). the proportion of cavities with gaps at the bottom and side walls was lower in those filled with af and pf (p < 0.05).conclusion : cavities filled with low viscosity composites presented the lowest amount of internal voids and gaps. glass - ionomer and high viscosity composite restorative materials showed the highest amount of interfacial gaps. only in the high viscosity composite restorations the amount of voids and gaps correlated with the cavity depth, width and volume. |
glaucoma is the second leading cause of blindness worldwide. moreover, in contrast to cataract, which is the leading cause of blindness, blindness caused by glaucoma is irreversible. since progression of the disease is prolonged and may remain unnoticed for many years, it has been referred to as a sneaky thief of sight. in fact, the term glaucoma describes a group of diseases with varying etiologies, which have 2 common clinical features : glaucomatous pattern of visual field loss and optic nerve neuropathy resulting in optic disc cupping (increased cup / disc ratio). primary if there are no signs of ocular pathology that elevate intraocular pressure (iop) above the normal range (role of the elevated iop in glaucoma is described in the next paragraph), or secondary glaucomas are further subdivided according by the type of the primary pathology (eg, uveitic, neovascular, traumatic, pseudoexfoliation glaucomas). another subdivision of glaucoma depends on whether the drainage angle is open (open angle glaucoma) or closed (closed angle glaucoma). formerly the definition of human glaucoma included elevation of iop over the upper limit of the normal range (most frequently > 21 mmhg). however, many individuals with elevated iop will never develop glaucoma (this apparently benign condition is termed intraocular hypertension), and quite a few people suffer from glaucoma while measurements of their iop never exceed 21 mmhg (this condition is called normal tension glaucoma, ntg). diagnosis of elevated iop is therefore neither sufficient nor necessary to diagnose glaucoma ; however, it is still regarded as a major risk factor for this disease. elevated iop might affect axonal transport, retrograde as well as anterograde, within the optic nerve. this disturbs delivery of neurotrophic factors such as bdnf and its receptor, trb, which may be required for survival of retinal ganglion cells (rgc). an interesting hypothesis has been proposed according to which the etiopathologic factor is not the high iop but the difference between iop and intracranial pressure (icp). the optic nerve is exposed to considerable forces acting across the lamina cribrosa. in a normal eye the lamina, which is 450 m thick, is exposed to a pressure gradient of 4 mmhg, which is one of the highest pressure gradients that any nerve in the body is constantly exposed to. moreover, some studies have shown that in patients with glaucoma icp is lower, whereas the lamina cribrosa is thinner, which means that their optic nerve is exposed to a higher damaging pressure. this mechanism may explain the phenomenon of normal tension glaucoma. in animal experiments, yablonski. (observations published only in abstract form) found that chronic lowering of intracranial pressure led to glaucomatous damage of the optic nerve, and that simultaneous lowering of iop prevented this damage. in the human retina, glaucoma mainly affects the rgc layer, and selective rgc death is regarded as the hallmark of glaucoma [1517 ]. there are several proposed mechanisms of this selective cell death, including excitotoxicity, nitric oxide toxicity, oxidative stress and trophic factors deficiency. although glaucoma is commonly considered as a retinal disease, there is substantial evidence that not only rgc and the optic nerve, but also upstream components of the visual pathway and visual cortex are affected. detailed information on how different components of the visual pathway are affected in glaucoma can be obtained with modern imaging techniques, in particular magnetic resonance imaging (mri) and spectroscopy (mrs). advances in visualizing visual pathways in the context of glaucoma were thoroughly reviewed by garaci.. here, we focus on novel results concerning use of mr techniques in the field of glaucoma research. mri makes it possible to visualize the bony structures of the skull including orbit, orbital apex, optic canal, as well as intraorbital masses, oculomotor muscles and retrobulbar adipose tissue. moreover, the bulb of the eye, the lens inside it, optic nerve, sheath, optic chiasm, tracts and radiations can be visualized. while the optic nerve and optic chiasm can be seen quite easily, other parts of the visual pathway may be more difficult to distinguish on mr images and may require more advanced techniques, as discussed later. generally the ability to see the structure on mr image is based on the difference in signal strength between them and the surrounding tissue, for example between nerves and cerebrospinal fluid or between white and gray matter. the optic nerve is a white - matter tract, which in the intraorbital part is surrounded by adipose tissue. this fat is characterized by high signal intensity, which makes the optic nerve easily discernible on mr images. glaucoma leads to loss of neurons, and its progression can be observed through measuring the diameter of the optic nerve. thinning of the optic nerve visible on mr images was most pronounced 15 mm behind the bulb and showed correlation with retinal nerve fiber layer thickness measured using optic coherence tomography. the other mri study also showed that the optic nerve diameter was significantly smaller in glaucoma patients (2.25 mm) in comparison with the control group (2.47 mm). the optic chiasm is surrounded by cerebrospinal fluid in the chiasmatic cistern, which makes it highly visible. changes in the optic chiasm in glaucoma reflect the decreased number of axons in the optic nerve. the optic chiasm was atrophic and its height was shorter in patients with glaucoma, especially when huge visual field defects were present. correlation between the height of the optic chiasm and visual field defects was even stronger than the correlation between vertical cup - disc (vc / d) ratio (which is the basic parameter for assessing the progression of glaucoma) and visual field defects. despite the relatively poor sensitivity and resolution of the technique and small thickness of the retina, it is also possible to investigate the retina and its neural connections with mri (figure 1). such studies can be performed using manganese as the dedicated contrast agent ; the technique is called memri (manganese - enhanced mri). mn ions are paramagnetic and sites of their accumulation can be visualized by mri because they shorten the t1 relaxation time of the surrounding water protons, resulting in positive enhancement of the mr signal. manganese ions, which behave as analogues of calcium ions, enter intraneuronal space by 2 mechanisms through calcium voltage - gated channels during activation of neuronal cell and by specific metal ion transporters without concomitant electrical activity. moreover, they are transported down axons via microtubule - based fast axonal transport and are able to cross synaptic clefts. in rats, mn injected intraocularly were taken up by rgcs and transported along microtubules in the optic nerve, and further through chiasms to the contralateral optic tract, the dorsal and ventral lateral geniculate nucleus, the superior colliculus and its brachium, the olivary pretectal nucleus, the nucleus of the optic tract, and the suprachiasmatic nucleus. when mncl2 was administered systemically during a visual task, it acted as a functional biomarker of intraretinal ion regulation. moreover, thickness of the retina layers measured with high - resolution memri technique is similar to the histopathological findings. one study showed accumulation of mn ions in the vitreous, retina and optic nerve head in a glaucomatous eye of a rat after intravitreal injection (figure 1). concentration of mn ions was higher in the glaucomatous eye than in the control eye, which could have been a reflection of decreased number of rgc cells, reduction in axonal density of the optic nerve and blockage of axoplasmic transport along the optic nerve. mncl2 is not approved for humans, but there is another manganese - based contrast agent that is already used in humans mangafodipir (sold under brand name teslascan) contains mn ions chelated by fodipir and is mainly used as a contrast agent in imaging of the liver and allows discrimination of tumors and healthy hepatic tissue. some recent studies also have shown that mangafodipir is useful as a contrast agent in imaging of retina and visual pathways in animal studies. diffusion of water molecules in tissues does not proceed equally in all directions usually movement in 1 direction is predominant. studies of water diffusivity provide information on cellular integrity, especially the integrity and connectivity of the white matter. conventional mri provides little information on diffusivity ; however, as diffusing protons move through intrinsic and extrinsic field gradients they lose transverse magnetization. there are 2 subtypes of diffusion mri diffusion - weighted mri (dw mri) and diffusion tensor mri (dt mri). dw mri applies when the observed tissue is dominated by isotropic water movement, and dt mri applies when the tissue is dominated by anisotropic water movement. one application of dt mri is tracking the nerve fibers (so called tractography). using dt mri, showed that in a rat model of ocular hypertension induced by laser photocoagulation of the episcleral and limbal veins, dt mri - derived parameters (ie, radial diffusivity and fractional anisotropy) were affected in the optic nerve. radial diffusivity was increased and fractional anisotropy decreased, suggesting that axonal density was reduced by around 10% when compared to control rats. moreover, the authors performed dt mri at various time points after photocoagulation and showed that radial diffusivity was increasing and fractional anisotropy was decreasing over time. mean diffusivity was increased and correlated with the stage of glaucomatous optic neuropathy, while fractional anisotropy was decreased and also correlated with the stage of glaucoma (figure 2). this technique, known also as bold (blood oxygen level - dependent) imaging is based on observation of changes of the local cerebral hemodynamics accompanying changes in neural activity. increased cerebral activity is followed by increase in blood flow, resulting in local lowering of deoxyhemoglobin to oxyhemoglobin ratio, because increased blood flow delivers more oxyhemoglobin than is needed. in particular, local changes in oxygenation of hemoglobin in the visual cortex corresponded with the visual stimulation of the eye, which made it possible to prepare a map of the cortical representation of the retina. further study was performed to find the relationship between visual field loss in poag (as measured with a standard automated perimetry) and visual cortex (v1) activation pattern evoked by a scotoma - mapping stimulus and measured with fmri in unilateral glaucoma. in that study it was found that altered patterns of neuronal activity in poag were consistent with visual field deficits. however, fmri indices of altered visual cortex functional response in poag may not have a simple and direct relation to the glaucomatous degeneration of the visual cortex. used fmri to investigate the impact of the glaucomatous neuropathy on the central normal visual field. these authors measured bold fmri changes in the glaucomatous eyes with asymmetric peripheral visual field damage, but without changes in central vision. bold signals corresponding to the central vision field in the primary visual cortex were decreased in comparison to the signals from the non - glaucomatous eyes, despite the fact that there were no changes in patients central visual fields. magnetic resonance spectroscopy (mrs) is the technique that enables non - invasive assessment of levels of resonance - visible metabolites in situ. in this technique measurements of areas under metabolite resonances in a magnetic resonance spectrum are directly proportional to the concentration of these metabolites. although mr spectra can be recorded for various nuclei (eg, phosphorus, fluorine, carbon c), in biomedical research proton (h) resonance spectra are of particular interest. the 3 most prominent resonance lines in the proton mr spectrum of the brain are those related to creatine and phosphocreatine (cr, involved in cellular energy metabolism), choline compounds (cho, associated with metabolism of cell membranes and involved in cholinergic transmission), and n - acetyl aspartate (naa, considered to be the marker of neuronal integrity). brain h mrs data are frequently reported as the metabolite ratios (naa / cr, cho / cr, etc.). more advanced mrs paradigms allow calibration of resonance signals in terms of molar concentrations and record up to 17 brain metabolites in animal experiments. although h mrs has many potential clinical applications in the area of neurodegenerative diseases (eg, parkinson s, alzheimer s disease, and multiple sclerosis), this technique has been rarely used in glaucoma studies. chan. recorded h mr spectra in a rat model of ocular hypertension induced by photocoagulation of episcleral and limbal veins with an argon laser. six weeks after initiation of intraocular hypertension, they found decreased cho / cr ratio in the visual cortex suggestive of a dysfunction in the cholinergic system of the visual pathway (figure 3). however, boucard. could not detect statistically significant differences of naa, cr and cho resonance signals in patients with poag compared to non - glaucomatous controls. different results from the animal and human studies may reflect a difference between the acute animal models (where quick degeneration is observed) and prolonged degeneration in humans subjects. also used mrs to analyze changes in lactate concentration in vitreous after induction of ocular hypertension in rabbits. they found a good correlation between rise in iop and increased lactate signal. performing localized mrs in the vitreous can be a useful tool in tracking pathological changes in experimental models of glaucoma and in human disease. glaucoma is a neurodegenerative disease that affects structure and functioning of the retina and optic nerve, and has an impact on morphology of chiasms and optic radiation. mr techniques seem to be useful tools for characterization of glaucoma - related changes in the visual pathway both in laboratory animals and in humans, and may prove useful as tools for monitoring the progression of glaucoma and assessing the efficacy of novel treatment strategies. | summaryglaucoma is the second leading cause of blindness. it affects retinal ganglion cells and the optic nerve. however, there is emerging evidence that glaucoma also affects other components of the visual pathway and visual cortex. there is a need to employ new methods of in vivo brain evaluation to characterize these changes. magnetic resonance (mr) techniques are well suited for this purpose. we review data on the mr evaluation of the visual pathway and the use of mr techniques in the study of glaucoma, both in humans and in animal models. these studies demonstrated decreases in optic nerve diameter, localized white matter loss and decrease in visual cortex density. studies on rats employing manganese - enhanced mri showed that axonal transport in the optic nerve is affected. diffusion tensor mri revealed signs of degeneration of the optic pathway. functional mri showed decreased response of the visual cortex after stimulation of the glaucomatous eye. magnetic resonance spectroscopy demonstrated changes in metabolite levels in the visual cortex in a rat model of glaucoma, although not in glaucoma patients. further applications of mr techniques in studies of glaucomatous brains are indicated. |
topological superconductivity is a state of matter that can host majorana modes, the building blocks of a topological quantum computer. many experimental platforms predicted to show such a topological state rely on proximity - induced superconductivity. however, accessing the topological properties requires an induced hard superconducting gap, which is challenging to achieve for most material systems. we have systematically studied how the interface between an insb semiconductor nanowire and a nbtin superconductor affects the induced superconducting properties. step by step, we improve the homogeneity of the interface while ensuring a barrier - free electrical contact to the superconductor and obtain a hard gap in the insb nanowire. the magnetic field stability of nbtin allows the insb nanowire to maintain a hard gap and a supercurrent in the presence of magnetic fields (0.5 t), a requirement for topological superconductivity in one - dimensional systems. our study provides a guideline to induce superconductivity in various experimental platforms such as semiconductor nanowires, two - dimensional electron gases, and topological insulators and holds relevance for topological superconductivity and quantum computation. |
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soft tissue sarcomas are a rare heterogeneous tumor group, representing < 1% (approximately 10,000 cases) of all new cancers diagnosed yearly in the united states.1 over 50 subtypes of malignant soft tissue sarcomas have been described in adults,2 leiomyosarcomas are one of the most common.2 leiomyosarcomas are tumors of distinct smooth muscle type cells and occur intramuscularly and subcutaneously.2 treatment choices for leiomyosarcomas are influenced by the site, grade, and extent of disease, and may include surgery, chemotherapy, and radiotherapy. surgical resection offers the best chance of cure and is sometimes coupled with radiotherapy.3 however, metastatic or unresectable sarcoma is rarely curable with reported median survival time of approximately 12 months and 5-year survival rates of 10%15%.4 chemotherapy may have substantial benefits for patients with metastatic or unresectable sarcoma and of the agents that have been utilized to treat soft tissue sarcomas, the most common are doxorubicin, ifosfamide, and, more recently, gemcitabine, both alone and in combination with taxanes.5 however, repeated and/or prolonged use of these agents is prohibited by their limited efficacy and/or associated toxicity. response rates of approximately 20%, 39%, 8%, and 16% have been reported for doxorubicin, ifosfamide, gemcitabine alone, and in combination with docetaxel, respectively.57 in addition, radiotherapy has been combined with chemotherapy as well as being used alone to treat unresectable sarcoma.3 vorinostat (zolinza, merck sharp & dohme, a subsidiary of merck & co., inc.), an orally active, potent, and competitive inhibitor of histone deacetylases (hdac),8 was approved by the us food and drug administration (fda) in october 2006 for the treatment of cutaneous manifestations in patients with cutaneous t - cell lymphoma (ctcl) who have progressive, persistent, or recurrent disease on or following two systemic therapies.9 in addition to efficacy in hematologic malignances, vorinostat has demonstrated promising efficacy in phase i trials in a wide range of solid malignancies.10 here we report on a female patient with leiomyosarcoma with an apparent response to vorinostat treatment after multiple previous chemotherapy treatments. in september 2005, a 43-year - old, asymptomatic female presented with an increasing mass (4 3 cm) in her left thigh which involved the subcutaneous tissue down to the muscular fascia and adductor canal. an immunohistochemical examination on the excised tumor confirmed it was positive for smooth muscle actin, heavy chain myosin, vimentin, and s100 protein. additional biopsies performed during the patient s therapy confirmed this initial diagnosis. once leiomyosarcoma was diagnosed the patient underwent computed tomography (ct) of the chest, abdomen, and pelvis which revealed multiple lung nodules and a tumor (2.2 cm rounded structure) in the myometrium of the left side of the uterine wall, which effaced the uterine cavity. the uterine tumor was c - kit estrogen/ progesterone receptor negative ; however, its origin was inconclusive. the patient initially (october 2005) received combination chemotherapy of doxorubicin (50 mg / m), ifosfamide (2000 mg / m), and mesna (2000 mg / m) every 21 days for a total of 6 cycles (finishing january 2006). a partial response based upon the ct findings was achieved. regretfully, disease recurred 6 months later and the above treatment regimen was resumed for 4 cycles (finishing october 2006). once more, the patient responded well until disease recurred again approximately 4 months later ; when combination chemotherapy with gemcitabine (800 mg / m) and docetaxel (75 mg / m) was commenced. however, the patient developed grade 4 mucositis and pancytopenia during the first cycle necessitating hospital admission and treatment was subsequently reduced to single - agent gemcitabine (1000 mg / m, once weekly for 2 weeks followed by 1 week rest). the patient responded well and received 5 single - agent gemcitabine cycles in total (finishing april 2007). disease again recurred approximately 4 months later and treatment with single - agent gemcitabine was resumed ; however, ct showed disease progression after 2 cycles and combination chemotherapy of doxorubicin, ifosfamide, and mesna was recommenced once more. confronted with a lack of established therapy options in this setting, the patient was offered the opportunity to either enter a clinical trial or receive vorinostat. vorinostat was recommended based on phase i results in patients with refractory solid tumors.10 vorinostat (400 mg / day) was started approximately 2 years after diagnosis (november 2007), stable disease (sd) was observed after 6 weeks and maintained. furthermore, some shrinkage was observed at 3 months in both her liver and lung metastases. the patient remained on vorinostat for 18 months (finishing april 2009) when she developed progressive disease and subsequently received single - agent ifosfamide, and mesna which failed to produce a response. the patient then received single - agent dacarbazine followed by single - agent paclitaxel, both of which failed to produce a response. after evaluating all of her treatment options, the patient selected doxorubicin therapy despite the cardiac risk ; however, after two cycles of weekly chemotherapy, with no response, treatment was stopped. the patient then chose to enter a hospice and died approximately 3.5 years after diagnosis (july 2009). in common with many patients with recurrent relapsing leiomyosarcoma, our patient had exhausted all treatment options for effective chemotherapy ; including first- and second - line combination chemotherapy of doxorubicin, ifosfamide, and mesna followed by combination gemcitabine and docetaxel which was stopped due to grade 4 adverse events (aes) and replaced with single - agent gemcitabine, followed by doxorubicin, ifosfamide, and mesna upon relapse. the best and longest duration of response, overall sd for over 1.5 years, was achieved with vorinostat (400 mg / day), with initial mild nausea which resolved, and with some metastases shrinkage. our case is the first to report an apparent effect of vorinostat in the treatment of leiomyosarcoma. vorinostat has been approved for the treatment of ctcl and has shown efficacy in a number of additional hematologic malignancies.1117 and promising efficacy in other solid tumor types such as breast cancer, lung cancer, and mesothelioma.10,1823 in a phase iib study in patients with persistent, progressive, or recurrent ctcl the objective response rate with vorinostat monotherapy was 29.7%, and 32.3% of patients (with baseline pruritus scores 3) had pruritus relief.11 the most common drug - related aes were diarrhea, fatigue, nausea, and anorexia, most of which were grade 2 ; aes of grade 3 and above included fatigue, pulmonary embolism, thrombocytopenia, and nausea.11 in an extension study, patients who had completed 6 months of treatment in the phase iib study were eligible to continue vorinostat treatment, until disease progression or unacceptable toxicity.24 fifteen of the initial 74 patients entered the extension study and six of these patients continued on vorinostat therapy for more than 2 years. the most common drug - related aes in the continuation study were diarrhea, nausea, fatigue, and alopecia and the incidence of grade 34 aes was low. to date, five patients have discontinued vorinostat and one patient continues to respond after 1445 days (4.0 years). both the clinical trials and post - trial clinical experience have confirmed the efficacy and tolerability of vorinostat. compared with conventional cytotoxic agents,5 vorinostat has a favorable tolerability profile with both short- and long - term use.11,24 thus unlike the cytotoxics, vorinostat has the potential for extended use. a variety of genetic changes have been observed in leiomyosarcoma, often including changes in tp53 and mdm2 expression, overexpression of cyclin - dependent kinase inhibitor 2a (cdkn2a ; p16), and a loss of gamma - smooth muscle isoactin expression.2527 these findings support a preclinical rationale for the potential clinical utility of targeted agents in the management of leiomyosarcoma. vorinostat is a small molecule inhibitor of class i and ii hdac enzymes, which catalyze the removal of acetyl groups from histone proteins on nucleosomes, with histone acetyltransferases (hats) facilitating the acetylation of these proteins.28 histone hypoacetylation results in closed chromatin structures and repression of important tumor suppressor genes.29 consequently, hat inactivation has been associated with tumorigenesis and aberrant hdac activity has been related to the development and maintenance of human tumors, including ctcl.30 in addition, hdacs have many other non - histone proteins as substrates, such as transcription factors (p53), -tubulin, heat shock protein 90 (hsp90) and various signaling proteins.28 there are limited preclinical data reporting the activity of hdac inhibitors in sarcoma.3139 ecke and co - workers observed that combined 5-aza-2deoxycytidine (a dna methylation inhibitor) and valproic acid (a hdac inhibitor) efficiently prevented medulloblastoma and rhabdomyosarcoma formation in patched mutant mice.32 nguyen and co - workers observed that the hsp90 inhibitor 17-aag and the hdac inhibitor ms-275 had synergistic, antiproliferative, and proapoptotic effects on synovial sarcoma in vitro.31 additionally, the hdac inhibitors ms-275, trichostatin - a, phenylbutyrate, laq824, and depsipeptide, enhanced the antineoplastic action of 5-aza-2deoxycytidine on ewings sarcoma cells.33 while sakimura and colleagues noted that depsipeptide inhibited chondrosarcoma cell growth, up - regulated the expression of aggrecan and alpha2 chain of type xi collagen (col11a2) mrna, and induced differentiation to a hypertrophic cell phenotype.37 two studies with vorinostat have reported growth inhibition of chondrosarcoma cells, significant inhibition of tumor growth in a xenograft model, and death of endometrial stromal sarcoma cells.34,36 this case, which demonstrates promising activity for vorinostat in leiomyosarcoma, represents one of the first clinical applications of an hdac inhibitor in soft tissue sarcoma. however, this is only one case report and more studies are needed to confirm the activity and efficacy of vorinostat in leiomyosarcoma. | leiomyosarcoma is a heterogeneous tumor group, representing < 1% of all new cancers diagnosed in united states. treatment choice is based upon site, grade, and extent of disease. however, prognosis for metastatic or unresectable sarcoma is very poor with reported median survival of 12 months. response to chemotherapy has been approximately 8% to 39% based upon the chemotherapeutic agent and whether used alone or in combination. vorinostat is an orally active, potent, and competitive inhibitor of histone deacetylases approved for cutaneous t - cell lymphoma. there are limited preclinical data illustrating the activity of histone deacetylase inhibitors in sarcoma. here is a case of a lady with leiomyosarcoma who has progressed through multiple chemotherapeutic agent who has achieved a partial response to vorinostat treatment. |
genetic technology has advanced at such a rapid pace that it is even difficult for those of us in the field of genetics to keep up with the latest techniques and methods for analyses. psychiatry in particular has a history of taking advantage of new advances in the field to explore differences that occur in people with mental illness. essentially, this has occurred because hypothesis pursuit in the field has continually led to failure over the years. thus, the underlying neural mechanisms for major mental disorders, such as schizophrenia, bipolar disorder, and autism remain mysteries, and preventive measures are nowhere in sight. the current thinking is that psychiatry will not be ready for hypothesis - driven research until searches of large genetic datasets produce consistent findings that then can lead to construction of new proposed pathways or mechanisms for disease. pharmaceutical companies have thus been liaising with current researchers in order to find clues from this work to target new drug development. it was therefore with great interest that it was heard and read that the most important research result in psychiatry has finally been published. one hundred and eight independent loci conferring elevated risk for schizophrenia have been found by an unusually large combination of case - control samples collected worldwide. questions asked are : (i) how elevated is the risk for each variant ? and (ii) are these additive, conferring higher risk ? creative statistical geneticists have been able to calculate a polygenic risk score that is valuable for correlational analyses in research studies, but is not likely to be of value for clinical prediction, given its likelihood of both false - positives and false - negatives. it has not yet been shown to be valid and reliable, nor do we understand the genetic architecture of the illness enough to be able to say how it is related to true risk. this fact, however, is not likely to be perceived that way by the public, and may unfortunately be taken up in the clinic as a new predictive tool. these data came about because of a new approach that was championed by the broad institute in boston, usa. it is paralleled by other collaborations in other complex genetic disorders such as alzheimer 's disease or colon cancer. thus, the larger the number of subjects, the more likely a significant result or set of results will emerge. this concept for psychiatry was first publicly proposed by dr edward scolnick of the broad institute in a closing session of the world congress of psychiatric genetics in new york city in 2007. it developed into one of the largest - ever psychiatric collaborations, called the pgc (psychiatric genomics consortium), now based at the broad institute for worldwide organization and analyses. one does not need a hypothesis with this design ; it just compares thousands of people and looks for any differences between patients and controls. it has worked most robustly for schizophrenia, but now what is the next step ? some research groups are sequencing large cohorts of patients and controls, while others are going back to examining individual families those with multiple ill members to note patterns of inherited sequenced segments and how they segregate with illness, a method used long ago when technology was not so advanced. yet these findings from families can lead to clues for pathways involved that will generalize to the larger population of people with illness, and hopefully lead to new targets for drug development. the findings from genome - wide association studies (gwas) consist of common variations in genes that by themselves may mean little. what is important is that they may form the basis for future studies of their mechanisms, how they relate to each other, and the pathways they involve, which may suggest targets for drug development. gwas are clearly not family - based, and thus it has not yet been determined whether the risk variants are associated with illness within families or are sufficient and/or necessary to lead to illness, nor whether they can be used to predict future illness within families. the most hopeful findings thus so far have come from the schizophrenia gwas, and not those in affective disorder. despite most of the elevated risk singlenucleotide polymorphisms appearing in noncoding regions of the genome, some promising pathways have evolved as candidates from these results, and these not surprisingly can be divided into those that affect nmda receptor modulation and related pathways, muscarinic acetylcholine receptors, -aminobutyric acid pathways, nicotinic-7 receptors, oxidative stress, and the immune system. the problem with large gwas collaborations is that the sample characteristics are lost. given that the illnesses screened are so very heterogeneous clinically, as well as demographically, it may not be realistic that the obtained polygenic risk factor identifies any one clear biological mechanism for a specific clinical disorder. the strategy used for such large gwas samples precludes carefully structured systematic ascertainment of samples with good diagnostic reliability across populations to be able to know whether a diagnosis in one center is the same as one in another. each cohort contributing to the larger one was collected, ascertained, and evaluated in different ways, and thus may have picked up different psychopathologies. there is certainly much that can be said for systematically collecting cohorts and obtaining reliability across them. unfortunately, in these times of the popularity of big data collecting, these principles and the quality of the work going into the sample collection is often lost. understanding how to predict response to medication makes up a major portion of this issue of dialogues in clinical neuroscience. perhaps the most successful use of new genetic technology will not come from gwas, but rather from the development of specific hypothesis driven dna markers that predict response to medications and also the risk of some dangerous side effects. the cytochrome p450 metabolic enzymes, important in drug metabolism, can be genotyped (cyp2d6 and cyp2c19) to determine slow- and fast - metabolizing variants, thus enabling the prediction of side effects in individuals at particular doses of antidepressants, antipsychotics, and other medications. moreover, these are established enough that the usa fda has issued warnings about genotypes for certain medication usage (http://www.fda.gov/drugs/scienceresearch/researchareas/pharmacogenetics/ucm083378.htm). studies still in progress, but of high interest, are those using genomics to determine prediction of antipsychotic weight gain and clozapine - induced agranulocytosis. pharmacogenetics will also be useful in determining outcome, with the most frequently used genes such as those encoding the serotonin transporter (slc6a4), the serotonin-2a receptor (htr2a), and p - glycoprotein (abcb11) and others that may be associated with lithium response. should scientists be concerned about the use of their data by the public and the implications drawn from them ? this is a much - debated question, leading to science, unfortunately, becoming entangled in politics, particularly when there is a clear gap between the knowledge that researchers have and the perceptions of the research by the public. an extreme example of the misuse of genetic information is the way the nazi era brought a pseudoscientific thinking that, given mental illness was genetic, cleansing the population of people carrying the genes would eradicate it worldwide. not only is this scientifically erroneous, but profoundly unethical unfortunately, thousands of psychiatric patients were exterminated in germany in the late 1930s because of this notion. currently, sequencing an individual 's genome carries the risk of stigmatizing that person and placing him / her under unnecessary scrutiny when applying for employment and in the social scene. the privacy of this information can only be maintained with proper government regulations and the stigma reduced by continual public education. if there is a gap between what researchers know and public knowledge, then misunderstandings, panic, and misuse of the information occur. in the research itself, ethical issues are debated and are difficult to deal with when attempts are made to combine samples collected worldwide in many ways. was informed consent obtained from each individual, and what really was their understanding of how their dna would be used ? some countries and ethical review boards have strict regulations about human dna being transported out of their respective countries. who owns these samples and has the rights to them ? is it each individual who contributed dna, is it the funding agency who made the project possible, the researchers who worked hard on obtaining each sample, the researchers in the laboratory who processed it, or the institutions who administered and oversaw the work ? biological psychiatry is replete with old findings of factors present in blood, urine, and even cerebrospinal fluid that were supposed clues to the cause of illness, predictive of it, or predictive of its outcome. a low platelet monoamine oxidase enzyme level was hypothesized in the 1970s to be causative of schizophrenia and also affective disorders, as was the endogenous hallucinogen, phenylethylamine. some of these were found to be artifacts of environmental and iatrogenic variables including general effects of longterm medication, and some were never replicated. when brain imaging was introduced into psychiatry, various indices, such as ventricular enlargement, were used to predict outcome, but none of these ever made it to clinical utility. nor has any brain imaging measurement been found to be predictive of a specific disorder. nevertheless, pet scanning, which is a difficult and expensive procedure to have patients undergo, does have promise for predicting receptor occupancy that will lead to response to specific medications that effect those receptors. it does not have predictive value for diagnosis of mental disorders, such as schizophrenia, despite its usefulness in alzheimer 's disease. psychodynamic issues were also raised, and at one time seemed popular candidates for the cause of schizophrenia, and other major psychiatric disorders leading to the family therapies of the 1960s and 1970s. a measles vaccine scare resulted from erroneously attributing autism to a vaccine side effect. at one time cooking in aluminum pots lastly, birth complications and flu during pregnancy as potential causative factors for later mental illness have caused scares among the patients of obstetricians, but have never found to be clearly causative in the majority of individuals who have these complications during pregnancy. even today, there is much debate about whether cannabis by itself can cause a psychotic illness. in genetics for example, there was much excitement in the late 1980s about a finding on the long arm of chromosome 5 that was linked to schizophrenia. this was followed in the 1990s by linkage to chromosomes 6p and 8p, which led to the candidate genes of dysbindin and neuregulin, respectively. sophisticated new technology has provided the medical profession with tools for diagnosis, prediction of illness, new medications, and personalized pharmacologic treatments. but with these come new problems for data interpretation, validity, and ethical use of the information generated. in general, it makes sense to adopt a cautiously optimistic approach to new findings generating excitement in psychiatric research, given that so many in the past are buried in the literature and forgotten, for good reason. | technology in genetics and brain imaging has advanced so rapidly that it is difficult to be knowledgeable about all the new tools being used in the pursuit of progress toward understanding and treating mental illness. while findings from new studies remain promising, caution is needed with regard to their current applicability to clinical use, both to predict who is likely to become ill and who is likely to respond to medication. a perspective on the past, using schizophrenia as an example, illustrates important findings that were published, had much visibility, and caused a flurry of new related studies, but then slowly disappeared, either to be abandoned as an artifact of the assay or study design, an epiphenomenon, or as simply nonreplicated findings not leading to further progress. remembering that good science is the pursuit of the truth and not joining the latest bandwagon fad of believers is an important principle to adhere to when participating in the politics of science. |
after many years of application in research, positron emission tomography (pet) is now emerging as a valuable clinical tool, particularly in the field of oncological imaging. this transition has resulted largely from the development of one particular pet radiotracer, fluorine-18 labelled fluorodeoxyglucose (fdg). the relatively long half - life of fluorine-18 (110 min) has meant that pet units no longer require their own cyclotron for isotope production as fluorine-18 from one cyclotron can be transported to several pet scanners at sites remote from the production facility. furthermore, flourine-18 imaging is not associated with the high count rates obtained with other shorter - lived isotopes, allowing lower cost sodium iodide (nal) detector systems to be used in preference to bismuth germinate (bgo). nal detector systems also benefit from greater energy resolution, allowing superior rejection of scattered photons and thereby enhancing imaging quality for whole body studies. uptake results from increased expression of hexokinase and of glut-1 glucose transporters on the cell surface. both of these metabolic changes result from oncogene mutations with the tumour cells, most notably the p53 gene - mutation, which occurs commonly in lung cancer. it is important to realise that the detection of a tumour focus on fdg - pet is determined by the degree of metabolic change rather than the size of the lesion (fig. the high glucose metabolic rate of tumours as compared to normal tissues allows tumour foci as small as 3 mm to be commonly identified by pet. figure 1 an illustration of how the detection of small tumours by pet is not determined by spatial resolution. the text on the left is displayed with high spatial resolution but is unable to detect the hidden message cancer. even though the text on the right is blurred (low resolution), the hidden message is revealed by labelling the letters that are functionally different (i.e. they carry the hidden message). an illustration of how the detection of small tumours by pet is not determined by spatial resolution. the text on the left is displayed with high spatial resolution but is unable to detect the hidden message cancer. even though the text on the right is blurred (low resolution), the hidden message is revealed by labelling the letters that are functionally different (i.e. they carry the hidden message). lung cancer is an important indication for pet imaging, accounting for approximately one quarter of the patients examined at the wesley pet centre. this article describes the technique and indications for pet in lung cancer and reports on its accuracy and cost - effectiveness. after a six - hour fast to ensure low blood glucose levels, patients are injected with 185370 mbq fdg and imaging of the chest, abdomen and pelvis is performed one hour later. the brain is not routinely imaged, as detection of small cerebral metastases is unreliable, due to masking by the high uptake seen in normal cerebral cortices. a dedicated pet system (nal or bgo) should be used, because the low sensitivity of coincidence gamma camera systems results in failure to detect small nodal metastases. image reconstruction should employ iterative (osem) techniques to further enhance the quality of final images. tumour uptake can be quantified by measurement of the standardised uptake value (suv). fdg - pet is highly accurate in the characterisation of solitary pulmonary nodules (spn), diagnosing malignancy with reported sensitivities of 89100% and specificities of 58100%. thus, fdg - pet is generally more accurate than per - cutaneous biopsy, which has lower sensitivity due to sampling error. in up to 50% of positive cases, identifies sites of metastasis that have not been suspected on the basis of other imaging. specificity values for fdg - pet are lower in populations with a high incidence of granulomatous diseases, such as histoplasmosis, which may produce false - positive results. false - negative studies can occur with alveolar cell carcinoma and carcinoid tumours, and therefore follow - up imaging is recommended following a negative study for spn. accuracy can be improved by reporting the pet data with ct data available for comparison. experience with fdg - pet, along with decision tree analysis techniques, have shown that the high accuracy of pet for spn translates to reductions in health care expenditure by avoidance of unnecessary biopsy procedures and thoracotomies. fdg - pet is more accurate for staging mediastinal nodes than ct (pet sensitivity 7782%, specificity 8198% ; ct sensitivity 5665%, specificity 4487%). pet will identify involved nodes that are too small to be reliably diagnosed by ct, whereas the absence of fdg uptake in nodes that are seen to be enlarged on ct indicates reactive adenopathy. distant metastases, especially in bone or adrenal glands, are also accurately depicted by fdg - pet. the high therapeutic impact of fdg - pet is shown by the fact that significant changes in management occur in 3251% of patients examined. patients classified as stage 1 by pet demonstrate improved survival as compared to those considered to be stage 1 on ct. this improvement results from the ability of pet to identify those patients most likely to benefit from surgery. furthermore, the high accuracy of pet has been shown to lead to savings in health care expenditure by allowing unnecessary thoracotomy to be avoided in those patients unlikely to benefit. emerging roles for fdg - pet in lung cancer include determination of prognosis, radiotherapy planning, therapy monitoring and diagnosis of recurrence. prognostic information can be obtained from measurements of uptake with high suv values implying a poor prognosis and a poorer response to radiotherapy. the use of pet for radiotherapy planning may improve the efficacy of treatment by ensuring that tumour revealed by pet, but undetected by ct, is not excluded from the treatment volume. the reduction in fdg uptake following radiotherapy correlates with the long - term therapeutic response, and pet is more accurate than ct in predicting the down - staging that has occurred with induction chemotherapy. fdg - pet is also useful in diagnosing recurrent tumour, particularly when ct is unable to distinguish between recurrent tumour and post - therapy fibrosis in the presence of a residual structural abnormality. fdg - pet is highly accurate in the characterisation of solitary pulmonary nodules (spn), diagnosing malignancy with reported sensitivities of 89100% and specificities of 58100%. thus, fdg - pet is generally more accurate than per - cutaneous biopsy, which has lower sensitivity due to sampling error. in up to 50% of positive cases, identifies sites of metastasis that have not been suspected on the basis of other imaging. specificity values for fdg - pet are lower in populations with a high incidence of granulomatous diseases, such as histoplasmosis, which may produce false - positive results. false - negative studies can occur with alveolar cell carcinoma and carcinoid tumours, and therefore follow - up imaging is recommended following a negative study for spn. accuracy can be improved by reporting the pet data with ct data available for comparison. experience with fdg - pet, along with decision tree analysis techniques, have shown that the high accuracy of pet for spn translates to reductions in health care expenditure by avoidance of unnecessary biopsy procedures and thoracotomies. fdg - pet is more accurate for staging mediastinal nodes than ct (pet sensitivity 7782%, specificity 8198% ; ct sensitivity 5665%, specificity 4487%). pet will identify involved nodes that are too small to be reliably diagnosed by ct, whereas the absence of fdg uptake in nodes that are seen to be enlarged on ct indicates reactive adenopathy. distant metastases, especially in bone or adrenal glands, are also accurately depicted by fdg - pet. the high therapeutic impact of fdg - pet is shown by the fact that significant changes in management occur in 3251% of patients examined. patients classified as stage 1 by pet demonstrate improved survival as compared to those considered to be stage 1 on ct. this improvement results from the ability of pet to identify those patients most likely to benefit from surgery. furthermore, the high accuracy of pet has been shown to lead to savings in health care expenditure by allowing unnecessary thoracotomy to be avoided in those patients unlikely to benefit. emerging roles for fdg - pet in lung cancer include determination of prognosis, radiotherapy planning, therapy monitoring and diagnosis of recurrence. prognostic information can be obtained from measurements of uptake with high suv values implying a poor prognosis and a poorer response to radiotherapy. the use of pet for radiotherapy planning may improve the efficacy of treatment by ensuring that tumour revealed by pet, but undetected by ct, is not excluded from the treatment volume. the reduction in fdg uptake following radiotherapy correlates with the long - term therapeutic response, and pet is more accurate than ct in predicting the down - staging that has occurred with induction chemotherapy. fdg - pet is also useful in diagnosing recurrent tumour, particularly when ct is unable to distinguish between recurrent tumour and post - therapy fibrosis in the presence of a residual structural abnormality. there is now a substantial body of evidence supporting the use of fdg - pet in lung cancer. the value of the technique is increasingly recognised by health purchasers. the high accuracy of fdg - selection of the most appropriate and effective therapy for patients with lung cancer. fdg - pet is accurate and cost - effective in the characterisation of spn and in the pre - operative staging of non - small cell lung cancer.fdg-pet has emerging roles in determination of prognosis, radiotherapy planning, therapy monitoring and diagnosis of recurrence. fdg - pet is accurate and cost - effective in the characterisation of spn and in the pre - operative staging of non - small cell lung cancer. fdg - pet has emerging roles in determination of prognosis, radiotherapy planning, therapy monitoring and diagnosis of recurrence. | the ability to demonstrate tumour foci that are undetected by conventional imaging has resulted in the emergence of positron emission tomography (pet) as a valuable clinical tool in oncology. this article describes the technique and indications for fluorine-18 labelled fluorodeoxyglucose (18fdg)-pet in lung cancer, demonstrating the high accuracy and cost - effectiveness of 18fdg - pet in the characterisation of solitary pulmonary nodules and in the pre - operative staging of non - small cell lung cancer. emerging roles in determination of prognosis, radiotherapy planning, therapy monitoring and diagnosis of recurrence are illustrated. |
precise preoperative marking of a discharging breast duct and intraductal lesions facilitates a minimal - volume microdochectomy. ultrasound (us) provides valuable guidance for localizing intraductal abnormalities in patients with nipple discharge, and several studies have reported the use of preoperative us - guided wire localization for cases involving problems with cannulation of the discharging duct. we herein report an alternative method of preoperative us - guided indigo carmine dye staining in a patient with no discharge on the day of surgery. a 35-year - old woman presented with spontaneously intermittent bloody discharge from her right nipple. she had no history of breast abnormalities, previous breast surgery, or radiation therapy. mammography showed an asymmetric tubular structure in the subareolar area and a 3.2 cm tortuous tubular structure in the upper outer quadrant of the right breast (figure 1a). us showed a solitary dilated duct containing two isoechoic intraductal lesions 2 cm from the nipple and an isoechoic lesion 5 cm from the nipple in the 10 o'clock region of the right breast (figure 1b). galactography demonstrated a dilated duct with several polypoid filling defects and complete obstruction at the site of the tortuous tubular structure (figure 1c). we performed a us - guided core needle biopsy for the intraductal lesions 2 cm from the nipple and the tubular lesion 5 cm from the nipple in the 10 o'clock region of the right breast, and all specimens were placed into one specimen bottle. histopathological examination of the biopsy specimens indicated invasive ductal carcinoma, including intraductal proliferation with a papillary growth pattern. magnetic resonance imaging for preoperative staging demonstrated a 4.4 cm area of linear and clumped nonmass enhancement that extended from the upper outer quadrant to the subareolar portion of the right breast (figure 1d). the surgeon recommended right mastectomy, but the patient desired to undergo breast - conserving surgery. the surgeon requested localization of the dilated duct and intraductal lesions for microdochectomy, and preoperative wire localization under galactographic guidance was planned. on the day of surgery, we inserted a needle under us guidance into the dilated duct in the 10 o'clock region of the right breast. under us guidance, a short bevel 25-gauge needle was used to avoid counterpuncture and contrast extravasation and a small amount of indigo carmine (0.8% indigotindisulfonate sodium ; korea united pharm., seoul, korea) was injected at low pressure (figure 1e). after injection, the targeted duct became more dilated, and dye was discharged from the right nipple. we performed us - guided hook wire localization for the mass at 5 cm from the nipple., the indigo carmine - stained duct was identified in the operative field (figure 1f). for the mass 5 cm from the nipple, histopathology of the excised specimen confirmed invasive ductal carcinoma with a negative margin. on follow - up mammography and us performed 8 months after the operation, tumor recurrence was not detected. the discharging duct and intraductal lesions can be identified before surgical excision through several methods, such as reviewing the preoperative galactogram, preoperative marking with blue dye staining, or wire localization under galactography guidance. in addition to these postoper - ative procedures, intraoperative intraductal blue dye injection, ductoscopic wire marking, and insertion of a lacrimal probe into the discharging duct can be performed. moreover, lesions causing intermittent discharge are difficult to localize ; the less discharge, the more difficult the cannulation. if no discharge can be provoked, duct cannulation is not possible, and it may not provide adequate guidance for surgery, leading to unnecessary wide excision of breast tissue or failure to remove the cause of nipple discharge. and hussain and lui reported several successful cases of an alternative method, namely preoperative us - guided wire localization, which was useful, when problems in cannulation of the discharging duct were encountered. we designed a convenient alternative method under us guidance for preoperative marking of the dilated duct and intraductal lesions in a patient with no nipple discharge. when a dilated duct is detected on us, a needle should be placed into the pathologic duct and us - guided indigo carmine injection should be followed. the oblique needle approach along the long axis of the dilated duct can facilitate the procedure by providing an elongated target. subtle adjustments with both hands, maintenance of proper needle alignment, exact placement of the needle into the pathologic duct, and careful dye injection with low pressure are factors for the success of this procedure. reported contrast medium extravasation during us - guided percutaneous galactography although the needle tip was seen inside the duct during contrast injection. it is assumed that the needle punctured the posterior duct wall when the syringe was pushed. similarly, it is important that the needle tip does not puncture the posterior duct wall during us - guided percutaneous indigo carmine staining. another important factor is careful monitoring of the patient and preparation of a vasopressor before using indigo carmine. although the procedure has clinically been considered safe, jo. recently reported two cases of hypotension after intradermal injection of indigo carmine into the periareolar area for sentinel node mapping. to date, no adverse reactions have been reported after intraductal injection of indigo carmine. in conclusion, us - guided percutaneous indigo carmine staining of the dilated duct and intraductal lesions can facilitate minimal - volume microdochectomy in patients with no nipple discharge. | spontaneous bloody nipple discharge from a single duct is a significant clinical problem. when performing preoperative marking of the discharging duct, it is sometimes difficult to identify the duct owing to intermittent discharge. precise preoperative marking of the discharging duct and intraductal lesions is very important to avoid unnecessary wide excision of breast tissue or failure to remove the cause of nipple discharge. we herein present a case of preoperative ultrasound - guided indigo carmine staining in a patient with no discharge on the day of surgery. when a dilated duct is visualized on ultrasound, the targeted duct can be localized using indigo carmine staining, and it is possible to perform a precise minimal volume microdochectomy. |
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