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aryl hydrocarbon receptor (ahr) is a ligand - activated transcription factor. together with its transcriptional regulators, basic helix - loop - helix per - arnt - sim (bhlh - pas) nuclear partner, and aryl hydrocarbon receptor nuclear transporter (arnt), it provides a powerful signaling system during a critical response to several environmental pollutants such as polyhalogenated aromatic and polyaromatic hydrocarbons. the ahr function is particularly well - characterized in response to the exogenous compound 2,3,7,8-tetrachlorodibenzo - p - dioxin (tcdd). moreover, several lines of evidence suggest that a battery of proinflammatory cytokine genes can be upregulated upon interaction of tcdd with ahr. tcdd treatment causes increased expression of tumor necrosis factor (tnf)-, interleukin (il)-1, il-2, interferon (ifn)-, il-18, il-6, chemokine (c - c motif) ligand 1 (ccl1), and plasminogen activator inhibitor-2 (pai-2) [49 ]. additionally, negishi. demonstrated that a synthetic antiallergic agent m50354, which is another ahr agonist, increased the levels of ifn- associated with reduced expression of gata-3, a key factor for th2 differentiation. in addition to its role in metabolizing exogenous compounds as part of an adaptive chemical response, there is growing evidence suggesting that ahr has normal physiological functions and that it likely has endogenous ligands. for example, there is evidence for the importance of ahr in normal development, liver functions, circadian rhythm, response to hypoxia, hormone signaling, and vascular regulation [1114 ]. we previously reported that spleen cells from ahr - null mice overproduce ifn- and il-12 when challenged with concanavalin - a (cona) or restimulated with ovalbumin in vitro. this observation agrees with previous findings indicating that ahr plays an important role in normal development and function of the immune system. moreover, recently are emerging new evidences that ahr also plays a role in normal physiology, including certain immune responses. in particular, th17 cells and dendritic cells (dcs) express high levels of ahr [18, 19 ]. for example, in influenza virus infection tcdd - induced ahr - activation diminishes the memory response but does not impair host resistance. in lethal streptococcus pneumonia infection model, the survival rate is slightly enhanced in mice lacking ahr. ahr/ mice infected with listeria monocytogenes, an intracellular bacteria, are more susceptible to infection but develop enhanced resistance to reinfection, even though their serum levels of inflammatory cytokines such as il-6, ifn-, and tnf- are comparable to wt mice. additionally, macrophages from ahr/ mice retain their ability to ingest listeria and inhibit parasite growth. these data suggest that ahr contributes to an optimal immune response, but its function appears to be distinct depending on the pathogen. thus, establishment of the role of ahr in some parasitic infections may extend our understanding of the biological functions of ahr. toxoplasma gondii is an opportunistic protozoan parasite that causes toxoplasmosis, which is clinically asymptomatic in most individuals but can be fatal in immunocompromised hosts. immunity to t. gondii is highly dependent on cell - mediated effector responses, that consist of high levels of type 1 cytokine production [2326 ]. the il-12/ifn- immune response axis plays a crucial role in determining resistance to t. gondii infection. deficiencies in ifn- production, ifn--receptor - mediated signaling pathway, cells that produce ifn- such as natural killer (nk) cells, cd4 and cd8 t cells, macrophage migration inhibitory factor (mif), or some other effector molecules such as nitric oxide (no), result in increased susceptibility to t. gondii. furthermore, deficiencies in il-12, its receptor, or its intracellular signaling pathway (stat-4) render mice extremely susceptible to acute toxoplasmosis with survival rates similar to those observed in ifn--deficient animals. on the other hand, exacerbated proinflammatory response can lead to immunopathology and death. for this reason the immune system has evolved an elaborated series of pathways to downregulate proinflammatory responses. since previous reports suggest that ahr participates in modulating th1/th2 balance and proinflammatory responses [10, 15 ], we analyzed the role of ahr in host control of experimental toxoplasmosis. we showed that ahr/ mice infected with me49 strain of t. gondii develop fewer cysts in the brain but, paradoxically, succumb significantly faster than wt mice. the increased mortality rate of ahr/ mice upon t. gondii infection was associated with higher levels of tnf- and ifn- and lower levels of il-10 and gata-3. these findings indicate that ahr plays an important role in downregulating inflammatory responses during toxoplasma gondii infection. ahr - deficient (ahr/) and wt (ahr+/+) mice were generated as previously described [15, 16 ]. these mice lack a functional ahr, as the exon1 is replaced from the translational start site onwards with a neomycin gene. ahr+/ males were matted with ahr+/ females to generate wt, ahr+/, and ahr/ mice. in the following experiments, we used eight- to 10-week - old male wt and homozygous mutant littermate mice (ahr/). all of the mice were maintained in a pathogen - free environment at centro de investigacin y estudios avanzados del instituto politcnico nacional animal facility in accordance with institutional and national guidelines for animal research. cysts from the avirulent me49 strain were harvested from the brains of c57bl/6 mice that had been inoculated intraperitoneally (i.p.) with 20 cysts, 1 to 2 months before harvest. for experimental infections, control inoculations with uninfected brain suspensions failed to elicit detectable inflammatory responses or significant increase in cytokine levels. soluble toxoplasma antigen (stag) from tachyzoites of t. gondii was prepared as described previously. to assess the disease progression, brains from t. gondii - infected wt and ahr/ animals were removed aseptically and homogenized in 2 ml of pbs at days 10, 15, 25, 57, and 60 postinfection. the cysts were counted in a 10 l brain - suspension at least three times, and the averages were multiplied by 200. parallel semiquantification of parasite - specific dna sequences was performed on the same brain samples in order to confirm the microscopic findings, as described previously. in brief, brains from t. dna was extracted from tissues using the qiamp tissue kit (qiagen, chatswort, ca, usa), and 50 and 25 ng of each sample was analyzed by polymerase chain reaction (pcr). pcr amplification was performed on parasite dna to amplify a 200- to 300-fold repeated fragment of 529 bp (primers tox4, 5-cgctgcagggaggaagacgaaagttg-3 and tox, 5-cgctgcagacacagtgcatctggatt-3). the 529 bp fragment this fragment is unique to t. gondii dna and is distinct from that of other parasites. the mouse gapdh gene (primers, table 1) was amplified in parallel as a control to monitor pcr inhibition and to control for dna integrity. twenty - five days after t. gondii infection peritoneal exudate cells (pecs) and spleen cells were obtained from wt and ahr/ t. gondii infected mice, in sterile conditions, and cultured as previously described. in brief, spleen tissues were minced and filtered to obtain spleen cells, which were then washed and resuspended in dmem culture medium supplemented with 10% fetal calf serum (fcs), 2 mm l - glutamine, 0.25 u / ml penicillin, and 100 mg / ml streptomycin (all from gibco, brl grand island, ny, usa). splenocytes were resuspended at 5 10 cells / ml in the same medium. one hundred l of the cell suspensions were transferred to 96-well flat bottom culture plates (costar, cambridge, ma, usa) and stimulated with either 100 l of con - a mitogen solution (2 g / ml ; sigma, st. louis, mo, usa) or with soluble toxoplasma antigen (stag) (2.5 g / ml). plates were then incubated at 37c, 5% co2 for 72 hours or 6 days with con - a or stag, respectively. fifty - four hours after seeding the plates stimulated with stag, 0.5 ci of methyl - h - tdr (specific activity 925 gbg / mmol. cells were harvested, pipetted onto a glass fiber filter paper (wallac), and analyzed by a liquid scintillation counter (betaplate, walac). pecs were prepared as previously described. in brief, 1 10 pecs were plated in 24-well plates. two hours later nonadherent cells were washed - off twice with complete dmem, and the remaining adherent macrophages (m) were replenished with complete medium. stag was added at a final concentration of 2.5 g / ml for 24 hours. in both spleen and pec cultures, after t. gondii infection, wt and ahr/ mice were bled from tail snips at various time points. sera from blood samples and supernatants from cell cultures described above were analyzed to measure production of il-2, il-4, il-10, il-12p70, ifn-, and tnf- by elisa (peprotech, mexico) using paired monoclonal antibodies and murine recombinant cytokines to make standard curves, as previously described. optical density (od) was measured after 5 minutes using an elisa microplate reader (spectramax 250, molecular devices, usa) at 405 nm. nitric oxide (no) levels in the serum were determined indirectly by measuring the total serum nitrite (no2) after reduction of nitrate (no3) to nitrite (no2) with nitrate reductase following the protocol described by granger. and adapting it to microwell plates (costar). briefly, 50 l of serum was incubated at room temperature with 50 l of substrate buffer (imidazole 0.1 mol / l, nadph 210 mol / l, flavine adenine dinucleotide 3.8 mol / l : ph 7.6, all from sigma - aldrich) containing nitrate reductase (aspergillus niger, sigma) for 45 minutes to convert no3 to no2. total nitrite was then mixed with an equal volume of griess reagent (1.5% sulfanilamide, 0.1% naphthylethylenediamine dihydrochloride, 2.5% phosphoric acid ; all from sigma), incubated for 10 minutes at room temperature in the dark, and the absorbance was measured at 570 nm in an automatic microplate reader (organon technika microwell system).values were quantified using serial dilutions of sodium nitrite. peripheral blood was collected at various time points from tail snips of all experimental mice infected with t. gondii. total ige production was measured by elisa, using a commercial kit (opt - eia elisa - set, bd - pharmingen). the presence of the liver transaminase enzymes alanine aminotransferase (alt) and aspartate aminotransferase (ast) was evaluated in sera from wt and ahr/ mice at 0, 3, 6, 14, and 25 days after t. gondii infection using alt and ast kits (spinreact, s. a. ctra. santa coloma, spain). at 25 days after t. gondii infection (when 40% the ahr/ mice die), the brains were removed and total rna was extracted using trizol reagent according to the manufacturer 's instructions (invitrogen, carlsbad, ca). cdna synthesis was performed with superscript one - step reverse transcription - pcr (rt - pcr, invitrogen). in brief, 3 g of total rna was mixed with 0.5 g oligo (dt) 12 to 18 primers, 10 mm of each dntp and 1x reaction buffer in a final volume of 20 l. cdna reactions were incubated at 65c for 10 minutes to denature the rna template and quench - cooled for 1 minute. 0.5 l of ssii - rt reverse transcriptase was added, incubated at 42c for 50 minutes and 70c for 15 minutes. 0.5 l of rnaseh was added and further incubated for 15 minutes at 36c. cdna samples were amplified for to 30 cycles using the red taq polymerase (invitrogen) and specific primers (table 1). after amplification, pcr products were separated by gel electrophoresis on 1.5% agarose gels containing sybr green i, a nucleic acid gel stain used at 1,000x (amresco), and visualized with the fla-5000 chemiluminescence detection system (fujifilm). the data were normalized to glyceraldehyde-3-phosphate dehydrogenase (gapdh) and analyzed using the multi - gauge image program. livers were removed and fixed in a solution that contained 10% formalin, 70% ethanol, and 5% acetic acid, embedded in paraffin blocks (all from sigma - aldrich). sagittal sections of livers (5 m thick) were obtained and mounted on slides and subsequently stained with hematoxylin and eosin (sigma - aldrich). photomicrographs of representative sections were taken with an axion - star microscope equipped with a built - in digital camera (zeiss). fluorescence - activated cells sorting (facs) analysis on cd4 + t cells, cd8 + t cells, and treg cells in the spleen and tlr-2 + and ccr5 + macrophages (m) from wt and ahr/ t. gondii - infected mice was performed. briefly, at 25 days after t. gondii infection, spleen cells or peritoneal adherent m were stimulated in vitro for 6 days or 24 hours with 2.5 g / ml of stag. the spleen cells were stained with fluorescein isothiocyanated (fitc) anti - cd8 antibody and phycoerythrinn (pe) anti - cd4 antibody, and treg cells were stained using a staining kit (mouse treg flow kit) containing fitc anti - cd4 antibody, pe anti - cd25 antibody, and alexa anti - foxp3 antibody, according to the manufacturer 's instructions. the m were stained with fitc anti - f4/80 antibody and pe anti - tlr2 or pe anti - ccr5 antibody. the cells were fixed, and the proportion of cells staining positive for the appropriate markers was evaluated (10,000 events / sample) using a flow cytometer (facs, becton dickinson, usa). nonspecific binding was blocked with fcblock, and the isotype controls were stained with rat antimouse igg conjugated with -fitc, -pe, or alexa (all from biolegend, san diego, ca). all statistical analyses were performed using prism 4 (gaphpad software, san diego, ca). comparisons between wt and ahr/ animal groups were made using a nonparametric mann - whitney 's u - test and student 's t - test as appropriate. for survival assays, to analyze the importance of ahr during acute toxoplasmosis, we first determined whether wt and knockout (ahr/) mice differed in their resistance to me-49 t. gondii infection. wt and ahr/ littermates were challenged i.p. with 40 cysts of t. gondii parasites, and we examined the course of the infection for 60 days. as shown in figure 1(a), ahr/ mice rapidly showed clinical signs of the disease that sustained for 5 days, while wt mice showed few symptoms. by day 7 after infection, ahr/ mice started to lose weight (figure 1(b)) and showed piloerection and prostrated behavior. t. gondii - infected ahr/ mice succumbed as early as day 11 after infection and reached 89% mortality rate by day 60 after infection. in contrast, the mortality rate in wt mice was significantly lower, 6.7% (figure 1(c)). interestingly, despite the evident signs of sickness and death in ahr/ mice, they developed fewer brain cysts compared to wt mice, even at day 60 postinfection (figure 2(a), p <.05). to further confirm our microscopic findings, we quantified the level of t. gondii - specific dna in the brains from both groups of mice at day 25 post infection using semiquantitative pcr based on the 529 bp repeat element (rep ; 200 to 300 copies / genome), as reported elsewhere. the level of parasite dna in the brains of infected wt and ahr/ mice correlated with the number of cysts, confirming fewer parasite burdens in the brains of ahr/ mice (figures 2(b) and 2(c)). taken together, these data suggest that ahr is critical in the host defense against t. gondii infection, and that the increased mortality rate in ahr/ mice is not due to an inability to restrict parasite replication. next, we compared the levels of cytokines (il-12, ifn-, tnf-, and il-10), nitric oxide (no), and ige, in sera from wt and ahr/ mice after t. gondii infection. no significant differences between ahr/ and wt mice in serum il-12 and ifn- levels were detected at any of the time - points examined (figures 3(a) and 3(b), resp.). however, significantly higher level of the inflammatory cytokine tnf- was detected after 30 days of infection in sera from ahr/ mice compared to wt mice (figure 3(c), p <.05). this observation was in accordance with a low level of il-10 observed after 15 days of infection in ahr/ mice (figure 3(d), p <.05). a higher level of total ige was also observed after 6 days of infection in ahr/ mice (figure 3(f), p <.05). interestingly, the serum nitric oxide level was higher in ahr/ mice than wt mice on day 25 post infection alone. we next determined the functional capacity of spleen cells from both ahr/ and wt mice to respond to t. gondii - specific stimulation. at day 25 post infection, proliferation of spleen cells from infected ahr/ or wt mice in the presence of stag or medium for 5 days was assayed by [3h]thymidine incorporation. as shown in figure 4(a), only primed - wt spleen cells were able to respond to stag stimulation. supernatants from the cell cultures were used to determine il-2, il-12, ifn-, and il-4 production. the unresponsiveness of spleen cells in ahr/ mice was in accordance with low levels of il-2 observed in the supernatants (figure 4(b), p <.05). interestingly, higher levels of ifn- were detected in supernatants of stag - stimulated spleen cells from ahr/ compared to wt mice (figure 4(d)). in contrast, no differences in il-12 (figure 4(c)) and il-4 levels (data not shown) were observed in the same cultures. it is well known that mononuclear phagocytes are important in controlling the early stage of t. gondii infection by early and continuous production of il-12, which is a key lymphokine that mediates host resistance to t. gondii infection [45, 46 ]. therefore, we asked whether il-12 production by mononuclear cells was altered in t. gondii - infected ahr/ mice. to assess this, we compared the ability of ahr/ and wt peritoneal exudate cells (pecs) to produce il-12 and ifn- in response to stag or medium alone for 48 hours. as seen in figure 5, pecs from mice lacking ahr produced greater amounts of il-12 and ifn- than wt mice (figures 5(a)5(b), resp.). from the above observations, we hypothesized that the immunopathology and death observed in infected ahr/ mice were due to overexpression of proinflammatory cytokines. to confirm this hypothesis, portions of the livers from infected wt and ahr/ mice 10 post infection livers of infected wt mice presented a small number of mononuclear inflammatory foci ; however, more number of small granulomas and inflammatory infiltrates were observed in livers from ahr/ mice at 10 days post infection (figure 6(a)). at day 25 post infection, inflammatory infiltrate was present, but granulomas were rarely observed, and when present, were smaller in livers from infected wt mice (figure 6(b)). in contrast, livers from t. gondii - infected ahr/ mice exhibited a large area of granulomas and had more mononuclear inflammatory infiltrates scattered by parenchyma and portal areas than t. gondii - infected wt mice. additionally, detection of alanine aminotransferase (alt) and aspartate aminotransferase (ast) confirmed the extent of damages induced by the inflammatory response in livers, as liver samples from infected ahr/ mice produced higher levels of ast and alt at 25 days after infection, when inflammatory infiltrates and granulomas were detected in livers of t. gondii - infected ahr/ mice (figures 6(c) and 6(d), resp.). given that gata-3 is a key factor for th2 differentiation, we investigated whether ahr deficiency had an effect on gata-3 mrna expression in spleen cells and brains from t. gondii - infected ahr/ and wt mice at 25 days post infection. comparable levels of ifn- mrna were detected in spleen cells and brains from ahr/ and wt mice. however, spleen cells and brains of ahr/ mice expressed lower transcript levels of both gata-3 and il-10 compared to wt mice (figures 7(a) and 7(b), resp.). upon establishing that deaths of ahr/ mice infected with t. gondii is likely due to a high proinflammatory response that may control parasite replication but, at the same time, cause severe systemic damage to the host, we asked whether ahr deficiency had a role on the immunophenotyping of t cell subpopulations. to test this, spleen cells were obtained at 25 days post infection and incubated with 2.5 g / ml of stag ex vivo for 5 days, and cd4 +, cd8 +, and cd4+/cd25+/foxp3 + (t regulatory lymphocytes - treg) lymphocyte subpopulations were quantified. as shown in table 2, the proportion of stag - specific cd4 + or cd8 + t cells (table 2) were comparable between ahr/ and wt mice. interestingly, a slight, but not significant, decrease in stag - specific treg cells was observed in ahr/ mice compared to wt mice (table 2). taken as a whole, these data show that ahr/ mice are capable of developing an adaptive immune response. macrophages are very important innate immune cells that respond promptly to t. gondii infection as well as to its soluble antigen. moreover, it is well known that ccr5 is one of the main receptors for stag that is involved in triggering the early production of il-12 and tnf-. hence, to determine whether ahr deficiency phenotypically and functionally alters these cell populations, we analyzed the expression of ccr5 and tlr2, another molecule that may be involved in t. gondii recognition, on peritoneal adherent macrophages isolated from ahr/ and wt mice 25 days after infection. ahr/ mice displayed at least 50% lower expression of tlr2 mainly on f4/80 + cells (figure 8(a)). in contrast, expression of ccr5 was unaltered in the same population (figure 8(b)). it has been demonstrated that the eicosanoid called lipoxina4 (lxa4) plays a role in the anti - inflammatory response against t. gondii. interestingly, lxa4 is an endogenous ligand for ahr, and the binding of ahr with lxa4 controls the expression of proinflammatory cytokines such as il-12 and ifn-. given that the expression of lxa4 is dependent on the expression of 5-lipoxygenase (5-lox) [33, 48 ], we asked whether expression of 5-lox may be affected in t. gondii - infected ahr/ mice. as seen in figure 8(c), the transcript levels of 5-lox in spleen cells were significantly lower in ahr/ mice compared to wt - infected mice (figure 8(c)). ahr has been suggested to play an important role in the immune response to virus and bacterial infections, where a functional innate immune response is pivotal for complete resistance to the pathogens [20, 22, 49 ]. however, in those studies the molecular mechanism associated with ahr was not too clear. thus, establishment of the role of ahr in response to parasitic infections may help in understanding the endogenous immune function of ahr. here, we showed that ahr deficient mice (ahr/) are more susceptible to t. gondii infection than wt mice. after peritoneal infection with 40 cysts ahr/ mice succumbed to t. gondii infection faster than wt mice ; however, ahr/ mice developed fewer brain cysts, despite higher serum levels of tnf-, nitric oxide (no) and ige and lower serum levels of il-10 compared to infected wt mice. the high mortality rates in ahr/ mice suggest that ahr is critical in the host defense against toxoplasmosis ; however, the lower number of cysts in the brain in conjunction with high levels of tnf-, and no in ahr/ mice suggests that the higher mortality rate is not caused by the inability to restrict parasite replication. resistance to experimental toxoplasmosis has been shown to be dependent on production of several proinflammatory cytokines (mif, il-1, il-12, tnf-, and ifn-) and no [25, 29, 30, 50 ]. furthermore, the powerful proinflammatory immune response together with ige, an antibody that has been correlated with early acute inflammation in toxoplasmosis [51, 52 ], and no restricts dissemination of the parasite and prevents death by parasitic infection. after parasite dissemination has been contained by ifn--dependent responses, the onset of the chronic phase of infection is characterized by continuous cell - mediated immunity. such potent responses are kept under tight control by anti - inflammatory cytokines such as il-10, which is required for preventing necrosis in the small intestine and death in both genetically resistant balb / c and susceptible c57bl/6 mice following infection with t. gondii. thus, these first results suggest that high production of inflammatory cytokines in conjunction with low levels of il-10 could reduce dissemination of brain cysts without preventing mortality, possibly because the inflammatory response causes systemic damage. to test the above hypothesis, we analyzed the t cell response by comparing the proliferative capacity and cytokine production in spleen cells from ahr/ and wt infected mice in the presence of stag. these observations were in line with previous report that show that embryonic fibroblasts from ahr/ mice exhibit a lower proliferation rate and impaired il-2 production associated with the fact that the il-2 promoter region contains distal regulatory elements that can be addressed by the ahr to induce il-2 and cooperate with the proximal promoter in this. we detected high levels of tnf- in serum, which could have contributed to low levels of proliferation in t. gondii - infected ahr/ mice. in accordance with our initial hypothesis, we found high levels of ifn- in the supernatant of spleen cells from infected ahr/ mice. somewhat to our surprise, no difference in il-12 levels was detected in supernatants of spleen cell cultures between ahr/ and wt mice. the levels of il-12 and ifn- in supernatants of ahr/ pecs were significantly higher than those in wt - pecs. these observations were consistent with the extensive histopathological damage in livers and high levels of alt and ast detected in sera. together, the high production of inflammatory cytokines in conjunction with low levels of il-10 may mediate the damage in ahr/ mice and may contribute to the high mortality rate observed in t. gondii - infected ahr/ mice. previous studies have suggested that ahr has a role in modulating the balance between th1 and th2 response [10, 15 ]. therefore, we asked whether high levels of proinflammatory cytokines favor th1 polarization in ahr/ mice. the mrna expressions of ifn-, il-10, and gata-3 were determined by rt - pcr in spleen cells and brains from infected ahr/ and wt mice. contrary to what we expected, we did not observe any significant difference in ifn- mrna levels in either spleen cells or the brains between ahr/ and wt mice. in contrast, reduced levels of il-10 and gata-3 were observed in ahr/ mice compared to wt mice. we suggest that the reduction of gata-3 and il-10 observed in t. gondii - infected ahr/ mice is responsible for potentiating the biological activity of ifn-, rather than favoring polarization of the immune response toward a th1 response. recent results suggest that ahr can regulate the generation of regulatory t cells (tregs), a main source of il-10, since ahr activation by tcdd induces differentiation of t - cell progenitor cells into tregs. given that infected ahr/ mice display low levels of il-10, one possibility is that tregs subpopulation is reduced in the absence of ahr. however, this idea was disproved by our observation that no significant differences were observed in the proportion of tregs cell subpopulations between ahr/ and wt mice. furthermore, no significant differences were noted in cd4 + and cd8 + t cell subpopulations between t. gondii - infected ahr/ and wt mice. these results suggest that ahr is not required for adaptive t cell response during toxoplasmosis, at least in those subpopulations analyzed. the production of cytokine il-12 is critical for the development of ifn- dependent resistance to t. gondii infection (aliberti j alan sher jem 196 no.9 2002).the signaling pathway through cc chemokine receptor 5 (ccr5) plays a critical role in triggering il-12 production, mainly by cd8+ subset of dendritic cells (dc) upon stimulation with stag [33, 47 ]. since macrophages also display ccr5 expression, we considered the possibility that overexpression of ccr5 on macrophages might be responsible for the high levels of il-12 secreted by pecs from t. gondii - infected ahr/ mice. however, in the present study, there was no significant difference in ccr5 expression on f4/80 + cells between infected ahr/ and wt mice. this observation shows that, at least in macrophages, ccr5 expression is not affected by the lack of ahr. toll - like receptors (tlrs), which are innate immune receptors, are also involved in the recognition of t. gondii profilin. tlr11 is the main receptor that plays a major role in il-12-dependent control of t. gondii, although other tlr family members also contribute to host resistance to this protozoan pathogen. thus, while tlr2 deficient mice display a normal il-12 production and resist t. gondii infection at conventional doses, they are susceptible when challenged with higher infective doses, arguing for a cooperative role of tlr2 in controlling the parasite. moreover, recently it has been described that both human and murine bone marrow - derived dc expressing high levels of tlr2 favor an anti - inflammatory response characterized by enhanced il-10 production [5961 ]. therefore tlr2 triggering mediates il-10 upregulation. here we showed that macrophages from t. gondii - infected ahr/ mice expressed significantly less tlr2 as compared to wt mice. this observation supports the notion that lower expression of tlr2 on macrophages contributes to reduce il-10, favoring the robust proinflammatory response observed in t. gondii - infected ahr/ mice. modulation of ccr5 on dcs involves ligation of g protein - coupled receptor formyl peptide receptor - like 1 (fprl-1). lxa4, an arachidonate - derived inhibitor of acute inflammation, has been suggested to bind to two receptors : fprl-1 (therefore could induce ccr5 downmodulation) and ahr. in vivo injection of stag triggers production of endogenous lxa4 in a 5-lipoxygenase-(5-lox-) dependent manner causing suppression of il-12 production by dcs. furthermore, upon infection with t. gondii, serum levels of lxa4 increase steadily in wt mice over the course of the acute phase and remain high during the chronic phase. moreover, induction of socs-2, an intracellular mediator of anti - inflammatory response, upon stag injection requires 5-lox and ahr. interestingly, 5-lox - deficient mice succumb to t. gondii infection at the early onset of chronic disease with excessive production of proinflammatory cytokines and substantially fewer brain cysts, suggesting that the excessive proinflammatory response in brains of 5-lox - deficient hosts is responsible for the mortality. in line with this, we found that 5-lox expression in spleen cells was significantly decreased in t. gondii - infected ahr/ mice. thus, one possible mechanism by which the absence of ahr may cause these enhanced inflammatory responses can be the lack of interaction between ahr and lxa4, through the absence of expression of 5-lox, which leads to a failure to control the magnitude of the inflammatory response induced by t. gondii infection., we presented here experimental evidence for a regulatory role of ahr during experimental toxoplasmosis using ahr/ mice. our data suggest that ahr is not required for adaptive t cell response to t. gondii infection but may play a constitutive role in the innate immune response to toxoplasmosis by dampening the inflammatory responses. these studies represent the first demonstration that ahr is critically involved during a protozoan infection.	the aryl hydrocarbon receptor (ahr) is part of a signaling system that is mainly triggered by xenobiotic agents. increasing evidence suggests that ahr may regulate immunity to infections. to determine the role of ahr in the outcome of toxoplasmosis, we used ahr/ and wild - type (wt) mice. following an intraperitoneal infection with toxoplasma gondii (t. gondii), ahr/ mice succumbed significantly faster than wt mice and displayed greater liver damage as well as higher serum levels of tumor necrosis factor (tnf)-, nitric oxide (no), and ige but lower il-10 secretion. interestingly, lower numbers of cysts were found in their brains. increased mortality was associated with reduced expression of gata-3, il-10, and 5-lox mrna in spleen cells but higher expression of ifn- mrna. additionally, peritoneal exudate cells from ahr/ mice produced higher levels of il-12 and ifn- but lower tlr2 expression than wt mice. these findings suggest a role for ahr in limiting the inflammatory response during toxoplasmosis.
perivascular stem cells (pvcs), originating from mesenchymal stem cells (mscs), have greater proliferation and differentiation potentials compared to those of bone marrow - derived mscs (crisan., 2008). among the pvc sources, human umbilical cords (huc) have some advantages over other tissues because they are easily accessible as a clinical waste product and have low inherent immunogenicity (nagamura - inoue., 2014). for these reasons, hucpvcs have drawn considerable interest as a promising material for regenerative medicine. in recent years, the therapeutic effects of hucpvcs on various diseases have been reported. tsang. (2013) showed that hucpvcs contributed to skeletal regeneration by generating a matrix and recruiting resident progenitors in a bone defect model. hucpvcs also contributed to the repair of injured lung tissue as well as protected against neurodegenerative diseases through paracrine effects (montemurro. thus, the identification and functional assessment of internal and external factors regulating pvc functions are critical for improving their therapeutic potential. potassium channels, the largest group of pore proteins, are widely distributed in various cell types (day., 1993 ; park., 2007). they selectively regulate the flow of potassium (k) ions across cell membranes (day., k channels have important roles in maintaining normal functions and physiological homeostasis in various cell types as well as regulating the cell cycle, differentiation, and maturation (kawano., 2002). although several studies have characterized the electrophysiological properties of mscs and pluripotent stem cells (heubach., 2003 ; li., 2005 ; wang., 2007 ; jiang., 2010 ; park., 2013 ; tarasov., 2017), the expression and function of ion channels in hucpvcs have not been studied. therefore, for therapeutic applications, we need to investigate not only the multi - lineage differentiation capacity, but also the electrophysiological properties of k channels in hucpvcs. in the present study, we investigated the expression pa tterns of big - conductance ca - activated (bkca) and vol tage - dependent k(kv) channels in undifferentiated hucpvcs and examined the effects of high glucose (hg, 25 mm) on the expression levels of bkca and kv channels. huc tissues (n = 3) were obtained from mothers undergoing caesarian sections, with written informed consent after approval by the institutional review board of kangwon national university hospital. the isolation and culturing of hucpvcs were performed as described previously (an., 2015a). briefly, hucs were rinsed with sterile phosphate - buffered saline (pbs ; sigma - aldrich) and incised along their length in dishes. both ends of the isolated vessels were ligated with black silk and then transferred into 100 mm culture dishes containing -minimal essential media (mem) supplemented with 10% fetal bovine serum (fbs ; hyclone), 1% penicillin - streptomycin (sigma - aldrich) and amphotericin b (0.3 g / ml ; sigma - aldrich). after 7 - 10 days, the vessels were removed from the dishes and the colonies were subcultured. once the cells reached 80% confluency, they were passaged by treatment with 0.05% trypsin - edta (sigma - aldrich). to evaluate the effect of high glucose on the expression of ion channels in pvcs, the cells were plated at a density of 410 cells in 35 mm dishes and cultured in -mem medium supplemented with low glucose (lg) (1.0 g / l, 5 mm) and hg (4.5 g / l, 25 mm) for 5 days. hucpvcs were dissociated into single cells using trypsin - edta and resuspended in 1% fbs - pbs. the cells were filtered through a 70 m cell strainer and reacted with the following antibodies conjugated with fluorochrome for 1 hr at 4 : cd31-phycoerythrin (pe), cd34-fluorescein - isothiocyanate (fitc), cd45-allophycoerythrin (apc), cd44-apc, cd90-apc, cd146-fitc, and ssea-4-fitc (all, bd biosciences). the dead cells were excluded with 7-aminoactinomycin d. stained cells were analyzed using the facscanto ii (bd biosciences) and data were analyzed by flowjo software (flowjo). total rna was isolated using the rneasy mini kit (qiagen) according to the manufacturer s instructions. briefly, total rna was reverse transcribed by using the topscript rt drymix kit (# rt200 ; enzynomics). transcripts were quantitated using topreal qpcr 2x premix (# rt501s ; enzynomics) and the abi steponeplus system instrument (applied biosystems). the expression levels of bkca and kv channel genes were normalized to glyceraldehyde 3-phosphate dehydrogenase (gapdh) and the relative quantification was performed using the comparative ct method according to the manufacturer s instructions (applied biosystems). we obtained pvcs from the vessels of huc tissues using a non - enzymatic (ne) isolation method (an., 2015b). to prevent cross contamination with hematopoietic and endothelial cells, both ends of the vessel were ligated and then plated into dishes. on days 7 - 10 post - plating the phenotypic expression of hucpvcs (passage 2) was analyzed by flow cytometry. the cells were positive for cd146 (93%), ssea-4 (10.1 %), cd44 (99.9 %), and cd90 (99%), and negative for cd31, cd45, and cd34, which precluded contamination by endothelial and hematopoietic cells (fig. these results suggested that the ne method enabled us to obtain a homogenous pvc population and that the expression pattern of surface markers on hucpvcs was in agreement with that of human pvcs reported in previous studies. both ends of the dissected vessel were ligated and plated into a 100 mm dish. perivascular stem cell (pvc) colonies were collected and subcultured when 80% confluent. pvc marker, cd146 ; msc markers, cd44 and cd90 ; endothelial and hematopoietic markers, cd31, cd34, and cd45. hucpvc = human umbilical cords perivascular stem cells ; msc = mesenchymal stem cells. we first investigated the expression patterns of bkca and kv channel subtypes in undifferentiated hucpvcs. we found comparable mrna expression of kca3a and kv1.3 subtypes in undifferentiated hucpvcs (fig. the expression levels of bkca1.1, bkca2.2, kca, kv3.3, and kv6.1 subtypes were relatively low (fig. high concentrations of glucose in the blood affected the growth and functions of endogenous stem cells. thus, we next asked if high glucose influenced the expression levels of ion channels. hucpvcs were plated at a density of 410 cells in 35 mm dishes and cultured in both lg and hg conditions for 5 days. hg - treated hucpvcs showed increased mrna levels of bkca3a, bkca4, kv1.3, kv1.6, and kv6.1 subtypes. we confirmed the upregulation of bkca4, kv1.6, and kv6.1 transcripts in hucpvcs exposed to hg using quantitative real - time pcr (fig. these results suggested that bkca and kv channels may have unique functions in the growth and differentiation of hucpvcs, which could be affected by hg exposure. (a) the mrna expression of bkca channel subtypes in hucpvcs cultured with lg and hg by rt - pcr. the expression levels of bkca3a and bkca4 subtypes were increased in hg - treated hucpvcs. (b) the mrna expression of kv channel subtypes in hucpvcs cultured with lg and hg measured by rt - pcr. the expression levels of kv1.3, kv1.6, and kv6.1 subtypes were increased in hg - treated hucpvcs. (c) representative images of hucpvcs cultured in lg (5 mm) and hg (25 mm) conditions for 5 days. real - time qpcr was used for confirmation of upregulated subtypes in hg - treated hucpvcs. in the present study, we showed for the first time the expression patterns of bkca and kv ion channels in hucpvcs, which could be altered by exposure to hg. in recent years, pvcs have been suggested as a promising source for cell - based therapy due to their greater regenerative potential (crisan., 2008). ion channels are detected in a variety of cell types and play fundamental roles in controlling cell proliferation and differentiation as well as maintaining homeostasis (kawano., 2002). therefore, we investigated the electrophysiological properties of hucpvcs to improve their proliferative and regenerative capacities. recent studies reported the electrophysiological properties of multipotent mscs and found that the expression patterns of ion channels were relatively heterogeneous among mscs. while similar expression levels of kv1.4, kv4.1, kv4.2, and kv4.3 subtypes were observed between adipose tissues (ad)- and bone marrow (bm)-derived mscs, expression of kv1.1, kv1.4, and kv7.3 subtypes varied across the tissues or among studies (heubach., 2003 ; li., 2005 ; bai., 2007 ; park., 2007 ; park., (2007) could not detect kv4.1 subtype in ad - mscs. however, park. the heterogeneity of kv ion channels in mscs may be attributed to variations among donors or the tissues from which mscs are isolated. our data showed different expression patterns of ion channel subtypes in pvcs compared with ad- and bm - mscs, indicating that pvcs are different subpopulations and are more homogeneous than mscs. culture conditions might also influence the expression of specific ion channel subtypes detected in mscs. for example, bm - mscs cultured in hg medium exhibited senescence and genetic instability by upregulation of autophagy and oxidative stress (chang., 2015). in addition, hyperglycemia impaired the proliferation and function of endogenous stem and somatic cells such as mscs, hematopoietic stem cells and mller cells (kim., 2015 ; manea., 2015 ; hadarits., 2016 ; qiu., thus, we assumed that the glucose concentration in the culture medium may affect the expression levels of ion channels in pvcs and found increased levels of bkca3a, bkca4, kv1.3, kv1.6, and kv6.1 transcripts in hg - treated pvcs compared to those of lg - treated pvcs. in summary, this is the first characterization of ion channels in hucpvcs, which provides fundamental information to improve the regenerative capacity of hucpvcs. further study will be needed to define the physiological roles of specific ion channels in the proliferation and differentiation of hucpvcs.	abstractpotassium channels, the largest group of pore proteins, selectively regulate the flow of potassium (k+) ions across cell membranes. the activity and expression of k+ channels are critical for the maintenance of normal functions in vessels and neurons, and for the regulation of cell differentiation and maturation. however, their role and expression in stem cells have been poorly understood. in this study, we isolated perivascular stem cells (pvcs) from human umbilical cords and investigated the expression patterns of big - conductance ca2 + -activated k+ (bkca) and voltage - dependent k+ (kv) channels using the reverse transcription polymerase chain reaction. we also examined the effect of high glucose (hg, 25 mm) on expression levels of bkca and kv channels in pvcs. kca1.1, kca3, kv1.3, kv3.2, and kv6.1 were detected in undifferentiated pvcs. in addition, hg treatment increased the amounts of bkca3a, bkca4, kv1.3, kv1.6, and kv6.1 transcripts. these results suggested that ion channels may have important functions in the growth and differentiation of pvcs, which could be influenced by hg exposure.
the liver plays a major role in the secretion of macromolecules into bile either by receptor - mediated or unspecific fluid - phase transcytosis. although the liver is composed of parenchymal (hepatocytes) and nonparenchymal cells (endothelial cells, kupffer cells, ito cells), only hepatocytes are involved in bile formation and biliary secretion of blood - derived molecules. as isolated hepatocytes in culture do not form a polarized epithelial layer where the apical and basolateral surfaces are accessible at opposite sides, transcytosis studies are confined to in vivo experiments or to the isolated perfused rat liver [1, 2 ]. using the latter system, biliary secretion of various ligands taken up by receptor - mediated and/or fluid - phase endocytosis has been investigated so far. in contrast to other polarized epithelial cells (intestine, kidney, placenta) receptor - mediated endocytic processes are mainly confined to the basolateral (sinusoidal) surface of hepatocytes. one of these sinusoidal receptors is a receptor for galactose - containing glycoproteins, so - called asialoglycoproteins. this galactose receptor or asialoglycoprotein receptor (asgpr) clears defective asialoglycoproteins (e.g., asialoorosomucoid ; asor) from the circulation by receptor - mediated endocytosis. the receptors recycle and asor is directed to lysosomes for degradation. however, about 0.53% of internalized asor is missorted into the transcytotic route and consequently secreted intact into bile [3, 4 ]. interestingly, the kinetics and amount of biliary secretion of different fluid - phase markers such as inulin or dextrans (mw 270 kda) differ considerably, an effect that is generally attributed to their different molecular weight and/or charge [5, 6 ]. even more surprising, horseradish peroxidase (hrp), following short pulse labeling, has a biphasic appearance in bile : the first, faster peak is assumed to occur by a paracellular route, while the second, slower peak takes place by transcytosis. transcytosis of the latter peak has been demonstrated by its complete absence in the presence of microtubule depolymerizing drugs. with respect to the fast hrp appearance in bile the following data argue against a paracellular pathway : we have previously shown that low - temperature perfusion (16c) results in a delayed appearance of the first peak in bile as compared to perfusion at 37c. furthermore, 2 minutes after hrp loading hrp containing vesicles were seen in the vicinity of the canalicular membrane. moreover, hrp is not an ideal fluid - phase marker as it is a mannose - containing glycoprotein and thus binds to mannose receptors (mr) on hepatic kupffer and endothelial cells with high affinity [1012 ]. in addition, hrp binds with low affinity (km ~ 4 m) to isolated hepatocytes [11, 13 ] and in these cells hrp uptake up to 0.2 mg / ml could be competed by mannan, indicative for receptor - mediated endocytosis via an mr. to study the potential role of hepatic mr on hrp biliary secretion we determined the influence of the mannose oligosaccharide mannan (mw ~100 kd) on hrp transport in the isolated perfused rat liver. male louvain rats (250300 g) were obtained from a local breeding farm and were kept under a controlled lighting schedule with a 12-hour dark period. animals had free access to food and water but were fasted overnight before the experiment. before surgical procedures, rats were anesthetized by an intraperitoneal injection of urethane solution (1 ml/100 g body - weight). fitc - dextran (70 kda) was extensively dialyzed against tris - buffered saline (ph 7.34) and finally against phosphate - buffered saline (ph 7.4) before use. asor was iodinated in the presence of iodogen (pierce) to a specific activity of approximately 1,4 10 cpm/g asor. following surgical removal, isolated rat livers were equilibrated for 30 minutes at 37c by single - pass perfusion with krebs - henseleith bicarbonate buffer (khb ; containing 10 mm glucose and 20 m taurocholic acid and gassed with a humidified mixture of 93% o2/7% co2, ph 7.4) at a perfusion rate of 3 ml / min and g liver [15, 16 ]. biliary secretion of the respective marker was analyzed by single - pass perfusion at 37c. hrp (0.08 mg / ml, 0.8 mg / ml or 8 mg / ml khb) or fitc - dextran (5 mg / ml khb) was perfused through the liver for 1 minute at 37c followed by marker - free perfusion for 40 minutes. where indicated, a twofold access of mannan over hrp was included in the perfusion buffer. single bile drops were collected and analyzed for the respective marker concentration. to determine the kinetics of biliary secretion of i - asor at 37c, the liver was perfused for 2 minutes with i - asor (1.32.3 10 cpm / ml khb) followed by marker - free perfusion for 40 minutes. single bile drops were collected and tca - precipitable (intact asor) and tca - soluble (degraded asor) counts in each bile drop were determined. fitc - fluorescence of fitc - dextran per biledrop was measured in a spectrofluorometer (fp-777, jasco, japan) using 495 nm and 515 nm as excitation and emission wavelengths, respectively. after subtraction of background fluorescence, values were converted to concentration (pg fitc - dextran / biledrop) with the help of a calibration curve. the enzymatic activity of hrp per bile drop was determined based on standard curve according to marsh.. following perfusion with i - asor single bile drops were subjected to precipitation with 10% tca (final concentration) and after centrifugation (1500 g, 15 minutes) radioactivity in the supernatant (tca soluble degradation products) and pellet (tca precipitable, i.e., intact i - asor) were determined by -counting (1282-compugamma cs, lkb). the bile flow was calculated by determining the time when each bile drop was formed normalized to 1 g liver (bile flow in l / min and g liver). the amount of marker / bile drop was determined using the respective calibration curve (hrp, fitc - dextran) or radioactivity. as one bile drop corresponds to 8 l bile, these data were converted to marker secretion (amount of marker / min and g liver) by division by 8 and multiplication with the respective bile flow. the appearance of the marker in bile was corrected for the dead space of the bile duct cannula. the cummulative hrp secretion was obtained by summing up the amount of hrp / bile drop over time. to analyze differences between the first and the second peak of hrp secretion, we defined the cumulative amount that was secreted between 0 and 10 minutes to correspond to the 1st hrp peak (secretion maximum 68 minutes). this amount was then subtracted from the total amount of hrp secreted within 40 minutes to obtain the total amount secreted during the 2nd hrp peak (secretion maximum 15 minutes). for comparison, the secretion kinetics of a bona fide fluid - phase marker, fitc - dextran, and a galactose - terminated ligand, asialoorosomuciod (asor), were analyzed. fitc - dextran (mw 70 kda ; 5 mg / ml) was added to the perfusate and perfused through the liver for 1 minute (= pulse) under single - pass conditions. to maintain constant bile flow 20 m taurocholic acid was always included in the perfusion buffer. the concentration of the marker was analyzed in single bile drops and data were related to bile flow and normalized to 1 g liver. as shown in figure 1(a), fitc - dextran appeared in bile 4 minutes after marker addition to the perfusate. the secretion rate increased with time reaching a peak at 8 minutes and then dropped to 0 at 40 minutes. the bile flow (figure 1(b)) revealed a transient decrease and subsequent increase when the perfusion medium was changed but then remained constant at about 1.75 l / min and g liver throughout the perfusion indicative for sufficient oxygen supply, atp generation, and intact liver function. asor is rapidly and efficiently taken up from the perfusate and after ligand - receptor dissociation in early endosomes the receptor is recycled and asor is sorted to lysosomes [18, 19 ]. the appearance of asor degradation products in bile reflects the kinetics of arrival in lysosomes. about 90% of asor degradation products are released into the perfusate and the remainder into bile. since 0.5%3% of internalized asor is missorted and secreted intact into bile, determination of tca - precipitable and tca - soluble radioactivity in bile correlates with transcytosis and transport to lysosomes, respectively [3, 4 ]. biliary secretion of asor at 37c was analyzed following a 2-minute pulse of i - asor with subsequent marker - free perfusion of the liver. single bile drops were collected and tca - precipitable (intact) and tca - soluble (degraded) radioactivity therein were determined (figure 1(c)). intact i - asor appeared in bile within 6 minutes after the pulse, while maximum biliary secretion was found about 17 minutes after uptake. 15 minutes after internalization tca - soluble counts appeared in bile, corresponding to asor arrival in lysosomes, immediate degradation and release of degradation products into the perfusate (not shown ; [3, 4 ]) and bile. the bile flow (figure 1(d)) remained constant at 1.8 l / g liver and min throughout this perfusion. taken together, biliary secretion of the fluid - phase marker fitc - dextran is faster than the ligand asor. hrp (0.8 mg / ml khb) was added to the perfusion medium for 1 minute followed by marker - free perfusion (figure 2(a)). although hrp appeared in bile roughly at the same time as fitc - dextran, the secretion rate revealed two peaks at 7 minutes and 15 minutes, respectively. these two peaks were seen irrespective of the hrp concentration perfused through the liver (0.08 ; 0.8 ; 8 mg / ml) and are in agreement with data from different laboratories [7, 8, 20, 21 ]. the bile flow, shown in figure 2(b), remained constant at 1.6 l / g liver and min throughout this perfusion. comparison of hrp secretion with secretion of fitc - dextran and asor revealed that hrp exhibits a similar kinetics as the fluid - phase marker (1st hrp peak) as well as that of the ligand (2nd hrp peak). next, we analyzed the influence of 1.6 mg / ml mannan on hrp secretion (figure 3). similar to the absence of mannan (compare to figure 2) two peaks of secreted hrp were seen, at about 8 minutes and 16 minutes, respectively (figure 3(a)). the bile flow was maintained at about 1.5 l / min and g liver (figure 3(b)). figure 3(c) demonstrates that the total cumulative secretion was reduced in the presence of mannan as compared to its absence (compare to figure 2(c)). to differentiate whether mannan influenced rather the first or the second peak, the amount of hrp being secreted between 0 and 10 minutes (i.e., during the fast, first peak) and between 10 minutes and 40 minutes (i.e., during the slow, second peak) was analyzed. as depicted in figure 4, the first hrp peak was reduced by 41% by mannan without any influence on the second peak. when hrp at 0.08 mg / ml was applied in the perfusate, the first peak was also decreased by mannan by 32% while the second peak was slightly increased by 14% (data not shown). however, due to the low hrp concentration in single bile drops individual experiments varied considerable. when the hrp concentration in the perfusate was raised to 8 mg / ml, no significant effect of 16 mg / ml mannan could be seen (data not shown). these data demonstrate that the fast pathway of hrp entry into bile has to occur at least in part by a receptor - mediated transcellular route. nevertheless, the kinetics of this route clearly differs from that of the ligand asor (see figure 1(c)). in our study, we have observed an effect of mannan on the first peak of hrp - secretion that indicates an interaction of hrp with mr on hepatocytes. the mr (180 kda) was first isolated from rat liver kupffer cells and binds glycoproteins with, for example, terminal mannose or fucose groups in a ca - dependent manner. the mr is found on monocyte - derived and tissue macrophages, cultured dendritic cells, on hepatic sinusoidal and lymphatic endothelial cells in the small intestine. its main function is the clearance of many serum glycoproteins, lysosomal enzymes, and denatured collagen containing the respective sugar moieties resulting in their rapid degradation [2428 ]. rat liver endothelial cells exhibit a high mr surface density and high affinity (km about 3060 nm) for ligands such as ovalbumin or n - acetylglucosamine containing glycoproteins. mrs are taken up by clathrin - mediated endocytosis due to a conserved lysine residue and a dihydrophobic motif in their short cytoplasmic tail [30, 31 ]. internalized mr - ligand complexes dissociate in the low ph environment of endosomes resulting in receptor recycling and ligand transport to and degradation in lysosomes [28, 30 ]. apart from being localized to kuppfer and endothelial cells receptors for mannose containing glycoproteins were also described in liver parenchymal cells [11, 33 ]. quantitative data from electron microscopy autoradiographic studies revealed that a minor fraction of mannose - terminated rnase was found in hepatocytes. similarly, aglycerase was taken up by hepatocytes in a mannose - dependent fashion. using mannosylated gold particles, kempka and kolb - bachofen demonstrated binding of the marker in coated pits followed by uptake, transcytosis, and secretion into bile by rat hepatocytes in situ. although these events in hepatocytes were rare as compared to liver sinudoidal cells, they were not completely absent. using isolated liver parenchymal cells, binding, uptake, and intracellular routing to lysosomes of invertase was shown that could be blocked by mannose. collectively, these data support the presence of an mr on the sinusoidal surface of rat hepatocytes. however, the mr on hepatocytes is clearly distinct from the mr on kupffer and endothelial cells and has not been identified, so far. antibodies specific for the 180 kda mr on endothelial cells did not reveal a protein with similar molecular weight in hepatocytes. binding of hrp to isolated hepatocytes at 4c was of low affinity thus preventing the identification of a specific plasma membrane protein. however, hrp uptake at 37c was saturable with a km of 8.3 mg / l and could be inhibited by mannan with an apparent ki of 2.5 mg / l clearly demonstrating the involvement of mr in hrp endocytosis in hepatocytes. receptor - mediated endocytosis can take place via clathrin - coated pits and vesicles (e.g., asor, polymeric iga), via caveolae (e.g., albumin in endothelial cells), and other less characterized mechanisms [38, 39 ]. irrespective of the uptake mechanism by which each endocytic vesicle is formed, it contains extracellular fluid and extracellular material (proteins, ions, etc.). consequently, fluid - phase endocytosis occurs concomitantly with receptor - mediated endocytosis. however, the contribution of distinct endocytosis mechanisms to fluid - phase uptake depends on the cell type under investigation. the kinetics of transfer of different markers, be they ligands or fluid - phase markers, do not necessarily allow a prediction of their intracellular route. fitc - dextran (fluid phase marker) and asor (ligand) utilize the same endocytic subcompartments on their transcellular route from the sinusoidal to the canalicular plasma membrane. this has been shown by low - temperature (16c) perfusion of isolated perfused rat livers and by subcellular liver fractionation. under this condition fitc - dextran and asor accumulate in early endosomes resulting in inhibition of transcytosis of both molecules and inhibition of lysosomal degradation of asor. nevertheless, they are secreted into bile with completely different kinetics (figures 1(a) and 1(c)). similar to the transcytosis of asor and fitc - dextran, low - temperature perfusion blocks the slow pathway of hrp secretion indicative for involvement of the same endocytic compartments (early endosomes) during transport to the canalicular membrane. consequently, a part of hrp has to be taken up into typical early endosomes (that accumulate marker at 16c), is then directed into transcytotic vesicles, and appears in bile at about 15 minutes (figure 5). in contrast, a proportion of hrp is internalized via an mr (mannan competeable uptake) into endocytic compartments distinct from asor - containing endosomes. such a distinct fast transcellular pathway is supported by the observation that hrp appearance in bile during 16c perfusion is only delayed but not blocked and that hrp - containing endosomes are observed within 2 minutes in the vicinity of the canalicular membrane. albumin and asor have been localized in distinct endocytic compartments early after uptake in isolated hepatocytes at low temperature. it is still a matter of debate whether the fast hrp appearance in bile is (additionally) due to paracellular transport as supported by stimulation of this peak under postcholestatic conditions and by reagents that lead to intrahepatic cholestasis [8, 9, 42 ]. all these treatments increase the permeability of the tight junctions and could therefore explain the stimulatory effect on the first hrp peak [42, 43 ]. however, the data presented in this investigation clearly demonstrate that a least part of the first hrp peak is reduced by mannan and is thus accounted for by receptor - mediated transcytosis. however, since mannose receptors are only involved in the binding and subsequent endocytosis of mannose containing glycoproteins to / into cells, it can be assumed that mannan has no influence on the paracellular permeability, though this has not been proven. we can not think of an experimental setup to unequivocally differentiate between the proportion of the transcellular and paracellular part of the 1st peak. such a setup is required to determine the effect of mannan treatment on the paracellular permeability. kuppfer cells and endothelial cells have an up to 10-times higher endocytic activity of fluid - phase marker uptake per se [34, 35, 44 ] and, in addition, mannose - containing glycoproteins are internalized by high affinity mr into endothelial cells at a very fast endocytic rate [28, 45 ]. consequently, the actual hrp concentration that will be in contact with hepatocytes in the isolated perfused rat liver is unknown. it can be assumed to be much lower as the amount of hrp added to the perfusion buffer due hrp removal by endothelial and kupffer cells. this may explain the discrepancies between our results and data by yamaguchi.. these authors observed saturation of hrp uptake in isolated hepatocytes already at 0.2 mg / ml hrp. in contrast, we could demonstrate mannan inhibitable hrp secretion at 0.8 mg / ml. thus, when hrp is applied at concentrations ranging between 1 mg / ml and 10 mg / ml as in morphological and perfusion studies [7, 8, 20, 21, 46 ] the proportion due to receptor - mediated endocytosis can not be ignored, although at such hrp concentrations uptake by isolated hepatocytes is mainly by a fluid - phase mechanism. bile is produced by hepatocytes at their canalicular surface due to activity of many solute transporters. the remainder is composed of bile acids (67%), phospholipids (22%), cholesterol (5%), and proteins (5%). the canalicular bile then passes through the bile ducts and is collected in the gallbladder for regulated secretion into the intestinal tract. ductular epithelial cells modify the canalicular bile by a series of secretory and reabsorptive processes resulting in its alkalinization and concentration. thus, ductular / gallbladder bile has a lower water content (about 87%) and a higher concentration of all the constituents listed above as canalicular bile. many biliary proteins (polymeric iga) are secreted via receptor - mediated transcytosis while others (albumin, igg) reach bile by a fluid - phase mechanism. though it has been shown that the total protein concentration is higher in ductular / gallbladder than in canalicular bile due to water absorption, additional absorptive / secretory processes may affect the concentration of individual proteins. with respect to hrp we may speculate that this marker is endocytosed by ductular cells by a fluid - phase mechanism as shown in isolated rat bile duct epithelial cells [50, 51 ]. such processes may result in a reduction of the amount of hrp in ductular bile. how ductular endocytosis may affect the concentration of the first and second hrp peak is unknown and can not be taken into consideration. in any case, we here demonstrate that hrp secretion in the isolated perfused rat liver can be inhibited by mannan indicative for the involvement of mr in hrp uptake by hepatocytes. the potential pathways for hrp secretion into bile are summarized in figure 5 : (1) a receptor - mediated, fast, transcellular route, (2) a slow, fluid - phase transcytotic route and (3) most likely also a fast paracellular route that is increased by intra- and extrahepatic cholestasis. based on previous data [8, 52 ] the slow pathway is identical to that taken by polymeric iga and asor, that is, clathrin - mediated uptake into early endosomes where sorting into transcytotic vesicles takes place followed by biliary secretion. this pathway is blocked by 16c in early endosomes and is entirely dependent on microtubules [8, 9, 52 ]. in contrast, the fast transcellular route is microtubule - independent and insensitive to inhibition by low temperature [8, 9 ]. this pathway may involve clathrin - coated pits and vesicles similar to the uptake of mannose containing glycoproteins in dendritic cells.	horseradish peroxidase (hrp) is often used as a fluid - phase marker to characterize endocytic and transcytotic processes. likewise, it has been applied to investigate the mechanisms of biliary secretion of fluid in rat liver hepatocytes. however, hrp contains mannose residues and thus binds to mannose receptors (mrs) on liver cells, including hepatocytes. to study the role of mr - mediated endocytosis of hrp transport in hepatocytes, we determined the influence of the oligosaccharid mannan on hrp biliary secretion in the isolated perfused rat liver. a 1-minute pulse of hrp was applied followed by marker - free perfusion. hrp appeared in bile with biphasic kinetics : a first peak at 7 minutes and a second peak at 15 minutes after labeling. perfusion with 0.8 mg / ml hrp in the presence of a twofold excess of mannan reduced the first peak by 41% without effect on the second one. together with recently published data on mr expression in rat hepatocytes this demonstrates two different mechanisms for hrp transcytosis : a rapid, receptor - mediated transport and a slower fluid - phase transport.
	the decaheme cytochrome mtrc from shewanella oneidensis mr-1 immobilized on an ito electrode displays unprecedented h2o2 reduction activity. although mtrc showed lower peroxidase activity in solution compared to horseradish peroxidase, the ten heme cofactors enable excellent electronic communication and a superior activity on the electrode surface. a hierarchical ito electrode enabled optimal immobilization of mtrc and a high current density of 1 ma cm2 at 0.4 v vs she could be obtained at ph 6.5 (eonset = 0.72 v). uv visible and resonance raman spectroelectrochemical studies suggest the formation of a high valent iron - oxo species as the catalytic intermediate. our findings demonstrate the potential of multiheme cytochromes to catalyze technologically relevant reactions and establish mtrc as a new benchmark in biotechnological h2o2 reduction with scope for applications in fuel cells and biosensors.
exome resequencing is increasingly becoming a standard tool for the discovery of genes underlying rare monogenic disease and the discovery of coding variants associated with common disease. although the cost of whole - genome sequencing has fallen dramatically over the last 23 years, it is still too expensive to be a useful approach for the identification of variants associated with different phenotypes in large cohorts. however, the combination of second - generation ' sequencing technologies (reviewed in refs 4,5) with robust and efficient methods of sequence capture has enabled the widespread targeting of the exome. exome resequencing studies are, however, currently being performed using designs based on an incomplete exome, and consequently many medically relevant genes are not being screened in ongoing large - scale disease studies. the two most widely used commercial kits for capturing the exome target exons from genes in the consensus coding sequence (ccds) consortium database, in addition to a selection of mirnas and non - coding rnas, are nimblegen sequence capture 2.1 m human exome array, http://www.nimblegen.com/products/seqcap/ and agilent sureselect human all exon kit, http://www.genomics.agilent.com. although the collaborative effort behind the ccds database has provided a high - quality set of consistently annotated protein - coding regions, there are still many annotated genes, with solid evidence of transcription, that are not yet part of this set. in addition, only 21% of ccds genes have an alternative spliced variant annotated. to address this shortcoming, we have designed and experimentally tested a more complete set of target regions for the human exome, based on the gencode annotation (release 2). the gencode collaboration is part of the encode project and responsible for the annotation and experimental validation of gene loci on the human genome. to generate the coordinates for the gencode exome, we extracted the coordinates for a total of 288 654 unique exons from 46 275 transcripts of 20 921 ensembl protein - coding genes (release 53) and 33 621 transcripts of 13 772 manually annotated protein - coding genes (havana, database version february 2009), together with an additional 1635 mirna genes (ensembl / mirbase). if the coordinates of any of these exons overlapped by one or more base pairs, regardless of strand, the overlapping exons were clustered together into expressed cluster regions (ecrs). any ecr that now overlapped as a result of this flank by at least 1 bp was merged. this resulted in 207 108 ecrs, covering 39.3 mb as the design target (35.2 mb of exonic sequence plus 4.1 mb of flanking sequence). the baits were created using the agilent sureselect design algorithm in three rounds of design using repeatmasker- and windowmasker - defined repeats in an attempt to avoid repetitive regions as well as to increase coverage of the target exons. each successive round of design was more permissive of repeat overlap (0, 20 and 40 bp). after sequencing, the underperforming baits were boosted at specific ratios to even out the coverage across all targets. depending on the placement of baits relative to repeat regions, the boosting was done either by direct replication or by shifting the booster bait either up or downstream by 30 bp. it was possible to design baits to 205 031 of the ecrs or 99% of the gencode exome target regions (table 1). the total size of these final bait regions is 47.9 mb. in all, 15 g of dna diluted in te was sheared to 100400 bp using a covaris s2 (covaris, woburn, ma, usa). sheared samples were quantified on a bioanalyzer 2100 (agilent, santa clara, ca, usa), and 7.5 l of cot 1 dna at 100 ng/l was added. this library was lyophilised in a vacuum concentrator to a pellet and resuspended in 3.4 l of ultrapure water. following agilent 's sureselect protocol, 10 g of sheared dna were end repaired and polya tailed, and illumina - sequencing adapters were ligated to the resulting fragments using the illumina (san diego, ca, usa) paired - end dna sample - prep protocol, except that the gel - size selection step was replaced with a purification using magnetic bead - based solid phase reversible immobilisation (spri) beads (following agilent 's protocol). the capture library was prepared by mixing 5 l of the oligo capture library, 1.5 l of ultrapure water and 1 l of 1:1 dilution of rnase block. in all, 500 ng of each sample library was hybridised to the appropriate bait set in pcr plates on a thermocycler at 65 c for 24 h (following the manufacturer 's protocol with the modification that no prehybridisation pcr was performed). the capture was performed according to the manufacturer 's protocol with streptavidin - coated dynal beads (invitrogen, paisley, uk), and captured samples were washed three times using sureselect wash buffers with a series of incubation steps. the samples were cleaned up using mini elute columns (qiagen, hilden, germany) and eluted in 50 l of pcr - grade water. eluted samples were amplified using a master - mix containing 2 m mgcl2, 0.2 m dntps, 0.5 pe.1, 0.5 pe.2 and 3 units of platinum pfx dna polymerase (invitrogen) per sample. samples were aliquoted into three individual wells of a plate and amplified using the following conditions : 94c for 5 min, followed by 20 cycles of 94c for 15 s, 58c for 30 s, 72c for 30 s and a final extension of 72c for 5 min. captured libraries were sequenced on the illumina genome analyzer 2 platform as paired - end 54-bp reads according to the manufacturer 's protocol. the presequencing preparation time is about 3 days, where sonication and library creation take 1 day, and hybridisation and amplification 1 day each. reads were aligned to the human genome (ncbi36) using the maq software package v0.7.1. base qualities were recalibrated using the genome analysis toolkit v1.0.3540 (http://www.broadinstitute.org/gsa/wiki/index.php/the_genome_analysis_toolkit) and duplicate fragments marked using picard v1.17 (http://picard.sourceforge.net/). snps were called using samtools v0.1.7 and gatk, and the intersection of the resulting call sets in the target regions (39.3 mb) with a sequence read depth of 8 were reported. to generate the coordinates for the gencode exome, we extracted the coordinates for a total of 288 654 unique exons from 46 275 transcripts of 20 921 ensembl protein - coding genes (release 53) and 33 621 transcripts of 13 772 manually annotated protein - coding genes (havana, database version february 2009), together with an additional 1635 mirna genes (ensembl / mirbase). if the coordinates of any of these exons overlapped by one or more base pairs, regardless of strand, the overlapping exons were clustered together into expressed cluster regions (ecrs). any ecr that now overlapped as a result of this flank by at least 1 bp was merged. this resulted in 207 108 ecrs, covering 39.3 mb as the design target (35.2 mb of exonic sequence plus 4.1 mb of flanking sequence). the baits were created using the agilent sureselect design algorithm in three rounds of design using repeatmasker- and windowmasker - defined repeats in an attempt to avoid repetitive regions as well as to increase coverage of the target exons. each successive round of design was more permissive of repeat overlap (0, 20 and 40 bp). after sequencing, the underperforming baits were boosted at specific ratios to even out the coverage across all targets. depending on the placement of baits relative to repeat regions, the boosting was done either by direct replication or by shifting the booster bait either up or downstream by 30 bp. it was possible to design baits to 205 031 of the ecrs or 99% of the gencode exome target regions (table 1). in all, 15 g of dna diluted in te was sheared to 100400 bp using a covaris s2 (covaris, woburn, ma, usa). sheared samples were quantified on a bioanalyzer 2100 (agilent, santa clara, ca, usa), and 7.5 l of cot 1 dna at 100 ng/l was added. this library was lyophilised in a vacuum concentrator to a pellet and resuspended in 3.4 l of ultrapure water. following agilent 's sureselect protocol, 10 g of sheared dna were end repaired and polya tailed, and illumina - sequencing adapters were ligated to the resulting fragments using the illumina (san diego, ca, usa) paired - end dna sample - prep protocol, except that the gel - size selection step was replaced with a purification using magnetic bead - based solid phase reversible immobilisation (spri) beads (following agilent 's protocol). the capture library was prepared by mixing 5 l of the oligo capture library, 1.5 l of ultrapure water and 1 l of 1:1 dilution of rnase block. in all, 500 ng of each sample library was hybridised to the appropriate bait set in pcr plates on a thermocycler at 65 c for 24 h (following the manufacturer 's protocol with the modification that no prehybridisation pcr was performed). the capture was performed according to the manufacturer 's protocol with streptavidin - coated dynal beads (invitrogen, paisley, uk), and captured samples were washed three times using sureselect wash buffers with a series of incubation steps. the samples were cleaned up using mini elute columns (qiagen, hilden, germany) and eluted in 50 l of pcr - grade water. eluted samples were amplified using a master - mix containing 2 m mgcl2, 0.2 m dntps, 0.5 pe.1, 0.5 pe.2 and 3 units of platinum pfx dna polymerase (invitrogen) per sample. samples were aliquoted into three individual wells of a plate and amplified using the following conditions : 94c for 5 min, followed by 20 cycles of 94c for 15 s, 58c for 30 s, 72c for 30 s and a final extension of 72c for 5 min. captured libraries were sequenced on the illumina genome analyzer 2 platform as paired - end 54-bp reads according to the manufacturer 's protocol. the presequencing preparation time is about 3 days, where sonication and library creation take 1 day, and hybridisation and amplification 1 day each. reads were aligned to the human genome (ncbi36) using the maq software package v0.7.1. base qualities were recalibrated using the genome analysis toolkit v1.0.3540 (http://www.broadinstitute.org/gsa/wiki/index.php/the_genome_analysis_toolkit) and duplicate fragments marked using picard v1.17 (http://picard.sourceforge.net/). snps were called using samtools v0.1.7 and gatk, and the intersection of the resulting call sets in the target regions (39.3 mb) with a sequence read depth of 8 were reported. a comparison of the coverage of the bait / oligonucleotide positions of the available ccds - based exome sets and the gencode exome with the set of gencode design targets (table 1) illustrates the increased coverage of our extended target set. the bait positions of the gencode exome cover 99% of the targets, which represents an additional 59 600 exons available for capture that are not present in either one of the ccds - based sets (supplementary data 3). comparison of exon and transcript coverage between bait / oligonucleotide locations of the available exome sets and the gencode exome, and three current reference gene sets (figure 1), shows that in all cases, the gencode exome covers a greater percentage of the reference gene sets. for example, there is an additional 9% of the exons from the ccds database and 12% of the exons from refseq covered by our expanded target set. the content present in the gencode exome exclusively consists of 38 933 cluster regions, which contain 5594 additional genes of the design target. the 4363 distinct ensembl-53-based genes of this set contain 1881 (43%) genes that have an official hgnc identifier, 711 (16%) that are linked to an omim entry and 1410 (32%) that have gene ontology annotation (supplementary data 4). in all, 41% (1809) of these genes have no external annotation of this kind and as such represent novel genes, which could prove to be an important source of variation.. the ratios of bases masked by repeatmasker, dust and trf against the total bases in the sets are nimblegen ccds : 0.027, agilent ccds : 0.021 and gencode exome : 0.027 (supplementary table 3). a comparison with a sequence uniqueness mask is given in supplementary table 4 and supports these findings. the list of 5594 genes and regions targeted by the gencode exome exclusively is available as supplementary data and on our ftp site (http://ftp.sanger.ac.uk/gencode/exome), as well as data for the full gencode exome and the initial design target. the 406 539 bait locations are supplied as a distributed annotation system data source as well (das.sanger.ac.uk/das/exome), which can be displayed in genome browsers like ensembl (version 53 ; http://tinyurl.com/browse-exome). to evaluate the performance of the gencode exome, dna from three hapmap individuals (na12878, na07000 and na19240) was subjected to sequence capture using both the agilent sureselect human all exon kit and baits designed to the gencode exome. in addition, to evaluate the performance using dna from clinical samples, dna from seven individuals recruited from a clinical neurological unit was subjected to sequence capture using baits designed to the gencode exome. 97% of reads could be successfully mapped back for both the gencode and the agilent ccds set. full details of the sequence yield and reads mapping back to target are given in supplementary table 2 (coverage was reported only using reads with a mapping quality of 10). the average fold coverage for the hapmap exomes for the ccds - based targets was 73-fold from 9.2 gb of sequence and for the gencode exome, 82-fold from 11.5 gb of sequence. the average fold coverage for the clinical samples was 58-fold from 7.5 gb of sequence. on average for the hapmap samples, 96% of targeted bases were covered at least once and 90% were covered at greater than or equal to eightfold for the ccds exome, with similar figures for the gencode exome of 92 and 83% (figure 2a). the clinical samples gave an average for the gencode exome of 95% of targeted bases covered at least once and 88% covered at greater than or equal to eightfold (supplementary figure 1). the results demonstrate that on average, the gencode - only regions perform equally to the ccds regions. an average of 22 271 snps, of which 2.6% were novel, were found for the hapmap gencode exomes compared with 18 554, of which 1.7% were novel, for the ccds - based exome (table 2 ; it should be noted that for most samples, only one lane of the sequencing machine was used. thus, the sequencing depth does not allow to identify all possible variants, slightly underestimating the number of identified variants). in this instance, novel is defined as not being present in dbsnp (version 130) or 1000 genomes project (1000 genomes project consortium, http://www.1000genomes.org, released on 26 march 2010). an average of 21 866 variants, of which 4.2% were novel, was found in the clinical samples. the clinical samples had been previously genotyped on the illumina 660 k chip that allowed the concordance rate of the variants found in common with exome sequencing using the gencode exome to be calculated at 99.8%. of the 62 sites, which were discrepant between array genotyping and sequencing, 47 were discrepant only in one sample, suggesting that the number of systematic genotyping errors is low. the ratio of stop codons gained is approximately in proportion to the size of the exome being captured, suggesting that the extra material in the gencode exome does not represent or select for a significant excess of pseudogenes (1.2:1 for the ccds - based exome in comparison with 1.8:1 for the gencode exome). the 22 002 snps found on average in the gencode exome - captured samples included a mean per sample of 9006 non - synonymous variants, 9424 synonymous variants and 91 stop - gained variants. therefore, on average, 268 synonymous variants, 256 non - synonymous variants and 2.6 stop - gained variants were found per megabase of the 35.2-mb targeted genomic sequence, corresponding to a total of 626.6 variants / mb. in the ccds - based exome - captured sample among the 18 554 coding snps found on average, there was a mean per sample of 7585 non - synonymous variants, 8880 synonymous variants and 45 stop - gained variants, corresponding to 512 variants / mb. the gencode gene set as the basis for our exome design provides a more complete set of targets, as it is a merger of the slower but thorough manual havana and the genome - wide automatic ensembl annotation. both havana and ensembl are part of the ccds consortium, and all of the agreed ccds structures at the time of construction have been incorporated into the new target set. the new design includes relevant genes for disease - associated variant and mutation discovery. among the genes in the new, expanded set are members of well - characterised gene families, which are associated with important medical conditions. mutations in ion channel genes are known to cause a range of channelopathies, including arrhythmias and inherited paroxysmal neurological disorders. also, the recently identified mll2 gene linked to the kabuki syndrome was not covered in the ccds exome but is now represented in the expanded gencode exome. deregulated protein kinase activity is frequently associated with, and members of the protein kinase family are commonly mutated in, cancer and are thus desired to be covered in cancer sequencing studies. furthermore, protein kinases are considered to be the targets for the development of new anticancer therapies. the sequence capture of the clinical samples for two genes that are targeted by the gencode exome only, abcb11 and xpc, (figures 2b and c) demonstrates that we have been able to design baits for genes that are unique to the gencode exome, and capture them efficiently, with a high degree of specificity. each exon is covered by a read depth of substantially more than eightfold, which is preferred for variant calling. abcb11 and xpc genes are already associated with disease, and provide examples of candidate disease genes that are missing from the existing exome - sequencing kits. indeed, use of the gencode exome has already allowed the identification of a pathogenic mutation in a gene causing an autosomal recessive dwarfism syndrome, which would not have been discovered using a standard ccds - based exome. the advent of the gencode exome represents a substantial improvement to the currently available designs for exome sequencing, allowing the capture of a more complete target. we estimate that we were able to call variants in 84% of the total gencode exome. this fraction of callable exome regions is likely to increase further with improving sequencing technology and sequencing depths. the gencode exome design is being used by the international cancer genome consortium (icgc ; http://www.icgc.org) for their exome - sequencing programme as part of their aim to obtain a comprehensive description of different tumour types and by the uk10k project (http://www.uk10k.org).	sequencing the coding regions, the exome, of the human genome is one of the major current strategies to identify low frequency and rare variants associated with human disease traits. so far, the most widely used commercial exome capture reagents have mainly targeted the consensus coding sequence (ccds) database. we report the design of an extended set of targets for capturing the complete human exome, based on annotation from the gencode consortium. the extended set covers an additional 5594 genes and 10.3 mb compared with the current ccds - based sets. the additional regions include potential disease genes previously inaccessible to exome resequencing studies, such as 43 genes linked to ion channel activity and 70 genes linked to protein kinase activity. in total, the new gencode exome set developed here covers 47.9 mb and performed well in sequence capture experiments. in the sample set used in this study, we identified over 5000 snp variants more in the gencode exome target (24%) than in the ccds - based exome sequencing.
mechanical ventilation is one of the supportive treatments used for various reasons such as the need to control patients respiration during surgery or treatment of severe head injuries and maintaining oxygenation when patients ventilation is inadequate. despite numerous benefits in using such mechanical ventilation machines, their long - term use may lead to several problems such as ventilator - associated pneumonia (vap), respiratory complications, gastrointestinal hemorrhage, cardiovascular complications, and bed sore. meanwhile, patients fight with the ventilator is one of the common complications of this supportive treatment. using a tracheal tube and a ventilator can lead to patients stimulation, anxiety, pain, and discomfort that result in many physiological and psychological complications. however, to reduce patients discomfort, sedation is routinely administrated with the goal of providing comfort to the patient. patients sedation with sedatives, narcotics, and tranquilizers can lead to facilitation of mechanical ventilation and treatment, decrease in patients physical and psychological discomfort, and anxiety control. on the other hand, in spite of having all the above - mentioned advantages, patients sedation has also some problems, and has thus been reported as an issue in treating these patients. studies show that using a high amount of sedatives to sedate the patient can lead to respiratory system depression and, consequently, prolonged patients connection to the ventilator, an increase in medication costs, medication dependency, respiratory infection, and therefore finally leading to high hospitalization costs. lesser consumption of such medications can also lead to anxiety, hyperactivity, pain, hypertension, and tachycardia and has unpredictable effects on patients function. with regard to the aforementioned points, striking a balance in the management of patients anxiety and pain to control the side effects and using a medication by which no respiratory depression occurs is one of the most challenging medical and nursing cares. meanwhile, using a specific and accurate program that can detect and reduce these complications and is accepted by all is among the main problems in injection of such medications. striking a balance in this regard or prediction and reduction of patients anxiety, restlessness, and pain without patients deep sedation are all controversial. research shows that using such scales of sedation and their scoring system can reduce the length of ventilation, and the incidence of pain and hospital infections. there are some scales such as patients comfort scale (cs), which was designed in 1992 to investigate patients stimulation and reaction to the environment of icus by assessing respiration, muscle tone, and other physical parameters. ramsay scale, designed in 1974, is the first scale to assess patients comfort and it only measures patients stimulation. meanwhile, research showed that application of rass is more clear and convenient and takes lesser time, leads to patients connection to the ventilator for a shorter time, and shorter duration of stay in the icu, compared to other scales. but some other studies show that application of this scale does not result in patients recovery, and moreover, it increases the risk of patients early and unsuccessful extubation and incidence of stress disorder. 2012), in a study conducted in germany on using sedation to have the body organs function at their best, showed that using rass leads to sedation in intubated patients, and a reduction in medication consumption and their complications including delirium. 2007), in a study conducted in 44 icus in france on the correct practice of sedating the patients connected to ventilator in the icu, showed that using scales such as rass led to reduced need for the sedative (43% vs 72%). on the contrary, albert and adam (2006) reported that using rass had a lesser effect on the number of days of hospitalization of the patient and the mechanical ventilation. back. (2008) reported in australia that using rass had no notable effect on ventilation, hospitalization length, and patients mortality. with regard to the existing controversy about the efficiency of rass in different studies and nurses need to use various evaluation scales for a better quality of care, the researchers thought that conducting the present study to investigate the efficacy of rass and sedation of the patients, connected to ventilators, with the cooperation of the physicians and nurses is essential. its effect on the length of mechanical ventilation, type and dosage of medications for patients sedation was also investigated in the present study. this is a two - group clinical trial in which the effect of sedation on the results of rass application among the patients connected to ventilator, as well as its effect on the length of mechanical ventilation, type and dosage of the consumed sedative among the patients in the study group were evaluated and measured. a total of 64 patients were selected by random allocation from three icus in al - zahra hospital in isfahan, iran during 20132014. after getting permission from the nursing and midwifery school, and handing her letter of introduction to the authorities of al - zahra hospital and explaining the research project to the head nurses of icus in al - zahra hospital, the researcher entered the selected icus. firstly, necessary explanations about the research and correct application of rass were given to all nurses in the wards by the researcher and an anesthesiologist, and the obscure points were clarified. then, following the ethical considerations including explanation of the method, maintaining the confidentiality of patients information, and obtaining consent from patients accompanying persons in all three icus in al - zahra hospital, the subjects were selected by convenient sampling. after the subjects meeting the inclusion criteria were selected, they were assigned to study and control groups through random allocation by use of random numbers table until there were 32 subjects in each group. subjects demographic characteristics were recorded by the researcher. in the control group, patients glasgow coma scale score was calculated and recorded, and the sedatives such as midazolam, morphine, and fentanyl, based on physician 's order (prn if needed) in each shift, were given [table 1 ]. basic dosage of sedation consumption validity and reliability of this scale in iran were established by tadrisi. in bagiatollah university of medical sciences among 120 patients (= 95%). before conducting the research, its validity and reliability were investigated among 10 patients in a pilot study under the supervision of the research supervisor in al - zahra hospital, and after obtaining his and the anesthesiologist approval, the research was conducted on 64 patients. in the study group, the score of rass was checked and recorded in different shifts every hour by the researcher or her co - worker and sedation was administrated accordingly. the type and dosage of sedatives were recorded and compared in each group. to score rass, three sequential steps of observation, reaction to auditory stimulation, and reaction to physical stimulations firstly, the patient was observed and scored from 0 to + 4 if conscious [rows 14 in table 2 ] ; but if unconscious, he / she was called loudly for several times, and if there was a response, he / she was scored from 1 to 3 [rows 57 in table 2 ]. at the last step, which was the step of painful stimulation, the patient 's sternum was pressed hard and the patient status was scored from 4 to 5 [rows 89 in table 2 ]. after determination of rass score, the medication dosage was determined with the goal of maintaining the patient in sedation scores between 1 and 0. if the patient obtained scores + 1+4, it showed inefficiency of the ventilator or patient 's inappropriate conditions or inefficiency of sedation. in such a case, firstly the ventilator and other equipments were checked, changeable causes (position and hypoxia) were modified, the environment was modified, and verbal assurance was given to the patient. if patient 's restlessness remained steady, the researcher informed the anesthesiologist about the patient 's condition and the anesthesiologist increased the primary dosage of sedation or changed the medication based on patient 's obtained rass score. ultimately, the patient was assessed by rass every hour to check whether the patient had obtained the favorite score (from 1 to 0) or not. dosage and type of medication were changed based on the low or high score of the patient, and medication was administered under the supervision of the researcher or an anesthesiologist. richmond agitation sedation scale the patients who scored negative scores less than 1 to 5 seemingly had higher sedation and, consequently, their medication was reduced based on the physician 's order to reach the favorite score. in the study group, patients constant monitoring was done by the researcher based on rass, and any changes were reported to the anesthesiologist. dosage of sedation differed based on the patients condition. for the patients who received scores 0 to 1, the level of sedation was appropriate and medication was continued without any change. using this scale in the study group was continued for 24 h a day from patients intubation to disconnection from the ventilator. the condition was investigated in the morning and evening shifts by the researcher and in the night shift by a trained co - worker, and was compared with that of the control group. in all working shifts, independent and paired t - test, with a significance level of 0.05, was used for comparisons. chi - square test with regard to sex (p = 0.78) and independent t - test with regard to age (p = 0.69) showed no significant difference between the two groups. in the study and control groups, subjects age ranged 1884 years with mean (sd) of 54.3 (22.1) years and 1989 years with mean (sd) of 56.4 (21.2) years, respectively. there was no significant difference in weight between the two groups (p = 0.08). results showed that the consumption of midazolam and morphine was not significantly different, but the mean consumption of fentanyl showed a significant difference (p = 0.03) between the two groups. mean length of connection to ventilator was significantly lower in the study group compared to the control group. as the consumed medication dosages and subjects weight were not identical in the two groups, to control the confounding effect of these variables, analysis of covariance (ancova) was adopted, and then again, the mean lengths of patients connection to ventilator were compared between the two groups. by controlling these variables, we found that administration of medication based on rass was effective on the length of patients connection to the ventilator. the researchers found that using this scale led to a reduction in patients stay in icu in the study group, compared to the control group [table 3 ]. comparison of mean age, weight, medications, and length of connection to ventilator in the two groups this clinical trial showed that using rass was effective on patients length of ventilation and consumption of some sedatives. (2012), in a study on using rass in cancer patients and giving them sedatives based on this scale as well as evaluating their level of satisfaction and quality of life, showed no significant difference in subjects demographic characteristics between the two groups, possibly due to random allocation of the subjects to the two groups. (2009), in a study on precise determination of validity and reliability of rass, showed that consumption of medications was less in the study group compared to the control group, which is in line with the present study findings, possibly due to similar methodology and the type of consumed sedatives. in a study with the goal of evaluating patients with rass and prescription of sedation based on this scale, showed that there was no significant difference between the study and control groups. they also reported that consumption of sedatives was significantly higher in the study group compared to the control group. the difference between their results and those of the present study can be due to different diagnoses of patients in the two groups and the reasons for taking sedatives, as well as the different methods used in the studies as this test was conducted mostly among the patients with high respiratory problems or distress that could have itself increased the score of rass and, consequently, consumption of sedatives. (2007), in a study with the goal of pain assessment and sedation in icu patients conducted during a week, reported no significant difference in subjects demographic characteristics between the two groups, they reported a significant decrease in consumption of midazolam and fentanyl after using the scales from the second to the sixth days (p < 0.05). they also claimed that consumption of morphine increased but that of fentanyl decreased in the study group. the reason of lower consumption of midazolam and increased consumption of morphine in the study group can be due to different methods used and the patients diagnoses. tanios., in a study on evaluation of the barriers in use of sedation protocols, showed that use of scales including rass led to less connection of the patients to the ventilator and their stay in icu as well as a reduction in consumption of the sedatives. the similarity of their results with those of the present study could be attributed to similar methods used, patients random allocation to two groups, and similar patients diagnoses. goodwin. (2012), in a study on functioning of body organs at their best, investigated the use of scales including rass in patients after coronary artery bypass graft surgery and acute respiratory distress syndrome to intermittently take sedatives and its association with disconnection of patients from the ventilator, and concluded that these scales led to a reduction in patients connection to the ventilator and their stay in icu. (2008), in a study conducted in australia on icu patients sedation, used scales including rass on 192 patients hospitalized in icu and reported temporary consumption of sedatives due to use of these scales. their results showed that use of the scales led to lower treatment costs and less consumption of sedatives including propofol and midazolam. the reason for lower consumption of midazolam can be the consumption of other sedatives based on diagnosis of the anesthesiologist or patients different diagnoses and signs, compared to the present study. the results showed that use of rass diminished the length of mechanical ventilation, but had no effect on the consumption of some sedatives. application of this scale in nursing interventions, in addition to speeding up patients recovery, can improve the ability of the nurses in evaluating patients and making decisions about them.	background : mechanical ventilation is one of the supporting treatments that are used for different reasons. to reduce patients inconvenience caused due to using tracheal tube and ventilator, sedation is routinely used. using scales for the sedation, for example, richmond agitation sedation scale (rass), may reduce dose of sedation and length of mechanical ventilation.materials and methods : this study is a randomized clinical trial on 64 patients selected from three intensive care units (icus) in isfahan, iran. through random allocation, 32 patients were assigned to each of the study and control groups. in the control group, patients level of consciousness and the amount of drug consumption in every shift, based on physician order, were recorded. in the study group, rass score was recorded every hour and sedation was administered based on that. the purpose of the study was to investigate of application of rass for drug consumption until weaning of the patient from the ventilator. independent t - test with significance level of 0.05 was used.results:results showed no significant difference in the mean consumption of midazolam and morphine after intervention, but there was a significant difference in fentanyl (p = 0.03) consumption (379 g in the control group vs 75 g in the study group) between groups after the intervention. the mean duration of being connected to the ventilator was significantly less in the study group (p = 0.03).conclusions : application of rass by nurses leads to a decrease in sedation consumption, connection to ventilator, and length of stay in the hospital.
the number of elderly patients with type 2 diabetes is continuously increasing and many of them suffer from poor vision due to senile changes or diabetic eye complications [1 - 4 ]. since type 2 diabetes is characterized by a progressive decline in insulin secretory function, many patients eventually require insulin therapy [5 - 7 ], which is a common problem in elderly patients with type 2 diabetes. furthermore, in elderly patients with type 2 diabetes, visual impairment may predispose inaccurate insulin dosing, which may result in erroneous glycemic control, including hyper or hypoglycemia. in this study, we examined whether the indicator magnifying window (imw) for insulin pens assists in effective insulin therapy for elderly patients with type 2 diabetes. fifty patients (30 men and 20 women) with type 2 diabetes were prospectively enrolled at the seoul national university hospital. the inclusion criteria required that subjects have type 2 diabetes, be over 60 years old, and self - inject insulin with insulin pens. written informed consent was obtained from all subjects prior to any study - related procedures. we asked the subjects to dial up the insulin pen (lantus solo - star pen ; sanofi, frankfurt, germany) to a randomly selected dose between 30 and 40 units. we checked whether they dialed the selected dose correctly and measured the time spent to complete the task. for each subject, we repeated the test three times with insulin pens with or without the imw (izumi planning co., tokyo, japan) (fig. if the subject dialed the selected dose correctly at all three trials, we concluded that the subject had set the dose correctly. after completing the test, we asked the subjects to assess the convenience of dosing when using the imw by checking a convenience score from 1 point (very inconvenient) to 5 points (very convenient) on the questionnaire. we also asked about their self - confidence in setting the designated doses, need for glasses in setting up the doses, preference for the use of the imw, and willingness to recommend the imw to other patients. a sample size of 49 patients was calculated to provide 90% power (=0.05) to simultaneously detect a 2-second difference in the time spent to complete the task with the imw, considering a 10% screening failure. the t - test was used to compare the time spent to complete the task and the convenience score. the chi - square test was used to analyze the dosing accuracy, self - confidence in setting the designated doses, and need for glasses in setting up the insulin doses. all the analyses were performed using ibm spss version 18.0 for windows (ibm, armonk, ny, usa). fifty patients (30 men and 20 women) with type 2 diabetes were prospectively enrolled at the seoul national university hospital. the inclusion criteria required that subjects have type 2 diabetes, be over 60 years old, and self - inject insulin with insulin pens. written informed consent was obtained from all subjects prior to any study - related procedures. we asked the subjects to dial up the insulin pen (lantus solo - star pen ; sanofi, frankfurt, germany) to a randomly selected dose between 30 and 40 units. we checked whether they dialed the selected dose correctly and measured the time spent to complete the task. for each subject, we repeated the test three times with insulin pens with or without the imw (izumi planning co., tokyo, japan) (fig. if the subject dialed the selected dose correctly at all three trials, we concluded that the subject had set the dose correctly. after completing the test, we asked the subjects to assess the convenience of dosing when using the imw by checking a convenience score from 1 point (very inconvenient) to 5 points (very convenient) on the questionnaire. we also asked about their self - confidence in setting the designated doses, need for glasses in setting up the doses, preference for the use of the imw, and willingness to recommend the imw to other patients. a sample size of 49 patients was calculated to provide 90% power (=0.05) to simultaneously detect a 2-second difference in the time spent to complete the task with the imw, considering a 10% screening failure. the t - test was used to compare the time spent to complete the task and the convenience score. the chi - square test was used to analyze the dosing accuracy, self - confidence in setting the designated doses, and need for glasses in setting up the insulin doses. all the analyses were performed using ibm spss version 18.0 for windows (ibm, armonk, ny, usa). we recruited 50 patients with type 2 diabetes (30 men and 20 women). the mean age was 696 years, duration of diabetes was 178 years, and duration of insulin treatment was 55 years. the mean hemoglobin a1c was 8.2%1.4% and the average near visual acuity of patients was 20/60 for each eye. forty patients (80%) and 39 patients (78%) dialed the designated dose correctly with and without the imw, respectively (p=0.671). time spent to complete the task was 8.24.3 seconds with the imw and 8.34.1 seconds without the imw (p=0.704). the convenience score with or without the imw was similar (4.31.0 points vs. 4.11.0 points ; p=0.418). all patients expressed self - confidence in setting the correct dose with the imw and 47 patients (94%) expressed self - confidence without the imw. three patients (6%) needed eyeglasses for near vision to set the designated insulin doses with the imw, while nine patients (18%) needed eyeglasses without the imw, which was statistically significantly different (p=0.004). seventeen patients (34%) reported that they would like to continue to use the device and 24 patients (48%) reported that they were willing to recommend it to other patients. various diabetes - care devices have been developed for patients with impaired vision, such as a talking blood glucose monitor, magnifiers for insulin syringes and pumps, and syringe - filling devices for the blind or partially sighted people with diabetes. however, the efficacy of these devices for diabetic patients had not been proven, except in the case of prefilled insulin pen devices. the insulin pen has been shown to exhibit distinct advantages over the syringe and vial in terms of accuracy and ease of use [11 - 16 ]. in this study, we examined the clinical usefulness of the imw for insulin pens in elderly patients with type 2 diabetes. since syringe or insulin pen magnifiers enlarge the scale of insulin syringes or pens, it was expected that they would be useful particularly for elderly diabetic patients. however, the imw did not improve the dosing accuracy or convenience score with regard to the use of insulin pens, which could be explained by the fact that all the patients had previously used insulin pens and felt comfortable with insulin pens. nonetheless, the imw significantly decreased the need for eyeglasses while setting up the insulin doses. overall, the imw may decrease the need for eyeglasses for insulin dosing with insulin pens, although other clinical benefits of the imw appear to be limited.	patients with type 2 diabetes who require insulin therapy are commonly elderly and have poor visual acuity. in this study, we examined the clinical usefulness of the indicator magnifying window (imw) for elderly patients with type 2 diabetes. we recruited 50 patients with type 2 diabetes over the age of 60 who had used insulin pens for glucose control. they were asked to set the insulin pen at randomly selected doses with or without an imw. we assessed dosing accuracy, convenience, self - confidence, need for eyeglasses, preference, and willingness to recommend the imw to other patients. although the imw did not improve the dosing accuracy or convenience, it significantly decreased the need for eyeglasses. overall, the clinical usefulness of the imw is quite limited in elderly patients with type 2 diabetes.
the symptoms of alzheimer s disease are numerous, many of which did not involve the degradation of cognitive functions. these symptoms include worsening of mood, psychotic symptoms, aggressive and impulsive behaviours, amongst many other. these symptoms are generally called behavioural and psychological symptoms of dementia (bpsd). aggressive and impulsive it is postulated that there exists a relationship between the progression of dementia and symptoms of aggression. a few studies have also indicated that aggressive behaviours may be connected with faster rates of progression in regards to cognitive disorders. nevertheless, not all kinds of aggressive and impulsive behaviours are connected with both the intensification of dementia and its further progression. some studies have revealed that such a relationship exists in cases of assaultive behaviour. in other studies in previously conducted studies, two potential relationships were revealed : 1) between aggressive behaviours and the intensification of dementia, and 2) between some forms of aggressive and impulsive behaviours and the prognosis of ad patients. there is research suggesting that acetylcholinesterase inhibitors (iaches) have a beneficial influence with regards to bpsd. the aim of this research was to assess the relationship between aggressive and impulsive behaviours and cognitive function disorders. the study was carried out on a group of patients with recognised ad (n=188), living in a nursing home in gdynia (poland), who underwent two years of naturalistic observation. all experimental procedures were approved by the ethics committee of the medical university of gdansk. the introductory procedure of qualifying subjects to the research group included acquiring the consent of each person to take part in the study and assessing criteria meant to exclude subjects from the study. exclusion criteria included having (during the examination or the interview) one of the following diseases : affective disease, schizophrenia, alcoholism, drug or psychoactive substance addiction, epilepsy, parkinson s disease, or mental retardation. other exclusion criteria included the presence (at the time of the examination) of consciousness disorders, motor system disorders, sight or hearing deficiencies (which would make it difficult to respond to commands and procedures included in the applied clinical scales) and the presence of serious somatic disease. primary selection to the research group also included completion of the mini - mental state examination (mmse). verification of recognised ad was done in cases of all those respondents who scored 24 or fewer points on the mmse scale. patients exhibiting clinical or radiological features which would suggest a vascular background to the disease were excluded. an additional selection criterion was a score equal to or higher than 4 points obtained using the hachinski scale. only individuals with a small or moderate intensification of disease were qualified to the research group, because procedures requiring the cooperation of patients were to be introduced as part of the study. a minimal result of 12 points on the mmse scale was treated as a baseline criterion. all in all, 48 inhabitants of the nursing home met the criteria for diagnosing dementia in ad and were qualified to take part in the research. all of the subjects gave their consent and scored above 11 points on the mmse scale. individuals accepted into the research group underwent double assessment : at the moment of being included into the research group and after two years. the baseline examination included assessment of the progression of agitation and aggressive behaviours with the use of the cohen - mansfield agitation inventory (cmai). a 29-element tool used to assess people in care institutions was applied for the purpose of that research. the behaviours listed in the inventory constitute four dimensions : verbal non - aggressive behaviour, physical non - aggressive behaviour, verbal aggressive behaviour and physical aggressive behaviour. a polish language version of the scale was used, and its reliability and accuracy were verified positively. the alzheimer s disease assessment scale (adas) its 11-element sub - scale was applied, which assesses cognitive functions (adas - cog). the results of the cognitive part range from 0 to 70 points, where zero corresponds to a lack of any difficulties, and 70 to deep dementia. we recorded the use of pro - cognitive drugs (administered for at least 90 days) and other psychotropic drugs administered for at least 14 days (during continuous treatment). individuals, who took psychotropic drugs within 14 days prior to follow - up were excluded from statistical analysis. the source of information about the patients was the personnel employed in the social care house. the degree of intensity of aggressive behaviours (the cmai scale) and dementia disorders (the adas - cog scale) obtained during the baseline study constituted the basis for the division of subjects into groups for the purpose of statistical analysis. the progression of the disease was also taken into account, assessed by means of the difference in adas - cog scale results obtained during baseline examination and follow - up studies. a test for two independent means and for two dependent means with longitudinal analysis was used. test results with a significance level equal to or smaller than 0.05 (p0.05) were considered significant, and remaining results (p>0.05) considered as insignificant. in order to verify assumptions about a normal distribution of the investigated variable in the general population (using the test for two means), the level of cognitive disorders according to the adas - cog scale equalled 20.405.24 points, with the intensity of aggressive and impulsive behaviours according to the cmai scale equalling to 52.4416.31. reasons for not performing the follow - up study in 17 cases were as follows : 1) death during the observation (n=6), 2) deterioration of physical state (n=2), 3) patient s refusal (n=4), 4) change of place of residence (n=2), and disqualifying medication (n=3). the characteristics of the investigated population in terms of age, intensity of cognitive disorders (adas - cog), and aggressive and impulsive behaviours during baseline study and after two years is presented in table 1. in order to determine the relationship between aggressive and impulsive behaviours and the intensity of cognitive function disorders, individuals with lower and higher levels of dementia (assessed by means of the adas - cog scale) were compared. the discriminative value was assumed to be the medial value of the adas - cog scale, equalling 21 point (table 2). in order to assess the influence of the rate of progression of cognitive function disorders on aggressive behaviours, the research group was divided into individuals with lower and higher rates of progression. the criterion used to divide the subjects into these groups was the medial value equal to 9 points. the rate of progression was defined by the difference between the overall scores in the adas - cog scale during the first and second study (table 3). during the observation, iaches were used for at least 12 months of continuous treatment in 19 subjects and included donepezil (510 mg, n=14), rivastigmine (39 mg, n=2), and in the case of 3 subjects, both donepezil and rivastigmine. subjects taking pro - cognitive drugs were compared to subjects not undergoing any treatment with reference to results obtained using cmai and adas - cog scales (table 4). fourteen individuals among those being analysed were treated for at least 14 days with neuroleptics or mood stabilisers (table 5). the following drugs were recorded : haloperidol (n=9), chlorpromazine (n=4), chlorprothixen (n=4), levomepromazine (n=2), promazine (n=8), risperidon (n=3), carmamazepine (n=4), and valproic acid (n=7). apart from that, some of the respondents took benzodiazepines (for a short period of time) and anti - depressants, a fact which was not taken into account during statistical analysis. treatment with neuroleptics or valproic acid did not significantly influence the intensity of aggressive and impulsive behaviours. the most evident effect of these drugs was observed in case of excitement, both physical and verbal (table 5). the presented research is the continuation of a previous study devoted to assessing the relationship between aggressive behaviours and cognitive function disorders. measurement of the intensity of cognitive disorders was based on the adas - cog scale, which ensured a sufficiently accurate assessment and allowed for monitoring of the level of cognitive function disorders during the observation period. the second applied scale (mmse) measured the intensity of cognitive function disorders and was used only during the stage of qualifying patients to the research group. the usefulness of this scale as a screening device is often underlined in literature. since the score of the mmse scale is dependent on education and age, the mungas. the second significant area of research dealt with the intensity of aggressive and impulsive behaviours in a population of individuals with ad. the cmai scale was applied, which was constructed especially for the purpose of assessing people with ad, including those living in nursing homes. an additional element, which seems to have made the results obtained more credible, was the application of a polish language version of the cmai scale (verified positively in terms of its reliability and accuracy). individuals with moderate and mild intensity levels of dementia disorders were qualified to take part in the research. this was enforced by necessitating the performing of an accurate assessment using the adas - cog scale. nevertheless, taking into account the quite high average age of the respondents, this could have been connected with the decreased natural dynamics of dementia disorders observed in the studied group. some sources suggest that the average rate of the deepening of dementia disorders equals to an annual increase in the adas - cog scale of 9.6 points. a decreased rate of the deepening of dementia disorders could have had an influence on decreasing the strength of this relationship with other assessed parameters. the current study confirmed results obtained earlier, which indicate a connection between the seriousness of cognitive function disorders and the intensity of aggressive behaviours. greater intensification of aggressive behaviours was observed in individuals with more severe cognitive deficiencies, and was seen to progress together with the progression of dementia disorders. however, this relationship was observed first of all in cases dealing with physical agitation and verbal aggression. no statistical confirmation was obtained pointing to the intensification of aggressive and impulsive behaviours assessed on the basis of the general score using the cmai scale during the observation. this refers both to individuals with a smaller intensification of cognitive function disorders and to those more affected by dementia. this may have been influenced by the presence of significant differences between the respondents and between subsequent categories of aggressive behaviours within the investigated groups. while verbal aggression intensified later on in patients with a lower baseline level of dementia, physical aggression increased in patients with deeper cognitive deficiency. on the other hand, almost all individuals with a higher baseline intensity of dementia demonstrated at the same time greater physical aggression in the baseline study, which makes the interpretation of further changes in this category much more difficult. the relationship between aggression and the progression of dementia has been confirmed by other studies. it is worth noticing the lack of intensification of agitation and verbal aggression, which may be connected with a general decrease in verbal activeness. the worsening of verbal contact may also lead to an increase in the physical component of aggression. apart from having a pro - cognitive influence, these medicines may also beneficially influence numerous bpsd symptoms, including aggressive behaviour. their effects can be explained by the positive impact they have on a decreased cholinergic transmission, which may be responsible for aggressive behaviours., verbal aggression was intensifying in people not being treated with iaches, on the other hand, a statistically significant increase in physical aggression was noticed among persons undergoing treatment. however, an intensification of physical aggression was also noticeable in the group not undergoing any treatment, and the lack of statistical confirmation could be due to the smaller number of individuals in that group. nevertheless, the potential influence of iaches on the spheres of drive and mood, not being analysed in this study, can not be ignored. because of the relatively small numeracy of the research group, there was no possibility to assess each iache separately. however, the majority of available data indicates a lack of significant differences amongst them. in the research group, taking neuroleptics and valproic acid was taken into account, as they were administered in order to reduce aggressive behaviours. differences in the intensity of symptoms of aggression were revealed, both in people who underwent treatment and those who did not. administering pharmacotherapy did not actually weaken the intensity, but decreased the progression of aggressive and impulsive behaviours. what is worth noticing, is a significantly greater progression of aggressive and impulsive behaviours in individuals who were not being administered treatment. this relationship was especially visible with reference to agitation and physical aggression. in individuals undergoing treatment, no significant intensification of aggression was observed. pharmacotherapy induced by aggressive behaviours was short - term, and lasted on average for eight weeks. despite treatment being periodical concentrating research only on the relationship between aggressive behaviours and the intensity of cognitive disorders should be considered as a major methodological limitation. aggressive and impulsive behaviours are probably conditioned by numerous variables, which were not included in this study. another factor having an influence on the results is the numeracy of the research group. taking into consideration the complex mechanisms leading to aggressive behaviours, and the meaning of subsequent variables differing from person to person, it seems necessary to conduct the research on a larger population in order to confirm the obtained data. there is a relationship between cognitive functioning disorders and the intensification of aggressive and impulsive behaviours. more severe forms of dementia are connected with greater intensification of aggressive and impulsive behaviours as the disease progresses.	summarybackgroundthe symptoms of alzheimer s disease (ad) are numerous, including worsening of mood, psychotic symptoms, aggressive and impulsive behaviours, and many others. it is generally assumed that there exists a relationship between the severity of dementia and aggressive symptoms. the aim of this study was to assess the relationship between aggressive and impulsive behaviours and cognitive function disorders in ad patients.material/methodsforty-eight ad patients living in a nursing home were included in the research group on the basis of nincds / adrda criteria. the subjects underwent two years of naturalistic observation. the intensity of agitation and aggressive behaviours was assessed on the basis of the cohen - mansfield agitation inventory (cmai). the alzheimer s disease assessment scale cog (adas - cog) was used to assess cognitive function. pharmacotherapy administered during the observation period was also taken into account.resultsthirty-one patients completed the two year long observation. individuals with more severe cognitive deficiencies demonstrated a greater intensity of aggressive and impulsive behaviours, as assessed using the cmai scale. aggression escalated together with the development of dementia disorders. the intensity of dementia disorders was most significantly connected with physical agitation and verbal aggression. the use of neuroleptics and mood stabilisers decreased the progression of aggressive and impulsive behaviours.conclusionsthere is a relationship between cognitive functioning disorders and the intensification of aggressive and impulsive behaviours. more severe forms of dementia are connected with greater intensification of aggressive and impulsive behaviours as the disease progresses. periodical administration of pharmacotherapy may reduce the development of aggressive behaviours.
a 3.5-year - old systemically healthy girl was referred with hazy corneas in both eyes since birth. she had undergone penetrating keratoplasty for ched elsewhere in le 1 year back. on examination, the child could identify objects kept close to her face in re and at 3 m in le. the right cornea 1 ] had diffuse haze with stromal edema (1007 - 1024 microns). vertical and horizontal corneal diameters were 10.5 and 11 mm, respectively. the rest of the anterior segment, adnexa, intraocular pressures (iop), and posterior segment (b scan) were normal. the left eye (le) showed an eccentric scar, resultant of resolved graft infection. preoperative operating microscope picture of right eye of case 1 showing diffuse corneal haze with corneal edema the patient was scheduled for dsek in right eye (re) under general anesthesia. donor corneal tissue (endothelial cell count : 3164 cells / mm) was manually dissected before surgery. the descemet 's membrane (dm) could be scored and removed using reverse sinskey 's hook. we found it difficult to implant the donor tissue with busin glide because of repeated iris prolapse from main wound and side port during graft insertion. it persisted even after decreasing the bottle height of irrigating anterior chamber (ac) maintainer. both sheet 's glide and endothelial side of donor tissue were covered well with cohesive viscoelastic substance and the donor tissue was slid inside the eye using sinskey 's hook. donor tissue was secured in position by filling anterior chamber with balanced salt solution (bss) followed by air. suturing of corneal wounds and repeated air injections were required to secure the graft in position. on 1 postoperative day, corneal edema was decreased and the graft was well - attached as seen under microscope. the graft was repositioned, ac was filled with air, and one venting incision was created away from pupillary axis. the corneal surface was massaged for 15 min to drain the interface fluid from venting incision. corneal edema resolved almost completely in 3 months although subtle stromal haze persisted [fig. 2 ]. the iop was 20 mm hg. at the last follow - up (20 months), one - year postoperative slit lamp picture of right eye of case 1 showing complete resolution of corneal edema a 2.5-year - old brother of case 1 had bilateral ched as evidenced by congenital hazy corneas. apart from corneal edema 3 and 4 ], rest of the ocular examination was essentially within normal limits. preoperative operating microscope picture of right eye of case 2 showing diffuse corneal haze with corneal edema preoperative operating microscope picture of left eye of case 2 showing diffuse corneal haze with corneal edema both eyes underwent dsek under general anesthesia within a gap of 4 months. the graft size and surgical steps including sheet 's glide assisted graft insertion, venting incision, and 15 min corneal massage were same as in first case. the cornea cleared within 6 weeks of surgery with no residual haze [figs. 5 and 6 ]. at last follow - up (16 months postsurgery), cornea was clear. the retinoscopy was + 3.0/2.0 40 (re) and + 5.0/1.5 130 (le). one - year postoperative slit lamp picture of right eye of case 2 showing complete resolution of corneal edema one - year postoperative slit lamp picture of left eye of case 2 showing complete resolution of corneal edema a 3.5-year - old systemically healthy girl was referred with hazy corneas in both eyes since birth. she had undergone penetrating keratoplasty for ched elsewhere in le 1 year back. on examination, the child could identify objects kept close to her face in re and at 3 m in le. the right cornea 1 ] had diffuse haze with stromal edema (1007 - 1024 microns). vertical and horizontal corneal diameters were 10.5 and 11 mm, respectively. the rest of the anterior segment, adnexa, intraocular pressures (iop), and posterior segment (b scan) were normal. the left eye (le) showed an eccentric scar, resultant of resolved graft infection. preoperative operating microscope picture of right eye of case 1 showing diffuse corneal haze with corneal edema the patient was scheduled for dsek in right eye (re) under general anesthesia. donor corneal tissue (endothelial cell count : 3164 cells / mm) was manually dissected before surgery. the descemet 's membrane (dm) could be scored and removed using reverse sinskey 's hook. we found it difficult to implant the donor tissue with busin glide because of repeated iris prolapse from main wound and side port during graft insertion. it persisted even after decreasing the bottle height of irrigating anterior chamber (ac) maintainer. both sheet 's glide and endothelial side of donor tissue were covered well with cohesive viscoelastic substance and the donor tissue was slid inside the eye using sinskey 's hook. donor tissue was secured in position by filling anterior chamber with balanced salt solution (bss) followed by air. suturing of corneal wounds and repeated air injections were required to secure the graft in position. on 1 postoperative day, corneal edema was decreased and the graft was well - attached as seen under microscope. the graft was repositioned, ac was filled with air, and one venting incision was created away from pupillary axis. the corneal surface was massaged for 15 min to drain the interface fluid from venting incision. corneal edema resolved almost completely in 3 months although subtle stromal haze persisted [fig. 2 ]. the iop was 20 mm hg. at the last follow - up (20 months), one - year postoperative slit lamp picture of right eye of case 1 showing complete resolution of corneal edema a 2.5-year - old brother of case 1 had bilateral ched as evidenced by congenital hazy corneas. apart from corneal edema [figs. 3 and 4 ], rest of the ocular examination was essentially within normal limits. preoperative operating microscope picture of right eye of case 2 showing diffuse corneal haze with corneal edema preoperative operating microscope picture of left eye of case 2 showing diffuse corneal haze with corneal edema both eyes underwent dsek under general anesthesia within a gap of 4 months. the graft size and surgical steps including sheet 's glide assisted graft insertion, venting incision, and 15 min corneal massage were same as in first case. the cornea cleared within 6 weeks of surgery with no residual haze [figs. 5 and 6 ]. at last the retinoscopy was + 3.0/2.0 40 (re) and + 5.0/1.5 130 (le). one - year postoperative slit lamp picture of right eye of case 2 showing complete resolution of corneal edema one - year postoperative slit lamp picture of left eye of case 2 showing complete resolution of corneal edema dsek has been performed in children with endothelial dysfunctions with an intention of providing better quality of vision ; minimizing the amblyopia and avoiding suture management. however, pediatric dsek can be different from adult dsek because of difference in ocular biomechanics (low scleral rigidity and positive vitreous pressure), low compliance to postoperative instructions, possibility of inadvertent trauma, and poor cooperation for examination. we herein describe the challenges we faced while performing dsek in three eyes of two siblings with ched. as reported before, iris prolapse was a significant intraoperative problem encountered while performing pediatric dsek in current series. we could not proceed with busin glide assisted graft insertion because of iris prolapse and apprehension of crystalline lens touch. hence, we used sheets glide similar to the one used by mehta., to insert the donor lenticule. however, it is important to enlarge the main wound to minimize the compression force on donor endothelium. first case had graft dislocation due to eye rubbing that required repositioning. because of obvious problems in postoperative care in children we performed corneal massage for 15 min after air injection and a venting incision in second and third case. a significant air bubble (6070% fill) was left in ac and pupillary block glaucoma was prevented by pharmacologically dilating the pupil immediately after surgery. alternatively, one can keep 8090% air fill and an inferior iridectomy. also, it is important to keep the children under strict monitoring for eye shield at least for first few days postoperatively. there were additional problems like poor visibility and difficult dm removal, which were related to basic pathology (ched) of our patients. there was 42% endothelial cell loss in first case, which may be attributed to intraoperative manipulation. the younger sibling could not cooperate for specular microscopy but had clearer corneas than first case. 7 ] that can be anticipated while performing pediatric dsek and the possible solutions to ensure optimum surgical outcome. possible difficulties and solutions in pediatric ek.	we performed endothelial keratoplasty (ek) in three eyes of two siblings (2.5 years, male and 3.5 years, female) with congenital hereditary endothelial dystrophy (ched) and report the intraoperative and postoperative difficulties. repeated iris prolapse, apprehension of crystalline lens touch due to positive vitreous pressure, and need for frequent air injections to attach the graft were intraoperative challenges in all three eyes. these were addressed by use of sheet 's glide instead of busin 's glide during graft insertion and suturing of main and side ports before air injection. one eye had graft dislocation on second postoperative day due to eye rubbing by the child. graft was repositioned with air and a venting incision was created. postoperative examination required repeated general anesthesia. corneal edema resolved completely in all three eyes. present case series highlights the possible intraoperative and postoperative challenges and their solutions in pediatric ek for ched.
arthritis is one of the most common diseases, which involves tempromandibular joints (tmjs). pain and dysfunction of the tmj are major clinical problems, especially in arthritis. up to 75% of patients with the rheumatoid arthritis (ra) have tmj involvement symptoms often noted include pain, tenderness and limitation of mandibular movement. pharmacologic therapy is one of the adjunective treatment in tmj inflammation, which is used in combination with other methods. intra - articular corticosteroid injection (iaci) is a very popular procedure, the main beneficial effect of iaci is pain relief. ia injection of corticosteroid is used to reduce the persistent pain, but this method requires multiple injections that increase the risk of complications. the use of iaci is controversial, they should be used only in patients with acute symptoms that do nt respond to conservative treatments. the duration of response is different according to the type of steroids, dose, subtype of arthritis, and accuracy of injection. sub dermal injection of a dissoluble and long - effect corticosteroid has been suggested as an alternative for ia injection. some of the considerable advantages of this method are imposing fewer traumas on joint structure and exact and clear place for the injection. several studies indicated that ia injection of corticosteroids have beneficial effects on tmj, but there is limited research in field comparison between local and systemic effects of corticosteroids in tmj. in this study, we aimed to investigate ia (local) versus intra - peritoneal (ip) (systemic) injection betamethasone l.a on tmj arthritis in rat. this experimental animal study was approved according to the guidelines of the ethics committee of isfahan university of medical sciences. before beginning the study, a pilot study using four rats was performed to confirm the stages and existence of inflammation from histological aspect. in main study 29 male wister rats, weighted 300 - 400 g was used. at first mixture of ketamin 10% (ulfasan, amsterdam, netherlands) and acepromazine 1% (ulfasan, amsterdam, netherlands) was injected intramuscular to create generalized anesthesia. after shaving the area over the left tmj one volume 50 ml of complete freund 's adjuvant (cfa) (biojan, mashhad, iran) was injected into the joint. cfa is composed of dead and dried mycobacterium tuberculosis in oil cfa is used to create artificial chronic inflammation ; according to the other studies the inflammation that caused by cfa, conforms ra the majority of inflammation was observed 1 week after cfa injection rats were divided randomly into five groups, 1 week after cfa injection : group a (control) : rats of this group were not treated (n = 5) groups b, c (n = 6 in each group) : the rats were injected with a single dose of mg / kg betamethasone l.a (exir, borujerd, iran) ia into left tmj and also saline was injected into the other joint. groups d, e (n = 6 in each group) : rats were received a single dose of mg / kg betamethasone l.a (exir, borujerd, iran) with using of ip injection method (for evaluation systemic effect). the joint felt 5 - 10 mm posterior to the lateral canthus of the eye while mandible was manipulated to provide movement of the condyle for a further positive identification of the joint. the needle was inserted from a posterior superior direction until the mandibular condyle was felt. rats were held with their abdominal exposed in the left hand and the needle was inserted deeply into the abdominal cavity in the lower right quadrant. control rats were sacrificed at 4 week, after induction maximum inflammation. while groups b and d were sacrificed 1 week after betamethasone l.a injection and groups c, e were sacrificed 4 week after drug injection. the animals sacrificed with intramuscular injection trought an overdose of ketamine 10% (ulfasan, amsterdam, netherlands). the head was separated and fixed for 48 h in formalins then processed to provide 4 m thick section of the joint and sections were stained with h and e for histological examination. synovial lining hyperplasia was graded from 0 to 2 : grade 0 : indicated the presence of 1 to 3 layers of cells.grade 1 : indicated the presence of 4 to 6 layers of cells.grade 2 : showed 7 or more layers of cells. grade 0 : indicated that dilation was not present.grade 1 : indicated the presence of dilated blood vessels that involved less than 1/3 of the synovial membrane length.grade 2 : involved 1/3 to 2/3 of the synovial membrane length.grade 3 : involved more than 2/3 of the synovial membrane length. grade 1 : indicated the presence of dilated blood vessels that involved less than 1/3 of the synovial membrane length. fibrin deposits and synovial adhesion were graded from 0 to 3 (similar to the dilated vasculature). the severity of inflammation was graded from 1 to 11 by adding the scores of the histological criteria then the data were collected and analyzed. non - parametric analysis of kruskal - wallis was used to compare the groups with other. mann - whiteney test used to compare groups b, c and d, e with each other. the joint felt 5 - 10 mm posterior to the lateral canthus of the eye while mandible was manipulated to provide movement of the condyle for a further positive identification of the joint. the needle was inserted from a posterior superior direction until the mandibular condyle was felt. rats were held with their abdominal exposed in the left hand and the needle was inserted deeply into the abdominal cavity in the lower right quadrant. control rats were sacrificed at 4 week, after induction maximum inflammation. while groups b and d were sacrificed 1 week after betamethasone l.a injection and groups c, e were sacrificed 4 week after drug injection. the animals sacrificed with intramuscular injection trought an overdose of ketamine 10% (ulfasan, amsterdam, netherlands). the head was separated and fixed for 48 h in formalins then processed to provide 4 m thick section of the joint and sections were stained with h and e for histological examination. synovial lining hyperplasia was graded from 0 to 2 : grade 0 : indicated the presence of 1 to 3 layers of cells.grade 1 : indicated the presence of 4 to 6 layers of cells.grade 2 : showed 7 or more layers of cells. grade 0 : indicated that dilation was not present.grade 1 : indicated the presence of dilated blood vessels that involved less than 1/3 of the synovial membrane length.grade 2 : involved 1/3 to 2/3 of the synovial membrane length.grade 3 : involved more than 2/3 of the synovial membrane length. grade 1 : indicated the presence of dilated blood vessels that involved less than 1/3 of the synovial membrane length. fibrin deposits and synovial adhesion were graded from 0 to 3 (similar to the dilated vasculature). the severity of inflammation was graded from 1 to 11 by adding the scores of the histological criteria then the data were collected and analyzed. non - parametric analysis of kruskal - wallis was used to compare the groups with other. mann - whiteney test used to compare groups b, c and d, e with each other. according to table 1 there were significant differences between 5 groups (p < 0/001). comparison the meanstandard deviations between the five groups inflammation is significantly lower in group c when compared with other groups. also, evidence of resorption in condylar head was seen in group c [figure 1 ]. photomicrograph of the group (c) 4 weeks after articular injection of betamethasone la showing evidence for bone resorption of the condyle. note active osteoclastic activity in the border of the condyle (h and e, 100) mann - whiteney test was used to find out which two groups have a significant statistical difference. all groups have significant statistical difference expect the groups d, e [table 2 ]. animal models are necessary for assessment tmj disorders, rats were selected in this study because they have condylar translatory movements similar to human condyle in this study administration of ia injection of betamethasone long acting showed a significant reduction in arthritis at 1 [figures 2 and 3 ] and 4 weeks after drug injection compared with ip injection [figures 4 and 5 ]. this might be due to local effects of betamethasone la as an anti - inflammatory agent in chronic arthritis of the tmj. betamethasone l.a is a corticosteroied that is injected directly inside the joints for decrease pain and stiffness in arthritis rheumatoied. therapeutic response started during several hours and continued about 1 - 4 weeks after ia and intra lesional injection. kopp. reported that favorite anti - inflammatory effects is seen with joint injection because the drug efficacy is increased and the effect of injection is limited to the affected area. in the present study, the most anti - inflammatory response was observed at 4 weeks after ia injection of betamethasone l.a in histological aspect. kopp. showed that ia injection of betamethasone (simple form) twice into the superior joint compartment of the tmj with a 2-week interval between injections, reduced the symptoms and signs in patients who had pain and tenderness to palpation in tmj. reported that betamethasone injections on tmj arthritis showed a significant improvement in subjective symptoms, clinical sign and bite forces at 1 or 2 years follow - up. stability of therapeutic effects was observed even after at 8 years follow - up. in the present study, although one injection was done, the condylar head showed resorption with osteoclastic activity at 4 weeks after drug injection. el - hakim. reported evidences of bone resorption of the condylar head at 6 weeks after ia injection of dexamethasone also bell showed that ia injection of steroids caused damage of tmj in monkeys. the results of this study was in agreement with el - hakim. and toller could not report any radiographical evidences that a single ia injection of corticosteroids cause damage to tmj, but he reported that multiple injections can cause damage to the tmj the differences between results of our study with toller can be due to this fact that the base of assessment in toller study was radiograophical evidence while the finding of this study and el - hakim. are based on histological findings during different times. the present study is the 1 research, which survey the local and systemic effect a single dose of betamethasone la on tmj structure while the other studies had used the multi injections of betamethasone (simple form). our results are based on histological findings at different time periods that are considered more realistic while the results of other studies were based on the symptoms and clinical signs in patients. photomicrograph of the group (b) 1 week after articular injection of betamethasone la showing mild hyperplasia of synovial cells (h and e, 100) photomicrograph of the group (d) 1 week after intra - peritoneal injection of betamethasone la showing distinct hyperplasia of synovial cells (h and e, 100) photomicrograph of the group (c) 4 weeks after articular injection of betamethasone la showing synovial cell adhesions (h and e, 100) photomicrograph of the group (e) 4 weeks after intra - peritoneal injection of betamethasone la showing evidence of hyperplasia synovial cells (h and e, 100) outstanding point in the study was condylar bone resorption 4 weeks after a single dose of ia betamethasone la injection but el - hakim. observed this effect at 6 weeks with a single dose of ia dexamethasone injection. the best therapeutic response was seen with ia injection of betamethasone l.a in comparison with ip injection. ia injection of betamethasone l.a is recommended in treatment tmj arthritis. the resorption of condylar head may be due to side effect of the local injection of corticosteroids.	background : pain and dysfunction of the tempromandibular joint (tmj) are major clinical problems, especially, in arthritis. the aim of this study was to compare the effect of intra - articular (ia) versus intra - peritoneal (ip) injection of betamethasone long acting on tmj arthritis in rat.materials and methods : in this experimental study, an inflammation in the left tmj of 29 rats was induced by injection of complete freund 's adjuvant. after 1 week, rats were divided into 5 groups : group a : rats of this group were not treated (n = 5) ; groups b, c : rats were injected with a single dose of mg / kg betamethasone l.a ia (n = 6) ; groups d, e : rats received a single dose of mg / kg betamethasone l.a ip (n = 6).rats in groups b and d after 1 week, and in groups c, e and a, at 4 weeks after drug injection were sacrificed. severity of inflammation was scored from 1 to 11 according to synovial hyperplasia, vascularity, fibrin deposits, and synovial adhesion. results were analyzed by using kruskal - wallis and mann - whitney (p < 0/05 was considered significant).results : significant differences were existed between groups b, d (p = 0/004) and groups c, e (p = 0/002). the least severity of inflammation and also evidence of resorbtion in condylar head was seen in group c.conclusion:the best therapeutic response was seen with ia injection of betamethasone l.a in comparison with ip injection.
plasmonic nanoparticles are key building blocks in nanotechnology, with unique features that allow exploitation of light since the optical properties of plasmonic nanomaterials are directly related to their morphology, a wide range of methods have been developed for the synthesis of (mainly) water - based dispersions of noble metal nanoparticles (nps) with well - defined morphologies and uniform sizes. upon the assembly of suitable building blocks, nanostructures with tailored plasmonic properties anisotropic nanoparticles are of particular interest because their plasmonic response can be tuned into the near - ir (nir), nanostars and nanorods being prominent examples of plasmonic performance. although the preparation of anisotropic nanoparticles with accurate size and shape control is usually carried out in water, processing them into self - assembled nanostructures can often be improved if the nanoparticles are dispersed in organic solvents. therefore, phase transfer of nanoparticles from aqueous to organic media has become an important strategy for different applications. previously reported methods were strongly limited to rather small particle dimensions (1 m), defects on the atomic scale do not play a significant role, but shape imperfections, a small degree of disorder within the assembly, and remaining organic residues may cause significant point - to - point intensity fluctuations. to avoid large errors and to statistically improve the signal, we averaged sers spectra over 100 points located on a 20 20 m grid. to probe the local homogeneity, we analyzed intensity fluctuations of single - point spectra presented as sers maps in figure 5 as well as figures s10 and s11. the lowest signal variation from the averaged value (di in %) was observed for the aunr sample with 12% (figure 5c). the aunp samples showed fluctuations ranging from 15% to 34% for increasing particle diameters (figure 5b and figure s10), whereas the sers signal from small, middle, and big aunss was found to scatter within ca. the lowest degree of homogeneity was found for ag 50 (figure 5d), with signal deviations up to 50%. these results are directly related to the degree of disorder within the assemblies as well as with larger interparticle contact area for smaller nanoparticles. the relatively high disorder of the ag 50 substrate accompanied by the presence of organics which could prevent the effective binding of the analyte are likely to be responsible for the high signal fluctuations on the ag substrate. ag particles are additionally sensitive to oxidation, as discussed above (figure s5), so the formation of a thin oxide layer can affect the sers performance. to probe homogeneity over large areas, sers maps were recorded from at least three different areas distributed over the substrate. the intrasample average signal variation did not exceed 5% for aunrs but amounted 7%, 10%, and 13% for au 30, 50, and 100, respectively. in small, middle, and big reproducibility of the sers response was studied by repeating sample preparation with different nanoparticle batches, finding average batch - to - batch intensity fluctuations lower than 5% for aunrs and 15% for aunps. when 4-mba sers spectra were recorded using 532 and 633 nm excitations, different results were obtained, as at 532 nm the most intense signals were obtained from ag 50, reaching intensities 2 orders of magnitude higher than those from aunp substrates (figure s12e). in line with the sers electromagnetic mechanism, recorded intensities were lower from ag 50 at 633 nm but higher from aunps (figure s12f). auns substrates did not show sers activity when excited with both high energy laser lines. as additional advantages of this system, we show that detection can be performed directly from solution and using a portable raman spectrometer. exemplarily, the spectrum and map for the aunr sample are shown in figure s13. the average sers intensity (at 785 nm) only varies by 2.5%. for other nanoparticle assemblies we recorded in solution the same average intensity deviations and point - to - point fluctuations as those measured in air. applicability outside the laboratory will also require using easy to handle standard analytical equipment for rapid detection instead of sophisticated scientific setups. we measured 4-mba on the aunr substrate using a portable raman spectrometer (bwtek, i - raman-785s) working at a fixed 785 nm excitation wavelength. plaser and tint were adjusted so that the 4-mba sers intensities in both setups were of the same order of magnitude without damaging the substrate, e.g., through high power exposition. to reach these conditions, plaser = 19 mw measured at sample and tint = 1 s we thus measured at three randomly chosen points distributed over the whole surface, and the average is shown in figure 5a, with a standard deviation of 7%. sers enhancement factors (ef) were calculated by means of the frequently used equationwhere isers and iraman represent the 4-mba signal intensities recorded from the probed number of molecules absorbed on the substrate (nsers) and from the number of molecules within the scattering volume (nbulk) of a solid powder, respectively. for determination of the scattering volume from the bulk material the depth of field approach presented by khan nsers (785 nm) was estimated to be 108 assuming formation of a 4-mba monolayer (more details in supporting information). iraman and isers were measured with 95 and 3517 cps, respectively, leading to an ef (785 nm) of approximately 3.7 10 in the case of the aunr substrate. we finally show that the obtained nanoparticle organosols can also be used for secondary coating with an amphiphilic polymer that has been previously reported as suitable for biological applications. after removal of excess ddt by centrifugation of the dispersion in chloroform, the reported polymer coating procedure (cf. materials and methods and supporting information section 9) was applied using dodecylamine modified polyisobutylene - alt - maleic anhydride (pma). the hydrophopic nanoparticles were thus wrapped with the polymer via hydrophobic interactions between the side chains of the polymer (dodecylamine) and the np ligands. the particles could then be transferred into water, where the charged polymer shell provides sufficient stability in biofluids. representative vis nir spectra and tem images (figure 6a, b) of pma - coated aunss confirm the colloidal stability of the particles upon polymer coating. tem images of negatively stained, polymer coated au nss confirm the presence of an organic layer wrapping the particles in water. a white shell surrounding the nps can be clearly appreciated for pma - coated particles (figure 6d and figure s16), whereas noncoated nss (figure 6c) do not display the characteristic white halo. (a) uv vis spectra of peg - sh stabilized au nss in water (red), after transfer into chcl3 with peg / ddt (green), and again in water after pma coating (blue). (b d) tem images of pma - coated au nss (b), negatively stained bare au nss (c), and negatively stained polymer coated au nss (d). a general route for transferring plasmonic nanoparticles from aqueous dispersions into chloroform and other organic solvents has been developed, which can be applied to ag and au nanoparticles of arbitrary sizes and shapes, including spheres, nanorods, and nanostars. the transferred particles are stable in chloroform for months and can be readily dried, purified, and redispersed in various organic solvents. this efficient phase transfer procedure is based on functionalization of the nanoparticles surface with a mixture of peg - sh and ddt. while ddt provides the required hydrophobicity for the nanoparticles to reach the organic phase, peg - sh provides enough stability to avoid aggregation during phase transfer. using ethanol : hexane 1:4 mixtures as spreading solvent, homogeneous extended self - assembled nanoparticle monolayers were obtained on the centimeter scale. whereas the self - assembly of plasmonic nanoparticles at liquid interfaces has been traditionally restricted due to limited size of nanoparticles available in organic solvents, limited stability, and complicated experimental procedures, the method proposed here allows us to obtain plasmonic nanoparticles in organic solvents with no restriction in size, shape, or surface chemical composition, readily forming nanoparticle assemblies. the close packing of the plasmonic nanoparticles favors plasmon coupling, thus providing enhanced plasmonic features. we compared the sers activity between the different plasmonic substrates fabricated by detection of 4-mba, finding that aunp and aunr self - assembled monolayers displayed optimum sers performance in air and in solution for 785 and 633 nm excitation lasers, with high signal reproducibility, robustness, and moderate point - to - point fluctuations. we also showed that high - end experimental setups are not necessary to obtain intense and reproducible sers signals from our assemblies. additionally, the obtained hydrophobic nanoparticles proved suitable for subsequent polymer coating, thereby broadening potential applications due to the obtained high stability under biological conditions.	we present a general route for the transfer of au and ag nanoparticles of different shapes and sizes, from water into various organic solvents. the experimental conditions for each type of nanoparticles were optimized by using a combination of thiolated poly(ethylene glycol) and a hydrophobic capping agent, such as dodecanethiol. the functionalized nanoparticles were readily transferred into organic dispersions with long - term stability (months). such organic dispersions efficiently spread out on water, leading to self - assembly at the air / liquid interface into extended nanoparticle arrays which could in turn be transferred onto solid substrates. the dense close packing in the obtained nanoparticle monolayers results in extensive plasmon coupling, rendering them efficient substrates for surface - enhanced raman scattering spectroscopy.
in 2011, in regensburg, southern germany, a 51-year - old german woman was hospitalized because of progressive headache, nausea, and vomiting. the signs and symptoms had started 2 weeks before, and intensity had been increasing ever since. at the time of admission, the patient showed cerebellar ataxia but no further neurologic deficits. she had no known chronic preconditions or recent hospital stays and had never taken immunosuppressant drugs. cranial computed tomography was performed and demonstrated a tumorous lesion (30 30 mm) in the right cerebellar hemisphere compressing the fourth ventricle. magnetic resonance imaging revealed a multicystic mass with little perifocal edema (figure 1). the patient s leukocyte count was elevated (27.4 10 cells), and a differential count indicated 84% neutrophils, 8% lymphocytes, and 4% eosinophils. aspartate aminotransferase (129 u / l), alanine aminotransferase (335 u / l), and gamma glutamyl transferase (196 u / l) levels were elevated, and total plasma protein concentration was slightly lowered (4.7 g / dl). kidney function test results, c - reactive protein levels, and gamma globulin levels were within normal limits. magnetic resonance (mr) and computed tomographic images of the brain of a 51-year - old woman infected with taenia crassiceps tapeworm larvae, germany. the 30 30 mm parasitic lesion with perifocal edema is located in the right hemisphere of the cerebellum and caused ataxia, headache, and nausea. this lesion can be misinterpreted as cerebral echinococcosis, racemose cysticercosis caused by a taenia solium tapeworm, or coenurosis. no residual parasitic masses, only the parenchymal defect in the cerebellum after resection of t. crassiceps tapeworm larvae, are visible. the tumor, which consisted of multiple spherical masses with diameters of 24 mm, was resected. because an intracranial parasitosis or tumor was suspected, serum, tissue, and fluid from the cystic lesion were examined. gross and histologic aspects of the excised tissue revealed typical structures for cestode larvae (figure 2). serum and tissue samples serologic test results for echinococcosis, which used crude and recombinant antigen elisas, and indirect hemagglutination test results were negative (11). commercial western blots for cysticercosis and echinococcosis (ldbio diagnostics, lyon, france) showed weak atypical bands of 47 kda and 30 kda, respectively (technical appendix figure 1). for the tissue samples, cestode - specific pcrs selective for the parasite s mitochondrial 12s rrna gene (13) and mitochondrial cytochrome c oxidase subunit i gene (14) were positive. after sequencing and conducting a blast search (www.ncbi.nlm.nih.gov/blast/blast.cgi) of the 380-bp and 450-bp amplicons, we found that the sequences showed 99% and 100% homology with the t. crassiceps tapeworm, respectively. histologic section through taenia crassiceps tapeworm larvae removed from the cerebellum of a 51-year - old woman, germany. a) section through parasite body showing multiple connected bladders (asexual buddings) at the caudal end. b) transverse section through the parasite s protoscolex showing numerous hooklets, similar to t. solium tapeworm larvae. original magnification 40. like the taenia solium tapeworm that causes cysticercosis, and in contrast to different tapeworms that cause coenurosis (t. [multiceps ] multiceps, t. [multiceps ] serialis) or echinococcosis, the t. crassiceps tapeworm has only 1 invaginated protoscolex, but it is on a very long neck (cysticercus longicollis). the hooklets of t. crassiceps tapeworms are larger than those of t. solium tapeworms and have a larger blade length than handle length. the small hooklets measure 123 m ; the large hooklets measure 167 m. a crude t. crassiceps elisa similar to an in - house echinococcus multilocularis assay was set up (11) by using laboratory - kept t. crassiceps tapeworm larvae from another human patient (1). serum samples from 10 healthy blood donors served as negative controls, and a standardized threshold index of 1.0 was calculated. because no serum from patients with proven t. crassiceps tapeworm infections was available to use as a positive control, we used serum from patients with histologically confirmed cystic echinococcosis (5 patients), alveolar echinococcosis (7 patients), and peripheral cysticercosis (2 patients). all serum samples were positive, showing indices of 1.29.1, 1.46.6, and 2.23.3, respectively. the patient s serum, however, had an index below the threshold (0.76). when 5-m cryosections from t. crassiceps tapeworm larvae were used for immunofluorescence tests, the patient s serum exhibited a weak tegumental signal (technical appendix figure 2). after surgery, the patient was given praziquantel (600 mg twice daily) and albendazole (400 mg twice daily) as described (1) for 3 months. the postoperative course was uneventful, the patient recovered rapidly, and there were no clinical or radiographic signs of recurrence after a follow - up period of 18 months. extended imaging investigations showed no further sites of infection. when the patient was asked about potential risk factors, she indicated that she had been living with her dog near a forest in a local rural area for many years. consumption of wild berries or mushrooms possibly contaminated by fox feces could not be excluded. the dog, which had not regularly undergone deworming, had access to the garden and the nearby forest. in recent years, more reports of human infection with t. crassiceps tapeworms have surfaced, possibly because of increasing numbers of immunocompromised persons. the patient described here showed no evidence of an impaired immune system, and the parasite was found in an immunologically privileged site. similarly, for patients with t. crassiceps larvae infection of the eye and for patients with neurocysticercosis caused by t. solium, immunosuppression does not seem to be a prerequisite for infection. t. crassiceps tapeworm larvae are apparently able to infect the same variety of human tissues as t. solium, but do so much more rarely. most infections, including the case reported here, have been reported from southern germany (1,6) and france (5,7,12). other infections of humans have been reported from neighboring switzerland and austria and from north america. similar to the distribution of alveolar echinococcosis in europe (another larval cestode disease for which the red fox is also the final host), a contiguous area with microfoci of transmission could hypothetically be possible. diagnosis depends on the radiographic image resembling a racemose cysticercus (because of the multicystic aspect of t. crassiceps tapeworm infections) and correct identification of the parasite by gross morphologic and histologic appearance by experienced pathologists or by molecular techniques. 12s rdna pcr proved to be a useful tool that is not widely used (13,14), and its use helped avoid the misdiagnosis of t. solium tapeworm neurocysticercosis. the diagnosis of t. crassiceps tapeworm infection is demanding for laboratories because no tests are commercially available. as described here, unusual serologic reactions displayed on tests for other larval helminthoses should raise the level of suspicion for a different causative agent. of note, the serologic diagnosis of neurocysticercosis caused by t. solium can be difficult ; commercial tests showed sensitivity < 72% (15). such a low sensitivity could hypothetically explain the negative elisa result for the patient reported here, for whom no peripheral tissues were infected, in contrast to the control serum used. thus, the true prevalence of human disease caused by t. crassiceps tapeworms could be underestimated, and future seroprevalence studies using elisa and immunofluorescence testing can possibly shed more light on this type of infection. the source of infection for this patient remains unclear, but her dog is probably the major risk factor (1,8,10). as a preventive measure, carnivorous pets should undergo regular deworming. cysticercosis and echinococcosis western blot patterns of the serum from the patient with taenia crassiceps infection, and immunofluorescence tests with cryosections of t. crassiceps larvae.	human cysticercosis caused by taenia crassiceps tapeworm larvae involves the muscles and subcutis mostly in immunocompromised patients and the eye in immunocompetent persons. we report a successfully treated cerebellar infection in an immunocompetent woman. we developed serologic tests, and the parasite was identified by histologic examination and 12s rdna pcr and sequencing.
transforming growth factor - beta (tgf-) was initially discovered in a hunt for autocrine factors that might enhance transformation of normal cells. surprisingly, it soon became apparent that this transforming factor regulated myriad diverse and often contradictory cellular responses, most famously as both a potent wound - healing factor and a putative tumour - suppressing antiproliferative factor. the rapid expansion of the tgf- family to include activins, nodals and bone morphogenetic proteins (bmps) led to an explosion of studies showing key roles for these factors in virtually every facet of developmental biology and homeostasis [5 - 11 ]. in the 1990s, efforts to identify tgf- superfamily receptors and intracellular mediators were anxiously pursued with the expectation that knowledge of the molecular components of the pathway would help illuminate how such diversity in biological responses was achieved. identification of the tgf- cell - surface receptors as a family of transmembrane serine / threonine (ser / thr) kinases, classified as type i or type ii receptors, revealed that engagement of distinct combinations of type i / ii receptor complexes, aided in some cases by ancillary proteins such as betaglyan or endoglin, provided for some diversity of responses. however, the genome contains surprisingly few very closely related receptors, challenging the notion that diversity of responses might be explained by a similarly diverse set of receptors. smads are direct receptor substrates that, upon phosphorylation, accumulate in the nucleus to regulate transcription through interactions with dna - binding partners. while non - smad pathways that were subsequently uncovered are important for aspects of cell behaviour such as polarity and motility, moreover, the limited set of ser / thr kinase receptors in fact funnel signals from multiple ligands to one of only two classes of receptor - regulated smads, r - smad2/3 for tgf--like ligands or r - smad1, 5 and 8 for bmp - like ligands, confounding efforts to explain complexity through a diversity of signalling pathways. the first member of the wnt (wingless - type mmtv integration site) family of secreted factors was described 30 years ago, roughly at the same time as tgf-. one arm of wnt signalling, the so - called canonical pathway, signals through -catenin, whose protein levels are controlled by a destruction complex comprising proteins that include adenomatous polyposis coli (apc), axin, dishevelled and glycogen synthase kinase 3 (gsk3) [16 - 18 ]. wnt stimulation induces stabilization of -catenin that in turn, and like smads, accumulates in the nucleus, where it promotes transcription in partnership with the dna binding factors lymphoid enhancer binding factor / t - cell - specific transcription factor (lef1/tcf). in fact, while the molecular components of morphogen signalling pathways including tgf-, wnt, notch, hedgehog and the hippo tissue size control pathway discussed below might bear little molecular resemblance, membrane and/or cytosolic regulation of a transcriptional modulator is a shared principle. similarly, the concept that cellular outcomes are significantly impacted by interactions with other signalling cascades is another common theme. the specific molecular components that mediate inter - pathway communication are varied and a description of these encompasses a huge literature. here, we will focus on some of the general features of pathway crosstalk using examples from the tgf- and wnt pathways, and then extend our discussion to recent advances on how these pathways intersect with the hippo tissue size and growth control pathway. there are myriad ways in which crosstalk is molecularly manifested, with points of regulation occurring throughout the signalling cascade from the extracellular space right through to the nucleus. here, a few illustrative examples of how signalling pathways are integrated will be discussed using tgf- and wnt as examples, with the details more extensively reviewed elsewhere [19 - 21 ]. perhaps the simplest form of signal integration occurs when activation of one signalling pathway regulates the transcription of the ligand or key components of the second pathway, thereby amplifying or attenuating signalling. for instance, in numerous developmental contexts, bmp and wnt ligands cross - regulate each other, thus controlling the extracellular milieu, or niche, and hence cellular responses [22 - 24 ]. however, this type of crosstalk at a distance probably does not provide sufficiently dynamic mechanisms to allow for the complex contextual responses that are required by sophisticated biological systems. for this, more intimate pathway integration is required, and many studies have uncovered a wealth of direct physical interactions between pathway components. one way in which these physical links modulate responses is via synergistic convergence of pathways on a common target. indeed, tgf--regulated smads and the wnt mediator -catenin can interact and converge on a common dna binding partner, lef / tcf to cooperatively induce expression of genes that control cell fate. for instance, simultaneous stimulation of smads and -catenin, whose pathways display overlapping activation in the xenopus spemann organizer, serves to localize and control expression of organizer - specific genes such as siamois and xtwn during gastrulation, while in mammalian cells, examples of cooperative smad/-catenin targets include msx2, myc and ctgf [27 - 29 ]. this type of cooperativity is particularly important for ensuring that expression of master regulators that initiate cell fate decisions is tightly constrained to the appropriate time and place. sharing, however, can also be a means of inhibiting pathway responsiveness in the context of integrated signalling. for example, distinct arms of a signalling pathway may use the same component. in the case of wnt, dishevelled proteins are important for both the canonical -catenin and the non - canonical -catenin - independent planar cell polarity (pcp) arms of the pathway [30 - 32 ], while in the tgf- family, the common smad smad4 is required for both the bmp and tgf- branches of the smad cascade. component sharing between distinct pathways is also well known, an example being the wnt negative regulator axin, a core component of the -catenin destruction complex, which has also been reported to associate with various smads to modulate tgf- signalling. competition for limiting levels of these common components has been proposed to mediate inhibitory crosstalk, such as the well - described ability of non - canonical wnt signalling to inhibit the canonical -catenin pathway and the antagonistic interactions between tgf-s and bmps. however, it is important to note that identification of a point of crosstalk does not equate to a universal mechanism of cross - regulation. for instance, -catenin is also a functional component of cell - cell adhesion complexes and is localized to the plasma membrane, but this function appears to be distinct from its role in wnt signalling. similarly, gsk3-mediated phosphorylation of -catenin is required for promoting -catenin degradation, and phosphorylation of gsk3 on a serine within its amino terminus by phosphatidylinositide-3 (pi3) kinase - activated pathways inhibits gsk3 activity. however, several studies indicate that there are distinct pools of gsk3 that compartmentalize its function and, thus, pi3 kinase - dependent inhibition is not universally manifested in all gsk3 activities [37 - 40 ]. degradation of key signalling components is another commonly reiterated theme in the context of pathway crosstalk. the e3 hect - domain ubiquitin ligases, smurfs, were originally characterized for their ability to mediate degradation of smads and receptor complexes in the tgf- pathway [41 - 43 ]. interestingly, smurf antagonism of smad signalling also synergizes with mitogen - activated protein (map) kinase signalling which, together with wnt / gsk3, leads to hyper - phosphorylation of smads, which then promotes smurf association and smad degradation. importantly, this link is critical for modulating smad activity in certain biological contexts, such as epidermal and neural patterning in xenopus and osteoblast differentiation in mammalian cells. smurfs can also associate with par6, a complex that functions both in tgf--induced epithelial to mesenchymal transitions and in the non - canonical wnt pathway to induce localized degradation of pcp pathway components, thus providing a key node that coordinates cell fate with planar polarity. in summary, these examples of multiple and diverse contacts between tgf- and wnt pathways point to a molecular framework of pathway integration that is structured to allow for maximal plasticity and versatility in cellular responses. in order to properly form tissues and organs, cells must not only integrate morphogenic signals provided by tgf-, wnts and other factors but also must incorporate information on the status of control pathways that govern overall cell and tissue growth. the hippo pathway, initially identified in drosophila, but well conserved in mammals, is one such pathway that acts as a major regulator of tissue growth and organ size [48 - 54 ]. activation of the hippo pathway, such as through cell - cell contact or upon polarization of epithelial cells, activates a cascade comprising the core kinases mst1/2 (encoded by the stk3 and stk4 genes) and lats1/2 (large tumor suppressor, homolog 1/2) that leads to phosphorylation of the related proteins taz (transcriptional co - activator with pdz - binding motif) and yap (yes - associated protein), resulting in their cytoplasmic retention. in the absence of hippo signalling, taz / yap accumulate in the nucleus and in complex with various dna binding factors, including teads (tea domain family members) and runx2 (runt related transcription factor 2), amongst others, that promote transcription of numerous target genes. thus, unlike the tgf- and wnt pathways that promote the nuclear activity of their respective mediators, smads and -catenin, activation of the hippo pathway serves to turn off the nuclear functions of taz / yap (see figure 1). however, similar to the tgf- and wnt pathways, a key transcriptional role for taz / yap, primarily in cooperation with teads, in regulating stem and progenitor cell maintenance and differentiation has emerged [55 - 66 ]. each of the indicated pathways converge on transcriptional modulators that act in the nucleus to regulate transcription of target genes by interacting with dna - binding partners, a selection of which are indicated (lowest cluster). extensive physical interactions between smads, -catenin and taz / yap (nuclear halo of components) describe a network of extensive crosstalk that provides for contextual transcriptional responses. in the presence of hippo pathway activity, taz / yap are sequestered in the cytosol, where they limit both tgf- and wnt--catenin activity. the upstream components and mechanisms whereby hippo pathway activation is linked to cell density sensing are still under intensive investigation, but convergence of this pathway with those of tgf- and wnt has been firmly established. taz / yap interact with the tgf--regulated smads and, when hippo is activated, the cytoplasmically localized taz / yap prevents smad nuclear accumulation and transcriptional activity. in epithelial cells, this is critically dependent on the assembly of the crumbs complex, which is a late event during establishment of apical - basal polarity and, thus, provides a sensor of cell density [70 - 76 ]. consequently, in dense cells, hippo activation and sequestration of taz / yap and, in turn, smads blunts transcriptional responses to tgf- to prevent epithelial - to - mesenchymal transitions. other components of polarity complexes, such as scribble and cadherins [77 - 79 ], have also been linked to regulation of taz / yap, indicating an intimate link between cell - cell adhesion, polarity and hippo pathway activity. similar to observations in cultured cells, during early embryogenesis in mice, taz / yap localization varies. in the blastocyst, taz / yap is nuclear in trophectoderm precursors where it induces cdx2 and the trophoblast fate, while in the inner cell mass it is cytosolic. importantly, this differential localization reflects and defines regions of active nuclear smad complexes in inner cells that are exposed to the tgf--like ligand called nodal. thus, interference with hippo activity in the inner cell mass leads to concomitant nuclear accumulation of both taz / yap and smads. in the nucleus, a second important function of pathway crosstalk is manifested as taz / yap cooperate with smads to promote activation of specific target genes. many of these cooperative targets control maintenance of stem cell pluripotency or induce differentiation. in human embryonic stem (es) cells, cooperation of taz with smad2/3 is required for tgf--like ligands to maintain pluripotency, while yap, in partnership with smad1, supports bmp maintenance of mouse es cells and is required for bmp - induced osteoblastic differentiation of mesenchymal stem cells. the composition of these smad and taz / yap - containing activation complexes, particularly with respect to the dna - binding partners that are responsible for recruitment to specific target genes, is an important question requiring further investigation. however, it is known that taz recruits the mediator complex and the tgf--regulated ctgf gene is induced by a yap - tead4-smad3-p300 promoter - bound complex. thus, by functioning to both control smad nuclear accumulation and synergize transcriptionally in the nucleus, taz / yap provides a mechanism to couple tgf- smad activity to hippo pathway activity and cell density sensing. intimate connections between the hippo and wnt/-catenin pathways have also been delineated, in which hippo activity antagonizes wnt signalling. genetic manipulation of hippo pathway components in both drosophila and mice showed that this crosstalk is evident in diverse tissue and organ contexts. taz knockout mice have polycystic kidneys [82 - 84 ] that display increased levels of cytoplasmic -catenin. heart - specific knockout of salvador,, an mst1 binding protein, or ablation of the expression of mst1/2 in the intestine, result in nuclear accumulation of yap and increased expression of wnt/-catenin target genes. moreover, during intestinal regeneration, transgenic overexpression of yap restricts wnt signals, while loss of yap leads to wnt hypersensitivity. in drosophila, a link between wingless, a wnt family ligand, and yorkie, the ortholog of taz / yap, in patterning of imaginal discs has also been established several molecular mechanisms have been proposed to explain how loss of hippo pathway activity might promote wnt signalling. we described a mechanism in which taz binds and inhibits phosphorylation of the cytoplasmically localized wnt/-catenin pathway component dishevelled to dampen wnt signalling. in the intestinal regeneration model, yap binding to dishevelled thus, hippo pathway activation by cell - cell contact, for example, enforces cytoplasmic localization of taz / yap, thereby attenuating wnt signalling through a cytoplasmic mechanism. in certain developmental contexts, such as trophectoderm cells in the blastocyst, or in cancer cells where contact - dependent growth inhibition is bypassed, the absence of hippo activity results in cells that display high levels of nuclear taz / yap. taz / yap can also interact with -catenin, and exactly as delineated for tgf-/smads, in the nucleus it can cooperate with -catenin to promote the transcriptional activation of a panel of target genes. thus, upon loss of hippo pathway activity, this nuclear activity reinforces wnt - induced gene responses. however, the recurring concept of context - dependence arises yet again, as this transcriptional enhancement can not be generalized to all gene targets, but rather is only relevant to a subset of genes. for instance, a recent report has shown that in -catenin - driven cancers, -catenin and yap cooperate with tbx5 to transcriptionally activate only a subset of genes, including several anti - apoptotic targets that are essential for cancer cell transformation and survival. moreover, additional complexity in cellular responsiveness can arise from many other points of communication between wnt and hippo pathways. the wnt/-catenin pathway induces expression of cd44, which binds to neurofibromatosis-2 (nf2 ; also known as merlin) that, in turn, promotes hippo activity to limit cell growth. also, a recent study reported that wnt signalling directly modulates taz, but not yap, stability via -catenin. in contrast, in the study of -catenin - driven cancers referred to above, only yap (but not taz) was identified as cooperating with wnt to contribute to transformation. thus, signalling outcomes may also depend on the relative levels of these two highly related proteins. shared components such as gsk3 and ck1/ that can phosphorylate -catenin, dishevelled, taz / yap, as well as trcp, the e3 ligase that promotes -catenin and taz / yap degradation, also provide for additional potential mechanisms of signal integration. thus, while it is clear that the hippo pathway targets taz and yap converge with tgf- and wnt signaling pathways, the impact of hippo activity on cellular responses to these important morphogens is very much dependent on context. regardless of its complexity, it is clear that pathway crosstalk is key for the diverse cellular responses observed during development and homeostasis. furthermore, as exemplified above for the tgf-, wnt and hippo pathways, even the nature, extent and response to this crosstalk can vary in a context - dependent manner. the experimental modus operandi for investigating cell signalling still tends to focus on a single pathway. this often leads to disparate observations on biological functions that are highly dependent on the cell type and model system under investigation ; for example, tgf- signalling regulating both es cell maintenance and cell fate determination. therefore, it is increasingly clear that understanding biological diversity requires understanding signalling pathways not only as isolated entities but also as integrated higher order networks that form the molecular framework required for contextual responses. the establishment of these contextual signalling environments is what is ultimately critical for allowing cells to robustly mount appropriate biological outcomes in space and time. moreover, the inherent dynamics of these systems also provide an adaptive reservoir for diseases, such as cancer, to circumvent targeted therapeutics and ultimately thwart our attempts to affect lasting cures. taking a network - wide view will undoubtedly provide insights that more adequately describe how a limited toolbox of extrinsic cues modulates cellular outcomes and will enhance our understanding of the complex physiological and pathophysiological processes that act to disturb these networks.	complete sequences of animal genomes have revealed a remarkably small and conserved toolbox of signalling pathways, such as tgf- and wnt that account for all biological diversity. this raises the question as to how such a limited set of cues elaborates so many diverse cell fates and behaviours. it is now clear that components of signalling pathways are physically assembled into higher order networks that ultimately dictate the biological output of pathway activity. intertwining of pathways is thus emerging as a key feature of a large, integrated and coordinated signalling network that allows cells to read a limited set of extrinsic cues, but mount the diverse responses that underpin successful development and homeostasis. moreover, this design principle confounds the development of effective therapeutic interventions in complex diseases, such as cancer.
the prevalence and severity of obesity is increasing dramatically among children and adolescents in many parts of the world, whereas prevalence rates are estimated to increase in the next decades. in children, excess body fat appears to be strongly associated with the clustering of risk factors, such as hyperglycemia, dyslipidemia, and hypertension, which play a key role in the pathogenesis of the metabolic syndrome (mets). obesity and the mets risk in children have been recently associated with systemic inflammatory markers, in particular c - reactive protein (crp) [3, 4 ], implying that low - grade inflammation can already exist in childhood and may be a potential link between the obesity and the mets. among behavioral variables, cardiorespiratory fitness has a protective role in mets and inflammatory factors ; however, it is not entirely clear if the interrelations among cardiorespiratory fitness, mets risk, and inflammation in children are independent or partly due to the mediating effect of obesity, since the existing data are limited and equivocal [5, 6 ]. recent evidence indicates that the prevalence rates of childhood obesity in greece remain high [1, 7 ] and often coexist with low cardiorespiratory fitness and an unfavorable cardiometabolic risk profile. for the greek pediatric population these data suggest an increased cardiovascular morbidity in adulthood, given that high - risk children and adolescents are likely to become high - risk adults. although the relationship among obesity and dyslipidemia in greek children has been thoroughly investigated [9, 11 ], there is a paucity of data regarding the clustering of metabolic risk factors, inflammation, and their relationship with cardiorespiratory fitness. the present study was undertaken in an attempt to investigate the prevalence of the mets and examine the associations among cardiorespiratory fitness, mets risk, and crp in 11-year - old children. this study sample consisted of 112 pupils (54 females, 58 males, mean age 11.4 0.4 years), registered in the sixth grade of 15 randomly selected primary schools in north attica, greece. subjects were healthy at the time of investigation and had no chronic or acute illnesses. prior to the commencement of the study, permission from the school principal was obtained and parental consent was secured after full explanation of study objectives and data collection procedures. the research ethics committee of democritus university of thrace approved the investigation and permission was granted by the greek ministry of education. measures of anthropometry and fitness were conducted in the school environment (during pe classes) by the same experienced investigator (the principal author), using the same order of testing procedures and under similar conditions. blood samples and blood pressure data were obtained at the same clinical biochemistry accredited laboratory by a medical practitioner. students were weighed on an electronic scale to the nearest 0.5 kg (seca beam balance 710), lightly dressed and barefooted. standing height was measured to the nearest 0.5 cm (seca stadiometer 208) with each subject 's shoes off, feet together, and head in the frankfort horizontal plane. body mass index (bmi) bmi values were also converted to standard deviation scores (sdss), whereas the iotf 's age- and gender - specific bmi cut - off points were used to calculate the prevalence rates for overweight / obesity. waist circumference, measured at the level of the umbilicus with a tape measure to the nearest 0.1 cm, was used as an indicator of central obesity. cardiorespiratory fitness was assessed by using the 20 m multistage shuttle run test, a standard field test included in the european fitness test battery. in brief, 510 subjects performed a series of runs across a 20 m track, changing direction at the end of each run to coincide with an audio signal that was getting progressively faster. subjects started running at a speed of 8.5 km / hr, and speed increased at various stages (0.5 km / hr every minute). each stage was made up of several shuttle runs, and pupils were instructed to keep pace with the signals as long as possible. the number of laps fully completed were recorded for each pupil and subsequently used to estimate cardiorespiratory fitness as predicted maximal oxygen uptake (vo2max) in ml / kg / min. based on gender- and age - specific cut - off values of vo2max participants were classified into two fitness groups. blood pressure (bp) was measured in the right arm, using a mercury sphygmomanometer with appropriate size cuffs and cuff bladders, after the subject had been sitting quietly for at least 5 min. the mean of two measurements at the first and the fifth korotkoff phase was recorded for systolic and diastolic bp, respectively. blood samples were obtained from each subject early in the morning, following an overnight fast. blood was centrifuged for plasma separation, and 1.5 ml aliquots were pipetted into plastic eppendorf tubes and stored at 70c until further analyses. plasma glucose, triglycerides, and hdl cholesterol (hdl - c) were determined using chromatographic enzymatic method on an automated mb ii analyzer (dade behring, marburg, germany). crp was measured by high - sensitive latex - enhanced immunoassay on a bn ii nephelometer from the same company. the mets was defined as meeting 3 of the following age- and gender - specific criteria : waist circumference 90th percentile, glucose 100 mg / dl, triglycerides 100 mg / dl, hdl - c < 50 mg / dl, and systolic or diastolic bp 90th percentile [17, 18 ]. we further computed a mets risk score as follows : for each of the mets components, an absolute z score was computed. for hdl - c the z score was multiplied by 1 to indicate higher metabolic risk with increasing value. the z scores of systolic and diastolic bp were averaged and then added to the rest of the z scores. this risk score is statistically more sensitive and less error - prone by comparison to dichotomous approaches. we also computed a nonobesity mets risk score, omitting the z score from waist circumference. because of a nonnormal distribution, crp values were logarithmically transformed prior to the analyses. continuous variables are presented as mean values standard error of means, while qualitative variables are presented as absolute and relative frequencies. relationships among cardiorespiratory fitness, mets risk score, and crp levels were summarized with pearson 's correlations. the independent effect of low fitness level on anthropometric indices, metabolic characteristics, mets risk score, and crp was assessed using one - way ancova. descriptive characteristics of the subjects by gender and for the entire sample are presented in table 1. anthropometric parameters are in line with recently published paediatric data [1, 7, 8 ], suggesting that the sample is representative of periadolescent children in greece. mean bmi values, mean plasma concentrations of the mets components, and mean bp recordings were within acceptable ranges and did not differ between genders. sds - bmi was significantly higher in males compared to females (t(110) = 2.52, p = 0.013). among all participants, 34.8% had one component, 12.5% had two components, 2.7% had three components, 1.8% had four components, and 0.9% had five components of the mets. hyperglycemia was the most common metabolic abnormality in both genders (27.7%), followed by hypertriglyceridemia (17.9%). elevated bp was more frequent in females, but differences did not achieve statistical significance (= 3.43, p = 0.064). the prevalence of the mets was 5.4%, without statistically significant gender differences (= 0.008, p = 0.928). pearson 's correlation analyses revealed that cardiorespiratory fitness was inversely associated with the mets risk score (r = 0.386, p < 0.001), and crp (r = 0.394, p < 0.001). mets risk score and crp were positively interrelated (r = 0.544, p < 0.0005). a high percentage of the study participants demonstrated cardiorespiratory fitness values considerably lower than the recommended fitness levels for metabolic health. in particular, ~72% of both genders did not achieve the acceptable fitness values for children of comparable age. children with lower fitness levels, compared to their peers with increased fitness levels, demonstrated significantly increased bmi (21.01 versus 17.91 kg / m, f(1,111) = 17.55, p < 0.001), sds - bmi (1.07 versus 0.14, f(1,111) = 13.43, p < 0.001) and waist circumference (72.89 versus 64.23 cm, f(1,111) = 19.82, p < 0.001). in addition, pupils in the lower fitness group showed significantly higher mets risk score (f(1,93) = 13.03, p < 0.001) and crp values (f(1,93) = 7.67, p < 0.007), compared to the high - fitness group (figure 1). adjustment for central obesity attenuated the observed differences in the mets risk score, which were no longer significant (figure 1(a)). differences in crp values between fitness groups decreased also when adjusted for waist circumference (figure 1(b)) ; however, they remained borderline significant (f(1,93) = 3.49, p < 0.067). analyses including bmi or sds - bmi instead of waist circumference yielded similar results (data not shown). the mets has a multifactorial aetiology, and the mechanisms underlying its onset are not completely understood. in the present study we investigated the prevalence of the mets and examined the independent associations among cardiorespiratory fitness, mets risk, and crp in greek children. using a paediatric definition based closely on atp iii [17, 18 ], we found that the prevalence of mets in our sample was 5.4%. this prevalence rate is similar to those reported in us and french adolescents (45.8%) [2123 ] but lower than the prevalence reported in iranian paediatric populations (14.1%). these data indicate that the mets is a common health problem among different paediatric populations, underlining the need for effective policies and appropriate intervention strategies at global, national, and regional level. we further found that the protective effect of cardiorespiratory fitness on the mets risk score was explained by the confounding effect from central obesity. our results are in agreement with previous findings, showing that cardiorespiratory fitness is not independently associated with markers of the mets. however, recent data from the european youth heart study (eyhs) [19, 25, 26 ] and the national health and nutrition examination survey (nhanes) 19992002 revealed that cardiorespiratory fitness is a significant and independent predictor of the mets in children, whereas the benefits of higher cardiorespiratory fitness seem to extend also to children at lower metabolic risk. differences in body composition and fitness levels are known to affect the metabolic profile in children and could explain at least part of these discrepancies among studies. compared to their north european and north american age - related peers, greek children exhibit higher rates of overweight / obesity and worse cardiorespiratory fitness performance [8, 19, 27, 2931 ]. these findings suggest that children in the present study did not achieve the required threshold in cardiorespiratory fitness, in order to profit from its beneficial effects with respect to the risk of mets in the face of obesity. indeed, nassis. have shown that exercise - related cardiorespiratory fitness enhancement in overweight girls can lead to marked improvements in insulin sensitivity, without changes in body composition. in addition, even if central obesity is an intermediate link between low fitness and mets risk, fitness will still be the principal cause, even if its association with mets risk disappears after adjustment for an intermediate variable in the causal chain (i.e., central obesity). in line with a previous report in children and young adults, we found that the association between cardiorespiratory fitness and crp was largely explained by their interrelation to obesity. reported no relationship between crp and cardiorespiratory fitness in 12 - 13-years - old schoolchildren. in contrast, results from the ten towns children 's study and more recent data from south african children reported strong correlations between fitness and crp. an explanation for this discrepancy may be the fact that in the ten towns children 's study a crude estimate of physical fitness (i.e., resting heart rate) was used, whilst in the latter study the investigators did not take into account the confounding effect of obesity. more studies are needed to improve our understanding of the interrelations among cardiorespiratory fitness, crp, and obesity in children. our findings indicate that the mechanisms linking cardiorespiratory fitness, mets risk, and crp are extensively affected by obesity. further, the favourable effect of cardiorespiratory fitness on mets risk may not become evident when fitness levels underlie a critical threshold. given that high levels of cardiorespiratory fitness improve the metabolic state of obese children, attenuate the proinflammatory milieu associated with obesity, and offer protection against the development of the insulin resistance syndrome from childhood into adolescence and young adulthood [3739 ], children should be encouraged to achieve the recently recommended physical activity levels (90 min / day of at least moderate intensity), in order to prevent clustering of cardiovascular disease risk factors and improve their metabolic and overall health profile. a root cause in the development of the mets is insulin resistance, which was not assessed in the present study. however, epidemiologic studies indicate that a substantial proportion of subjects with the mets do not have evidence of insulin resistance and the correlation between insulin resistance and individual components of the mets is weak to moderate. however, it is unlikely that our findings are invalidated by the use of the 20 m multistage shuttle run test since it has been extensively used in the literature and is recommended for paediatric studies as reliable, valid, and cost - effective [8, 13, 14, 30, 31 ]. further, a strict random sampling of all eligible pupils was virtually impossible, given that the study was dependent on the principals ' disposal and the willingness of individual children and their parents to participate. the prevalence of overweight / obesity, however, was similar to that reported for school - aged children from other greek regions [79, 11 ]. finally, the sample size and the age range of our subjects limit the generalization of the findings. within the above - mentioned limitations, it is concluded that the metabolic consequences of excessive body fat and low fitness are detectable early in life. taking into consideration the growing epidemic of childhood obesity, the public health authorities should seriously consider the design and early implementation of effective prevention strategies. health education programs aiming at the prevention of weight gain and the improvement of cardiorespiratory fitness in childhood could anticipate the metabolic abnormalities predisposed to or associated with mets risk and inflammation and thus contribute to the prevention of the mets and its complications later in life.	the aim of this study was to investigate the independent associations among cardiorespiratory fitness, metabolic syndrome (mets), and c - reactive protein (crp) in children. the sample consisted of 112 children (11.4 0.4 years). data was obtained for children 's anthropometry, cardiorespiratory fitness, mets components, and crp levels. mets was defined using criteria analogous to the adult treatment panel iii definition. a mets risk score was also computed. prevalence of the mets was 5.4%, without gender differences. subjects with low fitness showed significantly higher mets risk (p < 0.001) and crp (p < 0.007), compared to the high - fitness pupils. however, differences in mets risk, and crp between fitness groups decreased when adjusted for waist circumference. these data indicate that the mechanisms linking cardiorespiratory fitness, mets risk and inflammation in children are extensively affected by obesity. intervention strategies aiming at reducing obesity and improving cardiorespiratory fitness in childhood might contribute to the prevention of the mets in adulthood.
the human fibroblast growth factor (fgf) family includes at least 22 members with diverse biological functions, such as cell growth, cell differentiation, and wound healing. recent data has shown that this family may play important roles in defining and regulating the functions of some endocrine - relevant tissues and organs, as well as modulating various metabolic processes. although the physiological role of fgf-21 in humans is not understood, higher circulating levels are strongly associated with certain metabolic states, where fat is accumulated in the liver, triglycerides are high and hdl cholesterol is low (all features associated with hepatosteatosis and dyslipidemia). mounting evidence from animal - based studies suggests fgf-21 as a potent metabolic regulator with multiple beneficial effects on obesity and diabetes [8, 9 ]. serum levels of fgf-21, which has been suggested as a potential candidate for the treatment of diabetes, are increased in obesity. in addition, there is a positive association of serum fgf-21 levels with age, several parameters of adiposity including bmi, waist circumference, waist - to - hip ratio, fat percentage, insulin resistance, and adverse lipid profiles. fgf-21 is detected in fat and muscle, and the concept of fgf-21 as a novel hepatokine, adipokine and even a myokine were suggested. however, fgf-21 is also present in the exocrine pancreas and b - cells at drastically higher levels than in the liver, adipose tissue and muscle [1315 ]. in skeletal muscles, hepatic fgf-21 transcription is upregulated by fasting [16, 17 ], thus skeletal muscle can be an important source of fgf-21. in this study, we measured and investigated the correlation of fgf-21 concentrations in the sera of patients with primary and secondary mids to assess whether it is a feasible biomarker for human rcd. patients with mitochondrial diseases were excluded by the fact that these defects were the result of primary mids. the genetic defect was established in mtdna extracted from muscle and blood using established techniques. confirmation of the mtdna defect in each patient was sought but was not required for inclusion, assuming the clinical features were consistent and a pathogenic mutation had been confirmed within the pedigree. this approach was validated by a near-100 % rate of positive genetic tests where samples were available. the suspected mitochondrial diseases were referred to the medical genetics department at the special medical center in iran for investigation. some fa patients were recruited as candidates for secondary mids because of the link between fa and mid. a total of 30 control subjects with no family history of mids were randomly selected. human fgf-21 enzyme - linked immunosorbent assay (elisa) kits were obtained from biovendor laboratory medicine (modrice, czech republic). for the measurement of fgf-21. then, 100 l of diluted sera, calibrators and quality controls were added to 96-well microtiter plates coated with an affinity - purified polyclonal anti - human fgf-21 antibody. a calibration curve was constructed by plotting the absorbance values at 450 nm versus the fgf-21 concentrations of the calibrators, and concentrations of unknown samples were determined using this calibration curve. this assay was then used to measure serum fgf-21 levels in 47 iranian mitochondrial disorders. in our study, 32 patients had primary mids and 15 patients had secondary mids (patients with fa). our primary mid patients showed a pathogenic mutation or an abnormally low rc enzyme activity in muscle and had mitochondrial encephalomyopathy, lactic acidosis, myoclonus, epilepsy, ragged - red fibers mutation, point mutation, mtdna deletions and mtdna depletion. the mean concentrations of fgf-21 in serum are 70 pg / ml (range 15309 pg / ml) in healthy adults and 114 pg / ml (42244 pg / ml) in healthy children, respectively. interestingly, patients with primary mids [346.2 pg / ml (17.51707) ; n = 32 ] had significantly higher serum fgf-21 levels than the controls [87.1 pg / ml (32269.1) ; n = 30 ] (table 1). no significant difference in serum fgf-21 levels [fa patients (n = 15), 71.6 pg / ml (23.993.4) vs. controls [87.1 pg / ml (32269.1) ; n = 30 ] (table 1).table 1correlations of serum fgf-21 levels with primary mids and fagroupcontrolprimary midsfanumber of subjects303215gender (m / f)17/1317/159/6fgf-21 level in male (pg / ml)86.6261.469.2fgf-21 level in female (pg / ml)88.7422.475.1fgf-21 level in all (pg / ml)87.1346.271.6 correlations of serum fgf-21 levels with primary mids and fa there were gender differences in serum fgf-21 levels [men (n = 17), median 261.4 pg / ml (interquartile range 17.51707 ] vs. women (n = 15), 442.4 pg / ml (25.21,599.1) among patients with primary mids, and there were no gender differences in serum fgf-21 levels [men (n = 9), median 69.2 pg / ml (interquartile range 23.981.8) vs. women (n = 6), 75.1 pg / ml (36.993.4) in fa patients ] (table 1). mitochondria are vital components of all nucleated cells because of the presence of the respiratory chain (rc), and are the major sites of energy production. given the complexity of rc genetic inheritance and its function and regulation, a proper oxidative phosphorylation system (oxphos) requires the full assembly of functional proteins. mutations of the ndna and mtdna genes encoding the different rc sub - complexes and their regulatory factors can produce a wide range of oxphos diseases with extremely heterogeneous clinical manifestations. rc disorders are one of the most common causes of inherited metabolic disorders. over 100 genes govern the process of oxidative phosphorylation, and mutations in any of these can cause a mitochondrial respiratory chain defect. it has become clear that oxphos may be impaired by mutations in many mitochondrial and even non - mitochondrial proteins, or may be disturbed as a secondary effect of other biochemical defects of intracellular metabolism. oxphos defects caused by genes encoding non - oxphos mitochondrial proteins, such as frataxin, are responsible for fa. fa is an autosomal recessive neurodegenerative disease characterized by progressive ataxia, neuropathy, skeletal abnormalities and cardiomyopathy. in fa, mutations in the nuclear gene encoding frataxin, which is involved in iron homeostasis in mitochondria, result in severe deficiencies of iron defects of intracellular metabolism, particularly excess free radical generation, including nitric oxide or peroxynitrite, may cause secondary damage to the respiratory chain. other recessive nuclear mutations are known to affect structural subunits or assembly of mitochondrial respiratory chain complexes, and only fa was included in our prevalence figures. clinical experience in mitochondrial diseases suggest that ~75 % of adult - onset mitochondrial diseases are a consequence of primary mtdna mutations. the clinical presentation of rcd is highly variable in severity, age of onset, and the combination of organ systems involved. consequently, the diagnosis can be challenging, and there are very limited means of objectively monitoring disease progression. genetic disorders with impaired oxphos are extremely heterogeneous, as their clinical presentation ranges from lesions of single tissues or specialized structures, such as the optic nerve in mitochondrial dna - associated leber s hereditary optic neuropathy and in nuclear dna - associated dominant optic atrophy, to more widespread pathologies, including myopathies, peripheral neuropathies, encephalomyopathies, cardiopathies, or complex multisystem. increased concentrations of organic acids (particularly lactic acid and tca cycle intermediates) in blood, urine, cerebrospinal fluid, as well as in tissues, are frequently encountered in patients with oxphos deficiencies, especially in children and in those with the most severe biochemical defects. serum biomarkers for mids include lactate, pyruvate, amino acids, creatine kinase, and possibly serum creatine [18, 27 ]. the ratio of lactate to pyruvate is sometimes raised, especially in children and in adults with encephalomyopathies, but is frequently normal in patients with progressive myopathies. creatine kinase concentrations in serum are occasionally increased in people with mitochondrial diseases, mostly in the disorders that cause muscle damage. the sensitivity of fgf-21 in identifying mitochondrial disorders with muscle involvement compared to those for lactate, pyruvate, lactate to pyruvate ratio, and creatine kinase suggest that the measurement of fgf-21 in serum by elisa would be a useful first - line test in patients with suspected rcds. recent evidence has indicated that there is a direct association between daily physical activity and serum fgf-21 levels. there is also a positive association between mitochondrial myopathy and increased fgf-21 levels in the sera of mice. single rc - deficient muscle fibers induce the expression of the metabolic regulator fgf-21, with increased levels also seen in mouse plasma. these results strongly suggest that fgf-21 induction is a muscle - specific response, up - regulated especially in rc - deficient fibers, and that fgf-21 expression increases upon the disease progression, suggesting that their induction is related to the pathogenesis. suomalainen presented a major step forward in the investigative options for patients with mitochondrial rcds. they have focused their research on finding a biomarker to identify mitochondrial disease. in a mouse model with mitochondrial myopathy, they found that fgf-21 was induced in muscles and that concentrations in the serum were raised. fgf-21 in serum seems to be a sensitive and specific quantitative biomarker for muscle pathology in a wide range of mitochondrial disorders in adults and children, including deficiencies in one or more of the respiratory chain enzyme complexes in the skeletal muscle. fgf-21 concentrations in serum seemed to increase with increasing clinical severity of the mitochondrial disease and muscle pathology. tyynismaa and colleagues detected a threefold increase of fgf-21 levels in deletor mice plasma, compared to controls. these results raised an interesting possibility that fgf-21 could be secreted from the diseased muscle fibers upon rc deficiency. rcd of single or multiple complexes resulted in high concentrations of fgf-21 in serum, including complex i deficiency, which is the most common form. respiratory chain defects induce fgf-21 expression in mouse skeletal muscle, which leads to raised concentrations in serum. systemic availability of fgf-21 could play a role in the metabolic alterations that are often are associated with mitochondrial diseases. our data showed that serum fgf-21 levels in patients with primary mids were significantly higher than in control subjects, but showed no significant difference between patients with fa (as a secondary mid) than control subjects. among these primary mids these results support the role of fgf-21 as a key regulator of metabolism in humans, and suggest that serum fgf-21 levels are associated with mitochondrial diseases and can be potentially used as a biomarker for primary mids. although fgf-21 concentrations in serum were useful to differentiate patients with primary mids from healthy controls, fgf-21 concentrations were unable to diagnose fa in patients. this study provides the first clinical demonstration of the associations of serum fgf-21 levels with both primary mids and fa in humans.	mitochondrial respiratory chain deficiencies are a group of more than 100 disorders of adults and children, with highly variable phenotypes. the high prevalence of mitochondrial disorders (mids) urges the clinician to diagnose these disorders accurately, which is difficult in the light of highly variable and overlapping phenotypes, transmission patterns and molecular backgrounds. fibroblast growth factor 21 (fgf-21) is an important endocrine and paracrine regulator of metabolic homeostasis. the fgf-21 transcript is reported to be abundantly expressed in liver, but little is known about the regulation of fgf-21 expression in other tissues. fgf-21 could play a role in the metabolic alterations that are often associated with mitochondrial diseases. the aim of this study was to show the association of the fgf-21 biomarker with human primary mids and secondary mids in suspected patients in iran. serum fgf-21 levels were determined using elisa in 47 mitochondrial patients, including 32 with primary mids, 15 patients with friedreich ataxia as a secondary mid and 30 control subjects. serum fgf-21 levels were significantly higher in subjects with the primary mids (p < 0.05), compared to subjects without mids. however, serum fgf-21 levels did not show significant increase in subjects with fa as a secondary mid. there is an association between increasing concentrations of fgf-21 with mitochondrial diseases, suggesting fgf-21 as a biomarker for diagnosis of primary mids in humans. however, this biomarker is not appropriate for the diagnosis of fa.
gastroenteritis is the most common manifestation of human salmonella infections, especially the infection caused by salmonella typhimurium and salmonella enteritidis serotypes. epidemiological studies in different countries have shown an increase in salmonella enteritidis infections during recent years. according to the who, since 1990, salmonella enteritidis has been considered the most common cause of gastroenteritis worldwide, whereas previously, salmonella typhimurium had been the main cause of salmonella gastroenteritis. in most cases, salmonella gastroenteritis is self - limiting and usually heals without the need for antibiotic therapy, but it may progress to systemic diseases such as bacteraemia, meningitis and endocarditis, especially in patients with an immunodeficiency or neoplastic disease that are taking antibiotics and immune suppressor drugs. pregnant women and the elderly are more sensitive to salmonella infections. for effective epidemiological surveillance and control of salmonella species, many traditional ways of typing, such as biochemical and serological characterizations, are tedious and expensive with limited epidemiological value. moreover, these methods do not have an adequate discriminatory capacity for strains with identical serotype or biochemical characteristics. different molecular methods have been used by researchers for genotyping enteric bacteria, among which enterobacterial repetitive intergenic consensus (eric) -pcr is a powerful method for dna fingerprinting. in this method, repeated oligonucleotides are used as a starter of dna synthesis when there is no need for information on the target dna sequence, which makes it a powerful method with general applications. this method is faster, simpler and more economical than other genomic typing methods. in recent years, salmonella enteritidis has become a major public health problem, and the main cause of both human and animal salmonellosis in many countries. to analyse this problem, the objectives of this study were to determine the extent of genetic variation and clonality among food and clinical strains of salmonella enteritidis by examination of susceptibility to common antibiotics, plasmid analysis and eric - pcr patterns. in addition, we investigate the prevalence of salmonella enteritidis in children s stool samples. a total of 30 salmonella enteritidis isolates, including 15 isolates from humans and 15 from food, were used during this study. the human isolates were collected from 1950 patients with diarrhoea admitted to the children s medical centre hospital in tehran, from april to november 2014. all human isolates were recovered from stool of patients suffering from diarrhoea, except for one isolate which was obtained from the blood of a diarrheal patient. this patient was admitted to the hospital with acute gastroenteritis and salmonella serogroup d was obtained from blood after 3 weeks of initial isolation from stool. diarrhoeal cases were defined as patients with more than three non - bloody episodes of diarrhoea per day. the food isolates were taken from different food sources, including chicken, lamb, beef and duck meats were obtained from the microbiology department of the veterinary faculty, tehran university during the same period of time. the serogroup characterization of isolates was performed via slide - agglutination test with polyvalent and monospecific antisera against somatic and flagella antigens in phase i and ii according to the manufacturer s instructions (difco, franklin lakes, nj, usa). the identity of salmonella enteritidis isolates was confirmed by a multiplex - pcr as described previously. the disk - diffusion method was used to determine the susceptibility of strains as instructed by the clsi guidelines. the antimicrobial agents were as follows : amoxicillin (20 g), gentamicin (10 g), chloramphenicol (30 g), cotrimoxazole (25 g), nalidixic acid (30 g), ciprofloxacin (5 g), cefotaxime (30 g), ceftazidime (30 g), ceftriaxone (30 g), cephalexin (30 g), streptomycin (10 g), tetracycline (30 g), imipenem (10 g), meropenem (10 g), nitrofurantoin (30 g), colistin sulphate (25 g) (mast diagnostics, mast group ltd, merseyside, uk). plasmids were extracted using a qiagen mini plasmid kit (qiagen mississauga, ont, canada). a 0.8% agarose gel was used for plasmid horizontal electrophoresis ; moreover, standard e. coli ac11 strain with 68 kb, 7.2 kb and 1.9 kb plasmids was used as the control. the molecular weight of plasmids was determined by seqaid software (version 3.5, kansas state university, manhattan, ks, usa). one colony of each bacterial strain was purified using a commercial kit (bioneer, seoul, korea) and used as dna template. primer pairs for eric - pcr amplification were as follows : eric-1r (5'-atgtaagctcctggggattcac-3 ') and eric-2 (5'-aagtaagtgactggggtgagcg-3 ').the (gtg)5 amplification was performed using primers described previously. amplification was performed in a reaction mixture containing hot start taqplus master mix 12.5 l, 20 pmol of each primer, 3 l of dna, 2.5 l of pcr buffer and 5 l ultra - pure water (final volume 25 l). eric - pcr amplification was performed using a programme comprising one denaturation step at 95c for 5 min ; four cycles of 94c for 4 min, 53c for 4 min, 72c for 4 min ; 30 cycles of 94c for 30 s, 53c for 1 min, 72c for 1 min ; and one cycle at 72c for 7 min. the (gtg)5 amplification included one denaturation step at 95c for 5 min ; four cycles of 94c for 4 min, 50c for 4 min, 72c for 4 min ; 29 cycles of 94c for 30 s, 50c for 1 min, 72c for 1 min ; and one cycle at 72c for 7 min. gel images were analysed using gel compar ii version 4.0 (applied maths, sint - martens - latem, belgium). the dendrogram was generated according to the unweighted pair group method with arithmetic (upgma) (fig. 1). in general, 26 different salmonella serovars were recovered from children (14 boys and 12 girls). the distribution of salmonella serovars in stool samples was 14 (54%) serogroup d, 6 (23%) salmonella paratyphi c, 2 (8%) s. paratyphi b, 3 (11%) salmonella arizonae and 1 (4%) s. paratyphi a, respectively. all isolates belonging to serogroup d were identified as salmonella enteritidis by multiplex pcr. in the evaluation of 16 antibiotics, seven antibiotic - resistance patterns were observed among clinical and food isolates. the most common anti - biotype was ab1 (43.2%), which was indicative of resistance to nitrofurantoin. the second anti - biotype was ab2 (40.5%) which characterized a nitrofurantoin - nalidixic acid resistance phenotype. we did not observe any resistance to cephalexin, ceftriaxone, ceftazidime and cefotaxime, ciprofloxacin, imipenem or meropenem, chloramphenicol and gentamicin (table 1). plasmid profiling of 30 salmonella enteritidis isolates revealed seven different profiles (p1p7) with one to three bands ranging from 1500 bp to 68 000 bp. two plasmid profiles (p2 and p7) were common among clinical and food isolates ; in addition, 7 (47%) clinical isolates and 12 (80%) food isolates were p7. the second most common pattern was p2 (16.7 %), which included two other plasmids (1.96 kb and 2.6 kb) as well as 68 kb (table 2). genotyping of the 30 isolates showed five different banding patterns, with two major common types (ct1 and ct2) comprising 76.6% of total isolates, each of which consisted of both clinical and food isolates. the ct3 with two members only included clinical isolates with the single blood isolate in this category.. one single type (st1) was also detected, which corresponded to a food isolate. the isolates of duck meat origin were exclusively different from the rest of the samples with single type (st). in addition, this strain was isolated from north of iran and both (gtg)5 and eric - pcr successfully separated the strain as a single type (fig. 2 ; dendrogram 1). this method grouped 21 isolates of different origins (70%) in a single cluster (ct2) ; two other common types (ct1 and ct3) and one single type were also detected. this genotyping method showed more genetic similarity between human, and food isolates (fig. 2 ; dendrogram 2). the discriminatory power of eric - pcr and (gtg)5-pcr was assessed by simpson s diversity index, as presented by hunter and gaston. the predominant serotypes have changed over time and are now various in different geographic locations. in this study, the prevalence of salmonella enteritidis was 54%, which showed the dominance of this serovar in iran. recent reports from brazil, poland, malaysia, china and greece have shown the same serovar as the dominant serovar with the frequencies ranging from 34% to 86%, which reveals a coincident rise of this serovar around the world,,,,. in our study, all isolates were susceptible to cephalexin, ceftriaxone, ceftazidime, cefotaxime and ciprofloxacin. these results indicate that cephalosporins as well as fluoroquinolones are still appropriate drugs that can be used against invasive and systemic infection caused by salmonella. furthermore, all isolates were susceptible to monobactams (imipenem or meropenem), which shows that they can be considered as alternative drugs for clinical cases where more resistance to extended - spectrum antimicrobials is encountered. a previous report from iran showed 100% sensitivity to amikacin, ceftriaxone, xanofloxacin, levofloxacin, norfloxacain and imipenem. in our study, the most frequently antibiotic resistance was observed in nitrofurantoin (100%) and nalidixic acid (73%) among isolates of human origin. the isolates of food origin also showed their highest level of resistance to nitrofurantoin (86.7%) and nalidixic acid (13.3%), but they were significantly different (p < 0.005) from clinical isolates. the observed level of resistance to these two agents among human and food isolates was higher than that reported by previous studies in iran. the data obtained in the present study indicated 73% resistance to this antimicrobial agent. the increased resistance to nalidixic acid among salmonella enteritidis this increase might be due to point mutations in dna gyrase genes (gyra) or to activation of efflux pumps among this bacterial genus. morshed and peighambari showed that 77% of clinical isolates and 24% of food isolates were resistant to nalidixic acid, which is consistent with our results, which showed 73% resistance among clinical isolates and 13.3% among food isolates. among clinical isolates, 73.3% were multiple drug resistance phenotypes and were resistant to at least three or more antibiotic families, while among food isolates, only 20% had multiple resistance patterns. generally, in this study, antibiotic resistance rate was much higher than in food isolates. it seems that improper and indiscriminate use of antibacterial drugs in both human and veterinary medicine without careful evaluation of bacterial sensitivity, has led to a widespread resistance to antimicrobial drugs. horizontal transfer and clonal spread of resistance genes may occur among food - producing animals and humans. plasmid profiling has been used successfully for the typing of salmonella enteritidis strains, but it has some limitations. many salmonella enteritidis strains carry serotype - specific virulence plasmid with different sizes (50100 kb). in our study, all isolates successfully typed by plasmid profiling, and one single large plasmid (68 kb) was common in all the profiles, which can be proposed as a serotype - specific virulence plasmid. morshed and peighambari have reported six plasmid profiles (a f) between 49 salmonella enteritidis isolates of human and animal origin in iran. in their study, liebana. also reported a single 57-kb plasmid among 56% of their salmonella enteritidis isolated from the uk. he showed that the discriminatory power of this method was more than pulsed - field gel electrophoresis (pfge) and less than ribotyping. in addition, the same plasmid patterns can be observed among isolates with different chromosomal characteristics. using eric primers, between 30 strains of salmonella enteritidis isolates four main clusters had 60% similarity. the majority of isolates (23 ; 76%) fell in a major cluster (a) with two common types (95% similarity), while the remaining seven strains were classified into two distinct clusters with one single type. both clinical and food isolates existed in the clusters, which showed different plasmid profiles and antimicrobial resistance properties. the most common type was ct2 (40%), which contained six human isolates and six food isolates, respectively. in this type, food isolates had p7 plasmid patterns and resistance phenotypes (ab1), but the human isolates had different plasmid patterns and resistance phenotypes. the ct3 had two clinical members with identical resistance phenotypes (ab2) and plasmid patterns (p5) ; however, they had different origins (stool and blood). the result shows the importance of the treatment and control of salmonella infections in high - risk individuals, especially for children, which can lead to systematic infections. food isolates of duck meat origin were exclusively different from the rest of the samples with single type (st). this strain was isolated from the north of iran and both (gtg)5 and eric - pcr successfully separated the strain as a single type. the (gtg)5 analysis indicated ct2 as the most common type (70%) with 21 clinical and food isolates falling in this group. the diversity index of population was obtained to be 0.48 and 0.70 by (gtg)5 and eric - pcr analysis, respectively, which indicates the higher discriminatory power of the eric - pcr method. using eric - pcr, suh and song showed a high genetic homogeneity among 22 salmonella enteritidis isolates of human and chicken origin in south korea between 2001 and 2002. furthermore, several investigators have shown good discriminatory power and typing capacity of eric - pcr for salmonella enteritidis isolates from human and food origin,. in the study of campioni. performed with 128 salmonella enteritidis isolates of human and food origin over a 24-year period in brazil, eric - pcr and pfge exhibited a high genetic similarity, pfge being considered the reference standard method for the typing of salmonella enteritidis strains. have shown that eric - pcr exhibited a higher discriminatory power than pfge. in conclusion, salmonella enteritidis was phenotypically and genotypically homogeneous and was clonally dispersed among food and human populations in our study. these clones may continue to exist over a considerable period of time in tehran and spread in different time occasions. this supports the notion that infected animals and humans are important sources of contamination in the environment and the food chain. using combined genotyping techniques and greater number of isolates makes it more possible to trace the origin of infections in epidemiological investigations.	in recent years, salmonella enterica serovar enteritidis has been a primary cause of human salmonellosis in many countries. the major objective of this study was to investigate genetic diversity among salmonella enteritidis strains from different origins (food and human) by enterobacterial repetitive intergenic consensus (eric) -pcr, as well as to assess their plasmid profiling and antimicrobial resistance. a total of 30 salmonella enteritidis isolates, 15 from food samples (chicken, lamb, beef and duck meats) and 15 from clinical samples were collected in tehran. identification of isolates as salmonella was confirmed by using conventional standard biochemical and serological tests. multiplex - pcr was used for serotyping of isolates to identify salmonella enteritidis. antimicrobial susceptibility testing to 16 agents founds drug resistance patterns among salmonella enteritidis isolates. no resistance was observed to cephalexin, ceftriaxone, ceftazidime and cefotaxime, ciprofloxacin, imipenem or meropenem, chloramphenicol and gentamicin. the highest resistance (96.7%) was observed to nitrofurantoin. seven plasmid profiles (p1p7) were detected, and a 68-kb plasmid was found in all isolates. two different primers ; eric and (gtg)5 were used for genotyping, which each produced four profiles. the majority of clinical and food isolates fell into two separate common types (cts) with a similar percentage of 95% by eric - pcr. using primer (gtg)5, 29 isolates incorporated in three cts with 70% of isolates showing a single banding pattern. limited genetic diversity among human and food isolates of salmonella enteritidis may indicate that contaminated foods were possibly the source of human salmonellosis. these results confirmed that eric - pcr genotyping has limited discriminatory power for salmonella enteritidis of different origin.
isocitrate dehydrogenase (idh) is a member of the -decarboxylating dehydrogenase family of enzymes and catalyzes the oxidative decarboxylation of 2r, 3s - isocitrate to yield 2-oxoglutarate (-kg). somatic mutation of idh1 at the locus of r132 (idh1) occurs in > 70% of who grade ii - iii gliomas and secondary glioblastomas. acute myeloid leukemia (aml) is the only non - central nervous system (cns) tumor in which a substantial percentage of the mutations are present as well. in other cancer types mutation of idh1 a mutation - specific antibody was raised against the mutated enzyme (anti - midh1) and immunostaining is becoming an alternative screening tool for the presence of the mutation (dianova gmbh, germany) to be applied prior to dna sequencing. in immunostained glioma sections we discovered cells with microglial features which appeared to be positive for the mutated idh1. following up on this observation we initiated the present investigation aiming at proving the microglial lineage of these immunopositive cells. microglial cells are the resident macrophages participating in the active innate immune defense in the cns. phagocytosis, as a mechanism of innate immune defense, is typically inhibited in gliomas. because microglial cells largely arise from bone marrow they share functional and physical properties with cells of myeloid origin and the common origin more importantly, human glioma - infiltrating microglia / macrophages (gims) are known to substantially contribute to the tumor mass (fig. 1a). in the present study we specifically explored whether microglial cells / macrophages residing in glioma samples share the idh1 mutation with glial tumor cells. (a) gims as labeled by cd68 significantly contribute to the cellular components of a glioma. (e) gims double positive for cd68 and mutant idh1 are shown in overview. (f) gims double positive for iba1 and mutant idh1 are shown in overview. (g) gims double positive for cx3cr1 and mutant idh1 are shown in overview. the mutation - specific antibody enables robust detection of the mutation in routine biopsy samples. the specificity of the antibody is confirmed by specifically staining the idh1 mutant cells only while not cells in normal or non - neoplastic brains (fig. 1k) as identified by double positive cells of midh1 and glial fibrillary acidic protein (gfap, the classical marker for astrocytes), tumor samples generally contain reactive glial cells, vascular cells and phagocytic elements such as macrophages and microglia. we extended our investigations with a cohort of 60 patients with gliomas in which idh1 mutation had previously been determined by dna sequence analysis. the mutation was also confirmed at the protein level by using the anti - midh1 antibody for each case (fig. a cohort of control brains including ten non - neoplastic autopsy brains and resection specimens of six arteriovenous malformation (avm) were included in this study, and none of these controls appeared to be positive for the anti - midh1 (fig., we used cd68, iba1 and cx3cr1 as markers for microglial cells in double immunofluorescence labeling experiments with the anti - midh1. initially, we randomly counted 200 cd68 microglial cells in non - overlapping regions in each sample and found 26 ~86% cd68 microglial cells to be immunopositive for the mutant idh1 (fig. 1e and h). to further confirm the lineage of these cells we used additional microglial markers (iba1 and cx3cr1) in 25 samples, and found 16 ~68% iba1 (fig. 1f and i) and 20 ~56% cx3cr1 (fig. 1 g and j) microglial cells to be immunopositive for the mutant idh1. morphological signs of phagocytosis are largely not observed in cd68, iba1 and cx3cr1 microglia / macrophages. the findings demonstrate that part of the idh1 mutant cells are either true microglial cells or fusions of glial tumor cells and microgalia / macrophages. tumor progression of gliomas depends on both cellular proliferation and migration which are mutually exclusive phenomena at the level of individual cells. microglial cells fit well into the group of highly migratory cells (invasive tumor component) which display little mitotic activity. indeed, the large majority of mutant idh1 cells did not co - express the cell proliferation marker ki67 (fig. these non - glial cells harbour genetic aberrations of the tumor cells, reminiscent of tumor macrophage hybrids which have been supposed to be the origin of human metastatic cancers by fusogenicity. such fused cells would combine the migratory potential of macrophages and the proliferative potential of the tumor cells. we also examined the expression pattern of wild type idh1 (wt - idh1) in control brain samples and wt - idh1 is largely detected in neurons and microglia / macrophages (fig. it seems that the expression of wt - idh1 in normal or non - neoplastic brain is restricted to cells with high energetic demands, and the expression seems to be induced in other cells under pathological situations where cellular energy requirements have increased. (d to f) wtidh1 is expressed in microglia / macrophages present in samples of arteriovenous malformations (avm) (arrows). in summary, our findings demonstrate that the cell group of microglial cells / macrophages is an important target for idh1 mutation in glial tumors. the findings suggest that blood borne cells like microglia / macrophages serve as an additional tumor component contributing in particular to the tumor infiltrative behavior. all samples were known with idh1 mutation at locus r132h determined by dna sequence analysis. control brain samples were from autopsy brains without cns diseases (n = 10) and arteriovenous malformations (avm, n = 6). antibodies specifically targeting the idh1 r132h mutation (1 : 250 ; dianova gmbh, germany) ; cd68 (1 : 150 ; abbiotec) ; iba1 (1 : 100 ; bioss inc.) ; cx3cr1 (1:100 ; sigma - aldrich) ; gfap (1 : 50 ; dako), ki67 (1:100 ; dako) and wild type idh1 (wt idh1) (1 : 100 ; epitomics) were used for this study. corresponding secondary antibodies conjugated with ap, fitc and cy3 were used. the procedures for immunohistochemistry, double immunofluorescent staining and clsm were performed as previously published. all samples were known with idh1 mutation at locus r132h determined by dna sequence analysis. control brain samples were from autopsy brains without cns diseases (n = 10) and arteriovenous malformations (avm, n = 6). antibodies specifically targeting the idh1 r132h mutation (1 : 250 ; dianova gmbh, germany) ; cd68 (1 : 150 ; abbiotec) ; iba1 (1 : 100 ; bioss inc.) ; cx3cr1 (1:100 ; sigma - aldrich) ; gfap (1 : 50 ; dako), ki67 (1:100 ; dako) and wild type idh1 (wt idh1) (1 : 100 ; epitomics) were used for this study. corresponding secondary antibodies conjugated with ap, fitc and cy3 were used. the procedures for immunohistochemistry, double immunofluorescent staining and clsm were performed as previously published.	somatic mutation of isocitrate dehydrogenase 1 (idh1) at the locus of r132 (idh1r132h) occurs in > 70% of who grade ii - iii gliomas and secondary glioblastomas. to date it remains unknown whether the mutation is restricted to glial tumor cells. microglial cells are the resident macrophages in the central nervous system. tumor - infiltrating microglial cells / macrophages are major stromal cellular components of malignant gliomas and substantially contribute to the tumor mass. differential identification of the idh1r132h mutant cellular components is of particular importance for understanding of the mutation - associated tumor biology. here we discovered that a significant portion of cd68 +, iba1 +, cx3cr1 + microglial cells / macrophages also harbor the idh1r132h mutation. the findings provide novel insights for understanding the mutation - associated tumor biology relevant to clinical applications as a predictive and/or prognostic marker or therapeutic target.
insulin autoimmune syndrome (ias) was initially reported by hirata. in 1970 and is characterized by serious hypoglycemia and high levels of blood insulin and insulin autoantibodies in the absence of exogenous insulin administration. the cause of this disease is unclear, but insulin autoantibody formation is considered to be associated with autoimmune diseases such as graves disease, rheumatoid arthritis, or drugs containing sulfhydryl compounds, mostly penicillin, d - penicillamine, or methimazole. ias occurs frequently in east asian countries such as korea and japan and is associated with a genetic predisposition. -lipoic acid (ala) is a sulfhydryl - containing compound used to treat diabetic peripheral neuropathy and was first reported as the cause of ias in 2003. most of the cases were reported in japan, and only one case has been reported in korea. herein, we report a case of ias during which hypoglycemia occurred three times after associated ala treatments. a 67-year - old female visited the outpatient clinic of our institution complaining of numbness and coldness in both feet. she has been diagnosed with type 2 diabetes 5 years prior and was being treated with a sulfonylurea (gliclazide 30 mg). based on the examination results, she was diagnosed with diabetic peripheral neuropathy, and thioctacid (600 mg) was prescribed. she experienced repeated occurrences of hunger, hand tremor, cold sweat, and dizziness 3 to 4 hours after a meal since having taken the drug, but symptoms consistently improved after eating snacks. her past history was insignificant except an appendectomy 40 years prior. on physical examination, her height was 165 cm and body weight 60 kg. blood examination showed leukocyte count 4,700/mm, hemoglobin 13.0 g / dl, platelet 202,000/mm, blood urea nitrogen 17.0 mg / dl, cr 0.7 mg / dl, total protein 7.1 g / dl, albumin 4.6 g / dl, aspartate aminotransferase 26 iu / l, alanine aminotransferase 36 iu / l, alkaline phosphatase 116 iu / l, total cholesterol 166 mg / dl, triglyceride 115 mg / dl, high density lipoprotein 39 mg / dl, calcium 8.8 mg / dl, phosphate 3.3 mg / dl, sodium 135 meq / l, and potassium 4.2 meq / l. free t4 was 1.12 ng / dl (normal range, 0.93 to 1.7), thyroid stimulating hormone 2.25 iu / l (normal range, 0.27 to 4.2), adrenocorticotropic hormone 16.5 pg / ml (normal range, 6 to 56.7), and cortisol 13.4 g / dl. on admission, hemoglobin a1c (hba1c) was 6.6%, fasting blood glucose 208 mg / dl, and postprandial blood glucose 195 mg / dl. serum insulin, when measured by radioimmunoassay (ria, immunoradiometric assay, insulin - irma, biosource europe, nivelles, belgium) was normal at 15.33 and 19.41 iu / ml on fasting and postmeal, respectively (normal range, 2 to 25). however, c - peptide (immunoradiometric assay, c - peptide irma, izotop, budapest, hungary) was increased to 13.96 ng / ml on fasting and 18.24 ng / ml after meals (normal range, 1.07 to 3.51). sulfonylurea administration was discontinued, but the hypoglycemic symptoms persisted. after eating carbohydrate snacks between meals, daytime frequency of hypoglycemia decreased, but hypoglycemia with a level less than 50 mg / dl persisted at dawn with prolonged fasting. a 72-hour fasting test was attempted but was ended after 6 hours because the patient complained of severe hypoglycemia with a blood sugar level of 44 mg / dl. during a 6-hour examination, the blood sugar levels were 90, 70, and 44 mg / dl at baseline, 4 and 6 hours, and the serum insulin values measured by ria were within the normal range at 22.92, 22.16, and 27.47 iu / ml, respectively. the c - peptide values were increased to 15.60, 20.31, and 15.65 ng / ml, respectively. the antinuclear antibody was negative, while the insulin autoantibody value (ria, cobra 5010, biosource europe) was very high at 53% (normal range, < 7). because the patient continued to suffer from fasting hypoglycemia after suspending sulfonylurea administration, and serum c - peptide and insulin autoantibody levels increased, she was diagnosed with ias. as the sulfhydryl - containing thioctacid first administered 2 weeks before the occurrence of hypoglycemia, it was regarded as the causal factor and discontinued. prednisolone (10 mg) administration reduced the frequency of hypoglycemia and was able to be discontinued after 2 months because recovery was observed. at 4 months after diagnosis, the insulin autoantibodies were still high at 80.4%, but no signs of hypoglycemia were evident. at the 2-year follow - up, the patient visited the rehabilitation department of another hospital to treat diabetic peripheral neuropathy 2 years after the initial occurrence and was prescribed thioctacid. hypoglycemia recurred 10 days later, for which she visited our hospital. at that time, the patient was treated with voglibose (0.6 mg). the fasting plasma glucose was 98 mg / dl and the 2-hour plasma glucose after glucose load was 135 mg / dl, but serum insulin levels measured by enzyme - linked immunosorbent assay (elisa, roche elecsys insulin test, roche diagnostics, mannheim, germany) were increased to more than 1,000 u / ml for both fasting and 2 hours posttest. fasting and 2-hour posttest c - peptide values were 10.91 and 16.92 ng / ml, respectively. after taking prednisolone (10 mg) the dose of prednisolone was tapered by 5 mg and was discontinued 2 months later. before discharge, six months after discharge, most of the values were normalized, i.e., hba1c 5.7%, serum insulin 267 u / ml, and c - peptide 3.71 ng / ml, but insulin autoantibodies remained high at 78% without hypoglycemia. the patient was again prescribed thioctacid 2 years after the second occurrence, for which she revisited our hospital due to the recurrence of hypoglycemia. at that time her serum insulin level based on elisa was abnormal, measuring higher than 1,000 u / ml, while c - peptide was 21.06 ng / ml and insulin autoantibodies were 96%. according to the test results, the patient was prescribed prednisolone (5 mg), and after 4 months, serum insulin (measured by elisa) and c - peptide were reduced to 210 u / ml and 2.81 ng / ml, respectively, but the insulin autoantibodies remained high (88.9%). although the specific mechanism of ias remains unclear, insulin autoantibodies are assumed to combine with secreted insulin as blood glucose increases after meals to inhibit insulin action and further promote the secretion of insulin. excessive insulin combined with the autoantibody is dissociated as blood glucose is reduced and eventually induces hypoglycemia. in japan, over 200 cases of ias - related hypoglycemia have been reported ; in most cases, the patients were taking a sulfhydryl medicine 4 to 6 weeks before the occurrence of hypoglycemia. nevertheless, several cases have been reported in which symptoms were initiated in the absence of any specific drugs or sulfhydryls such as tolbutamide, diltiazem, loxoprofen sodium, and interferone-. a genetic predisposition may influence the occurrence of ias, showing a strong correlation with hla class ii and ias is frequently observed in patients with a specific hla allele. when considering ias cases in japan, 97% of patients were hla - dr4-positive and 43% were also drb1 0406-positive. additionally, dqa1 0301 and dqb1 0302 alleles were frequently found in these patients. the drb1 0406 molecule has a strong affinity to the amino acid ile - leu - gln motif, which is contained in the insulin chain. 17 (tsicslyqle) of the insulin chain has a strong affinity for drb1 0406, and this peptide has been shown to stimulate the t cells of drb1 040 6-positive patients. sulfhydryl drugs promote insulin s - s bonding dissociation and expose the peptide to the antigen - presenting cell to stimulate the t cells of drb1 0406-positive patients, resulting in the formation of insulin autoantibodies. in particular, japanese and koreans are known to have a higher hla - drb1 0406-related morbidity rate than caucasians, which is considered the main reason for the high incidence of ias in the japanese and korean populations. in the present case, ala is a relatively safe compound associated with a strong reductive reaction and thus is commonly used to treat peripheral neuropathy in diabetic patients. ala acts as a coenzyme related to oxidative decarboxylation of pyruvic acid and -ketoglutal acid in the mitochondria. oral ala contains two sulfur atoms and it is reduced to dihydrolipoic acid containing sulfhydryl which decreases the oxidative stress generated in peripheral cells. the two sulfhydryls in the dihydrolipoic acid dissociate the disulfide bond of the insulin molecule to form insulin autoantibodies. after the year 2000, ala began gaining public attention as a dietary supplement in japan, therefore becoming the main cause of ias, following methimazole. ias caused by ala has been reported a total of 20 times since first reported in japan in 2003 ; 18 cases in japan, one case in italy, and one case in korea (table 2). previously, hypoglycemia due to ias was mostly observed in patients without diabetes and was easily detected. however, ala is a compound frequently used for the treatment of peripheral neuropathy in diabetic patients. if a diabetic patient is determined to have hypoglycemia, oral hypoglycemic agent, or exogenous insulin administration is first suspected to be the cause. therefore, when a diabetic patient presents with ias following ala intake, the possibility of ala - induced hypoglycemia must be considered, otherwise the diagnosis may be delayed or the causative drug may be readministered. in the present case, the patient was instructed to stop taking ala after being diagnosed with ala - induced ias. however, this patient was twice more prescribed ala and experienced hypoglycemia that continued for several weeks after intake. among the 21 cases of ias caused by ala, this is the only case in which ias recurred due to repeated administration of ala. because koreans and japanese individuals significantly express the hla dr4 allele relevant to ias, if a korean diabetic patient taking ala has spontaneous hypoglycemia without any specific cause and has high levels of blood insulin and insulin antibodies, the physician should consider ala - induced ias. in the present case, the serum insulin value when the patient was initially hospitalized was 15.33 iu / ml before meals and 19.41 iu / ml after meals, both of which were within the normal range. however, after the second and third occurrences, the serum insulin measured over 1,000 iu / ml, well above the normal range. the initial serum insulin value was low, possibly because the insulin was measured using ria. in this method, the antibodies fixed to the test tube may be saturated with insulin when the serum insulin is too dense, and unbound insulin and signal antibodies may be combined, leaving only a small amount of signal antibodies after washing, thus causing an underestimation of the insulin level, referred to as the high - dose hook effect. namely, the highly concentrated insulin in the blood was assumed to be underestimated due to the hook effect when using ria during the patient 's first hospitalization. this possibility was overlooked at her first presentation, and we could not re - examine diluted sample or measure by other method like elisa. on the other hand, we used elisa to measure the insulin level on the second and third examination and confirmed a high insulin concentration in the blood. a majority of ias patients (80%) recover from hypoglycemia in 1 to 3 months. however, several patients have reported experiencing hypoglycemia for more than a year. to treat hypoglycemia, increasing the frequency of meals is helpful, but a steroid or -glucosidase inhibitor may be necessary. reportedly, azathioprine, 6-mercaptopurine, or plasmapheresis has also used in some cases. in the present case, snack intake between meals and oral steroid administration improved hypoglycemia. in summary, a 67-year - old female patient with diabetes visited our hospital for diabetic peripheral neuropathy and began taking ala but experienced hypoglycemia after 2 weeks. because there was no history of exogenous insulin administration and the serum insulin and insulin autoantibody levels we advised her to not take ala ; however, she was twice more prescribed ala at other hospitals to treat her diabetic peripheral neuropathy, which again provoked hypoglycemia. thus, she revisited our hospital to receive the same treatment as before and recovered both times. since koreans have a high incidence of hla - drb1 0406-related morbidity, and ala is a commonly used medicine to treat diabetic peripheral neuropathy, physicians should consider ias when a diabetic patient taking ala has hypoglycemia without any specific cause.	insulin autoimmune syndrome (ias) is characterized by spontaneous hypoglycemia caused by insulin autoantibodies in the absence of exogenous insulin administration. some drugs containing sulfhydryl compounds are known to initiate the onset of ias. a 67-year - old female who had diabetes for 5 years visited the outpatient clinic at our institution due to diabetic peripheral polyneuropathy. she was prescribed -lipoic acid (ala), which contains two sulfur atoms. two weeks later, she complained of recurrent hypoglycemic symptoms. we detected a high level of insulin and high titers of insulin autoantibodies. her human leukocyte antigen (hla) genotype included the drb1 0406 allele, which indicates a high level of susceptibility to ias. she was treated with prednisolone. after this episode, she experienced two more hypoglycemic events after taking ala for diabetic neuropathy in other hospitals. as ala can be used to treat diabetic peripheral polyneuropathy, physician discretion is advised based on the possibility of ias due to ala in diabetic patients.
most pulmonary arteriovenous fistulae have no symptoms and are detected as an abnormal shadow on the chest radiograph. a definitive diagnosis is made by means of pulmonary arteriography or three - dimensional computed tomography (3d - ct) angiography, although hypervascular lesions can mimic pulmonary arteriovenous fistulae. a pulmonary arteriovenous fistula (pavf) is often associated with various complications, and pregnancy could be a risk factor for these complications because of the increase in the shunt fraction. we describe a rare case of a young female with a pavf treated with video - assisted thoracic surgery (vats). the patient was a 20-year - old asymptomatic female. during a physical examination, no cyanosis, clubbing of the fingers nor skin telangiectasia was detected. abnormal shadows in the right upper and lower lung fields were detected on chest x - rays (fig. chest computed tomography (ct) revealed a 20 14 mm nodule with well - defined margins and smooth contours in the right upper lobe, and a 15 10 mm nodule with a similar form in the right lower lobe (fig. contrast - enhanced 3d - ct revealed two enhanced lung nodules which were connected with linear structures suggestive of feeding arteries and drainage veins, respectively (fig. although an arterial blood gas analysis showed no hypoxemia, with an arterial oxygen pressure (pao2) of 80 mm hg on room air, the right - to - left shunt fraction was 15.3% and abnormal uptake was detected in the brain and bilateral kidneys by the lung perfusion scintigraphy (fig., the patient was placed in the left lateral decubitus position at the first time, and the right lung was deflated. intraoperatively, pulmonary nodules were not palpable in the right upper and lower lobes, and there was no significant bruit. a nodule, which was non - tortuous in shape and covered with the visceral pleura, was detected in the right lower lobe (fig. the location of the other nodule in the right upper lobe was predicted based on the blood vessel. the histopathological findings of hematoxylin eosin stained sections revealed small and medium - sized vascular channels composed of arteries with mild and irregularly thickened muscle walls, and juxtaposed or seemingly anastomosing dilated veins (fig. 3). based on these findings, a diagnosis of pulmonary arteriovenous fistula was confirmed. it was difficult to preoperatively diagnose the pavf, because hypervascular lesions such as those due to inflammatory changes, can also present as strongly enhanced nodules after injection of contrast material. abnormal uptake in the brain and bilateral kidneys by the lung perfusion scintigraphy suggested a right - to - left shunt. the patient had an uneventful postoperative course and was discharged seven days after the operation. pavf is a relatively rare disease, which is considered to occur at a frequency of 0.02%. most of pulmonary arteriovenous fistulae are congenital and considered to be abnormal developments of the capillaries, and cases associated with rendu - osler - weber have also been reported. our case had neither a family history of rendu - osler - weber nor any symptoms. the causes of acquired or secondary pulmonary arteriovenous fistulae include chest trauma, thoracic surgery, hepatic cirrhosis, infections (actinomycosis, schistomiasis), metastatic carcinoma and systemic disease. this case had none of these causes, so we thought that her pavf might have been congenital. it was previously reported that when a single isolated pulmonary arteriovenous fistula is 2 cm or smaller, there are no significant symptoms appear. generally, when a pavf is 2 cm or larger, symptoms such as breathing difficulty, cyanosis, hypoxemia, finger clubbing and polycythemia, occur. therefore, treatment is considered to be required in most cases with a pavf larger than 2 cm in size. when the right - left shunt is 2030% or greater, the rate of occurrence of severe complications such as rupture of the fistula, hemoptysis, cerebral infarction and cerebral abscess, is considered to be approximately 30%. although the right - left shunt of this case was 15.3%, abnormal uptake was detected in the brain and bilateral kidneys by the lung perfusion scintigraphy. the existence of the right - left shunt was obvious, so it was thought that the present case was at high risk of the complications associated with pavf. moreover, pavf expand during pregnancy because of increases in the blood volume, cardiac output, and venous distensibility. females with known pavf should be maximally treated prior to becoming pregnant, and should be alert to the potential for complications due to the pavf during pregnancy. therefore, because our present patient was a young female, we thought that treatment should be recommended in this case in the event she might later choose to become pregnant. the abnormal uptake of the brain and bilateral kidneys in the lung perfusion scintigraphy disappeared after the surgical treatment. the excision was highly successful for the two ipsilateral isolated fistulae, and mortality rate is low in this surgery. embolization is an appropriate treatment modality in cases with multiple pulmonary arteriovenous fistulae which are not suitable for surgery. the complications of embolization include pleuritic chest pain, pulmonary infection, air embolism, the migration of coils and paradoxical embolism. therefore, surgical excision was preferable in the present case given her prognosis. pulmonary angiography or contrast - enhanced 3d - ct has been the standard diagnostic tool for pulmonary arteriovenous fistulae. however, it has limitations in detecting pulmonary arteriovenous fistulae presenting as small nodules and enhanced nodules. in this case, contrast - enhanced 3d - ct was effective for the diagnosis and lung perfusion scintigraphy was also supportive to obtain evidence of the right - to - left shunt fraction. in conclusion, surgical resection using vats for cases with a limited number of ipsilateral isolated pulmonary arteriovenous fistulae is therefore recommended due to its safety and the low recurrence and mortality rates. written informed consent was obtained from the patient for publication of this case report and accompanying images. a copy of the written consent is available for review by the editor - in - chief of this journal on request.	introductionwe herein describe a rare case of a pulmonary arteriovenous fistula (pavf).presentation of casethe patient was a 20-year - old asymptomatic female, admitted to our hospital because of an abnormal shadow in the right lung field on chest x - rays. chest computed tomography (ct) revealed two nodules with well - defined margins in the right upper and lower lobes. contrast - enhanced three - dimensional ct (3d - ct) revealed two enhanced solitary lung nodules which were connected with linear structures suggestive of feeding arteries and drainage veins, respectively. based on these findings, we made a preoperative diagnosis of pavf. we performed partial pulmonary resection of the right upper and lower lobes by video - assisted thoracoscopic surgery (vats). the histopathological findings revealed small and medium - sized vascular channels composed of arteries with mild and irregularly thickened muscle walls and juxtaposed or seemingly anastomosing dilated veins. based on these findings, a diagnosis of pavf was confirmed. the patient had an uneventful postoperative course.discussiona pavf is often associated with various complications, and pregnancy could be a risk factor for these complications because of the increase in the shunt fraction. females with known pavf should be maximally treated prior to becoming pregnant as complications of pavf during pregnancy can have devastating consequences. therefore, we thought that treatment should be recommended in this case in the event she might later choose to become pregnant.conclusionsurgical resection using vats for a limited number of ipsilateral isolated pulmonary arteriovenous fistulae is recommended due to its safety, low recurrence and low mortality rate.
malaria is a life threatening disease caused by a parasite that is transmitted to humans by the female anopheles mosquito. it remains a major public health problem, particularly in africa where over 90% of cases are recorded. it is estimated that 214 million new cases and 438 000 deaths occur worldwide yearly. most of those who die from malaria are children under the age of 5, and most of these children live in sub - saharan africa. in africa, malaria can be prevented by taking antimalarial drugs and by controlling the vectors that transmit the disease. this can be done in many ways such as spraying larvicides in the development sites of mosquitoes to kill larvae ; household residual spraying (irs) of insecticides and repellents to prevent mosquito bites. despite the efforts to combat malaria through vector control and treatment using antimalarial drugs, malaria remains a major problem in sub - saharan africa primarily due to drug and insecticide resistance and socioeconomic underdevelopment. the roll back malaria strategy recommends a combination of interventions for malaria control. who (2015) recommends vector control using insecticide treated bed nets (itns) as one of the best ways to prevent malaria. itns offer a double protection to those who sleep under nets and to persons who do not use nets by reducing the number of malaria infected mosquitoes in the community. recently pyrethroid treated long lasting insecticide nets (llins) were produced, which can last for at least 4 years before replacement. a llin is a factory - treated mosquito net made with netting material that has insecticide incorporated within or bound around the fibres. the net must retain its effective biological activity without retreatment for at least 20 who standard washes under laboratory conditions and three years of recommended use under field condition. llins have been associated with a sharp decrease in malaria in countries where malaria programmes have achieved high llin coverage. since adoption of itns as a preventive tool for malaria in 2002, several campaigns of free distribution of llins have been conducted all over the country, with priority given to pregnant women and children below five years. however, a study carried out in cameroon in 2012 suggested that 59.7% of households possessed at least one net while only 42.6% of households slept under these nets. the objective of the ministry of public health was to have 80% of the population sleeping under llins by 2015. in 2011, cameroon and its health partners distributed over eight million llins in all the ten regions of the country through the various health districts in an effort to reduce the burden of malaria in the country. the tombel health district (thd) in cameroon has abundant rainfall which entertains numerous mosquito larval developmental and adult resting sites. the quality of housing (mostly plank with no ceiling and screens on doors or windows) in the area exposes people to intense mosquito bites and makes them vulnerable to malaria attack. however, no information is available on the impact of the intervention (free distribution of llins) on malaria morbidity in this region, especially as the country has embarked on a second mass distribution campaign. this study therefore sought to investigate the effect of llins use on malaria prevalence in south west cameroon using tombel health district (thd) as a case study. thd found in the kupe muanenguba division is a cosmopolitan inhabited by people from different ethnic groups. it is made up of 7 health areas, baseng, ebonji, nyasoso, edibenjock, ndom, tombel, and ndibenjock. three of these health areas, tombel, nyasoso, and ebonji, were selected for the study by purposive sampling because of the high population in these areas and also because only health facilities in these health areas had trained laboratory personnel to run laboratory tests. the climate is warm and humid with 2 main seasons, long rainy (8 - 9 months) and dry (3 - 4 months) seasons, which induce abundant vegetation. a retrospective study was carried out using hospital records obtained from the tombel district hospital, presbyterian general hospital nyasoso, and integrated health centre ebonji for the period january 2010december 2013. because of lack of data collected before the distribution of llins in the thd, records for the two years before the intervention were compared to those of two years after distribution. before the start of the study, the study site was surveyed using our data quality assessment tools which tested mainly for completeness and accuracy of data ; thus, the study involved only health facilities that met the inclusion criteria used that included the following : patient must have done microscopic test to confirm malaria.the age and sex must be registered.the data for the study period must be available.a total of 31,657 patient records were consulted from the 3 health centers, and information on the number of inpatients and outpatients confirmed for malaria was retrieved for the pre- (2010 and 2011) and postdistribution (2012 and 2013) periods of llins. patient must have done microscopic test to confirm malaria. the age and sex must be registered. the kruskal - wallis test and chi square test were used to determine and compare malaria prevalence where necessary and a p value 5 yrs, and pregnant women) (table 2). malaria prevalence was higher in males (25.37%) than in females (23.37%) during the study period (table 3) but the difference was not statistically significant. in relation to seasonal transmission of malaria, the months of june august popularly known as the rainy season in cameroon had the highest percentage of malaria cases (32.7%). malaria is a major public health problem in cameroon and is responsible for 31% of all consultations and 44% of all hospitalizations in health facilities. the health centers chosen for the study used microscopy for diagnosing malaria in their laboratories which is the gold standard set by who for malaria diagnosis. the present study has revealed malaria prevalence of 27.4% during the study period 20102013 in the thd. this result shows that hospital records could be indicative of malaria prevalence in the country since prevalence rates tie almost with those of other studies in cameroon, which used blood smears. however, the malaria prevalence in patients observed in the study was 12% lower than that reported in a similar study conducted in ethiopia by alemu.. the prevalence of malaria in inpatients in the thd during the postdistribution period (13.7%) was lower than that of the predistribution period (19.4%) suggesting that the distribution of llins had an impact in reducing malaria prevalence in the thd. the prevalence of malaria in inpatients in the thd (33.10%) was also higher than the 29.80% reported by ndong., who carried out a similar study in the northwest region of cameroon, and also higher than that reported by a similar study conducted in ethiopia by ferede. one of the reasons could be the fact that the climate of tombel is warm and humid and favours growth of mosquitoes which constantly bite inhabitants as they sleep in their plank houses that most often have cracks in the walls and lack a ceiling that would serve as an additional barrier to protect them from infective bites. the prevalence of malaria obtained from records of the selected period (20102013) was highest in children 5 years category (22.5%). the prevalence of malaria obtained from records of the selected period (20102013) for the children 5 yrs, and pregnant women) and also with seasons. proper education on the importance of utilising llins should be given to inhabitants of the thd by health personnel and community health workers.another llin distribution campaign should be sustained by the cameroon government since the llins that were distributed in 2011 will be worn out and ineffective in preventing malaria by 2015. proper education on the importance of utilising llins should be given to inhabitants of the thd by health personnel and community health workers. another llin distribution campaign should be sustained by the cameroon government since the llins that were distributed in 2011 will be worn out and ineffective in preventing malaria by 2015. we can not attribute the reduction in malaria prevalence in the thd in 2012 solely to the distribution of llins in 2011, because other control measures such intermittent preventive treatment for pregnant women could have also contributed in reducing malaria morbidity in the thd, and our study design could not capture the contribution of each intervention.	malaria remains a major public health problem in africa, and its prevalence in cameroon stands at 29%. long lasting insecticide nets (llins) were distributed in 2011 to reduce malaria mortality and morbidity ; however, assessment of this intervention is scanty. the present study in the tombel health district (thd) investigated the impact of this distribution on malaria prevalence. a total of 31,657 hospital records from 3 health facilities in 3 health areas for 20102013 were examined. records for 2010 and 2011 provided predistribution baseline data, while those of 2012 and 2013 represented postdistribution data. 8,679 (27.4%) patients were positive for malaria. children below 5 years had the highest prevalence (40.7%). the number of confirmed cases was highest from june to august (peak rainy season). malaria prevalence was higher in males (25.3%) than in females (23.2%). malaria prevalence increased in thd from 26.7% in 2010 to 30.7% in 2011 but dropped to 22.7% in 2012 and then increased in 2013 to 29.5%. there was an overall drop in the total number of confirmed malaria cases in 2012 ; this decrease was significant in ebonji (p < 0.001) and nyasoso (p < 0.015) health areas. the distribution of llins led to a short lived reduction in malaria prevalence in thd. llin distribution and other control activities should be reinforced to keep malaria prevalence low especially among the 05-year group.
candida albicans, a type of the yeast candida, is a commensal of the digestive tract and skin (1). usually, it is harmless in healthy people (2). the occurrence of candida albicans found in the oral cavity of the neonates has been reported to be 45 % (3), in healthy children 45%65% (4), in healthy adults 30%45% (2), and in denture wearers 50%65% (5). it affects the skin (6), mouth (2), genitals (7), and rarely, it spreads in the form of an invasive systemic infection throughout the deep organ of the body such as the lungs (8), esophagus (9), liver (10), kidneys (11), and heart (12). many types of oral candidiasis have been described, among others, the acute pseudomembranous, the acute erythematous / atrophic, the chronic hyperplastic, the chronic erythematous / atrophic, the median rhomboid glossitis, and the angular cheilitis (14)., is a chronic inflammation of the oral mucosa underlying a partial or total denture (2, 15, 16) ; the palatal mucosa being the most affected (2). clinically, cec is presented as local or diffuse erythema and rarely nodular / papillary hyperplasia (17). the diagnosis of cec is based on a careful clinical assessment confirmed by a microbiological test to detect pathogenic candida albicans. the aim of this study was to evaluate the occurrence of cec in a group of lebanese denture wearers. inclusion criteria for patients in this study were : aged between 40 and 80 years old.wearing maxillary complete denture for more than a year. ninety - eight full acrylic maxillary denture - wearing patients (50 women and 48 men), meeting the inclusion criteria, were selected and divided into 2 categories : a) age between 40 and 60 years old, and b) age older than 61 years old. in order to make the diagnosis of cec, a meticulous clinical oral exam was carried out followed by a quantitative microbiological measurement from the patient s palatal mucosa in order to confirm the presence of candida albicans in its virulent form. the microbiological specimens were collected and transported by sterile devices (bbl culture swabs) from [becton - dickinson (new jersey, usa) microbiology system ]. in the laboratory, a mix of sabouraud s dextrose agar (dextrose 40 g / l, peptone 10 g / l), chloramphenicol 0.5 g / l, and actidione 0.5g / l was used for swabs culture ; the incubation time was set to 48 hours at 37c in aerobic conditions ; 0.5 ml of animal serum was added to separate candida albicans from other species by inducing filaments production. candida albicans colony counts expressed in colony forming unit (cfu)/ml collected from the palates were noted. statistical analysis was performed with chi - square tests and the statistical significance set at 0.05. our sample of lebanese population consisted of 98 full maxillary denture wearers, 50 females (51.02 %) and 48 males (48.97 %). the patient s age ranged between 40 to 75 years, with a mean 62.05 years ; out of the total 98 patients, 33 aged between 40 and 60 years whereas the rest were older than 60 (table 1). percent distribution of sample according to age and gender statistical analysis by x tests was done to determine if there is a relation between patient s gender and age and cec. concerning the gender, the clinical and microbiological assessments revealed a total of 68 patients (69.38 %) [25 males (25.51%) and 43 females (43.87%) ] presenting cec ; 30 patients (30.61 %) [23 males (23.46%) and 7 females (7.14%) ] had healthy palatal mucosa without any inflammation signs. when assessing the association gender - cec it showed a statistically significant relationship (p 60 years) and cec (p = 0.57114). twenty - seven out of the 33 patients (81.81%), aged between 40 and 60 years showed cec signs against 6 (18.18%) ; whereas for the second age group (> 60 years) 41 patients out of 65 (63.07%) showed cec signs while 24 (36.92%) had a healthy mucosa (table 3). statistics analysis (x2 tests) showing association between age groups and cec ; x2= 3.6193 ; p - value = 0.57114. (statistically significant at p < 0.05) ; cec + : presence of cec. cec - : absence of cec as for the distribution according to the type of the lesions, no statistically significant relation was found neither with the patient s gender nor age groups (tables 4 and 5). statistics analysis (x2 tests) showing association between gender and types of cec ; x2= 2.6188 ; p - value = 0.269983. (statistically significant at p < 0.05) statistics analysis (x2 tests) showing association between age groups and types of cec ; x2= 2.0721 ; p - value = 0.354845. (statistically significant at p < 0.05) out of the 98 patients, 5 (5.1%) [3 males (3.06%) and 2 females (2.04%) ] presented a local erythematic lesion, against 60 (61.22 %) [20 males (20.40%) and 40 females (40.81%) ] presenting a diffuse erythema, and 3 (3.06 %) [2 males (2.04%) and 1 female (1.02%) ] having the hyperplasic type (table 4). on the other hand, a significant proportion from the two age groups adhered to the diffuse erythematic type of cec. only a minority (8 patients) (8.16%) displayed local erythematic and hyperplasic types. interestingly, no patients from the younger group presented a hyperplasic type of cec (table 5). usually asymptomatic, the inflammatory erythema of the oral mucosa supporting the denture can be in some cases accompanied by pain or burning sensation (18). many researchers have studied in different ways (clinically and/or microbiologically) and in several races, the prevalence of cec reported to be ranged between 11 to 60% (19). according to a systematic review and meta - analysis by moosazadeh. (20), the prevalence of cec, based on clinical examination only, in 12 iranian studies varied from 1.91% to 54.6% ; the total number of persons examined was 2271 with an average age ranging from 32.68 to 87.5 years and a sample size floating between 40 and 343 individuals. similarly, in chilean (21) and finnish (22) clinical studies, cec was found to be the most frequent oral mucosal lesion among individuals older than 65 years with an incidence of 22.3% and 25%, respectively. (23) who compared the occurrence of cec in two populations, british and greek, concluded that a statistically significant difference exists between them with 27% and 39.7%, respectively. the same applies to the study of kossioni (24) who clinically examined 106 greeks and observed cec in 39.6% of them. in the present study, the prevalence of cec which was found to be 69.38% can not be compared with any of the findings cited above due to the difference in the assessment method which is contrary to the others based on both clinical and microbiological examinations. however, this result agrees with what was stated by budtz - jrgensen (65%) (25), webb. (65%) (26), pahlavanzadeh. (70%) (27), and tavakol and emdadi (75%) (28), who have adopted the same technique of patient s evaluation by searching the pathogen form of candida albicans in addition to the clinical examination. (30), concluded that the prevalence of cec was respectively 41.5% and 58% respectively. this difference might be related to a difference in the samples size or to the patient education with regard to a good oral and denture hygiene. despite the fact that both genders are affected by cec, women in the current study were more frequently concerned with 43 out of 50 (86%) included in the sample against 25 out of 48 men (52.08%) ; this supports what was reported in the literature (18, 19, 26, 31) and seems to be related to the women continuous use of the denture for aesthetic and social reasons. in our study, among the 98 patients examined, the diffuse erythematic type of cec was the most retrieved (61.22%) followed by the local erythematic type (5.10%) and the hyperplasic type (3.06%) ; this confirms the statements of scully (32) and burket., it was found irrelevant predictors of the disease ; all age groups were affected by the disease with a predominance of the patients older than 61 years. this result corroborates the one we noticed in a previous study aimed to evaluate the denture - related factors predisposing to denture stomatitis in a lebanese population (34). finally, our study aiming to evaluate the prevalence of cec is not without limitation ; because of the restricted number of patients assessed, exact conclusions must be delayed until future research validates our results. the prevalence of cec in lebanese denture wearers investigated by clinical and microbiological examinations is found to be high compared to other populations with a female predominance. among all the clinical types of cec,	objective : chronic erythematous candidiasis also known as denture - related stomatitis refers to inflammatory changes of the denture - bearing mucosa. the aim of this study was to evaluate the prevalence of chronic erythematous candidiasis in a lebanese population using clinical and microbiological examinations.materials and methods : ninety - eight patients wearing full acrylic maxillary denture (50 women and 48 men) were included in this study. a clinical oral assessment and a microbiological exam using swab samples collected from the palate of these patients were performed and the data obtained were analyzed statistically.results:sixty-nine point thirty - eight per cent (69.38%) of the patients examined, (68 out of 98 ; 25 men and 43 women), presented chronic erythematous candidiasis. the statistical analysis showed that patient s gender was a significant predictor of the disease while no statistically significant relationship with the patient s age was found.conclusion:within the limits of this study, the prevalence of chronic erythematous candidiasis is estimated to be high in lebanon. women were more affected than men.
diabetic retinopathy (dr) is the most common cause of vision loss, and a large number of diabetic patients experience significant vision impairment [13 ]. usually, in patients with type 1 diabetes mellitus (t1 dm) clinically overt changes in the eye fundus do not occur earlier than 5 years of the disease duration. however, after 10 years of the disease, diabetic retinopathy symptoms affect 50% of patients, and after 2030 years as many as 90% of t1 dm patients [2, 3 ]. current methods used in medical practice to identify the risk of dr development in children and adolescents with t1 dm include the examination of the eye fundus, vision acuity, and colour amblyopia [4, 5 ]. many authors emphasise that changes in the eye fundus caused by t1 dm are also accompanied by the development of albuminuria which in turn is a marker of diabetic nephropathy [13, 6 ]. however, these clinical markers of vascular damage point to the presence of certain tissue damage, which already gives clinical symptoms such as albuminuria or disturbances of vision. also, it is common knowledge among ophthalmologists that individual stages of diabetic microangiopathy change smoothly into more advanced. additionally, when no changes of the retina are seen during ophthalmoscopy, it does not rule out the presence of early histopathological changes in the vessel wall or the presence of abnormal vessel hemodynamics. apart from the dilatation of retinal blood vessels, the reduction of capillary basal membrane thickness, blood - retina barrier disruption, and a selective loss of pericytes in t1 dm patients may also occur [1, 6, 7 ]. therefore, there is a strong need for the development of new diagnostic tools which would enable early diagnosis of diabetic complications in juvenile patients with t1 dm. despite extensive research, the exact pathogenesis of diabetic retinopathy is still unknown. one of its mechanisms is a direct consequence of tissue hypoxia caused by hyperglycemia and the imbalance of local angiogenic factors expression [810 ]. for example, data from our previous studies underline the role of the tnf- and il-12 overexpression in the pathogenesis of dr in children and adolescents with t1 dm. recently, the role of several growth factors, including transforming growth factor - beta (tgf-), has been emphasised in the pathogenesis of dr [1113 ]. tgf- is one of the factors involved in the cellular growth, differentiation and migration, the formation and degradation of extracellular matrix components, chemotactic processes, and apoptosis. it comes in five isoforms, three of which, namely, tgf-1, tgf-2, and tgf-3 are encoded by different genes. the best known among them is tgf-1, which is produced by dendritic cells, leucocytes, and natural killer (nk) cells. many studies have shown that tgf-1 plays an important role in the pathogenesis of breast cancer, myocardial infarction, autoimmune diseases, osteoporosis, dr, and nephropathy in adults [1517 ]. in recent years, however, several studies (including ours) point to the role of tgf-1 in the pathogenesis of microvascular complications in children and adolescents with t1 dm [18, 19 ]. therefore, in the present study, we have decided to evaluate if serum tgf-1 concentrations may have an additional diagnostic role in predicting the occurrence of dr in juvenile patients with t1 dm. eighty - one adolescent patients (44 boys and 37 girls, age range 720 yrs) with t1 dm from the department and clinic of pediatrics, diabetology, and endocrinology at the medical university of gdask were enrolled into this study. all the patients were under intensive insulin therapy (0.83 0.21 iu of insulin per day / kg of body weight). diabetes was diagnosed according to the polish diabetes association guidelines which correspond with the guidelines of the european diabetes association [20, 21 ]. blood pressure was measured using a 24 h blood pressure monitoring (abpm) method. various sizes of the cuff were used according to age, weight, and arm circumference of the studied subjects. all the abpm reports which had less than 80% of technically correct measurements were excluded from the study. arterial hypertension was diagnosed when mean abpm values were above the 95th centile for the corresponding age, gender, and height on at least three separate measurements. this included visual acuity tests, intraocular pressure, and anterior segment estimation using the slit lamp (topcon sl-82, japan). after local administration of tropicamide (1% solution), the eye fundus was examined using the + 90 d lens (ocular instruments, usa). a digital camera (topcon imaginet 2000, japan) was used for the fluorescein angiography. the stage of retinopathy was diagnosed according to the guidelines of the international diabetic retinopathy division [4, 5 ]. twenty - four hour urine collection was performed three times during the period of 6 months for the evaluation of the daily albumin excretion. the urinary albumin was measured with immunoturbidometric assay using a tina - quant kit (boehringer mannheim gmbh, germany). albuminuria was diagnosed when at least two out of three urine samples displayed daily albumin excretion of between 30299 mg/24 h, collected within 6 months from patients with well - controlled diabetes with no clinical or laboratory signs of ketoacidosis [5, 23 ]. serum creatinine was measured using the crea assay system (boehringer mannheim gmbh, germany). glycated haemoglobin (hba1c) was measured with an immunoturbidometric method using a unimate 3 set (hoffmann - la roche ag, basel, switzerland). the levels of total cholesterol, hdl cholesterol, ldl cholesterol, and triglycerides were measured using cormay enzymatic kits (cormay, lublin, poland). serum c - reactive protein (crp) concentrations were determined using a highly sensitive testing method (hs - crp, dade behring, usa). the control group consisted of age and bmi matched 19 healthy children and adolescents (11 boys and 8 girls, age range 618 yrs). written informed consent was obtained from all the participants in the study or from their parents or guardians. this study was approved by the ethics committee of the medical university of gdask (nkebn/204/2009), and the investigation was carried out in accordance with the principles of the declaration of helsinki as revised in 1996. the serum concentrations of tgf-1 were measured using the cytometric bead array (cba) as instructed by the manufacturer 's manual (plex flex single set, becton dickinson, usa). the samples were read in an lsr ii flow cytometer using facs diva software (becton dickinson, usa). prior to reading, the cytometer was calibrated using the calibration beads included in the test pack (cytometer setup beads). based on the fss / ss images, the beads were gated, and fluorescence was read. the analysis was performed using an fcap array software (becton dickinson, usa). all statistical calculations were performed using a statistical computer program statistica version 9.0 (http://www.statsoft.com). quantitative data are presented as an arithmetic mean and standard deviation (sd). to verify whether a quantitative variable was drawn from the population of normal distribution, the shapiro - wilk test was performed. the significance of the differences between the control subjects and the diabetic patients was tested with the student t - test or u mann - whitney test when the variables were not normally distributed. due to multiple comparisons between the groups, bonferroni correction was applied in calculating the p value. to estimate the accuracy of the test, that is, the ability of the test to measure a characteristic in accordance with its actual status, two measures of quantifying the test accuracy were used : sensitivity and specificity. to determine the discriminating threshold value, when results of the test were measured on a continuous scale, receiver operating curve (roc) was calculated, with the axis of coordinates representing sensitivity and the axis of abscissa representing specificity. the maximum area under the roc curve (aucroc) (with values between 0 and 1) was a measure of discriminative power of the test, and the results were reported as means and 95% confidence interval (95% ci). the clinical characteristics of the studied children and adolescents with t1 dm as well as the control subjects are presented in table 1. the study included 31 t1 dm patients with npdr, aged 15.3 5.4 years and the mean duration of the disease of 9.5 4.9 years, and 57 t1 dm patients without retinopathy, aged 14.6 3.9 years and the mean duration of the disease of 6.3 3.5 years. in the npdr group, the control group consisted of 19 age - matched (14.2 3.5 years) healthy children. the patients with t1 dm and confirmed npdr had a longer duration of diabetes, higher serum hba1c levels, higher crp, higher urinary albumin excretion, and systolic blood pressure values compared to the t1 dm patients without retinopathy (p 0.05). however, the patients with t1 dm and without retinopathy had significantly higher serum levels of hba1c and crp, higher urinary albumin excretion, and higher systolic blood pressure values in comparison with the control group (p 0.05) (table 1). the patients with t1 dm and npdr displayed statistically significant higher serum levels of tgf-1 (1530 465 pg / ml versus 758 424 pg / ml, p = 0.003) as compared to the patients with t1 dm but without dr. however, the patients with t1 dm but without dr had significantly higher serum levels of tgf-1 compared to the healthy controls subjects (758 424 pg / ml versus 156 49 pg / ml, p = 0.001) (table 2). in order to determine the threshold values which could have a discriminative ability to predict the occurrence of dr in our t1 dm patients, we conducted an roc analysis. then, the area under the curve (aucroc) was calculated for the parameters such as age, duration of diabetes, systolic and diastolic blood pressure, albuminuria, serum hba1c, crp, creatinine, and serum tgf-1 levels. tgf-1 turned out to have to most discriminative power in predicting the occurrence of dr in the group of juvenile patient with t1 dm. the cut - off threshold value was calculated to be 443 pg / ml with aucroc of 0.80 (95% ci 0.660.94). sensitivity and specificity were 72 % and 88 %, respectively (figure 1). data from recent studies have shown that tgf-1 is a tgf isoform, which exhibits the strongest relationship with tissue fibrosis [12, 13, 17 ]. it has also been shown that in long - standing diabetes, tgf-1 overexpression can be the culprit of organ fibrosis, that is, kidney interstitial tissue in the course of diabetic nephropathy. in the retina, tgf-1 is produced by retinal pigment epithelial cells and pericytes, and it is known to be a key profibrotic factor. in proliferative diabetic retinopathy (pdr) and proliferative vitreoretinopathy (pvr), tgf- has been shown to be overexpressed in the vitreoretinal interface. nevertheless, data on the role of this cytokine in the pathogenesis of vascular diabetic complications in children with t1 dm are still scarce [18, 19 ]. (2000) found higher urinary tgf-1 concentrations in children with t1 dm compared to control subjects. also, data from our recent studies have shown increased serum levels of tgf-1 in children with nephropathy. what is more, there was a correlation between serum levels of this cytokine and the advanced glycation end products (ages) concentrations in the children and adolescents with t1 dm. however, we have not come across any reports concerning serum tgf-1 levels in juvenile patients with diabetic retinopathy. our hypothesis is that serum tgf-1 concentrations in patients with t1 dm may point to the occurrence of retinopathy. therefore, the aim of our study was to evaluate serum concentrations of this cytokine as well as to determine its threshold values having a discriminative ability in predicting the occurrence of dr in juvenile patients with t1 dm. the results of our study have supported our hypothesis and according to our calculations serum tgf-1 concentrations over 443 pg / ml may point to the presence of dr in juvenile patients with t1 dm. we have also found that juvenile diabetic patients with npdr are characterized by higher serum levels of tgf-1 in comparison to the patients without this complication. moreover, serum tgf-1 concentrations in the patients with t1 dm and npdr was over 10 times higher compared to the healthy controls and about 4 times higher in the diabetic juvenile patients without dr as compared to the healthy controls. according to our data, we conclude that there might be a relationship between tgf-1 overexpression and the presence of dr in juvenile patients with t1 dm. the main source of serum tgf-1 are blood plates. however, loukovaara. (2012) have also shown higher intravitreal tgf-1 concentrations in adult t1 dm patients with pdr or pdvr, which in turn may be associated with retinal angiogenesis and tissue fibrosis at the vitreoretinal interface. data from other studies also point to the role of tgf-1 gene polymorphism in the pathogenesis of dr. (2002) have confirmed a more frequent occurrence of 915g / c (r25p) polymorphism in patients with dr compared to control subjects. other researchers, in turn, have shown that the activity of urine tgf-1 in diabetic patients increases along with the damage to glomerular cells and renal tubules caused by the locally activated production of tgf-1 [2729 ]. this leads to the increase in the renal synthesis of laminin, fibronectin, and collagen. collagen is prone to glycation, which in turn increases the number of crosslinks in its structure. this results in an increased rigidity of its fibres, their reduced solubility, and reduced susceptibility to enzymatic digestion. in clinical practice, there is a strong need for new markers which point to the risk of development or the presence of a specific pathology. therefore, in the second part of our study, we calculated the threshold tgf-1 concentrations, which would enable us to predict the presence of early - stage diabetic retinopathy in juvenile patients with t1 dm. one of the statistical methods of determining the limit values of the parameters under investigation is the analysis of roc curves. the roc curve analysis helps to visualize and choose the optimal threshold values that will guarantee the highest decision accuracy [24, 25 ]. out of nine tested parameters (age, duration of diabetes, systolic and diastolic blood pressure, albumin excretion rate, serum hba1c, crp, tgf-1, and creatinine), it turned out that tgf-1 in serum had the most discriminative power in predicting the occurrence of dr in juvenile patients with t1 dm. the cut - off threshold value was calculated to be 443 pg / ml with aucroc of 0.80 (95% ci 0.660.94). the threshold serum values of tgf-1 were also calculated in other diseases by other investigators [30, 31 ]. the cut - point value with a diagnostic power was calculated to be 30.5 pg / ml with 80.5% sensitivity and 64.5% specificity. nevertheless, our study is the first one where an attempt has been made to define threshold serum tgf-1 concentrations having a discriminative power in predicting the occurrence of dr in patients with t1 dm. in summary, our results suggest that serum tgf-1 concentrations may be an additional parameter in predicting the occurrence of npdr in juvenile patients with t1 dm.	in the present study, we have decided to evaluate if serum transforming growth factor - beta 1 (tgf-1) concentrations may have diagnostic value in predicting the occurrence of diabetic retinopathy (dr) in juvenile patients with type 1 diabetes mellitus (t1 dm). the study included 81 children and adolescents with t1 dm and 19 control subjects. all study participants had biochemical parameters examined, underwent an eye examination, and 24-hour blood pressure monitoring. moreover, serum concentrations of tgf-1 were measured. the group of patients with t1 dm and nonproliferative diabetic retinopathy (npdr) had statistically significant higher serum levels of tgf-1 (p = 0.001) as compared to t1 dm patients without retinopathy as well as the healthy control subject. the threshold serum tgf-1 concentrations which had a discriminative ability to predict the presence of dr were calculated using the receiver operating characteristic (roc) curves analysis and amounted to 443 pg / ml. the area under the roc curve (aucroc) was 0.80, and its population value was in the range of 0.66 to 0.94. the sensitivity and specificity were calculated to be 72% and 88%, respectively. our results suggest that tgf-1 serum concentrations may be an additional parameter in predicting the occurrence of dr in juvenile patients with t1 dm.
on the recent years, an approach known as somatosensory electrical stimulation (ses), which consists in applying peripheral electrical stimulation below or at the motor threshold (mt) level [13 ], has been employed to treat patients with sensory - motor impairments such as spasticity [48 ]. although there are encouraging clinical results observed in stroke [3, 9 ] and spinal cord lesioned [10, 11 ] patients, several methodological aspects of using ses therapeutically remain unresolved. performed systematic reviews of ses parameters upon the primary motor cortex (m1) and suggest that there is insufficient evidence to determine their effects. additionally, there also seems to be a lack of consensus regarding the effects of ses over spinal circuits mainly due to methodological constraints, which are described in more details elsewhere. among ses underlying mechanisms, ward proposed that an increase in the frequency of stimulation upon the stimulated nerve would allow the ionic current to flow more deeply, leading to the recruitment of a larger pool of somatosensory receptors from different tissues adjacent to the ses stimulation site. thus, distinct effects upon excitability in m1 and/or spinal circuitries would be expected depending on the average output of recruited sensory fibers. however, to our knowledge, no previous studies have compared the effects of more than two ses frequencies upon corticospinal and spinal excitability. therefore, the aim of this study was to evaluate by means of transcranial magnetic stimulation (tms) and hoffmann 's reflex (h - reflex) in normal volunteers the effect of applying ses over the hand and forearm muscles at four different stimulation frequencies, conducted in two experiments. moreover, a group of stroke patients was also preliminary evaluated to ascertain ses effects at a low frequency over the forearm spastic flexors muscles on the wrist joint passive torque. the first two experiments were performed with two groups of right - handed volunteers without neurological disorders. the first was composed of fourteen volunteers (seven males and seven females ; age : 19.039.0 years ; height : 1.541.92 m ; body weight : 53.397.0 kg ; edinburgh handedness inventory : + 30.0+90.0) and the second comprised six volunteers (five males and one female ; age : 20.037.0 years ; height : 1.541.92 m ; body weight : 56.097.0 kg ; edinburgh handedness inventory : + 40.0+95.0). the first and second groups were subjected to 5 and 30 ses protocols, respectively, hereafter called experiments i and ii. both application times were arbitrarily defined. a third group composed of five chronic stroke patients (one female and four males ; age : 4570 years ; height : 1.561.70 m ; body weight : 61.089.0 kg) with spasticity, all right - handed (edinburgh handedness inventory : before stroke [+ 86.67+100.0 ] ; after stroke [100.0+23.33 ]), was also recruited for a pilot study concerning ses effects on spastic muscles (experiment iii). among the adopted criteria of inclusion, the main one was to present spasticity on the right forearm limiting wrist movements with a maximum score of 2 (more marked increase in muscle tone through most of the ranges of motion (rom), but affected part(s) easily moved) based on the modified ashworth scale. furthermore, cognition and skin sensitivity should be normal, which were evaluated by means of the mini mental state examination and pain and light touch sensation tests, respectively. the entire experimental protocols were submitted to the local ethical committee (process number : 082/08) and were conducted after each volunteer gave informed consent. the first and the second groups (experiments i and ii) were subjected to four different frequencies of ses : 3, 30, 150, and 300 hz. the frequencies were chosen based on previous reports in which they resulted in motor control improvement and/or neuromodulation [2, 2224 ] by using ses and other therapeutic modalities of electrical stimulation. the frequencies of stimulation were applied in a random manner in both experiments. in experiment i, the effects of the four frequencies of ses upon corticospinal and spinal excitability were evaluated in the same experimental session. each frequency of ses was applied for 5 and was followed by a rest interval of 10. motor evoked potentials (meps) and h - reflex were collected immediately before (baseline) and after (0) ses application and then 5 later (5). in contrast, in experiment ii, the volunteers were subjected to 30 of ses for each frequency and the protocol was conducted on four different days separated by a minimum interval of 72 hours. similarly to experiment i, meps and h - reflex were collected immediately before (baseline) and after (0) ses application and then 5 (5) and 10 (10) later. each single experimental session lasted for approximately 4 h. in experiment iii, the stroke patients were submitted to ses therapy only at 3 hz that was also applied for 30 in one single session, similarly to experiment ii. we justify such decision due to the time consumption (~4 h) of experiments i and ii and because previous authors report interesting m1 modulatory effects when ses was applied below 10 hz. additionally, the passive mechanical resistance of the right wrist joint in the extension movement was evaluated by means of an isokinetic system only before (baseline) and immediately after (0) ses therapy. the ses pulse was a constant amplitude current waveform (an unbalanced asymmetrical biphasic pulse) with duration of 500 s [26, 27 ]. ses was delivered using a custom electrical stimulator (fes - peb) built by velloso and souza. pulse intensity was set below the mt and was determined based on the volunteers ' reports of a tingling sensation in the stimulated area (forearm flexor muscles) without any pain or visible movement of the wrist and fingers. surface self - adhesive electrodes (5 5 cm ; model : cf5050, axelgaard manufacturing co., ltd., ses intensities were set between 27 ma (experiment i), 38 ma (experiment ii), and 35 ma (experiment iii). the surface electrodes were positioned over the right forearm, between the wrist (negative electrode) and the elbow (positive electrode) joints, parallel to the fcr muscle in the longitudinal direction (figure 1). because it has been suggested that forearm rotation around its longitudinal axis may modulate spinal reflexes, the volunteers remained seated in a comfortable chair with the right forearm maintained in a prone position throughout the experiments. a biopac system (model : mp150 ; a / d converter : 16 bits ; dynamic range : 10 v ; sampling frequency : 15 khz ; band pass filter : 4th order and 1005000 hz ; gain : 2000 ; biopac systems, inc., surface biopac reusable electrodes (ag / agcl ; diameter : 8 mm) were placed following seniam recommendations over the following muscle bellies : the flexor (fcr) and extensor (ecr) carpi radialis and abductor pollicis brevis (apb). the ecr and apb muscles were defined as controls in experiments i and ii. in experiment iii, we monitored one more muscle : the contralateral flexor carpi radialis (fcrc) from the nonaffected left forearm. therefore, in this particular experiment, the fcrc, ecr, and apb muscles were defined as controls. a tms butterfly coil (model : magpro ; magventure, denmark) was positioned over the left m1 in the optimal scalp position (hot - spot) to elicit fcr motor responses in the contralateral hemibody. the hot - spot position was achieved using a cap with marks (grid : 1 1 cm) according to the international 10 - 20 system for electroencephalography. the fcr hot - spot was defined as the site in m1 in which a single magnetic pulse set at a minimum intensity produced a mep response (amplitudes with more than 50 v in three out of six trials) in the relaxed muscle [3032 ]. then, the magnetic stimulus intensity was adjusted to 20% above the fcr resting mt. as previously mentioned, meps were collected from all the muscles studied immediately before (baseline) and immediately after (0) ses and up to 5 (experiment i) and 10 (experiments ii and iii) later after each session of ses and for each frequency. in each measurement, at least six (experiment i) and ten (experiments ii and iii) single tms pulses were delivered with an interpulse interval of 510 s. the surface electromyographic (semg) data acquisition was triggered by a hardware pulse provided by the tms system. changes in spinal excitability were monitored through the evaluation of the mean value between the maximum and the minimum fcr h - reflex responses obtained by means of a curve of recruitment collected before the beginning of each ses session. to elicit the h - reflex from the fcr, a rectangular monophasic pulse of 800 s was applied to a pair of surface electrodes, where one of the electrodes was placed over the median nerve (cathode : ag / agcl ; 4 mm diameter), ~4 cm above the elbow joint, and the other on the opposite limb (anode : ag / agcl ; 47.5 cm). the semg signal acquisition was started by a hardware pulse trigger provided by the stimulator. once the electrical pulse was applied, a data window of 100 ms was collected and saved. at least four semg signal data windows were obtained for further analysis due to the robustness of the collected h - reflex data. regarding the fcr h - reflex, experiment i provided suitable data only from seven volunteers (one man and six women ; age : 19.031.0 years ; height : 1.551.73 m ; body mass : 53.382.6 kg ; edinburgh handedness inventory : + 60.0+90.0) due to methodological constraints in recording it. similarly, also due to methodological constraints, it was possible to collect the fcr h - reflex only from two stroke patients and, therefore, we decided not to present these data in this study. visual feedback of the semg signals was provided throughout all the experimental sessions to certify that all the volunteers were relaxed. a custom - made device was built to evaluate the passive resistance of the wrist joint while being moved (figure 2). it consisted of a stepper motor of 100 kgfcm static torque controlled by a micro - step driver to make small steps of 0.036 that smoothed the movement. attached to the stepper motor, a load cell allowed measuring the torque applied to the wrist. the data was recorded using a spider 8 (hbm, hottinger baldwin messtechnik) system with 16 bits, 4800 hz sample rate, and an antialiasing filter set at 960 hz. the root mean square (rms) value of the force measured by a load cell was calculated and used as an estimate of the torque (gfcm) during 5 cycles of 35 degrees of wrist extension set at an angular velocity of 10 degrees per second to avoid the stretch reflex. surface emg signals collected from each muscle during experiments i, ii, and iii and at each interval of time (baseline, 0 and 5 or 10) were analyzed using an algorithm built in matlab 6.5 (mathworks, usa). this algorithm measured mep and h - reflex peak - to - peak (p - p) values, which represents corticospinal and spinal excitability. mep and h - reflex median values were then obtained from each six (experiment i) and ten (experiments ii and iii) semg signal data windows were from the muscles previously mentioned for each group, resulting in one value for each interval of time, that is, before (baseline) and after (0) ses application, as well as 5 (experiment i) and 10 (experiments ii and iii) later for each ses frequency and for each volunteer. the medians from mepp - p and h - reflexp - p values were then normalized by computing the ratio between the data for each interval of time (0, 5, and 10) and the corresponding baseline value achieved before ses multiplied by 100 [2, 3739 ]. all normalized mep data values were analyzed using a two - way repeated measures anova (factors : interval of time frequency) for each muscle individually. for the h - reflex data, a two - way repeated measures anova (factors : interval of time frequency) was used, as all measures were taken only from the fcr muscle. for experiment iii (tms and h - reflex), the analysis was performed using a two - way repeated measures anova (factors : interval of time muscle) while the isokinetic data evaluated by means of student 's paired t - test. the level of significance () figure 3 depicts the results obtained for normalized mepp - p and fcr h - reflexp - p values, respectively, immediately after (0) ses and up to 5 later for the four frequencies of ses applied during 5 and for the three muscles (fcr, ecr, and apb). the results obtained after ses set at all the investigated frequencies did not reveal any significant difference among frequencies (p > 0.155) and intervals of time (p > 0.087) in mepp - p data as compared to the baseline for all the muscles (figures 3(a), 3(b), and 3(c)). concerning the normalized fcr h - reflexp - p values (figure 3(d)), it also did not show any significant difference among frequencies (p = 0.469) and intervals of time (p = 0.177) from the baseline. figure 4 depicts the results obtained for normalized mepp - p and fcr h - reflexp - p values, respectively, immediately after (0) ses and up to 5 and 10 later for the four frequencies of ses applied during 30 and for the three muscles (fcr, ecr, and apb). the results obtained after ses set at all the investigated frequencies did not reveal any significant difference among frequencies for fcr (p = 0.443) and apb (p = 0.524) muscles. although there was a significant difference among frequencies for ecr (p = 0.006), they were not statistically different from baseline (p > 0.05). similarly to experiment i, there was also no significant difference among intervals of time (p > 0.235) in mepp - p data from the baseline for all the muscles (figures 4(a), 4(b), and 4(c)). our results did not also show any significant difference among frequencies (p = 0.638), intervals of time (p = 0.563), and interaction of interval of time frequency (p = 0.112) concerning normalized fcr h - reflexp - p values from the baseline. figures 5 and 6 depict the results obtained for the normalized mepp - p values and latency, respectively, for ses set at 3 hz and during 30. there were no significant differences among intervals of time in normalized mepp - p relative values from the baseline for all the three muscles, that is, the fcr (p = 0.092), fcrc (p = 0.172), ecr (p = 0.814), and apb (p = 0.864). latency measures also did not show any significant difference (p > 0.150) concerning baseline values. the resistance to passive extension (figure 7(a) ; p = 0.094) and flexion (figure 7(b) ; p = 0.774) movements of the wrist joint, evaluated by means of the isokinetic system, also did not show any significant difference for the comparison of measurements collected immediately after (0) (post) ses and the baseline (pre). it has been proposed that the use of ses in the clinical field can provide improvements similar to those obtained with intensive training. for instance, conforto. observed an increase in the force level of pinch movements in stroke patients after two hours of ses. dos santos - fontes. also suggested that ses might lead to long - lasting improvements of paretic arm performance in chronic stroke patients. furthermore, ses has been proposed as an alternative approach in minimizing spasticity [6, 7, 42 ], although there is also no clarity concerning its underlying mechanisms. hence, despite the above - mentioned examples concerning the positive effects of ses in a clinical setting, there is still no consensus regarding how some of the parameters used in ses (e.g., application time, frequency, intensity, waveform, and pulse width) are taken into account by the central nervous system (cns) [14, 43 ]. therefore, we decided to evaluate the effect of different frequencies of ses in the spinal and corticospinal excitability of healthy volunteers. in addition, we also performed a pilot study to evaluate the effect of ses applied for 30, in line with clinical practice, and chose a frequency of 3 hz applied over the spastic forearm flexor muscles of five stroke patients by means of tms and passive wrist movement assessment. nevertheless, what does the literature tell us about different frequencies of ses in the corticospinal modulation and spasticity ? excitatory as well as inhibitory effects in m1 were shown to be maintained from minutes to hours after ses therapy [4, 13, 37, 44, 45 ]. even though some authors have suggested that an increase in the stimulation frequency might lead preferentially to an increase in m1 excitability [13, 22, 38, 46, 47 ], others reported an opposite effect at the spinal level. we hypothesized that an increase in frequency would allow the ionic current to flow more deeply over the full forearm extent and around the median nerve, which innervates the skin of the palmar side of the thumb, the index, the middle finger, and the apb muscle. as a result, we expected to induce changes in corticospinal excitability of the fcr and apb muscles. however, we also considered the hypothesis of observing some effect in the ecr muscle given that type ii afferent fibers (nonadapting sensory fibers) in muscles are able to facilitate or inhibit antagonist muscles, although pierrot - deseilligny and burke state that the effect of this interaction on the upper limb is not well understood. nevertheless, in the present study, both short (5) and long (30) durations of ses applied at different frequencies did not lead to changes in spinal and corticospinal excitability of the muscle under stimulation nor at the muscles that share any kinesiologic property with the stimulated one. despite the discouraging results, some issues in addition to the frequency of stimulation must be taken in account and they are discussed as follows. we used a longer pulse width (500 s) than have other authors (100 to 300 s) [2, 6, 22 ], which are more selective. we decided on this pulse width due to the need of recruiting a wide range of diameters and modalities of sensory afferents fibers. as our main aim was to evaluate the effect of different frequencies of ses, an increase in the selectivity of recruitment could mask a possible source of a corticospinal excitability modulation derived from this parameter only. for instance, tinazzi. observed an increase and a decrease in the corticospinal excitability of ecr and fcr, respectively, after applying ses at 150 hz over the fcr muscle for 30. on the other hand, fernandez - del - olmo. performed a similar protocol and curiously, both studies used a narrower pulse (100 s), which is expected to be more selective for larger diameter fibers. a shorter pulse width is expected to achieve higher selectivity in discriminating between somatosensory, motor, and pain sensory nerves. a pulse width of 500 s may result in a greater summation of responses from receptors distributed close to the site of ses, which will be integrated and processed at different levels of the cns and likely transmitted to m1 as an averaged signal. thus, as a first hypothesis to explain the lack of ses modulation we suggest that this averaged input might not be able to induce any spinal or corticospinal modulation. another important issue concerns the intensity of the stimulation. some studies report divergent effects of ses set at different intensity levels in corticospinal excitability but also including paired associated stimulation (pas) protocols. in this context, pitcher. applied electrical stimulation above the mt and set at 3 hz over the first dorsal interosseous (fdi) muscle during 30 while pulses of tms were synchronized over the fdi hot - spot in a pas protocol. they observed a decrease in mepp - p for approximately 4050, suggesting that peripheral electrical stimulation at such low frequency mediates the recruitment of neural circuits that induce long - term depression (ltd). alternatively, aimonetti and nielsen showed that applying a conditioning electrical stimulus just below the mt over the median nerve, which supplies the fcr, produced a facilitation of the ecr for a very short term. in contrast, bertolasi. also showed that applying a conditioning electrical stimulus just above the mt over the median nerve produced an ecr inhibition. considered that the corticospinal excitability level appears to be modulated by a fine - tuning of ses intensity, which can vary from the perceptual to the motor threshold and be explained by the wide range and directions of neuronal responses obtained from the meps in tms experiments. their findings suggest that a stimulation intensity set at the perceptual threshold would not be capable of inducing any modulation in the corticospinal excitability. based on these remarks and on the methodological approach adopted in the present study to tune the ses intensity (see details above in section 2), as a second hypothesis we conjecture that, being provided at or very near the perceptual threshold for most of the volunteers, ses did not induce any measurable physiological effect for all the tested muscles, frequencies, and intervals of time. different authors adjust the intensity of ses during their experiments to avoid habituation [3, 41, 53, 54 ]. unlike most of authors, we decided to maintain the same stimulation intensity to avoid a bias in the evaluation of corticospinal modulation after ses. moreover, we evaluated two different application times of ses : a very short (5) and a long (30) one. therefore, habituation mechanisms must be taken into account even in the ses protocol of short duration. in line with this third hypothesis, the works of dobkin and dimitrijevi. supported the idea that a regular and constant pattern of ses can lead either to habituation / accommodation very quickly or to a failure in producing any change in m1 excitability, although this process does not seem to be rigid. thus, we suggest as a third hypothesis that a habituation effect induced by the maintenance of the stimulation intensity could be an additional variable of not inducing any spinal or corticospinal modulation from ses in the present study. even though we have faced methodological constraints to record the h - reflex from some healthy volunteers, ses seemed to fail to induce changes in the excitability of such measurement in fcr in experiments i and ii. despite the fact that the h - reflex is generally considered a monosynaptic response, it may be also modulated by other inputs which converge to common interneurons and that may receive inputs from spinal and supraspinal sources [7, 15 ]. therefore, ses seems to stimulate differently specific receptors which might cancel themselves reciprocally and produce a mixture of excitation and inhibition, which was previously assumed as one of our hypotheses of not observing any significant difference in corticospinal excitability. departing from the herein shown lack of ses effect upon the fcr h - reflex, some studies have shown that functional electrical stimulation (fes), which allows recruiting ia muscle spindle sensory neurons, is able to induce modulation of the h - reflex in neurological patients and healthy subjects as well. thus, it may be suggested that ia afferents, not likely recruited under the ses protocol adopted in this study, could be able to evoke a fcr h - reflex modulation. the purpose of this preliminary study was to evaluate for the first time, to our knowledge, the effects of ses set at a low frequency (3 hz) over the spastic forearm flexor muscles at the corticospinal level in chronic stroke patients. moreover, we intended to evaluate the carryover effects of ses in the passive mechanical resistance of the impaired wrist joint. although we did not observe any significant spinal and corticospinal modulation in experiments i and ii, we firstly hypothesized that a decrease in the corticospinal response provided by ses at a low frequency (3 hz) would be capable of contributing to decreasing temporarily such muscle overactivity. however, despite the negative results, different authors have provided some interesting arguments and results that preliminarily reinforce our previous hypothesis. as already cited, pitcher. observed that peripheral electrical stimulation at a low frequency results in a decrease in corticospinal excitability, although the hypotheses concerning the likely mechanisms under the effects of this electrical stimulation pattern in spasticity are still lacking. notwithstanding, liepert. suggested that the intracortical inhibition might be reduced in stroke patients, due to a decreased gabaergic (inhibitory) and/or an increased glutamatergic (excitatory) activity, respectively. the unbalance of those neurotransmitters might contribute to the manifestation of spasticity of supraspinal origin although its physiopathology is still under investigation. likewise, long - term (> 30) ses at low frequencies might be able to conduct to an increase in the recruitment of gabaergic circuitries and conduct to a depression in corticospinal excitability. pitcher. also support that the modulation of those neural circuitries may be frequency - dependent and might be optimized by 3 hz stimulation. sonde. also investigated ses set at a low frequency (1.7 hz) in the treatment of spasticity in individuals with stroke and despite the improvements in the motor pattern, they did not also observe any significant decrease in spasticity, as evaluated by means of the ashworth scale. even though the meps and wrist torque data did not reach statistically significant levels, the evaluation of the mechanical resistance by means of an isokinetic system, that is, a more accurate and sensible approach, four out of six patients presented relative decreases (5.5 to 14.3%) in passive wrist torque (flexion and extension), which might be seen as clinically significant. it is important to highlight that the ashworth scale does not provide sensitivity to minimal variations on the level of spasticity. a hyperexcitability of h - reflex has been considered as an index of spasticity [48, 61 ]. therefore, the lack of change in h - reflex measurements does not allow implying spinal instead of corticospinal excitability as the likely portion of the cns suitable to ses effects in stroke patients with spasticity. in summary, as we expected to observe a covariation between the mep behavior with a decrease in resistance to the passive extension movement of the wrist joint, we may also assume that a low frequency of ses set at 3 hz and the perceptual threshold might be unable to induce any neuromodulation in these patients. based on the work of ward, we proposed that an increase in the frequency of ses stimulation would allow the ionic current to flow more deeply and so a larger pool of somatosensory receptors from different tissues adjacent to the ses would be recruited. however, even though we must recognize small participant size samples in experiments ii and iii, the results provided by our experiments suggest that none of the investigated frequencies (3, 30, 150, and 300 hz) of ses along with all the other chosen parameters seem to be able to operate as a key in switching modulatory effects in the cns of healthy volunteers and stroke patients with spasticity.	somatosensory electrical stimulation (ses) has been proposed as an approach to treat patients with sensory - motor impairment such as spasticity. however, there is still no consensus regarding which would be the adequate ses parameters to treat those deficits. therefore, the aim of this study was to evaluate the effects of applying ses over the forearm muscles at four different frequencies of stimulation (3, 30, 150, and 300 hz) and in two intervals of time (5 and 30) by means of transcranial magnetic stimulation and hoffmann 's reflex (h - reflex) in healthy volunteers (experiments i and ii). a group of stroke patients (experiment iii) was also preliminary evaluated to ascertain ses effects at a low frequency (3 hz) applied for 30 over the forearm spastic flexors muscles by measuring the wrist joint passive torque. motor evoked potentials and the h - reflex were collected from different forearm and hand muscles immediately before and after ses and up to 5 (experiment i) and 10 (experiments i and ii) later. none of the investigated frequencies of ses was able to operate as a key in switching modulatory effects in the central nervous system of healthy volunteers and stroke patients with spasticity.
as there are already numerous publicly available glycan and carbohydrate databases, it is important to clarify how glytoucan is different. as reported from the acgg - db meeting (1) it was agreed that uniquely identifying glycan structures would be the main content of this repository. the user and time / date of registration would also be attached with the glycan structure information. therefore the ability to register glycan structures will be the main service provided, and a unique accession number will be generated to be used for reference in any lycan - related research or publications. we note that it was decided at this acgg - db meeting that in order to simplify development, glytoucan will be responsible only for glycan structures and minimal metadata (user and registration time / date). that is, other metadata such as experimental data, aglycon information and publication would be outside the scope of this repository, and it would be the responsibility of curated databases such as unicarbkb (2) and bcsdb (3) to cover such information. other attributes such as mass and motifs are attached to each entry as they can be computed from the structure. however, glycan structures need not consist purely of monosaccharides as glytoucan also covers substituents on the monosaccharides that are not on the reducing end of the glycan. thus the major substituents such as sulfates, phosphates, etc. are handled in version 1.0. the first release of the international glycan structure repository was recently completed to enable this registration of glycan structures through either a web browser or a programmable interface. the repository was pre - registered with structures available in major public glycan databases that provided their data : at the time of this writing, links are available for glycomedb (4), bcsdb (3), glycoepitope (5), and progress is currently being made to link with unicarbkb (2). for entries in other databases that contained duplicate glycan structures (e.g. because of the inclusion of aglycons), these became single entries in glytoucan, and the linked db section would list all entries in the original database containing that glycan structure. for the current version, there were instances where structures could not be registered because they could not be converted to glycoct (6). in this case, we put these entries on hold for the next version, which will use the web 3.0 unique representation of carbohydrate structures (wurcs) (7) as the base format and should be able to handle such cases. the site also provides a variety of methods to query and browse the relationships of glycan structures based upon logic inherent among the registered structures. herein, we describe the functionality developed in the frontend of glytoucan, backend web services, and linked data endpoint. the aim of glytoucan is to simplify the identification of glycan structures and to help link related research within the many life sciences databases available worldwide. in order to facilitate the integration of various life sciences databases, we chose to use semantic web technologies, in particular resource description framework (rdf) as the base technology. as such, a triplestore (rdf database) is incorporated into the glytoucan architecture, and an endpoint is made available as a url where the rdf data (triplestore) can be accessed using the sparql query language. figure 1 illustrates the overall architecture of glytoucan, which consists of three major parts : the front - end web interface, the backend which also includes a relational database, and the triplestore. as glytoucan was an international collaboration between the us and japan, the us developers first developed the backend system, which was used by the japanese developers to develop the web interface, and then converted to the triplestore. a schematic of the glycan repository architecture, which comprises of the front - end web pages, a rest api interface to the relational database, and a sparql interface to the triplestore. it should be noted that detailed documentation is available on the user guide website (http://code.glytoucan.org). this site not only includes a detailed explanation of all functionalities, but also linked data information and links to source code. it will primarily cover the process by which a accession number can be searched for, registered via a graphical user interface, accessed using a program and finally queried through our publicly available linked data endpoint. glytoucan offers a variety of methods to search and browse through the glycan structures registered and other preloaded data available. in the cases where there are a vast number of search results, it is possible to filter based upon common structural attributes. structures from glycomedb (4), bcsdb (3) and glycoepitope (5) are a few of the databases that were pre - registered in glytoucan. the browsing functionality shows the wealth of information that was gathered from the currently available data in these public glycan databases. this section will explain the browsing options available, as well as the three search methods : graphical, sequence text and motif. glycan list link which is a quick method to view the glycan structures contained within the entire repository. the top text input field can be used to screen for a specific motif or monosaccharide name. the mass range can be quantified by inputting the minimum and/or maximum values, or by altering the scrollbar. the ranges for these filtering options, such as the maximum mass or monosaccharides to choose from, are retrieved from the entire scope of the glycan structures currently registered. if a monosaccharide is chosen, it is then possible to filter for a specific cardinality of that monosaccharide. in the results list, the format can also be altered to display the glycan sequences in either glycoct (6) or wurcs (7). moreover, the results can be sorted according to accession number, contributor, mass or date entered. this same browsing and filtering functionality is available in the search results of the motif search, described next. the browsing functionality can filter out the results based upon motifs, monosaccharide names, monosaccharide cardinality and mass range. it is also possible to sort the results in order of accession number, contributor, mass or date entered. the motif search method (https://glytoucan.org/motifs/search) initially displays a list of the 61 predefined motifs, which are defined as commonly found glycan substructure patterns in the literature. clicking on a motif in this list displays the listing of the glycans registered in glytoucan that contain the selected motif. some motifs are very common and can thus return a very large number of glycan structures containing the selected motif. therefore, multiple filtering options allow users to easily find their glycan structure of interest. in the cases where a more specific glycan structure needs to be specified, it is possible to build a specific glycan substructure via the glycanbuilder interface (8) (https://glytoucan.org/structures/graphical) as displayed in figure 4. once the structure building is complete and the search button is pressed, it is converted into glycoct (6) format and used to search the repository. regardless of whether the structure has been registered already, it and any superstructures (registered glycan structures that contain the input glycan structure), if the structure used to search for is not registered yet, it will indicate so by displaying the glycan structure overview page displays the core structural information as well as any related data such as motifs found and monosaccharide compositions. a listing of the public databases under the linked db section is also displayed with links to reference the structure information directly. the methods to register a structure are by building one graphically from glycanbuilder (8), pasting a sequence text into a form or uploading a file. the registration confirmation screen shows an example of registering a glycoct (6) text sequence. the text search method (https://glytoucan.org/structures/structuresearch) is similar to the graphical interface, however, the input can be specified using commonly utilized sequence formats such as glycoct (6), kcf (9) or linearcode (10). after submission, the format is converted to glycoct (6) as necessary, after which the results screen is displayed, similar to the graphical search method. from the results listing, more details about a specific glycan structure can seen by clicking on an accession number, which will display the glycan structure overview page. the glycan structure overview page displays all data specific to the glycan structure and any related information. the core structural information is at the top of the page, where a graphical representation is displayed (graphical representation display options can be changed using the preferences menu). below the image, sections describing motifs contained and monosaccharide compositions are displayed. further below, a listing of the public databases under the linked db section is shown with links to the database site where more (curated) information can be referenced. from the glytoucan website, there are currently three different methods to register structures into the repository : graphically, text and file upload. text and file upload are limited to glycoct (6), linearcode (10) and kcf (9) glycan structure text sequence formats. for the sake of brevity we will review the simplest method which is via the graphical interface using glycanbuilder (8). in order to access the registration functionality, it is first necessary to sign in by clicking on the sign in button. login is required in order to associate a user to the structures that are registered. for each glycan structure glycan structures can be drawn on the glycanbuilder (8) interface and once the submit button is pressed, the graphical form of the structure is converted into the glycoct format (6) and submitted to the backend system. the backend system checks for duplicates and generates the accession number for the newly registered glycan structure. structures with ambiguous linkages and fragments are all accepted, however, checks such as conflicting linkages are made to ensure that chemically impossible structures are not registered. we note here that structures with ambiguous linkages will be registered if the glycoct (6) string comparison does not match any other registered structures, even if other ambiguous structures could potentially match it. this will become an important feature for users to be able to browse ambiguous structures and find more defined structures that match it (e.g. a structure registered as structures will be made browseable in the next release using a visualization tool currently being developed, as all subsumed structures and their relationships will be added to the triplestore, thus allowing users to find these relationships easily through a visualization interface. it is possible to utilize the backend server for other methods of access using a standard programmable interface, which will be described later. after registering the glycan structure, multiple server - side processes are executed in order to analyze and link the structure to previously registered glycan structures or glycan motifs (a commonly known glycan substructure pattern, explained below in the motif search section). lastly a confirmation page will be shown which displays the details found for the registered structure, such as the calculated mass, motif and monosaccharide content. following the registration process, another batch process (labeled gs2virt in figure 1) is executed which retrieves the structure and accession number from the relational database and converts the data into rdf format. these data are stored into a separately available triplestore and linked to content such as conversion to other textual formats. the glytoucan backend server provides a method for system or application developers to access the information stored in the repository. using the provided library, or application programming interface (api), it is possible to search, retrieve and register new glycan structures. the complete specification is available online and describes in detail the functionality of each method (http://api.glytoucan.org/documentation/apidoc.html). as an example, we will request the glycoct (6) format of an already registered glycan structure utilizing the accession number. this is done with a simple call to a web location (uniform resource locator ; url) using the accession number as a parameter by embedding it within the url. the accession number for an n - glycan core (man3glcnac2) structure is g00026mo. as the api is based on the rest (representational state transfer) protocol, a request in the following format can be made via a url to retrieve the sequence (and other information) of the glycan to retrieve an xml - formatted response : http://api.glytoucan.org/glycans/g00026mo. this request will return the following response : the xml - formatted response of a glycan structure request, where the structure is represented in glycoct (6) format in the tag. registration of a glycan structure can be completed by inputting the information in glycoct (6), and the xml output will indicate the relevant accession number generated. it is thus possible to create separate applications or websites utilizing this api to register structures and extract information in the repository. as described in the previous section, the registered structures are batch - processed in order to generate and enrich the rdf data. these data are then made freely available through the linked data endpoint at http://ts.glytoucan.org/sparql and can be accessed with standard sparql queries. for readers interested in using the sparql interface, we provide an explanation and examples of how to retrieve / download the data in glytoucan in the supplementary materials. in this manuscript, we describe the first version of the glycan structure repository, called glytoucan, which allows users to obtain unique accession numbers for any glycan structure. it is possible to register a variety of glycan structure information : glycan compositions, glycans with ambiguous linkages, ambiguous fragments and fully - defined structures. based on the accession numbers in glytoucan, existing databases can simply use these numbers when referring to glycan structures. it will then be very simple for glytoucan to link back to the databases that have stored these same accession numbers, and display the linkages in search results. comparison of glycan structures with specific sequence formats is no longer needed when comparing glycan data across databases or when searching for glycans in publications. in preparation for the next release, one of the major items proposed is to have more user - editable content, such as the ability to make comments and rate entries. thus, nearly all of the data in glytoucan such as the structures, motifs and monosaccharides would be able to be commented on or rated by registered users. the contents of entries themselves will not be editable, however, a community - driven framework for data quality check would improve content quality and give direction to new functionality requirements. adding relationships between glycan structures also adds value to the data set. enrichment with sub / super - structure, subsumed structures and isomeric relationships is simply a matter of creating algorithms that analyze the structures already registered ; however, performance analysis will be necessary as volume could be highly resource - intensive. once this is ready, it will be possible to show ambiguous siblings or sub / super structure content while browsing through search results. finally, the post - registration processes are currently run at a batched interval in order to process newly submitted glycan structures, thus there may be some delay between the time when a glycan structure is registered and when its data are viewable from a browser. ideally this should be a real - time process, however, the complexity involved and potential resource bottlenecks require more evaluation on how this is to be implemented. this is a high - priority item that will be covered after the first release. national bioscience database center (nbdc), japan science and technology agency (jst) and national institute of general medical sciences [8p41gm103490 ] ; biotechnology and biological sciences research council [bbf0083091 and bb / k016164/1 to s.m.h ]. funding for open access charge : japan science and technology agency (jst) grant code tg2608 and soka university.	glycans are known as the third major class of biopolymers, next to dna and proteins. they cover the surfaces of many cells, serving as the face of cells, whereby other biomolecules and viruses interact. the structure of glycans, however, differs greatly from dna and proteins in that they are branched, as opposed to linear sequences of amino acids or nucleotides. therefore, the storage of glycan information in databases, their curation, has been a difficult problem. this has caused many duplicated efforts when integration is attempted between different databases, making an international repository for glycan structures, where unique accession numbers are assigned to every identified glycan structure, necessary. as such, an international team of developers and glycobiologists have collaborated to develop this repository, called glytoucan and is available at http://glytoucan.org/, to provide a centralized resource for depositing glycan structures, compositions and topologies, and to retrieve accession numbers for each of these registered entries. this will thus enable researchers to reference glycan structures simply by accession number, as opposed to by chemical structure, which has been a burden to integrate glycomics databases in the past.
iran, because of extent, geographical situation and climatic variety, is one of the damageable countries of the world. natural disasters, for example earthquake, are an unexpected event that cause damage and destruction to human life and health, and the injured persons without others assistance are not able to meet their need. earthquakes in iran and neighboring regions (e.g., india, turkey and afghanistan) are closely connected to their position within the geologically active alpine - himalayan belt[25 ] [table 1 ]. this crisis happens in an especial situation that changes all of the daily affairs of disastrous society, such as people earning, city services, communication system and community public needs and people health. earthquake information management system (eims) is a system that records, collects, keeps, retrieves and analyzes inputs and alters the reports and required earthquake information (ei) and renders it to the right people and organization to manage earthquake outcomes. deadliest earthquakes by year, 19952005 information is not an end in itself, but a means to better decisions in policy design, planning, management, monitoring and evaluation of programs and services, including damage of disasters reduction. unfortunately, information systems in most countries are inadequate in providing the needed management support. earthquake loss estimates are forecasts of damage and human and economics impacts that may result from future earthquakes. the earthquake loss estimation methodology is a system that uses mathematical formulas and information about building stock, local geology and the location and size of potential earthquakes, economic data and other information to estimate losses from a potential earthquake. eims uses arc gis (geographical information system) to map and display ground shaking, the pattern of building damage and demographic information about a community. once the location and size of a hypothetical earthquake is identified, eims will estimate the violence of the following : ground shaking, the number of buildings damaged, the number of injured persons, the amount of damage to transportation systems, disruption to the electrical and water utilities, the number of people displaced from their homes and estimated cost of repairing projected damage and other effects.[1013 ] an estimate of losses from future earthquakes is essential to preparing for a disaster and facilitating good decision making at the local, regional, province and national levels of government. an eims can estimate earthquake losses, providing vital tools for the following : 1)land - use planning and facility site decisions (e.g., a map - based analysis of the potential intensity of ground shaking from a postulated earthquake that identifies those parts of the community that will experience the most violent shaking and the buildings at greatest risk of damage).2)prioritization of retrofit or abatement programs (e.g., an estimate of building damage that provides the basis for establishing programs to mitigate or strengthen buildings that may collapse in earthquakes by providing estimates of damages and casualties).3)regional, province and local emergency response and contingency planning (e.g., estimates of casualties and of damage to buildings and utilities).4)medical and relief agency preparedness and response (e.g., estimates of casualties and homelessness).5)assistance planning. land - use planning and facility site decisions (e.g., a map - based analysis of the potential intensity of ground shaking from a postulated earthquake that identifies those parts of the community that will experience the most violent shaking and the buildings at greatest risk of damage). prioritization of retrofit or abatement programs (e.g., an estimate of building damage that provides the basis for establishing programs to mitigate or strengthen buildings that may collapse in earthquakes by providing estimates of damages and casualties). regional, province and local emergency response and contingency planning (e.g., estimates of casualties and of damage to buildings and utilities). medical and relief agency preparedness and response (e.g., estimates of casualties and homelessness). assistance planning. in this research, our important questions were : when can eims be useful?what are the essential substructures in eims?what is the usage of eims?what are the stages for formulating eims?how do we use eims to prepare for earthquakes ? when can eims be useful ? how do we use eims to prepare for earthquakes ? in this study, the management network for information related to earthquake in india and afghanistan was compared with iran to determine the total score of eims in them., several recommendations and a model were proposed to decrease weaknesses, improve the efficiency of eims process and, therefore, reduce damages and loss and expedite relief to victims after earthquakes. the statistical population consisted of the eims on network in india, afghanistan and iran. countries such as iran, india and afghanistan were chosen because these countries have high rate of disasters, especially earthquakes. to perform this study, the researcher made forms and questionnaires. the forms contained items to define standard characteristics of the information management system that was extracted from jcaho ; joint commission on accreditation of healthcare organization, ahima ; american health information management association and cchsa ; canadian council on health services accreditation, and then made synthetic forms. the questionnaire was designed to determine viewpoints and opinions of experts to set weights for every characteristic of the eims. in the first phase of data collection, validity of forms and data included ei sources, method of recording, storing, retrieving, analyzing, interpreting, distributing of ei, national and international levels usage, and so on. standard characteristics of the information management system were selected as criteria. in the second stage, for comparing characteristics of eims, experts opinions were selected to set weights (the relative importance of each criterion from 1 was low until 10 was high) by brainstorm decision criteria and measured mean of experts opinions to set weights for each of them in table 1. then, scales (positive = 4, moderate = 3, not access = 2, negative = 1) and scores (score = ratioscale) for selected countries were calculated. 1)information users and addressers specified,2)time, form and mechanism of information distributed specified,3)ei must be valid and reliable,4)fast access to ei. information users and addressers specified, time, form and mechanism of information distributed specified, ei must be valid and reliable, furthermore, the data received are often not helpful for management decision making because they are incomplete, inaccurate, untimely and unrelated to priority tasks and functions of crisis management. essential sub - structures in eims : 1)science of crisis management,2)information technology,3)geographic information system,4)information system,5)mass media,6)cell phone,7)capital,8)human resources. science of crisis management, information technology, geographic information system, 1)impossibility of fast and easy retrieval, extract and access of information for managers and all related users;2)extract integrated data from different resources;3)prevent to implement parallel and repeated activities by various organizations;4)decrease cost and time;5)assessing and monitoring function and plans before and after earthquakes;6)recognizing training needs functional forces;7)formulate prevention, action and rehabilitation according to outcomes of eims evaluating. impossibility of fast and easy retrieval, extract and access of information for managers and all related users ; extract integrated data from different resources ; prevent to implement parallel and repeated activities by various organizations ; decrease cost and time ; assessing and monitoring function and plans before and after earthquakes ; recognizing training needs functional forces ; formulate prevention, action and rehabilitation according to outcomes of eims evaluating. formulating eims 's stages : 1)institute joint commission from related governmental and non - governmental sectors and organizations;2)determine elementary sources;3)determine and formulate general concepts and purposes;4)recognize necessary data items;5)recognize and determine informatics resources;6)recognize and determine registration, collection and storage methods and administrators;7)recognize and determine retrieval and analyze methods and administrators;8)recognize and determine, distribute and issue of information methods and administrators;9)create methods to connect systematically among these administrators;10)notice to render feedback process in eims to ensure continuous improvement system;11)dynamic and flexible plans and functions must be design. institute joint commission from related governmental and non - governmental sectors and organizations ; determine elementary sources ; determine and formulate general concepts and purposes ; recognize necessary data items ; recognize and determine informatics resources ; recognize and determine registration, collection and storage methods and administrators ; recognize and determine retrieval and analyze methods and administrators ; recognize and determine, distribute and issue of information methods and administrators ; create methods to connect systematically among these administrators ; notice to render feedback process in eims to ensure continuous improvement system ; dynamic and flexible plans and functions must be design. loss estimates can provide the basis for developing mitigation policy, for developing and testing emergency preparedness and response plans and for planning for post - disaster relief and recovery. loss estimates provide public and private sector agencies with a basis for planning, zoning, building codes and development regulations, and policy that would reduce the risk posed by violent ground shaking and ground failure. loss estimates can also be used to evaluate the cost - effectiveness of alternative approaches to strengthening potentially hazardous structures. before an earthquake : preparing to respond, understanding the scope and complexity of earthquake damage is essential to effective preparedness. these estimates can serve as the basis for developing emergency response plans and for organizing tests and exercises of response capability. about eims : in india, findings showed that the disaster information management system (dims) was launched by sristi sristi ; http://www.sristi.org society for research and initiatives for sustainable technologies and institutions on the 18th of january 2002 at the indian institute of management, ahmedabad, gujarat, india. however, the relief work suffered immensely due to lack of information and proper planning. when we tried to get answers to important questions that were cropping up for instance, whether there is a database on the distribution of available resources and expertise with individuals, institutions and corporations all we got in response was a blank. this pointed to the urgent necessity of building a system for disaster mitigation and for documenting experiences of individuals and organizations, which might act as a knowledge resource and help in better coordination in case of future disasters. through this initiative, we are trying to develop a database - driven information system for disaster management authorities (dma) in various states, ngos and other organizations. we appealed to ngos, relief workers, dmas and individuals to share their experiences and volunteer services and resources to the online database maintained at our website. the database currently contains more than a thousand volunteers who have offered to volunteer their services and resources in time of emergency. about 700 organizations and institutions indexed on the site, besides other resources and web links. the dmis is a voluntary activity run with contributions in terms of time and services by sristi volunteers, ngos and, above all, civil society institutions across the world. all the information shared with us is accessible to all, except where the volunteer has chosen to limit accessibility only to the relevant authorities. about eims : in afghanistan, findings showed that afghanistan is in danger of many natural disasters. disaster management is a legal attempt that needs miscellaneous information, different locations and tenses, and this information must have a true format to be in access of key staff in deciding. the temporary project of management information for natural disasters in kabul province and kunduz province in afghanistan has been doing well for 8 months. also, it updates and gives information to governments. this project has increased afghanistan 's ability in crisis management nationally, supporting educated people and city renewing services by building governmental organizations in management information for natural disasters. natural disaster management in these two provinces is mostly based on estimating the amount of damages and concentrated rescue operations, and there is no system to avoid or decrease the amount of damages by natural disasters. before or after disaster, management is very weak in these countries and they are dependent on un or ngo. information source in the process of collecting eims includes human forces and geographical informative systems that are a dangerous area and shows locations with high potential danger. in this system, the satellite has an important part in recognizing crisis locations distance. to record collected information, a team of information system unit is educated to record in puts portions, information and management of stations of informative system of disaster management, and they will record the information. structure of dims special earthquake is not useful in many parts of the country. the most important problem is lack of exact information (not in time). in iran, the eims showed absence of timely reporting of most of the data before, during and after earthquakes, defective, insufficient and inaccurate registration of data, declaration and publishing different and contradictory population statistical reports by related organizations and weakness to use reliable information to support the prevention systemic planning. in order to make suitable ei in iran, we need to provide and support managers. to improve current situation of eims, we need to design a midified model of eims. the modified model contains ; responsible organizations, their functions, and flow - work that were approved by the delphi technique. table 2 denotes the highest sum of score relative to india and the lowest relative to iran. the weakness issues are respectively concluded : ei stores systematically, no parallel and repeated activities by various organizations and accessibility of ei easy and fast in eims criteria in iran. in the range of ranks, afghanistan and india were classified in the very good range and iran in the moderate range. responsible journalism can also help clear inaccurate rumors and influence public 's attitude toward preparing for disasters. moreover, press coverage of old disasters may be a good source of data where official records do not exist. however, the present study has revealed that the press has largely failed in terms of disaster mitigation and preparedness guidance. it seems that media is more interested in giving disastrous news than informing the public. turkish media had been more influential in urging both the public and the officials in getting ready for the coming earthquakes. the japan meteorological agency (jma) as a governmental organization responsible for issuing ei and tsunami forecasts had developed an early earthquake notification system in japan. at present, jma issues the following kinds of information successively when a large earthquake occurs : (1) prompt report of occurrence of a large earthquake and major seismic intensities caused by the earthquake in about 2 min after the earthquake occurrence, (2) tsunami forecast in around 3 min, (3) information on expected arrival times and maximum heights of tsunami waves in around 5 min and (4) information on a hypocenter and a magnitude of the earthquake, the seismic intensity at each observation station, the times of high tides in addition to the expected tsunami arrival times in 57 minutes. to issue the above information, jma has established an advanced nationwide seismic network with about 180 stations for seismic wave observation and about 3400 stations for instrumental seismic intensity observation, including about 2800 seismic intensity stations maintained by local governments. beginning in the late 1950s in the world, planners started to develop and use computerized models, planning information systems and decision - support systems to improve performance. they have found tools to enhance their analytical, geospatial technologies that differ from one country to another. they use it in many fields ; the governments apply urban information systems in all aspects of the planning process, including data collection, storage, data analysis and presentation, planning and policymaking, communication with the public, policy implementation and administration. the united states is the pioneer in this field ; they began working with urban information systems in the 1970s. canada and australia have developed systems ; also european countries like france, germany and holland have been successful in applying these technologies. turkey is a latecomer in this field because of financial problems, the other priorities and the lack of technical expertise and different mentalities of the administrators. but the first initiative of local governments like bursa and ayden cities began to use urban information systems in the mid-1990s, and then the other three metropolitan municipalities, istanbul, ankara and izmir, made studies about digitizing the maps, plans and creating inventories about their cities. in this section, first, turkey examples and their studies about urban information systems are explained, then the other world examples are described for their different uses and applications. in the earthquake management section the preparedness and mitigation phases have more emphasis. because it is believed that besides advice and instructions given to the public, raising awareness for the earthquake risk is necessary for the management levels and the residents, the technological tools help in this process. but, the management, including protection and recovery, is completed with the interrelated system. the first effort to systematically collect and analyze data in developing countries should be undertaken by national program managers. based on the investigation of the current situation in india, afghanistan and iran, we need to have eims because of the following reasons : 1)relevance of the information must be gathered;2)continuous improvement of data must be concerned;3)control and manage natural disasters (rapid availability and retrieval ei);4)timely reporting and feedback must be rendered;5)analyze information and render reports and define strengths, weaknesses, threats and opportunities;6)monitoring healthcare services status and services needs;7)coordinating activities between government and non - government sectors and other related sectors to use ei;8)determining causes of deaths and health priorities and planning to decrease mortality after earthquake in the future;9)using outcomes for determining cause of earthquake mortalities and other related problems to prevention in the future;10)formulating strategies to diseases incidence prevention and decrease of controllable deaths after earthquakes on wards. relevance of the information must be gathered ; continuous improvement of data must be concerned ; control and manage natural disasters (rapid availability and retrieval ei) ; timely reporting and feedback must be rendered ; analyze information and render reports and define strengths, weaknesses, threats and opportunities ; monitoring healthcare services status and services needs ; coordinating activities between government and non - government sectors and other related sectors to use ei ; determining causes of deaths and health priorities and planning to decrease mortality after earthquake in the future ; using outcomes for determining cause of earthquake mortalities and other related problems to prevention in the future ; formulating strategies to diseases incidence prevention and decrease of controllable deaths after earthquakes on wards. after an earthquake, a rapid response to a damaging earthquake will reduce loss of life, lessen complications from injuries and secondary damage and loss and expedite relief to victims. reliable and up - to - date information can have an impact on the destruction factors and prevent them. because of the financial and human damages of disasters, establishing a general, scientific and practical management network is necessary. figure 1 demonstrates a proposed model that shows the process of relationships between organizations related to eims in iran. in this model, duty and function of every organization these duties are classified according to registering and collecting earthquake data, storing and processing, analyzing and distributing and using issued ei. the proposed model that shows process of relationships between organizations related to eims in iran	context : damages and loss of life sustained during an earthquake results from falling structures and flying glass and objects. to address these and other problems, new information technology and systems as a means can improve crisis management and crisis response. the most important factor for managing the crisis depends on our readiness before disasters by useful data.aims:this study aimed to determine the earthquake information management system (eims) in india, afghanistan and iran, and describe how we can reduce destruction by eims in crisis management.materials and methods : this study was an analytical comparison in which data were collected by questionnaire, observation and checklist. the population was eims in selected countries. sources of information were staff in related organizations, scientific documentations and internet. for data analysis, criteria rating technique, delphi technique and descriptive methods were used.results:findings showed that eims in india (disaster information management system), afghanistan (management information for natural disasters) and iran are decentralized. the indian state has organized an expert group to inspect issues about disaster decreasing strategy. in iran, there was no useful and efficient eims to evaluate earthquake information.conclusions:according to outcomes, it is clear that an information system can only influence decisions if it is relevant, reliable and available for the decision - makers in a timely fashion. therefore, it is necessary to reform and design a model. the model contains responsible organizations and their functions.
to facilitate rapid differential diagnosis of vhf agents, we established the greene masstag panel vhf version 1.0, which comprises the following targets : ebola zaire virus (zebov), ebola sudan virus (sebov), marv, lasv, rvfv, cchfv, hantaan virus (hntv), seoul virus (seov), yfv, and kfdv. oligonucleotide primers were designed in conserved genomic regions to detect the broadest number of members for a given pathogen species. we developed a software program that culls sequence information from genbank, performs multiple alignments with clustalw, and designs primers optimized for multiplex pcr. the program uses a greedy algorithm to identify conserved sequences and create the minimum set of primers for amplification of all sequences in the alignment. primers are selected within standard design constraints whenever possible (melting temperature 55c65c, guanine - cytosine content 40%60%, no hairpins) ; degenerate positions are introduced in cases where template divergence requires more flexibility. although degeneracy is not tolerated in the five 3 nucleotides, masstag pcr allows up to 4 nonneighboring variable positions per primer. primers are checked by the basic local alignment search tool for potential hybridization to sequenced vertebrate genomes (table 1). zebov, ebola zaire virus ; sebov, ebola sudan virus ; marv, marburg virus ; lasv, lassa virus ; rvfv, rift valley fever virus ; cchv, crimean - congo hemorrhagic fever virus ; hntv, hantaan virus ; seov, seoul virus ; yfv, yellow fever virus ; kfdv, kyasanur forest disease virus ; fwd, forward ; rev, reverse. primers were designed on lassa lineage iv sequences (15) and the recently identified outlier sequence lassa av (af256121). because only released mass tags are analyzed, staggering the size of amplification products created in multiplex reactions is unnecessary ; thus, primers are selected for efficient and consistent performance irrespective of amplicon size (typically 80200 bp). before committing to synthesis of tagged primers, the functionality of candidate multiplex primer panels is examined in a series of amplification reactions that use prototype templates representing individual microbial targets. primers that fail to yield a single, specific product band in agarose gel analysis are replaced. target sequence standards for evaluation are cloned into pcr2.1-topo (invitrogen, carlsbad, ca, usa) by using pcr amplification of cdna templates obtained by reverse transcription (rt) of extracts from infected, cultured cells or by assembly of overlapping synthetic polynucleotides. the agents assayed in the vhf panel have rna genomes ; thus, assay sensitivity was determined by using synthetic rna standards. synthetic rna standards were generated from linearized target sequence plasmids by using t7 polymerase (mmessage mmachine, invitrogen). after quantitation by uv spectrometry, rna was serially diluted in 2.5 g / ml yeast trna (sigma, st. louis, mo, usa), reverse transcribed with random hexamers by using superscript ii (invitrogen), and analyzed by masstag pcr as previously described (14). qiaquick 96 pcr purification cartridges (qiagen, hilden, germany, with modified binding and wash buffers) were used to remove unincorporated primers before tags were decoupled from amplification products by uv photolysis in a flow cell and analyzed in a quadrapole mass spectrometer by using positive - mode atmospheric pressure chemical ionization (apci - ms, agilent technologies, palo alto, ca, usa). the sensitivity of the 10-plex vhf panel with synthetic rna standards was 4 and 8. these results confirm earlier work in respiratory diseases that show that masstag pcr offers a rapid, sensitive, specific, and economic approach to differential diagnosis of infectious diseases. small, low - cost, or mobile apci - ms units extend the applicability of this technique beyond selected reference laboratories. given the capacity of the method to code for up to 32 genetic targets, we are expanding the hemorrhagic fever panel to include additional viruses (dengue and south american hemorrhagic fever viruses) and are exploring the inclusion of bacterial and parasitic agents that may result in similar clinical signs and symptoms and, thus, have to be considered in differential diagnosis.	viral hemorrhagic fevers are associated with high rates of illness and death. although therapeutic options are limited, early differential diagnosis has implications for containment and may aid in clinical management. we describe a diagnostic system for rapid, multiplex polymerase chain reaction identification of 10 different causes of viral hemorrhagic fevers.
anemia in chronic kidney disease (ckd) has long been a substantial problem for both patients and providers. currently there are more than 8 million american adults with ckd stage 3 or greater (glomerular filtration rate [gfr ] less than 60 ml / min/1.73 m) and anemia. current prevalence is estimated to range between 18% in stage 3 disease to near 60% in stage 45 (gfr less than 30 ml / min/1.73 m).1 the incidence and prevalence of end - stage renal disease (esrd) continues to increase in the united states, and it is well documented that anemia worsens as gfr declines due to less native erythropoietin production. erythropoietin regulates the proliferation, differentiation, and survival of erythroid progenitor cells and thereby controls erythrocyte levels.2 multiple factors contribute to anemia in the patient with esrd including decreased erythrocyte half - life in the uremic milieu, decreased gut absorption of iron and chronic blood loss in the tubing set with each dialysis treatment. in addition, it is unclear if anemia in patients with renal disease is entirely due to decreased production of erythropoietin or if there is a derangement in oxygen - sensing capacity leading to a secondary decrease in erythropoietin production.3 however, anemia in esrd patients has been clearly shown to increase the incidence of cardiovascular complications,4 reduce quality of life (primarily by decreasing physical capacity), and impair cognitive function.5 erythropoietin - stimulating agents (esas) are considered the standard of care for treatment of anemia in esrd patients.6 the introduction of recombinant human erythropoietin (rhuepo) to clinical practice in 1989 in the united states revolutionized the treatment of chronic anemia in this patient population. prior to the introduction of rhuepo, patients were transfused only when they became clinically symptomatic. however, this practice put many patients at risk for secondary complications including volume and iron overload, hyperkalemia, allosensitization, transfusion reactions, and blood - borne infections.7 prior to 1989, dialysis patients were transfusion - dependent and suffered the debilitating symptoms consistent with hemoglobin levels chronically in the 67 g / dl range.8 the initial response to erythropoietin treatment was dramatic, with improvements in quality of life as well as the physiologic benefits associated with fewer transfusions and improved hemoglobin levels.6,9,10 consequently, the use of erythropoietic - stimulating agents to correct anemia in the esrd population exploded over the next few years. observational studies noting that patients with higher hemoglobin levels at dialysis initiation had decreased hospitalization rates and mortality spurred a more aggressive approach to correcting anemia.11 not surprisingly, the concept of trying to achieve near - normal hemoglobin levels through higher dose esas was particularly appealing to nephrologists who now had a tool available to help reach this goal. however, in recent years, the optimal target level of hemoglobin has been the subject of a great deal of debate within the nephrology community. several large, randomized trials investigating dosing strategies attempting to normalize hemoglobin levels have increased morbidity and mortality. these trials also demonstrated increased thromboembolism rates (both arterial and venous) and stroke with higher doses of esa administration.12,13 the first study to incite concern about normalizing blood counts was the aptly named normal hematocrit cardiac trial.14 this study enrolled over 1200 patients known to have heart failure or ischemic heart disease and hematocrit levels of 27%33% on epoetin treatment. the patients in the normal hematocrit group received escalating doses of epoetin to achieve and maintain levels of 42% 3%, while the low hematocrit group received doses adjusted to maintain hematocrit levels of 30% 3%. the study had a planned duration of 3 years, but was stopped after 14 months due to concerns when interim analysis demonstrated more primary end - point adverse events, including death or first nonfatal myocardial infarction (mi) in the normal hematocrit patients. further data analysis of this study failed to demonstrate a clear relationship between either hematocrit level or epoetin dosing and mortality.13 target hemoglobin levels for patients with anemia secondary to ckd remained nebulous. a cochrane meta - analysis of 19 randomized controlled trials comprising almost 2700 patients demonstrated that those with hemoglobin targets above 13 g / dl were associated with higher all - cause mortality than those with targets 11 g / dl was associated with decreases in transfusion rates, number of units transfused, and hospital rates and lengths of stay as well as increases in all measured quality of life parameters ; no data on survival benefit was examined.16 in an effort to clarify the appropriate hemoglobin goal for esa use, two randomized controlled trials were conducted in ckd patients not yet on dialysis to examine the effect of normalizing hemoglobin on cardiovascular end points and death. the first was the correction of hemoglobin and outcomes in renal insufficiency (choir) trial, randomizing 1432 patients to hemoglobin targets of 11.3 g / dl versus 13.5 g / dl. the trial was terminated early by the data and safety monitoring board due to a trend towards increased composite cardiovascular events in the high hemoglobin target group and with no statistical probability of showing an appreciable difference in quality of life assessment scores. subsequently, the authors concluded that the higher hemoglobin target conferred increased risk with no benefit as compared to the lower hemoglobin target of 11.3 g / dl. the cardiovascular risk reduction by early anemia treatment with epoetin beta (create) trial followed, with 603 ckd patients randomized to hemoglobin targets of 1315 g / dl versus 10.511.5 g / dl. ultimately the findings demonstrated no significant difference in cardiovascular events and left ventricular mass index after 3 years, but progression to dialysis and hypertensive episodes were more frequent in the high hemoglobin group and the authors concluded that early and complete correction of hemoglobin levels with epoetin therapy confers no benefit.12,13 secondary analyses of both major trials have since been published with some interesting findings, including the suggestion that patients randomized to a higher hemoglobin level who achieved that target did better than those who did not, and there may be a negative outcome associated with the higher epoetin dosing in poor responders.17 it appears those at greatest risk of adverse events are the subset of patients on increasingly higher doses of esa, but are consistently unable to reach target hemoglobin goals. multiple explanations have been hypothesized, but it may indicate that a standardized, protocol - driven approach to esa management focused on increasing doses of esa to meet hemoglobin goals may harm some patients. those that, updated national kidney foundation / kidney disease outcomes quality initiative guidelines and recommendations for clinical practice concerning hemoglobin targets were published in 2007. they were modified after the choir and create trials had been released and the us food and drug administration (fda) had released new prescribing information and advisories regarding esa use. recommendations were for a hemoglobin target of 1112 g / dl and not greater than 13 g / dl, with emphasis on the need to weigh risks and benefits of esa use in individual patients.18 fda recommendations were to keep hemoglobin between 1012 g / dl and to minimize esa use where possible, which is accomplished primarily by aiming for higher transferrin saturation and ferritin goals and increased use of parenteral iron formulations. during this time, the trial to reduce cardiovascular events with aranesp therapy (treat) trial was ongoing. this international trial enrolled more than 4000 patients with ckd, anemia, and type 2 diabetes mellitus. anemia was defined as a hemoglobin 10 g / dl ; in those patients who fall below this threshold, it is suggested that a patient - specific risk and benefit analysis be undertaken prior to esa administration. this risk - to - benefit analysis should consider transfusion risks, symptoms attributable to anemia, prior response to iron therapy, and the rate of hemoglobin decline. in all ckd patients with anemia, iron supplementation 13 g / dl. there were serious adverse events reported in 20% of patients, including one possible drug reaction, but this was found to be consistent with routine observations in this patient population. the phase iii clinical trial program for peginesatide involved four randomized, controlled trials with either epoetin or darbepoetin alfa as comparable therapy. two of the studies were in ckd patients, pearl-1 (peginesatide for the correction of anemia in patients with chronic renal failure not on dialysis and not receiving treatment with erythropoeisis - stimulating agents) and pearl-2, and two were in dialysis patients, emerald-1 (efficacy and safety of peginesatide for the maintenance treatment of anemia in patients with chronic renal failure who were receiving hemodialysis and were previously treated with epoetin) and emerald2. a total of 2600 patients were recruited for the trials at more than 400 centers across the united states and europe. the mean follow - up duration for patients on the study drug in the four trials was 1.3 years. the primary end points in all trials were anemia correction or hemoglobin level maintenance, and this objective was achieved in all trials. an initial analysis of a composite cardiovascular end point met the prespecified noninferiority criteria, but in a subanalysis of the two pearl studies, there was an increased risk of developing the cardiovascular composite in patients receiving peginesatide rather than the comparator esa (hr, 1.32). the data was published recently, and the cause of the increased incidence of adverse events in the peginesatide group when compared with darbepoetin in the ckd population remains unclear. the initial study findings in all the phase iii trials indicated that peginesatide met the adjudicated composite safety end point (cse), which was composed of death, stroke, myocardial infarction, congestive heart failure, unstable angina and arrhythmia (hr, 1.06 ; 90% ci : 0.911.22) (figures 2 and 3). in the subgroup analysis of cse events in the emerald trials to maintain treatment of anemia in dialysis patients, the frequency of cse events was balanced between peginesatide and the comparator (hr, 0.95 ; 90% ci : 0.791.13). when this same subgroup analysis was conducted using data from the pearl trials in nondialysis patients, the frequency of cse events was higher in the peginesatide group (21.6%) versus the comparator group (17.1%) (hr, 1.34 ; 90% ci : 1.031.73).43 the hr in the nondialysis patients was driven primarily by higher rates of death, unstable angina, and arrhythmia events in the patients treated with peginesatide. the percentages of patients experiencing stroke, myocardial infarction, and congestive heart failure were similar between treatment groups. there is further investigation of this data ongoing, but this subanalysis prompted filing of a new drug application (nda) with the fda to be limited to the dialysis patient population only, rather than the broader category of ckd. there are several potential advantages to using peginesatide in the dialysis population, and with competitive pricing, it has the potential to become popular among the esa market (figure 4). the lack of antierythropoietin antibody development with the use of peginesatide may be one of its greatest strengths. at the time experiments were being conducted to determine dosing in peginesatide, there was a collaborative clinical study in europe to investigate whether it would be possible to rescue patients with pure red cell aplasia induced by erythropoietin administration, many of whom developed a severe transfusion - dependent anemia. given the fact that peginesatide does not share structural homology with human erythropoietin, they postulated that patients with antierythropoietin antibodies would respond favorably to the drug. of 14 patients enrolled, 13 of them achieved a hemoglobin > 11 g / dl without requiring further transfusions. one patient developed antipeginesatide antibodies in addition to preexisting antierythropoietin antibodies and peginesatide had to be stopped.44 peginesatide s long half - life can be financially advantageous and may promote its future growth. its monthly administration may improve compliance while consuming less human resources in the outpatient dialysis setting. in addition, hospitalized patients would be less likely to require administration of the medication. the reduced number of injections could translate into decreased need for cold storage capacity, reduced risk of wastage, and risk of loss in the event of case of cold - storage interruption. phase iii trial data also suggest that for patients requiring very high epogen doses at baseline, perhaps due to antibody - mediated interactions, they required significantly less dose of peginesatide to achieve comparable hemoglobin. this may translate into improved outcomes for the subset of patients requiring very high dose esa to achieve adequate hemoglobin response who are at increased risk for cardiovascular complications as noted in the treat subanalysis.45 one potential concern regarding peginesatide is antibody development against the peg moiety. it has been shown that peg can induce specific as well as nonspecific recognition by the immune system.46 this response can, of course, increase the rate of clearance of pegylated proteins, including peginesatide.40 a recent review demonstrated that 22% to 25% of a cohort of 350 healthy blood donors had peg antibodies, potentially due to increased exposure of the general population to peg - containing compounds in cosmetics, pharmaceuticals, and processed foods.47 increased consumer use will determine if these antibodies are clinically relevant neutralizing antibodies and if they will impact efficacy of the drug. for many practitioners, financial pressure due to the cms bundle will cause them to consider switching patients to peginesatide due to cost of savings offered by this medication. entering the market at a very competitive price point, combined with decreased administration time and need for less cold storage space may persuade many medical directors to consider peginesatide. with the increasing pressure to streamline the costs associated with medication administration on dialysis and simultaneously decrease the need for expensive blood transfusions in this population, peginesatide may be a useful and cost effective tool in the anemia arsenal. if administered at the recommended dose of 0.04 mg / kg monthly, omontys is estimated to cost approximately $ 302 wholesale, which is significantly less than a comparable dose of aranesp (darbepoetin alfa) administered weekly or procrit (jannsen pharmaceuticals, inc., tutisville, nj, usa) (epoetin alfa) three times weekly, both of which are estimated to cost around slightly more than $ 700 monthly.48 time and widespread use will determine if it is possible to titrate hemoglobin adequately with monthly dosing, or if there will ultimately be an added burden due to financial censure for hemoglobin falling above or below goal ranges with use of this long - acting preparation. treatment of anemia remains a costly and challenging problem for renal patients and those who care for them, with many questions unanswered regarding the best approach to esa administration and how to juggle the delicate balance between adequate hemoglobin and quality of life considerations and the concern for worsening anemia, transfusion rates on the rise and the significant costs associated with anemia management in the outpatient setting. as economic pressures to streamline costs and provide efficient and effective care increase, new strategies to help our patients maintain adequate hemoglobin levels are a welcome addition. in patients who are iron - replete and responsive to esa administration, peginesatide may represent a new and exciting addition to the treatment algorithm for chronic anemia in esrd. peginesatide had been approved for use in the dialysis patient population and was marketed under the trade name omontys by affymax, inc, and takeda pharmaceutical company ltd. however, at the time of submission of this manuscript affymax has suddenly decided to voluntarily recall the product due to a risk of severe allergic reactions and anaphylaxis. they noted 0.02% of patients had been victims of a fatal anaphylactic reaction within 30 minutes of drug administration in more than 25,000 patients who had received the drug after its fda approval. it is currently unclear if affymax will plan to enter the market again with a similar product that has a better safety profile in the future.	anemia in chronic kidney disease is a prevalent and expensive problem in the united states, and it is well documented that anemia worsens as glomerular filtration rates decline. the complications of severe anemia in this patient population contribute significantly to their overall morbidity with increased cardiovascular complications, decreased quality of life, and increased dependence on transfusions to maintain adequate hemoglobin levels. erythropoietin - stimulating agents (esas) have revolutionized the treatment of anemia in this population, but there has been a great deal of controversy surrounding the quest for the ideal hemoglobin target. in addition, there are economic and practice management implications where anemia treatment is concerned, with ongoing refinement of centers for medicare and medicaid services - bundled payments. one of the newest additions to the arsenal used to fight anemia in end - stage renal disease patients is peginesatide (omontys), a synthetic, pegylated, peptide - based esa that acts by stimulating the erythropoietin receptor. the role of peginesatide in the future treatment of anemia in chronic kidney disease remains uncertain, with new safety concerns being brought to attention as it emerges on the market, prompting a national recall.
a daily intake of 800 g retinol equivalents was recommended as a safe level of vitamin a for pregnant women. according to who, 7.8% of pregnant women in africa have night blindness and 15.3% have low serum retinol concentrations. vitamin a deficiency (vad) during pregnancy is associated with increased maternal mortality and increased infant mortality rates during the first year of life. according to who, anemia globally affects 41.8% of pregnant women all over the world and 57.1% of pregnant women in africa. maternal anemia during pregnancy increases the maternal mortality and has many adverse effects on fetal outcome including small for gestational age, premature rupture of membranes, preterm delivery. vitamin a is known to play a role in hematopoiesis, and anemia is a common consequence of vad. the aim of the present study was to measure serum retinol concentrations of a cohort of poor egyptian mothers and correlate it with their hemoglobin concentrations (hb%) and cord hb% of their respective newborns. this cross - sectional study included 200 full term newborns and their respective mothers who were recruited from the reception room of a university gynecology and obstetrics hospital, cairo, egypt, during the period from june 2011 to december 2011. this hospital provides antenatal and obstetric care for pregnant women at an urban metropolitan in cairo of low socioeconomic standard. the study was approved by the local ethical committee of the faculty of medicine. online statistical calculator was used for sample size calculation guided by confidence level of 95% and error of 5%. we included only apparently healthy mothers, aged 1939 years, with singleton pregnancy and proper spacing in - between pregnancy (a gap of more than 18 months from birth to subsequent conception) who delivered through uncomplicated spontaneous vaginal delivery. all included mothers were of low income (less than 53.43 us dollars per capita per months). we excluded all women with grand multiparity (more than 5 deliveries), complicated pregnancy including multiple pregnancy, established diagnosis of maternal anemia during pregnancy, preeclampsia, renal disease, antepartum hemorrhage (placental abruption placentae, placenta previa, vasa previa), history of fever, and signs of acute infection as well as mothers who had delivered through instrumental vaginal delivery or cesarean section. we also excluded all women who had vitamin a supplementation or history of exposure to teratogens. we excluded neonates who were delivered before 37 weeks and had birth weight below 2500 grams. neonates with congenital anomalies, birth trauma were also excluded as well as neonates with family history of hemolytic anemia or maternofetal incompatibility (positive coombs ' test or high reticulocyte count). during the 6 months period, among 2058 deliveries 1554 mothers were excluded, 96 refused participation in the study, and 208 newborns were excluded. causes of exclusion of mothers included inappropriate age for the study (87), grand multiparity (194), improper spacing in - between pregnancies (110), complicated pregnancy (204), exposure to teratogens (5), vitamin a supplementation (117), delivery by caesarean section (693), and instrumental vaginal delivery (144). causes of exclusion of newborns included prematurity and low birth weight (70), presence of congenital anomalies and birth trauma (69), maternofetal incompatibility (22), and family history of hemolytic anemia (47). detailed history was obtained from each woman including parity and symptoms suggestive of vad : recurrent urinary and respiratory infections and symptoms of dry eye (eye discomfort, eye dryness, foreign body sensation, photophobia, and night blindness). mothers were asked to recall everything consumed (including foods and fluids) a day representing their usual intake starting from first meal or beverage on awakening until midnight of the reporting day. data were analyzed and vitamin a intake was calculated using the diet analysis program, 1995 (lifestyles technologies, inc. physical examination was done with stress on eye examination for dry eyes using 3 simple noninvasive tests. the tests were carried out in sequence, starting with tear film break - up time (tbut), followed by examination of the cornea by fluorescein staining and the schirmer i test without topical anesthesia. china) were inserted into the lower conjunctival sac at the junction of the lateral and middle thirds, avoiding touching the cornea, and the length of wetting strips in millimeters was recorded after 5 minutes. the cutoff point used for diagnosis of dry eye was < 10 mm per 5 minutes. for the newborns, apgar scores were recorded at 1 and 5 minutes to exclude the presence of perinatal asphyxia. five ml of maternal blood was collected by venipuncture immediately before delivery of the newborn. cord blood was collected at delivery from the placental end of the cord ; about 5 ml of mixed arterial and venous blood were collected. one specimen was collected on edta tube for cbc (for both mothers and newborns), reticulocyte count and coombs ' test (for newborns only). according to who, maternal anemia was considered when hb% is below 11 gm%. the other specimen was collected in an autoclaved glass vial for serum retinol concentration measurement. the vials were immediately wrapped in aluminum foil to avoid photooxidation of vitamin a, were stored at 4c, and were allowed to clot. after centrifuging the blood samples, the serum was carefully pippeted off into another vial and stored in a dark container at 20c until analysis. measurement of serum retinol concentration was done by high - performance liquid chromatography (hplc) using reversed - phase column and diode - array detectors. according to the who, we used maternal serum retinol level 0.7 mol / l as a cutoff value for maternal vad. the data were coded and analyzed with the statistical package for social sciences (version 17 ; spss inc, chicago, il, usa). description of quantitative variables was presented as mean and sd, and that of categorical variables was presented as frequency and percentage. unpaired t - test was used to compare parametric quantitative variables between the 2 groups : mothers with vad and mothers without vad. chi square () test was used to compare categorical variables between both groups. pearson 's correlation test was used for correlating maternal serum retinol concentrations with different variables. for all analyses, the age of the included ranged between 19 and 37 years with a mean of 26.4 3.9 years. four mothers (2%) gave history of night blindness and had signs of dry eyes. the maternal retinol intake ranged between 217.7 and 1300 g / day with a mean of 943.4 433.1 g / day and a median of 435.5 g / day (390890). one hundred and forty - one mothers (70.5%) had retinol intake 800 ug / day. the mean maternal hb% was 10.7 1.2 g% with a range between 6.6 and 13 g% and 50% of the mothers were anemic with a mean hb% of 9.6 1 gm% and 50% were nonanemic with a mean hb% of 11.6 0.4 gm%. anemic mothers had a lower mean serum retinol concentration (1.2 0.7 mol / l) compared to nonanemic mothers (1.9 0.7), but the difference was not statistically significant, p = 0.07. the maternal serum retinol concentrations ranged between 0.31 and 3.6 mol / l with a mean of 1.6 0.8 mol / l. forty - seven mothers (23.5%) had vad with a mean serum retinol concentration of 0.56 0.14 mothers with vad had a significantly lower mean retinol intake compared to mothers without vad, with a significant positive correlation between maternal serum retinol concentration and retinol intake (r = 0.44 and p = 0.001). no significant differences were found between both groups regarding age and parity (table 1). mothers with vad had a significantly lower mean hb% (8.95 1.63 gm%) compared to mothers without vad (10.11 0.83 gm%), p = 0.007, with a significant positive correlation between maternal serum retinol concentrations and maternal hb% (r = 0.487 and p = 0.001) (figure 1). mothers with vad had a significantly higher frequency of anemia (95.7%) compared to mothers without vad (35.9%), p = 0.001. the relative risk for anemia among mothers with vad was 2.7 (ci = 2.123.3). the mean gestational age of included newborns was 38.1 1.0 weeks with a range between 37 and 40 weeks. the mean birth weight was 3330 307 gm with a range between 2890 and 3950 gm. the mean ofc was 34.5 0.9 cm with a range between 33 and 36 cm. the mean length was 46.6 0.9 cm with a range between 47 and 50 cm. the mean hb% of all newborns was 16.8 1.3 gm / dl with a range between 14.0 and 19.0 gm / dl. newborns delivered to mothers with vad had significantly lower mean values of hb%, mcv. mch and mchc compared to newborns delivered to mothers without vad (table 2) with a significant positive correlation between maternal serum retinol concentrations and cord hb% (r = 0.531 and p = 0.001) (figure 1). no significant differences between the two groups regarding gestational age, anthropometric measurements, wbcs, and platelet counts of the newborns (table 2). newborns delivered to mothers with vad had a significantly lower mean cord serum retinol concentration (0.43 0.1 mol / l) compared to newborns delivered to mothers without vad (1.19 0.42 the cord serum retinol concentration of all newborns had a significantly positive correlation with the serum retinol concentration of their respective mothers (r = 0.952 and p < 0.001). in egypt, vad during pregnancy represents a major public health problem. in a recent study, el - khashab. in other developing countries, vad was found among 15.8% (in nigeria) and 18.8% (in bangladesh) of pregnant women [16, 17 ]. the frequency of mothers with retinol intake below the recommended intake (70%) is higher than that reported from other developing countries (53%). the positive correlation between the maternal serum retinol concentrations and maternal vitamin a intake is documented in many previous studies. the higher frequency of vad in the present study may be explained by inclusion of only women of low income families. the frequency of anemia among included pregnant females (50%) is similar to that reported from west and central africa and other developing countries. significantly lower mean hb% among pregnant women with vad compared to healthy women and significant positive correlations between maternal serum retinol and maternal hb% were reported in previous studies [2022 ]. women with vad had 1.8 times greater risk of being anemic than did the women without vad. vitamin a supplementation was found to improve hemoglobin concentrations and reduces maternal anemia for women who live in areas where vad is common. the mechanisms of anemia resulting from vad and how vitamin a supplementation can improve hemoglobin have not been elucidated. first, modulation of erythropoiesis as retinoic acid was found to stimulate erythropoietin gene transcription. the second mechanism is the anti - infective role as infection is associated with decreased serum iron levels, suppressed erythropoiesis, and lower hemoglobin concentration. it has been suggested that vitamin a is required for the mobilization and utilization of iron for hemoglobin synthesis. vitamin a maintains iron homeostasis by the modulation of liver hepcidin expression and regulation of iron regulator protein-2 (irp2). in cases of vad, iron is trapped in the liver and spleen and is not effectively released for erythropoiesis by bone marrow. the nonsignificant difference between anemic and non - anemic mothers regarding serum retinol concentrations indicates that vad is not the only cause of anemia during pregnancy. causes of anemia during pregnancy include iron deficiency (most common cause), other micronutrient deficiencies (zinc, copper, vitamin b12, and folic acid), hemoglobinopathies (sickle cell disease and thalassemia), and human pathogens in certain geographical populations such as hookworm, malaria, and human immunodeficiency virus [31, 32 ]. because the present study aimed at correlating maternal vad with maternal anemia and neonatal hb% and the lack of financial support, we could not assess iron status for all included women. maternal vad during pregnancy is associated with maternal anemia and lower hb% of the newborns at birth. vitamin a supplementation during pregnancy is recommended especially in low income countries to decrease the frequency of anemia.	background. vitamin a deficiency (vad) during pregnancy represents a major public health problem in developing countries. anemia is a common consequence of vad. we aimed to measure serum retinol concentrations of a sample of poor egyptian mothers and correlate it with their hb% and cord hb%. methods. this cross - sectional study included 200 healthy mothers and their healthy full term newborns. maternal and cord blood samples were collected for cbc and measurement of serum retinol concentrations. results. forty - seven mothers (23.5%) had vad and 50% were anemic. mothers with vad had a significantly lower mean hb% and a significantly higher frequency of anemia (95.7%) compared to mothers without vad (35.9%). the relative risk for anemia among mothers with vad was 2.7 (ci = 2.123.3). newborns of mothers with vad had a significantly lower mean cord hb% compared to newborns of mothers without vad. maternal serum retinol concentrations were positively correlated with maternal hb% and cord hb%. conclusion. maternal vad during pregnancy among poor mothers is associated with maternal anemia and lower hb% of newborns at birth. vitamin a supplementation is highly recommended for this vulnerable group.
ks arises as multiple patch, plaque, or nodular lesions, which are generally located on the skin or mucosas but can also involve visceral organs (reviewed in). early - stage ks lesions resemble an inflammatory, granulation - type reaction, and they are characterized by abnormal angiogenesis, leukocyte infiltration, endothelial cell activation, edema and by the proliferation of endothelial - like, spindle - shaped cells which are considered to be the ks tumor cells (ks cells). the development of ks lesions is triggered by inflammatory mediators and growth factors, whose production can be induced or enhanced by the human herpesvirus-8 (hhv-8), a viral agent infecting nearly all ks patients (reviewed in). among the growth factors expressed in ks lesions, specifically, upon its production by ks cells or infiltrating leukocytes, fgf-2 stimulates the locomotion and growth of both endothelial and ks cells by paracrine and autocrine mechanisms, respectively. consistently, fgf-2 can directly promote the development of ks - like lesions in vivo [3, 4 ], and it mediates the formation of angioproliferative lesions induced by ks cell injection in nude mice. despite rare and slowly progressive in general population, ks becomes frequent and aggressive in hiv - infected individuals (acquired immune deficiency syndrome- [aids- ] associated ks). in this regard, previous results indicated that the tat protein of hiv-1 can act as a progression factor in aids - ks. in particular, tat, a transactivator of hiv-1 gene expression which is essential for virus replication, is released by hiv-1 acutely infected t cells (reviewed in). by engaging cell surface receptors belonging to the integrin family, extracellular tat induces endothelial or ks cell locomotion and adhesion. at the same time, tat competes fgf-2 binding to the heparan - sulphate proteoglycans of the cell surface and extracellular matrix (ecm). in doing so, tat can retrieve sequestered fgf-2 into a soluble, biologically active form which, in turn, promotes endothelial or ks cell growth. while early - stage ks lesions have a polyclonal nature and may regress, late nodular ks lesions can evolve into a true sarcoma. noteworthy, at this stage of disease progression, ks lesions undergo a rapid growth in the presence of a low mitotic index. this suggested that vascular cell survival could be prevalent in ks maintenance and progression. in this context, the protein levels of bcl-2, a potent antagonist of programmed cell death (apoptosis), were found to be increased in late - stage, as compared to early - stage, lesions from all forms of ks [812 ]. coexpression of bcl-2 and endothelial cell markers [9, 10 ] indicates that bcl-2 is upregulated in activated endothelial cells lining the newly formed, abnormal blood vessels characterizing ks lesions. consistently, bcl-2 up - regulation is associated with the reduction or absence of endothelial cell apoptosis [8, 9 ]. at the present time, however, little or nothing is known about the mechanisms of bcl-2 induction in ks. since either tat or fgf-2 has been shown to modulate bcl-2 expression by a wide variety of cell types [1321 ], herein we evaluated the effects of fgf-2 and tat, alone or combined, on bcl-2 expression in animal and in vitro experimental models previously employed to study aids - ks pathogenesis. recombinant hiv-1 tat protein (from the iiib isolate) was obtained, purified, tested for biological activity, and handled as previously described. bovine serum albumin (bsa, fraction v), heparin (sodium salt, from porcine intestinal mucosa), gelatin (denatured collagen i, from bovine skin), and the chemicals employed for protein extraction were from sigma (st. matrigel, a reconstituted basement membrane, and endothelial cell growth supplement (ecgs) were obtained from bd bioscience (bedford, ma). the phosphate buffered saline (pbs) solution, rpmi 1640 growth medium, and its supplements were from invitrogen (paisley, scotland, uk). recombinant fgf-2 and/or tat protein (1 g and 10 g, resp.) were suspended in 0.2 ml of pbs-0.1% bsa, mixed with an equal volume of matrigel and then injected subcutaneously into the lower back of balb - c nu / nu female mice (46-week old, charles river breeding laboratories, calco, italy), as previously described [3, 4 ]. control animals were inoculated with the same volume of matrigel and the buffer (pbs-0.1% bsa) employed to suspend fgf-2 or tat. seven days later, mice were sacrificed, and, at this time, the sites of injection were evaluated for the presence of macroscopic lesions [3, 4 ]. tissue samples were taken and fixed in formalin or frozen in oct compound (miles laboratories, naperville, il). slides obtained from formalin - fixed paraffin - embedded blocks were examined at the microscope after hematoxylin - eosin staining. frozen tissue sections were fixed in acetone and stained with 0.4 g / ml of anti - bcl-2 rabbit polyclonal antibody (santa cruz biotechnology, santa cruz, ca) by the peroxidase - anti - peroxidase method (dako) [3, 4 ]. the percentage of positive cells was determined from the mean of 5 high - power fields (40x magnification) and expressed as the mean (minimum and maximal range) of values obtained. the care and use of mice were in accordance with the european community guidelines. human umbilical vein endothelial cells (huvecs) were obtained from lonza (verviers, belgium), seeded on surfaces precoated with gelatin, and cultured in rpmi 1640 medium supplemented with 15% fetal bovine serum (fbs), sodium pyruvate, l - glutamine, mem essential and nonessential amino acids, heparin (1 g / ml), and ecgs (45 g / ml). experiments were performed in the absence of ecgs or heparin. at the end of each experiment, huvecs were washed in ice - cold pbs, scraped off the flask, and lysed in 50 nm tris ph 7.5, 1 mm phenylmethylsulfonyl fluoride (pmsf), 2 mm egta, 10 mm dtt, 5 g / ml aprotinin, 200 g / ml leupeptin, and 0.15% triton x 100. the protein content in the cell lysates was assayed with the bradford reagent (bio - rad, hercules, ca). eight g of proteins from each experimental condition were subjected to 12% sodium dodecyl sulfate - polyacrylamide gel electrophoresis followed by transfer onto nitrocellulose membrane (amersham pharmacia biotech, little chalfont, buckinghamshire, uk). filters were rinsed in blocking buffer (5% nonfat dry milk, 0.1% tween-20 pbs), probed with anti - bcl-2 monoclonal antibodies (2 g / ml, santa cruz), incubated with horseradish peroxidase - conjugated goat anti - mouse secondary antibodies (amersham), and developed with the use of the enhanced chemiluminescence (ecl) method (amersham). intensity of the bcl-2-specific band was quantified by densitometry, using an imaging densitometer gs-700 and a multi - analyst software (bio - rad). to verify equal loading of protein in each lane huvecs were seeded on plates coated with gelatin or with 10 g / ml of poly-2-hydroxymethyl - methacrylate [poly(hema) ]. cells were exposed to fgf-2, tat, or their suspension buffer and then collected. the first was the programmed cell death elisa kit (roche) which measures the amount of nucleosomes released in the cytoplasm of dying cells. briefly, huvecs were lysed, and the cytoplasmic fraction was recovered after centrifugation, and nucleosomes assayed as indicated by the manufacturer. the second method was the terminal deoxynucleotidyl transferase - mediated dutp nick end labeling (tunel) assay. briefly, huvecs were spun on slides by cytospin, fixed in 4% paraformaldehyde, and the dna strand breaks of apoptotic cells were identified in situ by tunel kit (roche), according to the manufacturer 's instructions. fluoresceinated nucleotides incorporated in polymers by the terminal deoxynucleotidyl transferase - based enzymatic reaction were detected by immuno - histochemical staining using a mouse antifluorescein isothiocyanate (fitc) monoclonal antibody and the alkaline phosphatase - anti - alkaline phosphatase method (dako). the percentage of positive, apoptotic cells was determined from the mean of 4 high - power fields. total rna was extracted from huvecs with the rna assay mini kit (qiagen, hilden, germany) and further purified by three - round digestion with pancreatic dnase i (10 iu / sample). purified rna (0.5 g) was retrotranscribed with the reverse transcription system kit (promega, madison, wi, usa) by incubating the reactions with hexanucleotide random primers for 10 min at room temperature, 30 min at 42c, and 30 min at 53c. after heat inactivation of the rt reaction, each sample was subjected to a low (pre - plateau) number of cycles of pcr (28 to 30) for -actin with primer ba-1 [5-catgtgcaaggccggcttcg-3, nucleotide (nt) 1137 to 1156 ], located in the first exon of the human -actin gene, and primer ba-4 (5-gaaggtgtggtgccagattt-3, nt 1475 to 1494) designed in the second exon of the gene (nt enumeration as in genbank m10277). these primers yield a 226 bp cdna amplicon ; contaminant dna is revealed by the appearance of a 357 bp genomic amplicon. the amount of cdna to be used for bcl-2 pcr was determined after cdna normalization by pre - plateau -actin pcr. bcl-2 primers were 5-tcgccctggtggacaaca-3 (nt 1957 to 1975) and 5-tgacttcacttgtggctcaga-3 (nt 2183 to 2163) (nt enumeration as in genbank m13994). conditions for bcl-2 amplification were as follows : 4 min at 94c, followed by 35 cycles of denaturation at 94c for 1.5 min, annealing at 58c for 1.5 min, and extension at 72c for 2 min. negative control reactions lacking dna template were always performed in parallel to control for sample to sample contamination. pcr - amplified products were separated onto a 1.8% agarose gel, blotted on nylon membrane, and hybridized to a 32 [p]-labeled oligonucleotide probe internal to the amplicon by standard techniques. after extensive washing, filters were exposed to x - ray films and the intensity of the bands quantified by instant imager (packard instruments, downers, il, usa). analysis of the results from animal studies was performed according the test on equality of proportion by stata program. statistical evaluation of the in vitro data was performed by the analysis of variance model (anova) with tukey - kramer adjustment for multiple comparisons, using the sas software, version 9.1 (sas institute, cary, nc, usa). a p - value of < 0.05 was considered as significant. our previous work tested the dose response effects that fgf-2 or tat (0.1100 g) has on the induction of histological features characterizing ks lesions in vivo [3, 4, 24 ]. starting from those findings, we employed the tat and/or fgf-2 concentrations particularly effective at promoting the development of macroscopic ks - like lesions in mice. specifically, nude mice were injected with 1 g of fgf-2 and/or 10 g of tat. after 7 days, mice were sacrificed and the sites of injection were examined for the presence of angioproliferative lesions [3, 4 ]. tissue samples were excised and processed for histology and bcl-2 immuno - histochemistry. as shown in figure 1, specifically, 19 out of the 43 mice injected with fgf-2 (= 44%, p < 0.001) developed ks - like lesions. this was associated with the appearance of spindle - shaped cells (figure 1(a)), which are the histological hallmark of human ks lesions, and with the induction of bcl-2 expression (figure 1(b)). in particular, 32% (range 1949) of the cells present in the lesions were positive for bcl-2. noteworthy, the simultaneous inoculation of fgf-2 and tat further increased both lesion development and bcl-2 expression. in particular, out of the 31 mice injected with combined fgf-2 and tat, 22 (= 71%, p < 0.05) developed macroscopic lesions (figure 1(c)), in which 47% (range 2267) of the cells were bcl-2 positive (figure 1(d)). in contrast, none of the 17 mice injected with tat alone, and none of the 70 mice injected with the protein suspension buffer (pbs-0.1% bsa), developed macroscopic lesions (figures 1(e) and 1(g), resp.). for both experimental conditions, bcl-2 was not expressed at the sites of injection (figures 1(f) and 1(h), resp.). to confirm these in vivo results, the effects of fgf-2 and tat on bcl-2 expression, huvecs were exposed for 24 hours to fgf-2 and/or tat (controlled by their suspension buffer), and bcl-2 protein levels were examined by western blot analysis. as shown in figure 2, fgf-2 increased bcl-2 protein expression, while tat alone had no effect. however, when tat was combined with fgf-2, bcl-2 protein levels further increased, confirming the in vivo data. bcl-2 can protect endothelial cells from anoikis, the process where adherent cells die when they lose contact with the ecm [25, 26 ]. in view of our in vivo and in vitro results, additional work was performed in order to evaluate fgf-2 or tat effect on endothelial cell anoikis. to this end we seeded huvecs onto plates coated with poly- (hema), a compound preventing cellular adhesion. then, we incubated the cells for 6 h with fgf-2, tat, or their suspension buffer. the entity of cell death was determined by measuring, in huvecs cytoplasm, the content of nucleosomes, histone complex of low - molecular - weight dna fragments resulting from the cleavage of apoptotic cell genome. adherent huvecs plated onto gelatin were the negative control for cell death [25, 26 ]. as expected, huvecs did not adhere onto poly- (hema)-coated surfaces, and this was followed by a dramatic increase of nucleosome content in the cytoplasm of nonadherent, as compared to adherent, huvecs (p < 0.0001) (figure 3(a)). noteworthy, exposure of nonadherent huvecs to 1 or 10 ng / ml fgf-2 reduced the amount of cytoplasmic nucleosomes by 30% and 50%, respectively (p < 0.0001) (figure 3(a)). although tat alone had no effect, when it was combined with 1 ng / ml of fgf-2, it decreased the amount of cytoplasmic nucleosomes to levels as found in huvecs treated with 10 ng / ml fgf-2 (figure 3(a)). thus, tat increased fgf-2 capability of rescuing endothelial cell apoptosis (p = 0.0102). specifically, in nonadherent conditions, 1 ng / ml of fgf-2 diminished the number of apoptotic huvecs by 55% when employed alone (p = 0.0490) and by 80% when combined with 10 ng / ml of tat (p = 0.0025) (figure 3(b)). to investigate whether the increase of endothelial cell survival promoted by fgf-2 and tat in nonadherent conditions was accompanied by a modulation of bcl-2 expression, bcl-2 mrna levels were evaluated in huvecs under the different experimental conditions described above. results from rt - pcr assays indicated that bcl-2 mrna levels decreased by 53% in nonadherent huvecs, as compared to the adherent control (p = 0.0396) (figure 4). when nonadherent huvecs were exposed to 10 ng / ml of fgf-2, bcl-2 mrna expression was rescued to levels as expressed by adherent huvecs (p = 0.05) (figure 4). although tat alone had no effect, when it was combined with 1 ng / ml of fgf-2, the expression of bcl-2 was restored to the levels detected in adherent huvecs (p = 0.0480) (figure 4). in healthy tissues, undesired angiogenesis is switched off through the induction of endothelial cell apoptosis [25, 28 ]. this can be triggered by conditions, such as the lack of angiogenic growth factors, or the inhibition of endothelial cell adhesion onto the ecm, which downregulate endothelial cell expression of antiapoptotic molecules [25, 28 ]. among the latter,, bcl-2 is overexpressed by endothelial cells of the newly formed blood vessels nourishing the growing tumor [29, 30 ]. in agreement with the fact that bcl-2 expression can lead to inappropriate endothelial cell survival and abnormal angiogenesis [25, 28 ], clinical evidence suggests that the deregulation of bcl-2 expression could play a role in the maintenance and progression of ks. in fact, bcl-2 protein levels augment in late - stage, as compared to early - stage, ks lesions, and this parallels a decrease in vascular cell apoptosis [812 ]. a role for bcl-2 in ks progression is supported also by the efficacy of the bcl-2 inhibitor paclitaxel in ks patients, confirming previous work performed with animal models of ks. here, we evaluated whether fgf-2 or hiv-1 tat, two key aids - ks pathogenetic factors, could affect bcl-2 expression in vivo and in vitro. results indicate that fgf-2 injection into mice results in the induction of bcl-2 expression and that this associates with the development of angioproliferative, ks - like lesions. tat protein is known to enter cells and activate the expression of cellular genes. in particular, tat was shown to up - regulate bcl-2 expression by monocytes, albeit at higher concentrations than the ones we used here. however, the possibility that in our experimental model tat could directly activate bcl-2 expression is excluded by our results indicating that tat alone is not capable of promoting ks - like lesions in mice, rescuing endothelial cell apoptosis or inducing bcl-2 expression by these cells. the contrasting data that others obtained in monocytes could depend on the different cell type they utilized. the fact that tat effects are cell - type dependent is strongly supported by previous findings indicating that tat promotes apoptosis in other cell systems [3439 ]. in contrast, when tat is combined with fgf-2, it strongly enhances fgf-2 effects on bcl-2, this paralleling a dramatic increase in the formation of angioproliferative lesions in mice. these findings are consistent with all the other activities that tat exerts on endothelial cells, which require fgf-2 presence in order to respond to tat either in vitro or in vivo [3, 4, 7 ]. in this regard, tat and fgf-2 synergy is likely to depend on fgf-2 capability of inducing the expression of the v3 and 51 integrins, which, in turn, function as tat receptors [3, 4, 7, 40 ]. on the other side, tat can maintain fgf-2 into a highly active and readily available form, thus amplifying its biological effects. previous studies indicated that the cross - talk between the integrins and growth factor receptors triggers a signalling cascade which is relevant for endothelial cell survival and growth [25, 28 ]. in this regard, it has to be reminded that signalling pathways leading to endothelial cell survival are activated upon tat binding to the integrins, being further enhanced when fgf-2 is present in the environment. in this context, our previous work indicated that induction of ks - like lesions by combined fgf-2 and tat is reproduced by combined fgf-2 and the integrin - binding tat rgd peptide (tat 6580), but not by mutated tat kge peptide. in agreement with these findings, here we have reported that the induction of bcl-2 expression by fgf-2 and tat can prevent the death of endothelial cells resulting from the inhibition of their adhesive interaction with the ecm. altogether, these results suggest a pathway for bcl-2 induction and a molecular mechanism by which the simultaneous presence of tat and fgf-2 could promote ks maintenance and progression in hiv-1-infected individuals. since tat and fgf-2 are expressed in aids - ks lesions together with bcl-2 [1, 3 ], these findings appear to be of biological relevance. finally, as bcl-2 diverts vascular cells from apoptosis and prolongs their survival [21, 29, 30, 42 ], its expression in advanced ks, coupled with a deregulated cellular proliferation by hiv, hhv8, and cellular factors [1, 2 ], may result in ks maintenance and/or the progression of the early angio - hyperplastic ks lesions into a true sarcoma. this hypothesis is supported by our recent observation that both fgf-2 and tat can increase in ks cells the activity of the telomerase, an enzyme allowing cells to escape replicative senescence and to proliferate indefinitely [43, 44 ]. our results indicate that fgf-2 and hiv-1 tat synergize at inducing bcl-2 expression and that this is accompanied by the inappropriate survival of endothelial cells and by the development of angioproliferative lesions. these findings suggest a molecular mechanism for the deregulated bcl-2 expression observed in aids - ks lesions and confirm that bcl-2 may represent a key target for therapeutic intervention of ks.	kaposi 's sarcoma (ks) is a vascular tumor frequently occurring in human immunodeficiency virus- (hiv-) 1-infected individuals. our previous work indicated that the angiogenic fibroblast growth factor (fgf)-2 and the tat protein of hiv-1, both expressed in ks lesions of hiv - infected patients, synergize at inducing angioproliferative, ks - like lesions in mice. here we show that the development of angioproliferative lesions promoted in mice by combined tat and fgf-2 associates with an increase in the levels of expression of the antiapoptotic bcl-2 protein. upregulation of bcl-2 expression by combined fgf-2 and tat occurs also in vitro, and this protects human primary endothelial cells from programmed cell death. as bcl-2 is expressed in human ks lesions in a fashion paralleling the progression of the disease, these findings suggest a molecular mechanism by which tat and fgf-2 cooperate in ks maintenance and progression in hiv - infected individuals.
cognitive impairment represents a common mental health problem in community dwelling and institutionalized older adults, and the prevalence increases with age. several measures have been developed to assess cognitive functioning in this population with some measures having a greater emphasis on memory and language functioning in analog conditions and others focusing more on functional adaptive skill use. occupational therapists often work within multi - disciplinary settings and are frequently asked to assess the functional and cognitive status of their patients. the advantage of using a measure like the cam is that it covers a greater range of cognitive impairment compared to most cognitive screens, making it potentially more useful as a screening measure. in addition to providing a global measure of cognition, the cam also assesses practical skill domains. for example, the cam allows for assessments of working memory and simple mathematical ability (e.g., can the person make change) that would allow for specific recommendations (e.g., providing assistance to the affected person in managing finances). the cam also allows for hierarchical grading of cognitive skills, which may be useful in monitoring change over time. thus, the cam may be a promising measure to be used in geriatric settings. although the cam is frequently used internationally by ots in practice and in training settings [35 ], the psychometric properties of the cam have not been extensively validated. one of the most common screening measures for cognitive impairment is the mini - mental state exam (mmse), a scale developed in neurology to be used as a screener in a number of settings, including medicine, neurology, and psychiatry. the mmse has proven to be a useful tool because its simplicity allows providers from any discipline to screen for cognitive impairments. the national comorbidity study replication (ncs - r) reports lifetime prevalence rates of major depression for community dwelling adults over the age of 60 to be as high as 10.6% and prevalence rates for dysthymia at 1.3%. it is likely that this is a conservative figure as older adults with depression often present with less sadness and report experiencing more physical or cognitive symptoms, and thus the depressive symptoms may go unrecognized. depression can complicate differential diagnosis as it can exacerbate or mimic symptoms of cognitive impairment. research has revealed depression is linked to impaired cognition in the areas of short - term memory, attention [1012 ], information processing, recognition, and executive functioning [10, 12 ]. however, monsch. have suggested that skills such as psychomotor speed that decline with aging may be responsible for more of the variance in the decline in scores on cognitive measures, rather than the depression itself. given that cognitive screening measures have varying demands on different types of cognitive abilities and that older adults may differ from younger adults on the extent that depression affects each area of cognitive functioning, the validity of screening measures for use among older adults with depression may be reduced. however, despite the widespread use of cognitive screening measures among individuals with depression, few studies have compared the psychometric properties of these instruments for use with older adults with depression. if the presence of depression negatively impacts cognitive assessment measures, one would expect to see impaired scores across similar cognitive screening measures. however, if the cognitive measures are differentially affected, then perhaps some other factor may be responsible for the different levels of agreement between those measures. for example, it may be that individuals with depression show variability in response over time due to the effects of fatigue, effortful processing, a perception of task difficulty, and so forth. clinically, this would be important to take into account before choosing a screening measure to use with individuals with depression. the purposes of this study were to (1) investigate the relationship between two measures of cognitive status (the mmse and the cam), (2) examine if depressive symptoms moderate the relationship between these measures, and (3) examine group differences on areas similarly measured in both screening instruments. participants included 113 adults (25 men and 88 women) over the age of 60 (m = 81.00, sd = 8.57, range 6195), predominantly european american, with 43.8% of the sample having an education level of 12 or more years. eligible participants were those who completed an evaluation at the geriatric assessment center (gac) which is affiliated with three university systems in the midwest. the gac is a collaborative multi - disciplinary assessment team consisting of a nurse, occupational therapist, pharmacist, geriatrician (physician), psychologist, and a social worker. typical reasons that individuals would contact the gac include : self - referral for concern over memory loss or depression ; concern for partner 's worsening memory, depression / isolation, anxiety, or overmedication ; and functional capacity issues (e.g., adult child concern for the capacity for parent(s) to live independently / manage finances / maintain driving, mobility concerns). a comprehensive evaluation by this team includes a 2 - 3 hour home visit and a 4-hour clinic visit. because some sections of the mmse (e.g., following a written command, writing a sentence, copying a design) and the cam (e.g., subsections requiring the examinee to look at a flash card and perform the arithmetic problem) are not appropriate for participants with sensory impairment, they would not be given to individuals who are visually impaired or illiterate, and because a section might be refused by a participant, some participants were dropped from the final analyses, resulting in a sample of 103 older adults (23 men and 80 women). three measures were administered to each participant as part of a standard comprehensive assessment : the mini - mental state exam (mmse), the cognitive assessment of minnesota (cam), and the geriatric depression screen, short form (gds - sf) [14, 15 ]. the mmse usually takes approximately 1015 minutes to administer and has a maximum score of 30. the examination includes measures of orientation, recall and recent memory, abstract thinking, attention and calculation, and object identification. research on this measure indicates good reliability with interrater correlations of r =.82 and test - retest reliability ranging from r =.89 to r =.98 [6, 17 ]. validity studies on this measure vary depending on the population measured and the cut score utilized. for the commonly used cut score of 24, research using the mmse and comparing it to dementia status as a criterion indicates sensitivity rates of < 85% and specificity rates of < 98% in community samples when compared to dementia status as diagnosed by trained neuropsychological examiners [18, 19 ], 89% sensitivity rates with corresponding specificity rates of 81% in general practice samples, pooled sensitivity and specificity rates of 71% and 95.6% in hospitalized patients, and 84.9% and 60.8% in memory clinics. for this study, a member of the multi - disciplinary team who was trained in its administration and scoring administered the mmse in the home at intake (day 1). the cam is a 29-item standardized assessment of cognitive functioning developed primarily for use with individuals with brain injury, but also has data on a nonaffected sample. the administration time takes substantially longer than a typical screen to administer (approximately 45 min compared to 1015 min), yet it is more manageable in integrated settings than a complete neuropsychological battery which could take a minimum of 6 hours to complete. the cam provides information on a range of cognitive skills including : attention span (distractibility), orientation, recent and remote memory, basic language (register, process, and respond to information), mathematical and practical (monetary) skills. it also assesses temporal awareness, visual memory and sequencing, auditory memory and sequencing, discrimination skills (object identification and matching), mental flexibility, and safety and judgment. very little data exits on the psychometric properties of the cam outside from what is found within the examiner 's manual. rustad and degroot indicate an interrater alpha of 0.90, adequate test - retest reliability, and good concurrent validity for level of impairment when compared against professional clinical judgment by occupational therapists. this measure was administered on day 2 in the clinic by an occupational therapist and an occupational therapy student trained in cam administration and scoring. the gds - sf is a 15-item depression inventory specifically designed for use with older adults. this depression inventory has a maximum score of 15, with scores higher than five considered to be indicative of depression. the items assess overall mood, changes in energy and activity level, and attitudes about the future. the gds - sf, also called the gds-15, was found to be as good as the longer version of the gds for assessing depression among older adults with dementia. this measure was administered on day 1 in the home setting by one member of a three to four person team trained in administration and scoring. the team consisted of an occupational therapist, nurse, pharmacist, or psychologist and students from one of those disciplines (i.e., 1 - 2 professionals and 2 students per team). pearson product moment correlations were calculated for total scores of the two cognitive measures (cam and mmse) to determine the overall level of agreement of these screening measures for a group of older adults. subgroups were established based on the presence or absence of depressive symptoms to determine whether the presence of depressed mood, as measured by the gds - sf, differentially altered the relationship between the two measures. t - tests were conducted comparing participants with depressed mood to participants without depressed mood using the scores on the gds - sf. the nondepressive symptom group (non) consisted of those with gds scores of 04. the depressive symptom group (dep) consisted of gds scores of 515. the pearson product moment correlation described above was computed for each group and coefficients were then compared using fisher 's z transformation and examined for significant group differences. the two cognitive measures were examined for items that sampled the same basic skills (i.e., recall and orientation) (refer to table 1). the strength of the relationship between these subscales for both measures was examined using pearson correlation coefficients and a fisher 's z was then conducted to analyze for significant differences. then, the two groups identified earlier as dep and non were compared for the association of depressive symptomology through examinations of these two groups on each of these subscales using the same statistical procedures. additionally, a reliability coefficient was run on each of the two cognitive measures to provide an index of each measure 's reliability in this population. a cronbach 's alpha was conducted on both the total scores of each measure and on the orientation subsection to provide an index of internal consistency. it was not possible to conduct a cronbach 's alpha on the recall subsection due to the fact that both measures only have one score / subscore available for the recall section. according to convention, of.70 or higher a cronbach 's alpha was calculated for total scores of the cognitive measures and for the orientation subsection. the mmse was found to have good internal consistency =.79 for total score and.82 for orientation. the cam also performed well, yielding an =.87 for the total score and =.76 for orientation. pearson product moment correlation was calculated for total scores for 103 participants on the two cognitive measures with r =.74, p <.01, indicating a strong correlation. the depressive symptom group (dep) consisted of 36 older adults (11 men and 25 women ; mage = 76.9, sd = 9.6, and 52.2% with 12 + years of education). the nondepressive symptom (non) group consisted of 67 older adults (12 men and 55 women, mage = 83.1, sd = 6.7, and 45.6% with 12 + years of education). analyses on the two measures among each subgroup (i.e., dep and non) yielded an r =.72, p <.01 for non and r =.79, p <.01 for dep. a fisher z was then conducted, indicating no significant group difference (z = 0.67, p =.50). when comparing dep to non groups, an independent samples t - test indicated no significant group differences on sex, t = 1.47, p =.145, but did indicate a significant group difference on age, t = 3.62, p < therefore, partial regression correlation coefficients, holding age constant, were calculated for each subanalysis and are described below. secondary analyses indicated the effects of depressed mood on the overall relation yielded an r =.72, p <.01 for non and r =.79, p <.01 for dep. a fisher z was then conducted, indicating no significant group difference (z = 0.67, p =.50). analyses were conducted to examine the agreement between the two cognitive measures on specific subsections for orientation and recall (see table 3). for orientation, the correlation for all participants was r =.65, p <.01, following standard conventions, a correlation in this range suggests that the two measures have a moderate agreement on this subtest. when comparing dep and non groups using partial correlation coefficients (holding age constant), a fisher z indicated no significant group differences, z = 1.08, p =.28. for recall, the correlation for all participants yielded an r =.67, p <.01, suggesting that the two measures have moderate agreement on this subtest, as well. when comparing dep and non partial correlation coefficients (holding age constant), a fisher z indicated no significant group differences, z = 1.42, p =.16. cognitive impairment is a common problem in older adults and has been linked to several negative outcomes including poor treatment adherence [2528 ] and increased disability, suggesting a need for early recognition in medical and mental health treatment. multi- disciplinary teams are often asked to provide more specific and detailed answers and recommendations than can be obtained through brief cognitive screening measures. for example, functional recommendations are often sought by rehabilitation professionals (can ms. smith resume daily activities ?), social security disabilities services (e.g., can mr. jones perform work - related activities ?), and by families seeking help with planning (e.g., is my aging parent able to manage his / her finances independently ?). cognitive screening measures like the mmse, though frequently used, are primarily gross cognitive screening tools and may have several limitations when utilized for purposes beyond detecting global cognitive impairment (e.g., mmse has poor utility for use with post - stroke medical patients). the cam is a promising measure for use in geriatric populations because of the wide range of abilities assessed and its utility with brain - injured populations. previously only one small study has examined the psychometric properties of the cam using the mmse as a comparison. rustad and colleagues found a moderate correlation (r =.44, p =.05), however the sample size reported in this study was small (n = 16), and additional examinations are needed to determine whether this relationship will exist in other samples. this study examined the utility of the cam as a screening measure and examined its relationship to the mmse in a relatively large sample of older adults in an outpatient clinic setting. both measures evidenced adequate / good reliability when evaluated using a cronbach 's alpha. when examining the orientation subsection alone, reliability estimates remain adequate / good. the overall relation suggests good agreement between the two measures of cognitive status on orientation. the data suggest that when depressive symptoms are taken into consideration, overall scores were not affected ; however there was a tendency for depression to affect cognitive functions such as recall and orientation. for example people in the dep group scored lower on the recall subsection on the mmse than they did on the cam. interestingly they dep group scored higher on the orientation subsection on the mmse than they did on the cam. higher scores on recall on the cam could be affected by the difference of the type of words used in recall, however both measures use commonly found objects. attention could clearly be affected by the fact that the cam takes longer than the mmse to administer, and this could in part account for differences in scores on orientation. alternatively, the differences in orientation may be an artifact of the sample, since there is no evidence in the research literature to suggest that differences in orientation should exist between cognitive screens. unfortunately, neither measure has an alternate form available for use with those individuals with visual impairment or those with illiteracy. these same challenges exist for the majority of cognitive screens and many neuropsychological test instruments that are currently available (i.e., many rely heavily on visual and verbal abilities ; e.g., boston naming test) constituting a problem for the field of neuropsychology as a whole. in a recent survey of neuropsychologists in the usa and canada, rabin and colleagues reported on the most common assessment used. of the 20 neuropsychological assessment instruments used, the authors are not aware of any published cognitive screening instruments that have been designed specifically for individuals with visual impairment or illiteracy. while this study is the first large sample study to examine the cam and the mmse, the drawback to utilizing clinical data collected from an assessment center is that we can not control for ethnic diversity and the clinicians may not have always been able to administer the assessments on the days planned. patient cancellations and rescheduling of appointments may have led to a longer delay in administration (e.g., one week in between administration of assessment measures rather than one day). since participants with vision or reading disabilities were excluded from the study, results may only generalize to healthy and literate older adults. in addition, because the sample was predominantly european - american, we can not assume generalization to more diverse samples. in addition, because these data are cross - sectional, we can not assume that the relationship between the two measures will remain stable over time, particularly in the presence of cognitive impairment. future research should include follow up data to assess whether the utility of these measures changes over time and with advanced impairment. in addition, in order to investigate the prognostic utility of the cam, longitudinal studies are required to evaluate its ability to predict both mild cognitive impairment and dementia as compared to assessment by a trained neuropsychologist. this study finds the two measures to have good reliability and agreement in assessing cognitive impairment, but that each measure has a distinct advantage. the mmse offers a shorter administration time, which may be beneficial in primary care settings where time constraints preclude a longer assessment for cognitive impairment but detailed function recommendations may not be needed. whereas the direct clinical usefulness of some of the information from specific tasks of the cam test (i.e., the problem solving task that involves completing multiple errands in a day assesses executive functions that include planning, organization, and sequencing ability) along with the opportunity to obtain a larger sample of behavior in all tasks assessed, are some of the strengths of the cam. from an evidence - based perspective, the decision to select an assessment tool should be tied to the goal or purpose of the assessment. if the purpose is only to obtain a quick simple screen (obtain a brief snapshot of the person 's cognition to evaluate for risk of cognitive impairment or to establish a baseline), then a clinician could choose to use the mmse. if the purpose is to make clinically useful recommendations (e.g., focus on functional adaptive skill use), the longer assessment will be more helpful. the cam is useful for geriatric teams where the goal is to obtain a more comprehensive measure of cognitive impairment. given that the cam allows for assessment of more practical aspects of cognitive impairment and offers more extensive assessment information than the mmse, including hierarchical grading of cognitive skills, the cam may be more ideal for settings such as home health care or rehabilitation settings where the goal would be to have more information about practical skills and activities of daily living. however, the longer duration of testing may increase issues associated with fatigue during assessment. thus, examiners would need to incorporate breaks as necessary or administer over sessions to minimize test fatigue. therefore, our finding that the two measures are so closely related means that clinicians can adjust or tailor their screening based on the information they need (goal driven assessment). in addition, having strongly correlated measure means that those who work mainly with the mmse can look at the cam and have a good idea about the individual 's cognitive capacity and those who work with the cam can look at the mmse and have a good idea about the individual 's cognitive capacity (i.e., aids in communication within the scientific community). as gerontology is an interdisciplinary field, it is often useful to have tools that would aid in communication about a patient between disciplines. in sum, the cam appears to be a promising measure for use with older adults with cognitive impairment and when examiners are interested in functional capacity recommendations. more research is necessary exploring the psychometric properties of the cam to explore its full utility with diverse populations. in addition, studies comparing the cam to other measures of functional capacity may be warranted.	cognitive impairment represents a common mental health problem in community - dwelling and institutionalized older adults, and the prevalence increases with age. multidisciplinary teams are often asked to assess cognitive and functional impairment in this population. the cognitive assessment of minnesota was created by occupational therapists for this purpose and is frequently used, but has not been extensively validated. this study examined the performance of the cam and compared it to the mmse with 113 outpatient clinic patients over the age of 60. subgroups were established based on scores on a depression inventory to determine if the presence of depressed mood altered the relationship between the measures. both measures demonstrated good internal consistency. the overall correlation between the two measures was high, statistically significant and remained high regardless of depression status. we offer recommendations about the utility of each measure in screening cognitive functioning for older adults.
http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=gse31519 (link to geo series)http://www.ncbi.nlm.nih.gov / geo / download/?acc = gse31519&format = file&file = gse31519%5fcomplete%5fdataset%2etxt%2egz (direct link to normalized complete dataset in geo supplement)http://www.ncbi.nlm.nih.gov / geo / download/?acc = gse31519&format = file&file = gse31519%5ftnbc%5fsampleinfo%5fbcr%2etxt%2egz (direct link to sample information in geo supplement). http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=gse31519 (link to geo series) http://www.ncbi.nlm.nih.gov/geo/download/?acc=gse31519&format=file&file=gse31519%5fcomplete%5fdataset%2etxt%2egz (direct link to normalized complete dataset in geo supplement) http://www.ncbi.nlm.nih.gov/geo/download/?acc=gse31519&format=file&file=gse31519%5ftnbc%5fsampleinfo%5fbcr%2etxt%2egz (direct link to sample information in geo supplement). breast cancer is a heterogeneous disease of different subtypes and separate analyses by subtype are mandatory. triple negative breast cancer (tnbc) represents an aggressive disease and the use of currently available molecular prognostic signatures is limited. reasonable sample sizes of tnbc for molecular analyses may be obtained by pooling several microarray datasets. however, because of significant inter - laboratory variation such studies require precise control of dataset bias. the set of 579 tnbcs in gse31519 includes : (i) 67 cel files in gse31519 (gsm782523gsm782589), (ii) 489 re - analyzed geo samples linked in gse31519, and (iii) 23 re - analyzed arrayexpress samples., cel files were downloaded from geo and the affy package from bioconductor was used to generate mas5 values. next, mas5 values corresponding only to the 22,283 probesets from the u133a array were compiled. subsequently, normalization of mas5 data was performed using the command line version of the program cluster 3.0 (michael eisen ; updated by michiel de hoon ; http://bonsai.hgc.jp/~mdehoon/software/cluster/command.txt). the following three steps were performed in the following order:1.log2 transformation of mas5 values2.median centering of arrays3.magnitude normalization of arrays. these three steps correspond to the following commands : cluster.com filename -lcluster.com filename -ca mcluster.com filename -na cluster.com filename -l cluster.com filename -ca m cluster.com filename -na in step 3 of these procedures (magnitude normalization) the expression values of all (22,283) probesets from the u133a array are multiplied by a scale factor s so that the magnitude (sum of the squares of the values) equals one. the normalized data are available under the following link : http://www.ncbi.nlm.nih.gov / geo / download/?acc = gse31519&format = file&file = gse31519%5fcomplete%5fdataset%2etxt%2egz http://www.ncbi.nlm.nih.gov/geo/download/?acc=gse31519&format=file&file=gse31519%5fcomplete%5fdataset%2etxt%2egz all 579 samples in the dataset are triple negative according to the following predefined cutoffs for esr1 (205225_at) < 0.0075, pgr (208305_at) < 0.0078, and her2 (216836_s_at) < 0.0135. a major concern of the pooling procedure are systematic technical differences between individual datasets (batch effects). many adaption methods as e.g. z - normalization often do not eliminate but rather blur such effects. second, we controlled for biased genes which still show associations with the dataset vector. we calculated a comparability metric c for each of the datasets to identify the most comparable samples. this metric c is derived from the sum of the squared differences of the mean () within a specific dataset and among all datasets, respectively, normalized by the standard deviation () calculated for all genes (g) on the array : cdataseti=g=1ng, datasetig, totalg, total2. the metric is based on the assumption that overall the mean of a gene expression within a dataset should be similar between different datasets and gives an estimation to what extent the arrays in a specific dataset differ from the combined overall cohort. all datasets were sorted according to this metric and the top 15 datasets with the lowest values (normalized c 0.03), corresponding to 394 samples in total, were used as the discovery cohort (fig. 1). all probesets were checked for dataset bias (i.e. differential expression by dataset of origin that would indicate laboratory - bias or sampling differences compared to the rest). wallis statistic comparing the expression of each probeset with the primary dataset vector across the 394 tnbcs. thus an enrichment of biased probesets can be monitored in any downstream application e.g. cluster analyses,,. cutoffs for exclusion of probesets due to strong dataset bias may be derived from the distribution of the kruskal this effect originated from the inclusion of two datasets which were obtained from fine needle aspiration (fna) samples. such samples generally contain relatively higher amounts of blood and lower amounts of stromal tissue as compared to surgical biopsy samples. breast cancer is a heterogeneous disease of different subtypes and separate analyses by subtype are mandatory. triple negative breast cancer (tnbc) represents an aggressive disease and the use of currently available molecular prognostic signatures is limited. reasonable sample sizes of tnbc for molecular analyses may be obtained by pooling several microarray datasets. however, because of significant inter - laboratory variation such studies require precise control of dataset bias. the set of 579 tnbcs in gse31519 includes : (i) 67 cel files in gse31519 (gsm782523gsm782589), (ii) 489 re - analyzed geo samples linked in gse31519, and (iii) 23 re - analyzed arrayexpress samples., cel files were downloaded from geo and the affy package from bioconductor was used to generate mas5 values. next, mas5 values corresponding only to the 22,283 probesets from the u133a array were compiled. subsequently, normalization of mas5 data was performed using the command line version of the program cluster 3.0 (michael eisen ; updated by michiel de hoon ; http://bonsai.hgc.jp/~mdehoon/software/cluster/command.txt). the following three steps were performed in the following order:1.log2 transformation of mas5 values2.median centering of arrays3.magnitude normalization of arrays. these three steps correspond to the following commands : cluster.com filename -lcluster.com filename -ca mcluster.com filename -na cluster.com filename -l cluster.com filename -ca m cluster.com filename -na in step 3 of these procedures (magnitude normalization) the expression values of all (22,283) probesets from the u133a array are multiplied by a scale factor s so that the magnitude (sum of the squares of the values) equals one. the normalized data are available under the following link : http://www.ncbi.nlm.nih.gov / geo / download/?acc = gse31519&format = file&file = gse31519%5fcomplete%5fdataset%2etxt%2egz http://www.ncbi.nlm.nih.gov/geo/download/?acc=gse31519&format=file&file=gse31519%5fcomplete%5fdataset%2etxt%2egz all 579 samples in the dataset are triple negative according to the following predefined cutoffs for esr1 (205225_at) < 0.0075, pgr (208305_at) < 0.0078, and her2 (216836_s_at) < 0.0135. a major concern of the pooling procedure are systematic technical differences between individual datasets (batch effects). many adaption methods as e.g. z - normalization often do not eliminate but rather blur such effects. second, we controlled for biased genes which still show associations with the dataset vector. we calculated a comparability metric c for each of the datasets to identify the most comparable samples. this metric c is derived from the sum of the squared differences of the mean () within a specific dataset and among all datasets, respectively, normalized by the standard deviation () calculated for all genes (g) on the array : cdataseti=g=1ng, datasetig, totalg, total2. the metric is based on the assumption that overall the mean of a gene expression within a dataset should be similar between different datasets and gives an estimation to what extent the arrays in a specific dataset differ from the combined overall cohort. all datasets were sorted according to this metric and the top 15 datasets with the lowest values (normalized c 0.03), corresponding to 394 samples in total, were used as the discovery cohort (fig. 1). all probesets were checked for dataset bias (i.e. differential expression by dataset of origin that would indicate laboratory - bias or sampling differences compared to the rest). wallis statistic comparing the expression of each probeset with the primary dataset vector across the 394 tnbcs. thus an enrichment of biased probesets can be monitored in any downstream application e.g. cluster analyses,,. cutoffs for exclusion of probesets due to strong dataset bias may be derived from the distribution of the kruskal this effect originated from the inclusion of two datasets which were obtained from fine needle aspiration (fna) samples. such samples generally contain relatively higher amounts of blood and lower amounts of stromal tissue as compared to surgical biopsy samples. we calculated a comparability metric c for each of the datasets to identify the most comparable samples. this metric c is derived from the sum of the squared differences of the mean () within a specific dataset and among all datasets, respectively, normalized by the standard deviation () calculated for all genes (g) on the array : cdataseti=g=1ng, datasetig, totalg, total2. the metric is based on the assumption that overall the mean of a gene expression within a dataset should be similar between different datasets and gives an estimation to what extent the arrays in a specific dataset differ from the combined overall cohort. all datasets were sorted according to this metric and the top 15 datasets with the lowest values (normalized c 0.03), corresponding to 394 samples in total, were used as the discovery cohort (fig. 1). all probesets were checked for dataset bias (i.e. differential expression by dataset of origin that would indicate laboratory - bias or sampling differences compared to the rest). to assess dataset bias, we used kruskal wallis statistic comparing the expression of each probeset with the primary dataset vector across the 394 tnbcs. thus an enrichment of biased probesets can be monitored in any downstream application e.g. cluster analyses,,. cutoffs for exclusion of probesets due to strong dataset bias may be derived from the distribution of the kruskal this effect originated from the inclusion of two datasets which were obtained from fine needle aspiration (fna) samples. such samples generally contain relatively higher amounts of blood and lower amounts of stromal tissue as compared to surgical biopsy samples. r code enabling to programmatically access the data and to fully reproduce the results presented in the paper. r code enabling to programmatically access the data and to fully reproduce the results presented in the paper.	heterogenous subtypes of breast cancer need to be analyzed separately. pooling of datasets can provide reasonable sample sizes but dataset bias is an important concern. we assembled a combined dataset of 579 affymetrix microarrays from triple negative breast cancer (tnbc) in gene expression omnibus (geo) series gse31519. we developed a method for selecting comparable datasets and to control for the amount of dataset bias of individual probesets.
jaundice- many infants are affected by this disease, some are even born with it, but it is rarely mentioned, though a great many die of it. neonatal cholestasis syndrome (ncs) accounts for almost 30% of pediatric hepatobiliary diseases. it is defined as prolonged elevation of conjugated bilirubin beyond 14 days of life (i.e., the conjugated fraction > 20% of total bilirubin). neonatal cholestasis includes a wide spectrum of clinical conditions ranging from congenital malformations of hepatobiliary tree, infections and inborn errors of metabolism. there is no data from central india, on the etiology and clinical profile of neonatal cholestatic disorders in this region. the objective of the study is to study the proportion, clinical presentation and etiology of ncs at a tertiary referral center in central india. details of all patients treated as in patients or outpatients in the department of pediatrics from 2007 to 2012 were collected from the medical record section of the hospital. the case records of all patients with ncs were collected by two methods- collection of details of all patients who underwent hepatobiliary iminodiacetic (hida) scan for suspected neonatal cholestasis and then getting the their detailed records from the record section. collection of case records of all patients treated in department of pediatrics with following international classification of diseases (icd) codes : icd 9 and 10 -k71.0 (toxic liver injury with cholestasis), k87 (disorders of gall bladder, biliary tract and pancreas in diseases classified elsewhere), k83.9 (unspecified diseases of biliary tract), r16 (hepatomegaly and splenomegaly) and r17 (unspecified jaundice). we excluded all patients whose records were incomplete or in whom a final diagnosis could not be made. detailed information including history, routine blood investigations, thyroid function tests and liver function tests was collected. serological tests for toxoplasma, cytomegalovirus, rubella and herpes simplex were done in all patients. specific tests like tandem mass spectrometry, serum ferritin and lip biopsies were done in suspected cases of congenital metabolic disorders. the data was collected by two authors one each from departments of gastroenterology and pediatrics. it was entered in microsoft excel sheet and analysed using appropriate statistical methods using spss software version 16. all patients who had full records available formed the study group. the study group was analysed on the basis of age, sex, presenting complaints and final etiological diagnosis of ncs. the case records of all patients with ncs were collected by two methods- collection of details of all patients who underwent hepatobiliary iminodiacetic (hida) scan for suspected neonatal cholestasis and then getting the their detailed records from the record section. collection of case records of all patients treated in department of pediatrics with following international classification of diseases (icd) codes : icd 9 and 10 -k71.0 (toxic liver injury with cholestasis), k87 (disorders of gall bladder, biliary tract and pancreas in diseases classified elsewhere), k83.9 (unspecified diseases of biliary tract), r16 (hepatomegaly and splenomegaly) and r17 (unspecified jaundice). we excluded all patients whose records were incomplete or in whom a final diagnosis could not be made. detailed information including history, routine blood investigations, thyroid function tests and liver function tests was collected. serological tests for toxoplasma, cytomegalovirus, rubella and herpes simplex were done in all patients. specific tests like tandem mass spectrometry, serum ferritin and lip biopsies were done in suspected cases of congenital metabolic disorders. the data was collected by two authors one each from departments of gastroenterology and pediatrics. it was entered in microsoft excel sheet and analysed using appropriate statistical methods using spss software version 16. the study group was analysed on the basis of age, sex, presenting complaints and final etiological diagnosis of ncs. during the study period, a total of 1,31,440 children attended outpatient and inpatient services at our center. history of consanguity was noted in nine cases and family history of neonatal jaundice was present in one case. the clinical features noted were- jaundice (100/100,100%), failure to thrive (73,73%), organomegaly (68, 68%), acholic stools(38,38%), abdominal distention (52,52%) and poor feeding (29, 29%). patients with biliary atresia were advised for kasai portoenterostomy and liver transplantation as per the disease severity. patients with sepsis, cytomegalovirus and herpes infection were managed with intravenous antibiotics, antifungals and antivirals. patients with other causes were managed symptomatically and referred to higher centers for further management. fat - soluble vitamin supplementation, nutritional support and early referral to centers where ncs can be managed are required. in our study, the proportion of ncs patients was 1.2 per 1000 patients. the median age of presentation was 78 days (2.6 months) and male to female ratio was 1.17:1. two major studies from india and bangladesh reported the median age of presentation to be 3.5 months. the male to female ratio in these studies was also similar to our patient group. the earlier presentation in our study may be secondary to increased awareness over the years. the presenting clinical features in our patients were jaundice, failure to thrive and organomegaly. ahmad., from rawalpindi reported jaundice, listlessness, oraganomegaly, failure to thrive and feeding difficulty as the main clinical presentations. however, we found feeding abnormalities to be less common in our group. in our study, three studies from indian subcontinent had noted neonatal hepatitis to be the major cause for ncs. in a study by bazlul karim., from bangladesh, neonatal hepatitis (22 ; 35.5%17 with torch), followed by biliary atresia (16 ; 25.8%) and idiopathic neonatal hepatitis (15 ; 24.2%), were the commonest causes of cholestasis. ahmad., from rawalpindi reported biliary atresia to be commoner in their group of patients. the difference in etiological spectrum in our study compared to other studies may be secondary to referral bias or lack of definitive diagnostic tests like liver biopsies in majority of the patients. the present study is one of the few studies on neonatal cholestasis and represents a single - center experience over a period of 6 years. moreover, our center is one of the few centers in central india where hida scan is available and hence, patients from other hospitals also get referred for imaging. however, with our collection method, it is highly likely that we might have missed a few cases of ncs at our center. we retrospectively traced the records and it is likely that a few patients in whom the icd coding was not properly entered or hida scan was not done could have been missed. although complete work up was done in most patients, data on long - term outcome and follow - up is not available. we would like to suggest maintaining of national / regional registries of this disease to ascertain regional variations in etiological spectrum and standardize treatment. the mean age of presentation and clinical features were similar when compared to other studies. however, we found biliary atresia to be the commonest cause of ncs at our center. early referral of neonates with conjugated hyperbilirubnemia to centers with expertise and facilities for early diagnosis and treatment is important. we would like to recommend maintenance of national / regional registries of these cases to ascertain regional variation.	background : neonatal cholestasis syndrome (ncs) is a major cause of morbidity and mortality in infants. the disorder has rarely been studied in centers from central india.objectives:to study the prevalence, clinical presentation and etiology of ncs at a tertiary referral center in central india.materials and methods : the study was carried out at a tertiary referral center in central india. the study is a descriptive study. the records of all patients with suspected ncs treated in the department of pediatrics from 2007 - 2012 were analyzed.results:one hundred and sixty - eight children had a provisional diagnosis of ncs. the complete records of 100 children were available for the study. the median age of presentation was 78 days (range 15 - 270 days). the male : female ratio was 1.17:1. the clinical features noted were- jaundice (100/100,100%), failure to thrive (73,73%), organomegaly (68, 68%), acholic stools (38,38%), abdominal distention (52,52%) and poor feeding (29, 29%). the etiology as confirmed by investigations is as follows- neonatal hepatitis (20,20%), idiopathic neonatal hepatitis (18,18%), biliary atresia (41,41%), sepsis (14,14%) and others (7,7%).conclusions : the proportion of ncs in our group of patients was 1.2 per 1000 patients. jaundice, organomegaly and failure to thrive are the common presentations. biliary atresia, neonatal hepatitis and idiopathic neonatal hepatitis were the common etiological factors at our center.
differential diagnosis of parkinson s disease (pd) and atypical parkinsonian syndromes is based mainly on clinical examination (hughes. 2002). transcranial sonography (tcs) could serve as easily accessible, quick and safe diagnostic tool for movement disorders neurologist. recent studies indicate that midbrain abnormalities determined by tcs might be useful findings in uncertain cases of parkinsonism (becker. becker. postulated that the relative increase of glial cells concentration and microstructural changes in the sn may result in increased tissue echogenicity. further studies concentrated on the role of iron in the hyperechogenicity of sn (berg. 1999b, 2002 ; zecca. berg. injected rat brains with ionic iron, ferritin, 6-ohda together with desferrioxamine, zinc and buffer into the sn. a dose - dependent increase of sn echogenicity was observed in the case of ionic iron injection only. in another study, zecca. the correlation was observed between sn echogenicity and the concentration of iron measured by atomic absorption spectroscopy as well as between sn hyperechogenicity and h- and l - ferritins. accurate quantification of iron levels in control and pd brains (wypijewska. 2010) shows that total iron level remains unchanged and only labile iron level increases. the concentration of labile iron is almost 2,000 times smaller than the concentration of total iron. in most recent studies (berg. 2010), the role of activated microglia in substantia nigra hyperechogenicity was emphasized again. the aim of this study was to assess the influence of iron - loaded ferritin and glial tissue on echogenicity of the brain structures. experiment was performed on esaote mylab 70xvision ultrasound scanner equipped with 134 mhz linear array probe working in b mode. the cause of hyperechogenicity was tested on an animal model (fig. 1). fresh, i.e. max 8 h after animal death, porcine brains were used. no method of tissue fixation was applied for the experiment. to obtain the homogeneous structure, special attention was paid to remove all air bubbles, so free spaces were filled with usg gel. in this study we used porcine brains as a medium similar to human brain tissue for experimental procedures. the experimental model we used was maximally simplified and, simultaneously, designed to mimic conditions similar to those in real patients. it is not possible to localize sn or any other particular anatomical structure in such conditions. in spite of that, as described further in the study, we were able to assess repeatable conditions and well established localization for experimental procedures. ferritin (sigma aldrich horse spleen ferritin) of about 1 cm volume, containing 4,000 200 g / g of iron (the concentration of iron was assessed by us with atomic absorption), apoferritin (ferritin void of iron, sigma aldrich horse spleen ferritin) also of about 1 cm volume, containing 4.0 0.4 g / g of iron and 1 cm volume water were injected, respectively, into our models and ultrasound examination was performed for each injection separately. injection depth was 1.52 cm. in the next experiment, samples of glial tissue obtained from human glioma (approx. 0.2 cm samples of astrocytoma gii and glioblastoma multiforme giv, stored at 70, without tissue fixation procedures before the experiment) after neurosurgical operation were inserted into porcine brain and usg was performed.fig. 1animal model scheme and its photographic registration. skin and fat on top first bright layer animal model scheme and its photographic registration. skin and fat on top first bright layer region of interest was always marked either with the top of injection needle in case of injections or with metal grid for experiment with glial tissue. the localization of the injection needle seen as the regular linear structure producing intensive echo could be unequivocally determined. in the first stage of the experiment, the change of tissue echogenicity after ferritin, apoferritin and water infusion was assessed. the echogenicity shift at the site of the injection needle tip was visually evaluated at the time and directly after the infusion. then the pictures of the roi were also assessed by the same experienced sonographist, to obtain better reliability. the tcs with glioma tissue insertions was performed as next part of our experiment with the use of the same porcine brain model. for better localization, tumour samples were inserted in the centre of the metal grid and then tightly covered with brain tissue as well as fat and skin layer. the change of tissue echogenicity after injections and glial tissue insertions was assessed qualitatively by experienced physician during model examinations. experiment was performed on esaote mylab 70xvision ultrasound scanner equipped with 134 mhz linear array probe working in b mode. the cause of hyperechogenicity was tested on an animal model (fig. 1). fresh, i.e. max 8 h after animal death, porcine brains were used. no method of tissue fixation was applied for the experiment. to obtain the homogeneous structure, special attention was paid to remove all air bubbles, so free spaces were filled with usg gel. in this study we used porcine brains as a medium similar to human brain tissue for experimental procedures. the experimental model we used was maximally simplified and, simultaneously, designed to mimic conditions similar to those in real patients. it is not possible to localize sn or any other particular anatomical structure in such conditions. in spite of that, as described further in the study, we were able to assess repeatable conditions and well established localization for experimental procedures. ferritin (sigma aldrich horse spleen ferritin) of about 1 cm volume, containing 4,000 200 g / g of iron (the concentration of iron was assessed by us with atomic absorption), apoferritin (ferritin void of iron, sigma aldrich horse spleen ferritin) also of about 1 cm volume, containing 4.0 0.4 g / g of iron and 1 cm volume water were injected, respectively, into our models and ultrasound examination was performed for each injection separately. injection depth was 1.52 cm. in the next experiment, samples of glial tissue obtained from human glioma (approx. 0.2 cm samples of astrocytoma gii and glioblastoma multiforme giv, stored at 70, without tissue fixation procedures before the experiment) after neurosurgical operation were inserted into porcine brain and usg was performed.fig. 1animal model scheme and its photographic registration. skin and fat on top first bright layer animal model scheme and its photographic registration. skin and fat on top first bright layer region of interest was always marked either with the top of injection needle in case of injections or with metal grid for experiment with glial tissue. the localization of the injection needle seen as the regular linear structure producing intensive echo could be unequivocally determined. in the first stage of the experiment, the change of tissue echogenicity after ferritin, apoferritin and water infusion was assessed. the echogenicity shift at the site of the injection needle tip was visually evaluated at the time and directly after the infusion. then the pictures of the roi were also assessed by the same experienced sonographist, to obtain better reliability. the tcs with glioma tissue insertions was performed as next part of our experiment with the use of the same porcine brain model. for better localization, tumour samples were inserted in the centre of the metal grid and then tightly covered with brain tissue as well as fat and skin layer. the change of tissue echogenicity after injections and glial tissue insertions was assessed qualitatively by experienced physician during model examinations. as seen in fig. 1, all layers of animal model (i.e. fat plus skin layer, porcine brain tissue, plastic holder wall) were clearly depicted. figure 2a, b, c shows the same echogenicity of porcine brain before and after injections of iron - loaded ferritin (a), apoferritin (b) and water (c). we were unable to notice any change in tissue echogenicity except subtle transient distortions caused by liquid flow, which were the same as typical for any liquid infusion. right same model with glioma inserted. needle (see interference pattern) marked tissue insertion. hyperechogenicity compared to plain model can be clearly seen aleft plain animal model. right same model after ferritin injection. right same model with glioma inserted. needle (see interference pattern) marked tissue insertion. hyperechogenicity compared to plain model can be clearly seen on the other hand, in the second part of our experiment a marked hyperechogenicity in the area of glioma sample insertion was noticed (fig. by definition, tissue echogenicity describes its ability to create echo during ultrasound examination. in our study, we were focused only on brightness change caused by different factors. in clinical trials assessing substantia nigra and other basal ganglia echogenicity in parkinsonian patients, the area of hyperechogenic region our experiment has shown that ferritin overloaded with iron is not able to cause brain tissue hyperechogenicity. it is important to note that the concentration of iron in the ferritin injected in our experiment was even higher than that in human substantia nigra. should the hyperechogenicity of the sn in pd patients depend on iron overload, injections of iron - loaded ferritin would have to produce such an effect. having in mind the dense structure of the experimental model, physical rules of liquid diffusion and the fact, that photographic registration was performed immediately after infusions, it appears to be improbable, that the infused liquids could leave the region of interest and penetrate the surrounding tissue at the time of experiment. our findings are, at least partially, in contradiction to findings of berg. the measurements were obtained immediately after experimental injections, so, most probably, we were able to observe the effect of injected substances in situ and in real time before any biochemical interactions between injected substances and surrounding tissue occurred. in the study by berg., experimental solutions were injected into the sn of the living rat brain. echogenicity of the injected sn increased with the concentration of iron injected. at 1.5 g the echogenicity of the whole brain hemisphere increased. in our opinion, this massive increase of ionic iron in sn and its consequence is far from conditions met in sn of parkinsonian patients. one has to underline that after zinc injection, hyperechogenicity of the whole hemisphere was also seen. we do not know, however, about the exact cause of this hyperechogenicity (no histopathological data available). the question remains what was going on within the injected hemisphere during the week between injection and further experimental procedures. this subject needs further research. having this in mind our own findings and findings of berg. there is also a relatively high percentage (upto 80%) of patients with lewy bodies dementia and corticobasal syndrome having hyperechogenicity of the sn (walter. 2006). the role of iron metabolism disequilibrium in these particular neurodegenerative diseases remains unclear, whereas neuronal loss and reactive gliosis are obvious. one should mention that in physiological conditions there are, except for the sn, other iron - rich structures in the brain such as globus pallidus (gp) and red nucleus (rn). these are non - detectable (gp) or variably detectable (rn) by tcs. on the contrary, it is obvious that glioma samples can not be treated as the equivalent of microglial cells that gradually replace neurons in parkinson s disease. the exact structure, number and concentration of cells are different. keeping in mind these limitations, one has to pay attention to striking sensibility of tcs in depicting brain tissue structure changes, especially connected with glial cells. tcs has been used for many years as an useful diagnostic tool in neuro - oncology. this method is especially useful in post - operative follow - up of patients with high - grade gliomas being, in experienced hands, one of the most sensitive techniques in identification of residual tumour (becker. gii and giv glioma samples, we have used for the experiment, were clearly visible. applying this finding to parkinson s disease there is a neuroinflammatory glial response present in parkinsonian brains from the very beginning of neurodegenerative process. the relative number and activity of glial cells appear to be stable during disease progression (ouchi. 2005), which corresponds well to the stable area of sn hyperechogenicity observed in 5-years follow - up (berg. we hypothesize that gliosis is, at the present level of knowledge, the simplest and most satisfactory biological explanation of sn hyperechogenicity in neurodegenerative diseases. the role of other considerable factors such as changes in protein conformation or alfa - synuclein aggregation remains to be elucidated. to support our hypothesis the physics of ultrasound examination ultrasound echo is created due to reflection and scattering phenomena. as reflection occurs only on boundaries, magnitude of scattering produced echogenicity is proportional to the number of objects that actually interacts with ultrasound wave. therefore, the ratio between the object size and the ultrasound wave length is the most important factor. as proteins and iron nanoparticles are much smaller than ultrasound wave length, other factors like the change in cell properties or arrangement seem to be much more significant. keeping in mind all limitations and inaccuracies of animal model used, it seems to be more probable that hyperechogenicity of sn is caused rather by structural changes within the brain tissue than by an increase of iron concentration. although our comparative studies are informative, still more experiments are needed to elucidate hyperechogenicity of sn in pd phenomena, particularly role of iron.	the aim of the present study was to assess the origin of the substantia nigra hyperechogenicity in parkinson disease patients. the cause of hyperechogenicity was tested on an animal model. fresh porcine brains were injected consecutively with ferritin, apoferritin and water. then, glioma samples were inserted into animal model. the echogenicity of the region of interest was assessed before and after experimental procedures. we observed the same echogenicity of porcine brain before and after injections of iron - loaded ferritin, apoferritin and water. increased echogenicity of glioma samples compared to surrounding porcine brain tissue could be clearly seen. we postulate that the relative gliosis might be, at least partially, responsible for the increased echogenicity of the substantia nigra in parkinson disease patients. keeping in mind all limitations and inaccuracies of animal model used, it seems that hyperechogenicity of substantia nigra is caused rather by structural changes within the brain tissue than by increased iron concentration.
adherence with treatment is an important factor which can influence the outcome of that treatment (hayden., 2005). adherent patients may have better treatment outcomes than non - adherent patients (vermeire. poor adherence to treatment has been identified across many healthcare disciplines including physiotherapy (vasey, 1990 ; friedrich., 1998 ; one study found that 14% of physiotherapy patients did not return for follow - up outpatient appointments (vasey, 1990). another suggested that non - adherence with treatment and exercise performance could be as high as 70% (sluijs., 1993). adherence has been defined as : the extent to which a person 's behaviour corresponds with agreed recommendations from a healthcare provider (who, 2003). within physiotherapy, the concept of adherence is multi - dimensional (kolt., 2007) and could relate to attendance at appointments, following advice, undertaking prescribed exercises, frequency of undertaking prescribed exercise, correct performance of exercises or doing more or less than advised. many factors related to the patient, the healthcare provider and the healthcare organisation are thought to influence patient adherence with treatment (miller., 1997). within physiotherapy identification of barriers may help clinicians identify patients at risk of non - adherence and suggest methods to reduce the impact of those barriers thereby maximising adherence. firstly, to identify important barriers to adhering with musculoskeletal outpatient treatment. secondly, to discuss strategies that may help clinicians to overcome these barriers. the following databases were searched from their inception to december 2006 : amed, cinahl, embase, medline, pubmed, psycinfo, sportdiscuss, the cochrane central register of controlled trials and pedro. osteopath, chiropractor, sports, pain, joint, muscle, musculoskeletal, and outpatients. the references of primary studies identified were scanned to identify further relevant citations. studies were included if they : (1) were rcts, prospective studies, ccts or cross - sectional surveys which were peer - reviewed and published in the english language, (2) investigated patients with mechanical musculoskeletal dysfunctions, (3) related to treatment or therapeutic exercise administered by physical or exercise therapists and (4) identified barriers or predictors of adherence. studies were excluded if they investigated non - attendance at initial appointments, asymptomatic populations, in - patient populations, life threatening conditions / reduced mortality, non - musculoskeletal conditions or systemic musculoskeletal conditions being managed primarily by drug therapy or a multidisciplinary team approach. firstly, one investigator (kj) identified potentially relevant studies by scanning their titles and abstracts. secondly, remaining citations were examined independently by two investigators (kj & smc) and agreement reached on articles which did not meet the selection criteria. finally, both investigators (kj & smc) independently reviewed the full text of remaining articles against the selection criteria and consensus was reached for their inclusion in the review. in the event of disagreement, a third reviewer (jkm) arbitrated. the quality tool used in this review was modified from tools used in previous systematic reviews (borghouts., 1998 ; 2003). since adherence was the focus of this study, loss to follow - up was eliminated as an item of assessment from the quality tool. criterion meeting the quality standard were given a score of 1, while those not meeting the standard were given a zero score. high quality, while those scoring < 7 were considered low quality (borghouts., 1998 ; scholten - peeters., 2003). multiple publications derived from a single cohort were awarded one quality score based on the information available from all the publications (scholten - peeters., 2003). two reviewers (kj & smc) independently assessed and scored the included studies. where there was disagreement a third reviewer (eg) made the final decision. a standardised template was used to extract data regarding the study population, study design, predictor variables, outcome measures, study quality, data analysis and results. inter - observer agreement of quality assessment was determined by calculating percentage agreement and a kappa co - efficient (viera and garrett, 2005). information extracted is presented in table format to highlight methodological quality, similarities and differences between the studies. qualitative conclusions are based on levels of evidence (see table 2) which have been used in previous reviews (karjalainen., 2001 ; verhagen., 2004). where possible, the significance of factors affecting adherence and the levels of evidence were derived from multivariate analyses. significant associations of p < 0.05 or relevant estimated odds ratios or risk ratios were used ; these were defined as meaningful when 0.5 or 2.0 (ariens., 2000). 745 articles were excluded and 88 citations were retained for the second screening. using inclusion and exclusion criteria a further 24 articles were excluded. of the remaining 64 articles, following discussions, six of these were included, with two further papers referred to the third reviewer (eg) for arbitration. in total, 22 articles reporting on 20 independent cohort studies were selected for the review. the reviewers scored 286 items and disagreed on 29 items (10%). the overall inter - observer agreement (= 0.72) represents substantial agreement between the reviewers (viera and garrett, 2005). (2000, 2003), had their quality assessment scores combined to prevent bias in assessing the levels of evidence. the quality scores ranged from six to 11 indicating that all but one study were of high quality. the most common methodological shortfalls related to description of the source population (item a), the study size (item d) and failing to present univariate analysis (item m). the main characteristics of the study populations, barriers and outcome measures for each cohort are outlined in the supplementary electronic file. of the 20 studies, seven recruited from osteoarthritis / rheumatoid arthritis populations attending physiotherapy (stenstrom., 1997 ; schoo., 2005), part of a health organisation (shaw., 1994 ; castenada., 1998), post - surgical patients (fekete., 2006) or exercise trials (minor and brown, 1993 ; rejeski., 1997) ; four studies investigating lower back pain recruited from general outpatient populations (sluijs., 1993 ; alexandre., 2002 ; kolt and mcevoy, 2003) or a tertiary rehabilitation agency (kenny, 2000) ; three studies recruited from a sporting population (laubach., 1996 ; taylor and may, 1996 ; milne., 2005) ; two studies investigated fibromyalgia patients (oliver and cronan, 2002 ; dobkin., 2006) ; one study investigated an anterior cruciate ligament post - operative population (brewer., 2003), 2003) ; one study recruited patients with temporo - mandibular joint pain (funch and gale, 1986) and one study recruited patients from an upper limb rehabilitation centre (chen., 1999). all studies investigated at least one aspect of treatment adherence including attendance at appointments, adherence with home exercises and in - clinic adherence. only one study (stenstrom., 1997) did not report multivariate analysis. there was strong evidence that low levels of physical activity at baseline (4 trials,728 participants) or in previous weeks (2 trials, 883 participants), low self - efficacy (6 trials, 1296 participants), depression (4 trials, 1367 participants), anxiety (2 trials, 159 participants), helplessness (2 trials, 792 participants), poor social support or activity (6 trials, 2286 participants), greater perceived number of barriers to exercise (3 trials, 857 participants) and increased pain levels during exercise (2 trials, 159 participants) were barriers to treatment adherence. there was also strong evidence that low in - treatment adherence with exercise (3 trials, 287 participants) was a barrier to longer term exercise adherence. there was conflicting evidence that age and greater pain at baseline were barriers to treatment adherence. limited evidence was found for a range of other variables with one good quality study supporting each of them. this systematic review summarised the results from 20 high quality studies and found strong evidence that low levels of physical activity at baseline or in previous weeks, low in - treatment adherence with exercise, low self - efficacy, depression, anxiety, helplessness, poor social support or activity, greater perceived number of barriers to exercise and increased pain levels during exercise are barriers to treatment adherence. the results of this review are in line with others which have found that non - adherent individuals were likely to have lower levels of prior activity, lower exercise self - efficacy, greater number of barriers and low levels of social support (martin and sinden, 2001 ; jackson., 2005). these reviews vary from our own in that psychological variables such as anxiety, stress and helplessness did not emerge as predictive. in the review by martin and sinden (2001) few studies investigated whether psychological variables predicted adherence of non - clinical populations of older adults to exercise intervention. in the review by jackson. (2005) there was conflicting evidence for depression and anxiety in patients attending cardiopulmonary rehabilitation (cpr). one reason for this could be that these traits are more likely to be present in women, who are less likely to be referred to cpr. therefore these symptoms may be less likely to emerge as predictors of non - adherence in cpr (benz scott., 2002). this review was conducted in accordance with guidelines from the centre for reviews & dissemination (crd, 2001), however the possibility of publication bias can not be excluded (altman, 1991). unpublished studies and studies from lesser known databases or published in languages other than english may have been missed. motivation to adhere with treatment and therefore the barriers may vary between different pathology types and populations (shaw., we have not attempted to analyse these differences. whilst all studies included in this review were rated as high quality the studies had sample sizes ranging from n = 34 (laubach., 1996) to n = 695 (sluijs., 1993) with only five (25%) studies exceeding 300 subjects. whilst there are no universally agreed methods of calculating sample sizes for multivariate analysis, smaller studies with large numbers of predictive variables may allow less confidence in the findings (tabachnick and fidell, 2001). for example, low socioeconomic status (ses) emerged as a predictor of non - adherence with cpr (jackson., 2005) and may warrant further investigation in populations with musculoskeletal disorders. in addition, much of the research has focussed on patient factors and little research has investigated the barriers introduced by health professionals or health organisations (miller., 1997). further research to investigate potential barriers such as ses, health professional factors and health organisation factors would be appropriate. the most commonly used measures of adherence were attendance at appointments, adherence with home programmes and in - clinic adherence. whilst attendance at appointments is standardised it provides no information about patient attitude and behaviour towards rehabilitation e.g. adherence with home exercise programmes or within clinic adherence (kolt., 2007). patient self - reports using paper diaries were the most common measure of adherence with home programmes. however, poor real time compliance with diary completion and recall accuracy may lead to data of questionable validity (stone. it is possible that the use of electronic diaries with compliance enhancing features may improve the quality and accuracy of data collected (broderick and stone, 2006 ; green., 2006). the most common measure of in - clinic adherence was the therapist - rated sports injury rehabilitation adherence scale (siras). however patients and practitioners may disagree on the level of patient adherence (donovan, 1995 ; carr, 2001) and this variation between patient self - rating and therapist - rating of patient adherence leaves scope for considerable inaccuracy (kolt and mcevoy, 2003). the use of therapist - rated adherence measures in conjunction with exercise diaries to corroborate patient self - reports (kolt and mcevoy, 2003) may improve assessment of adherence (shaw., 2005). worsening pain during exercise was a barrier to adherence with exercise (minor and brown, 1993 ; dobkin., 2006) indicating that strategies to minimise initial pain are important. in most cases the appropriate use of simple analgesics, heat or ice coupled with passive physiotherapy treatments, e.g. acupuncture, manual therapy, etc may help to alleviate pain sufficiently to allow patients to adopt more active treatment strategies (moffett and mclean, 2006). in the rare case of a patient with severe pain an analgesic review with their gp or consultant may be required in order to allow participation in rehabilitation. clinicians need to gain a clear understanding of the patient 's pain experience and beliefs about pain (eccleston and eccleston, 2004) and counter those which are mal - adaptive. clinicians should reinforce messages which reduce fear or anxiety about pain, e.g. that the presence of pain should not prevent most patients from safely participating in therapeutic exercise (waddell., 2004) and may lead to reduction in symptoms (guzman., 2002), improved function and return to work (van tulder., 2000). those who participate in regular exercise are also less likely to experience progressive problems (mclean., 2007). patients should be encouraged to start exercise gently and advised to progress to moderate or even high intensity levels of exercise over a period of time (pernold., 2005). this evidence could counter the fears held by many pain sufferers that movement could be damaging or lead to re - injury. low levels of physical activity at baseline (minor and brown, 1993 ; rejeski., 1997 ; stenstrom. 1997 ; oliver and cronan, 2002) and low in - treatment adherence with exercise (alewijnse., 2003 ; schoo., 2005 ; dobkin., 2006) were barriers to treatment adherence. physiotherapists need to recognise and be ready to mitigate the many barriers to initiating and adhering to exercise programmes ; these include poor programme organisation and leadership, poor education, poor history of exercise, perceived physical frailty, perceived poor health and readiness to change (duncan and mcauley, 1993 ; courneya and mcauley, 1995 ; boyette., 1997 ; hellman, 1997 ; rhodes., 1999). firstly providing explicit verbal instruction, checking the patient 's recall and supporting this with additional written instructions may be effective at improving exercise adherence (schneiders., 1998). secondly, employing motivational techniques such as counselling sessions, positive feedback, reward, written treatment contracts and exercise diaries may also be helpful (friedrich., 1998). setting goals and drawing up action plans and coping plans which have been agreed collaboratively between the clinician and patient may be effective with patients who intend to participate in exercise (bassett and petrie, 1999 ; evans and hardy, 2002 ; ziegelmann., 2006). identifying potential barriers to exercising can support the development of action plans to initiate an exercise programme, whilst coping plans can help to overcome the difficulties that may arise over time and help patients to maintain that exercise programme (gohner and schlicht, 2006 ; ziegelmann., 2006). low self - efficacy was identified as a barrier to treatment adherence (shaw., 1994 ; taylor and may, 1996 ; stenstrom., 1997 poor self - efficacy could explain a patient 's low confidence in their ability to overcome obstacles to initiating, maintaining or recovering from relapses in exercise (sniehotta., 2005). low self - efficacy could be identified by clinicians using simple questions such as how confident are you that you can (b) return to exercise, despite having relapsed for several weeks ? strategies to address low self - efficacy should be specific to the individual 's stage of exercise behaviour or perceived obstacles (scholz., 2005). the use of strategies such as agreeing realistic expectations (jensen and lorish, 1994), setting treatment goals (evans and hardy, 2002), action planning (sniehotta., 2005), coping planning and positive reinforcement (gohner and schlicht, 2006) may help increase patient self - efficacy and adherence., 1997 ; oliver and cronan, 2002), anxiety (minor and brown, 1993 ; dobkin., 2006) and helplessness (sluijs., 1993 ; physiotherapists should be sensitive to the presence of anxiety, depression and helplessness and ensure that these patients are referred to relevant healthcare services for appropriate management as required. simultaneously ensuring that pain is being effectively managed may be helpful in reducing anxiety or depression which is pain related. additionally it may be helpful to reinforce the message that exercise is an effective way of countering both low mood and negative thoughts, whilst simultaneously improving pain and function (lim., 2005). greater social support and encouragement for exercise in this group of patients may provide motivation, role models and guidance that may be important (castenada., 1998). low levels of social activity (funch and gale, 1986 ; minor and brown, 1993 ; sluijs., 1997 ; oliver and cronan, 2002) and social or familial support (shaw. some patients believe they would more readily exercise if accompanied by someone else during their activity (milroy and o'neil, 2000 ; campbell., 2001). the support provided by the physiotherapist, the development of the patient practitioner relationship and positive feedback from the physiotherapist may also increase adherence (sluijs. for example group based rehabilitation, exercise referral schemes, expert patient programmes and exercise classes based in the community may be an ideal way of providing some patients with the social stimulation and long term encouragement to continue their exercise progression. for other patients, actively involving partners in the rehabilitation process to encourage and motivate the patient may help (fekete. envisaging a greater number of barriers to participating in exercise predicted non - adherence with treatment (sluijs. barriers included transportation problems, child care needs, work schedules, lack of time, family dependents, financial constraints, convenience and forgetting. physiotherapists need to be aware of difficulties that patients foresee in relation to adhering with a proposed treatment plan and act collaboratively with their patients to design treatment plans which are customised to the patient 's life circumstances (turk and rudy, 1991). the addition of coping plans may help patients to overcome difficulties that may arise and allow them to maintain the treatment programme (gohner and schlicht, 2006 ; ziegelmann. there was limited evidence for many barriers and a lack of research into other potential predictors, e.g. socioeconomic status and the barriers introduced by health professionals or health organisations. adherence has been identified as a priority in physiotherapy research (taylor., 2004) therefore further high quality research is required in order to investigate the predictive validity of these barriers within musculoskeletal settings. poor attendance at clinic appointments is an objective measure with quantifiable cost implications to the health service. the extent to which patients actually carry out a programme of exercises recommended by a physiotherapist is an important research question which is methodologically more difficult to answer. these two different aspects of adherence may be related to different barriers and may require different strategies to overcome them, therefore these different aspects of adherence may be better addressed individually. this review identified 20 studies investigating barriers which predicted non - adherence with musculoskeletal treatment. strong evidence was found that low levels of physical activity at baseline or in previous weeks, low in - treatment adherence with exercise, low self - efficacy, depression, anxiety, helplessness, poor social support or activity, greater perceived number of barriers to exercise and increased pain levels during exercise are all barriers to treatment adherence. identification of these barriers during patient assessments may be important in order to adopt appropriate management strategies which help to counteract their effects and improve treatment outcome. the results of this review suggest that physiotherapists should be concerned about the attitudes, beliefs and barriers facing their patients and act collaboratively with their patients to design realistic treatment plans which are customised to the patient 's life circumstances. there was conflicting evidence regarding age and pain at baseline and limited evidence for many other barriers. in addition there is a lack of research investigating barriers introduced by health professionals and health organisations. further high quality research is required to increase our understanding of all the factors which contribute to patient non - adherence.	poor adherence to treatment can have negative effects on outcomes and healthcare cost. however, little is known about the barriers to treatment adherence within physiotherapy. the aim of this systematic review was to identify barriers to treatment adherence in patients typically managed in musculoskeletal physiotherapy outpatient settings and suggest strategies for reducing their impact. the review included twenty high quality studies investigating barriers to treatment adherence in musculoskeletal populations. there was strong evidence that poor treatment adherence was associated with low levels of physical activity at baseline or in previous weeks, low in - treatment adherence with exercise, low self - efficacy, depression, anxiety, helplessness, poor social support / activity, greater perceived number of barriers to exercise and increased pain levels during exercise. strategies to overcome these barriers and improve adherence are considered. we found limited evidence for many factors and further high quality research is required to investigate the predictive validity of these potential barriers. much of the available research has focussed on patient factors and additional research is required to investigate the barriers introduced by health professionals or health organisations, since these factors are also likely to influence patient adherence with treatment.
cerebral palsy (cp) is a broad term that describes a set of conditions that is associated with major physical impairments and other developmental deficits and arises in the early stages of brain development. cp is the most common type of physical disability affecting children in developed countries with an estimated prevalence of 2.0 to 2.5/1000 children. despite the nonprogressive nature of the condition, the nature of functional impairments may change as the child develops. as a consequence, evolving limitations in everyday activities may be experienced, with possible impact on the individual 's overall health and well - being [4, 5 ]. the type and distribution of movement disorder in cp may be categorized as spastic, dyskinetic, hypotonic, or mixed. spastic patterns are the most common and may be further differentiated as diplegia, quadriplegia, hemiplegia, or monoplegia, relating to the limbs involved [6, 7 ]. individuals with cp have been an important target population requiring rehabilitation services. the traditional focus of health care services for this population has been primarily directed at rehabilitation interventions that address the underlying motor and other developmental impairments, such as abnormal muscle tone, decreased attention span, poor dexterity, or difficulties with perceptual concepts, as well as limitations in essential daily self - care skills and mobility. in the last decade, however, increasing interest has been attributed to the quality of life (qol) and participation of children with cp. researchers and clinicians are concerned with the extent to which children with cp have the opportunity to be involved and enjoy leisure activities at home and in the community and the extent to which they report a good qol [8, 9 ]. qol is a broad concept encompassing many components of overall health and well - being (e.g., physical, psychosocial, economic, and cultural). it is influenced by the context of the culture and value systems in which the individual lives and relates to the individual 's goals, expectations, standards, and concerns. the qol of children who live with a neurological condition can be impacted at different levels, including physical (e.g., physical health, independence in basic functional activities), psychological (e.g., mental status, positive self - perception), and psychosocial dimensions (e.g., forming friendships, leisure time, finding a partner). identifying the factors that are associated with better or poorer qol is important as this can be used to guide program planning and the allocation of resources, thus optimizing the well - being of these children. studies have shown that individuals with disabilities do not necessarily have a lower qol and a diminished perception of their well - being or general dissatisfaction with life [13, 14 ]. recent studies are exploring a variety of factors that may contribute to a good qol in children with neurodevelopmental disabilities ; however, most studies explore aspects related to body function and activity limitations. participation in leisure activities includes participation in sports, arts, entertainment, social, self - improvement, and religious activities. engaging in leisure activities may be influenced by the child 's personal factors, environmental factors, and underlying health condition. by participating in leisure activities, children may develop competencies, achieve mental and physical health, gain an understanding of their strengths and abilities, and form lasting meaningful friendships and relationships. it is through participating that children make contributions to their community, learn about themselves and the expectations of society, and develop skills needed to become successful and autonomous in their home, school, and community [16, 17 ]. for these reasons, participation is increasingly considered as one of the primary aims of pediatric rehabilitation and is believed to contribute to child health, development, and qol [16, 18 ]. researchers have shown that multiple factors may contribute to a perceived good qol in children with physical disabilities, including the child 's participation in a variety of leisure activities. participation in leisure is an objective, tangible outcome that can be incorporated and measured in rehabilitation and health programs, while qol is a subjective outcome. although frequently assumed, the association between leisure participation and qol has not been clearly delineated. the objective of this study was to examine the association between involvement in leisure activities and qol in children between 6 to 12 years of age with cp. this study used a cross - sectional design involving a historical cohort of children with cp seen by a single neurologist over a 10-year period (19912001) in a variety of settings (private office, hospital, neonatal clinic, suburban private clinic). this sample is representative of children with cp from a local community who are routinely sent to a pediatric neurologist for diagnosis, etiological determination, and referral for rehabilitation services. exclusion criteria were children presenting progressive disorders, disorders of noncerebral origin, and specific syndromes. parents who could not easily read or converse in english or french were excluded from participation. a total of 63 children and their parents completed the leisure participation and qol questionnaires. twenty - one participants who could not actively participate in the completion of the leisure participation questionnaire were excluded. those participants were children with more severe cognitive impairment and therefore who could not actively contribute to the completion of these self - report questionnaires. demographic and clinical characteristics of the participants and also of the children who could not complete the questionnaires (nonparticipants) are described in table 1. scientific and ethical approval was obtained through the mcgill university health centre, montreal children 's hospital 's institutional review board. information letters describing the study were sent to parents of children with a diagnosis of cp between the ages of 6 and 12 years. informed signed consent a 3-hour visit was scheduled at the montreal children 's hospital to conduct required assessments. participants completed a series of developmental evaluations administered by a neurologist and a physical therapist and/or occupational therapist. as part of this study, the neurologist assessed the level of gross motor function for each participant using the 5-level gross motor function classification system (gmfcs). the gross motor function measure (gmfm-66) was administered by a physical or occupational therapist during the visit. parents (and children when feasible) completed questionnaires assessing leisure participation and qol and also completed a demographic questionnaire. the pediatric quality of life inventory (pedsql) generic core scales were used to assess quality of life in the physical, emotional, social, and school domains. this questionnaire includes 23 items, and the parent proxy - report version was used in this study. the child - report version was also used when feasible, but, due to smaller numbers, the parent report was used for analysis in this paper due to the small sample of children who were able to complete the child version independently. the psychosocial well - being summary score represents the way the child feels about their social life, their school functioning, and their emotional well - being. the physical summary score represents the ease in which one can get around and do basic activities, without pain while maintaining a good energy level the validity of the pedsql generic core scales has been demonstrated through known groups comparisons and correlations with other measures of disease burden. the children 's assessment of participation and enjoyment (cape) was used to assess participation in leisure activities. this 55-item child self - report questionnaire was designed for children and youth with and without disabilities between the ages of 6 and 21 years. it provides information on five dimensions of participation (diversity, intensity, where, with whom, and enjoyment) and five types of activities (recreational, active - physical, social, skill - based, and self - improvement). parental assistance was sought when children had difficulty completing this questionnaire ; however, children actively contributed in the completion of the cape. for the purposes of this study, intensity (how often does the child participate in each activity) and diversity (how many different activities a child engages in) scores in the five different activity types were used. participants (children with cp) who could not actively participate in completion of the cape were excluded from analysis. pearson correlation coefficients were computed to examine the association between leisure participation (cape) and qol (pedsql). a multiple linear regression model was estimated with qol (psychosocial and physical summary scores) as the dependent variable and leisure participation as the independent variable. models were also derived including age and level of motor function using gmfm scores. diagnostic tests were used to check for violation of the assumptions inherent in linear regression models. due to the lack of evidence on specific leisure domains that may predict qol, forced entry regression was used to determine the significant predictors of psychosocial and physical qol. this method is likely not influenced by random variation in the data and is therefore appropriate for using theory testing. when reporting their child 's qol, about half of the parents reported that their child had low well - being (more than one standard deviation below normative means) in the physical and psychosocial well - being domains. well - being in emotional functioning and school functioning was within normative means for about 60% of the children as reported by their parents (table 2). intensity scores measure how often the child has engaged in a given activity in the past four months (ranging from once in the past four months to everyday or more). the intensity of recreational activities (e.g., doing crafts) was higher than the other four activity domains, followed by social activities (e.g., talking on the phone) and self - improvement activities (e.g., reading) (table 3). a detailed description of participation in leisure activities and qol for this population has been published elsewhere [2224 ]. the relationship between involvement in leisure activities (i.e., diversity and intensity of participation in activities) and parent - proxy report of child 's qol was tested. intensity of participation, but not diversity, in active - physical activities such as team sports, bicycling, water and snow sports, and other individual physical activities was significantly correlated with physical well - being (r = 0.34, p = 0.007). intensity and diversity of involvement in skill - based activities, that is, of activities such as dancing, arts, and music classes done with an instructor, were negatively correlated with physical well - being (r = 0.39, p = 0.001 for intensity and r = 0.41, p = 0.001 for diversity, resp.). intensity of participation in active - physical activities also accounted for better psychosocial well - being (r = 0.31, p = 0.01) of children with cp. in a multiple regression model, diversity and intensity of participation in five domains of leisure accounted for 32% (p = 0.002) of the variation in physical well - being however, when age and gross motor functioning (gmfm score) were included in the model, this value increased to 48% (p < 0.001) of the variance in the physical well - being domain (table 4). this study describes the association between participation in leisure activities and qol in school - aged children with cp. results indicate a positive association between engagement in physical activities and both physical and psychosocial well - being. these findings suggest that school - aged children with cp who participate more actively in physical activities get around more easily to do basic activities, without pain and with a good energy level, and feel better about their social life, school functioning, and their emotions, according to parent - report. inversely, it is possible that children who generally feel better in these domains naturally participate more in active physical activities. skill - based activities and physical well - being were negatively related, suggesting that other factors may play a role in this relationship. for instance, motor functioning alone accounted for a high variance in the outcome variable in the multivariate model. indeed, child attributes such as the severity of motor dysfunction, age, and gender were previously described as important predictors of physical well - being, a component of a child 's qol [22, 32, 33 ]. studies have shown that, with increasing age, children tend to decrease their participation in out of school leisure activities ; therefore, it is important to motivate them to maintain and increase their activity level as they develop. for instance, children and families who do not have the environmental supports and adaptations they require may not be able to participate in leisure activities of their own choosing, which is related to the child 's qol. furthermore, children with more severe motor limitations often attend segregated school environments where they are exposed to more intense rehabilitation services and adapted leisure activities as compared to children in an integrated school setting. children in special schools may have the opportunity to participate in activities such as adapted horseback riding, adapted arts, and swimming programs. half of the parents of school - aged children with cp in our study reported their children as having poorer physical well - being (< 1 sd of the normative mean) compared to parents of typically developing peers. this finding is probably related to their motor limitations, which may also explain our result that higher levels of participation in skill - based activities were associated with lower physical well - being. skill - based activities (e.g., dance lessons, karate lessons) may be challenging for children with disabilities, especially if the demands of the particular task are not adapted to the child 's capabilities. families should be assisted in choosing appropriate and meaningful activities and learning about available adapted sports and recreation programs in their community to foster a sense of mastery and competence. in this regard, it is interesting to observe that children in adapted or segregated schools may have more opportunities to participate in skill - based activities as adapted skill - based activities may be provided by the school or referred to by professionals in the school, in spite of their higher motor limitations. physical activity has substantial benefits to the health and well - being of children and adolescents. active - physical pursuits are known to improve cardiovascular and emotional health, motivating children to stay physically active and contributing to a sense of well - being [34, 35 ]. participation in leisure activities also fosters friendships and other social relationships, often creating a social support network that may contribute to the individual 's overall well - being. in congruence with the findings of our study, a recent study with 120 school - aged children with cp using different measurement tools reported a significant relationship between the intensity of participation in leisure activities with physical well - being, emotional well - being, and social support and peers. this particular study measured leisure participation more globally ; however, we applied a measure with five subscales of different types of leisure activities. our study indicates that it is the active - physical leisure activities specifically that are most strongly related to aspects of qol. associations between emotional and psychosocial well - being and participation in leisure have also been reported when children engage in everyday activities or social activities [32, 36 ]. qualitative studies have shown how children and adolescents with disabilities place a high value on participating in leisure activities. youth with cp and their parents reported that participation in chosen and enjoyable activities has a positive impact on their well - being [3742 ]. a recent analysis of the association between leisure participation and qol in children with neurodevelopmental disabilities found evidence from quantitative, qualitative, and mixed - methods studies supporting the relationship between participation in leisure and different domains of qol [32, 33, 3538 ]. similarly to our study, others have found a positive association between engagement in active physical activities and physical well - being in children and youth with cp [32, 35, 43 ]. however, the measurement of both constructs (qol and participation) is very variable across studies, limiting interpretability for clinical practice. our study, however, is the first to report on the relationship between psychosocial and physical domains of qol, specifically with respect to leisure participation in school - age children with cp. the cape measures the activities that the child actually does (i.e., performance), which may or may not be in line with their preferences or personal choices. a strong linear relationship between cape diversity and intensity scores may account for a limited capacity of the model to represent the sample and limit the size of regression coefficients in the regression models. a moderate correlation between levels of actual involvement in active - physical pursuits with the level of preferences for these types of activities was previously shown, implying that children may be participating in activities that they do not necessarily enjoy. programs and interventions should consider the preferences of children and families in order to individually tailor the level of engagement in activities to their preferences (i.e., through adapted programs or those that have no environmental barriers) and thus foster better overall well - being. interrater reliability between parental and child report of qol was previously described for this sample and shown to be very good for the physical domain (icc = 0.72, p < 0.0001), but less for the psychosocial aspect of qol (icc = 0.54, p < 0.0001). unfortunately, 21 children were either too young or had cognitive and/or language deficits that limited their ability to self - report. whereas the cape can be completed with the help of an interviewer and uses pictorial and verbal depiction of the items, the self - report version of the pedsql requires more ability to be completed. therefore, we chose to use the pedsql parent - report version in the analysis. this is a frequently reported challenge in measurement of subjective constructs in children with complex disabilities. children may place a higher value than their parents on engagement in leisure activities, and an association between the child 's self - report leisure participation and their qol may be stronger than findings using parental report. the fact that only children who could self - report were considered in this study clearly indicates a need to explore other measurement alternatives for children with more severe impairments in order to promote qol for children across all severity levels. due to the cross - sectional design of this study for instance, children who participate in more leisure activities may experience better qol than children who participate in fewer or less frequent activities. the opposite may also be true, such that children who experience a higher sense of physical and psychosocial well - being may engage in more leisure activities. our findings indicate that leisure participation in active - physical activities is positively associated with both physical and psychosocial well - being in school - aged children with cp. meaningful and adapted leisure activities appropriate to the child 's skills and preferences may foster qol. further studies are needed to explore the temporal and possible causal connections between these aspects of children 's lives. qol and participation are important complex, multidimensional conceptual constructs that encompass many subjective aspects, including individual preferences, the influence of environmental factors, but also objective aspects such as the availability, accessibility, and affordability of preferred activities, the school setting, and the family 's involvement in specific activities. assessment of both constructs and interventions aiming to improve the qol of children with cp should include the promotion of meaningful and adapted leisure activities appropriate to the child 's skills and preferences, especially active - physical activities.	the objective of this study was to examine the association between leisure participation and quality of life (qol) in school - age children with cerebral palsy (cp). leisure participation was assessed using the children 's assessment of participation and enjoyment (cape) and qol using the pediatric quality of life inventory (pedsql). pearson correlation coefficients were calculated to examine the association between cape and pedsql scores, and a multiple linear regression model was used to estimate qol predictors. sixty - three children (mean age 9.7 2.1 years ; 39 male) in gmfcs levels i v were included. intensity of participation in active - physical activities was significantly correlated with both physical (r = 0.34, p = 0.007) and psychosocial well - being (r = 0.31, p = 0.01). intensity and diversity of participation in skill - based activities were negatively correlated with physical well - being (r = 0.39, p = 0.001, and r = 0.41, p = 0.001, resp.). diversity and intensity of participation accounted for 32% (p = 0.002) of the variance for physical well - being and 48% (p < 0.001) when age and gross motor functioning were added. meaningful and adapted leisure activities appropriate to the child 's skills and preferences may foster qol.
actinomycosis is an indolent infectious disease characterized by pyogenic response and necrosis, followed by intense fibrosis. several syndromes have been associated with actinomyces infection, including cervicofacial, pulmonary, abdominal, female genital and disseminated actinomycosis. clinical presentations of thoracic actinomycosis have changed considerably over the last decades with the reduction of its incidence. the presenting signs of actinomycosis are unresolved pneumonia or pulmonary infiltrate or a mass on routine chest radiograph. the infection typically spreads without regard to anatomic barriers, but involvement of the major bronchi is exceptionally uncommon. a 43-year - old man was admitted to our hospital with one - month history of productive cough. he had a 3-year history of mitral stenosis and an operation for mitral valve replacement. laboratory tests revealed a leukocyte count of 11,600/l and hemoglobin of 13.2 g / dl. differential cell count disclosed the following values : neutrophils 76% ; lymphocytes 17% ; monocytes 5% ; eosinophils 1.7% ; basophils 0.3%. chest radiograph showed focal consolidation of the right lower lobe (figure 1). computed tomography of the chest demonstrated patchy air - space consolidation and atelectasis of the right lower lobe (figure 2). gram stain showed many gram positive cocci ; afb smear and culture for mycobacterium tuberculosis were negative ; there were no fungi. fiberoptic bronchoscopy showed subtotal occlusion of the right superior segment of the lower lobe by a yellow - white, stony - hard mass, which was surrounded by inflamed and edematous bronchial mucosa (figure 3). histologically, the biopsy specimen revealed an inflammatory cellullar infiltration and colonies which were formed of a radiating network of filaments staining intensely with hematoxylin (figure 4). the gram stain also showed a thin, filamentous, gram - positive, branching organism. the patient was initially treated with 10 million units of intravenous penicillin daily for two weeks, followed by oral amoxacillin / clavulanic acid. after 3 months of therapy, the clinical manifestations and radiologic findings species of actinomyces are gram - positive, anaerobic microorganisms belonging to the resident flora of the oropharynx and gastrointestinal tract and are found in 3050% of normal saliva specimens. the term actinomycete, which is of greek origin, means ray fungus ; however, actinomycosis is due to a gram - positive anaerobic bacterium. bollinger described the micro - organism in 1877 as the causative agent of lumpy jaw in cattle and named it actinomyces bovis. in 1891, wolf and israel found a similar micro - organism from pulmonary abscess and named it actinomyces israelii. although most cases of human actinomycosis are due to a. israelii, less common agents are actinomyces naeslundii, actinomyces viscosus, actinomyces odontolyticus, actinomyces meyeri and propionibacterium propionicum. the disease is classically divided into three types, depending on the anatomic sites involved ; cervicofacial, abdominopelvic and thoracic. cervicofacial infection is the most frequent manifestation, with thoracic actinomycos is accounting for approximately 20% of cases. direct extension may occur from disease in either the head and neck or abdominal cavity. the organisms may then spread from the lung to the pleura, mediastinum and chest wall without regard for normal anatomic barriers. the reason for this is unclear but may relate to the proteolytic activity of the bacteria. the peak incidence is reported to be in the mid - decades, with cases in individuals younger than 10 and older 60 years being less frequent. nearly all series have reported males to be infected more frequently than females, at an approximately 3:1 ratio. plausible but unproven explanations for this discordance include poorer dental hygiene and increased oral trauma in males. studies of the occurrence of actinomycosis estimated a yearly incidence of 1:100,000 in the netherlands and germany in the 1960s and 1:300,000 in the cleveland area during the 1970s, making this disease uncommon but not rare. oral and cervicofacial diseases are frequently associated with dental procedures, trauma and oral surgery. the typical lesion consists of abscesses filled with neutrophils and surrounded by dense fibrous tissue. actinomyces organisms tend to grow in dense microcolonies or granules that may reach 4 mm in size. these are often called sulfur granules because they are usually yellow, although they do not contain much sulfur. the micro - organisms are slender branching gram - positive bacilli embedded in the matrix of the granules. similar structures can be formed by groups of other bacteria (streptomyces, staphylococcus and streptococcus) or hyphae. aggregates of nocardia occainsionally form granules similar to actinomyces, though usually lacking the peripheral clubbing. nocardia, however, differs from actinomyces in that it is an aerobic and modified acid - fast positive organism. actinomycos is has been called the most misdiagnosed disease and there is no disease which is so often missed by experienced clinicians. also, the finding of actinomyces by sputum cytology and/or culture, unless obtained directly from the bronchus, can not be used as a definitive diagnosis due to its frequent presence as a commensal of the oral cavity. the radiographic findings are dependent on the chronicity of the disease. in acute infections, the pattern has been reported as consisting of non - segmental air - space disease indistinguishable from other pneumonic process. there is peripheral and lower lobe predominance, possibly reflecting the role of aspiration in the pathogenes is of this disease. the other case, evaluating unresolved pneumonia, led to the diagnosis of endobronchial - lipoma with superimposed actinomycosis. common causes of endobronchial obstruction are bronchogenic carcinoma, bronchial benign tumors and endobronchial tuberculosis. since the microorganisms of the associated flora are not always susceptible to penicillin g, some investigators favor aminopenicillin + / clavulanic acid. clindamycin, tetracyclines and erythromycin are alternative drugs in the case of penicillin allergy. in general, a treatment course of 312 months is recommended for pulmonary actinomycosis, considering the difficult penetration of antibiotics in areas of dense fibrosis. high cure rates of actinomycosis on appropriate medical treatment have been reported in all recent series, with a mortality of only 1 out of 48 cases. in conclusion, endobronchial actinomycosis must be considered in the differential diagnosis of an endobronchial lesion, especially unresolving pneumonia. a presumptive clinical diagnosis should be attempted, since special requirements for successful culturing are needed. when sulfur granules are noted on routine histologic or cytologic examination of material obtained at bronchoscopy, additional stains will confirm the diagnosis of actinomycotic infection by revealing non - acid - fast filamentous micro - organisms stained positively with gram stain.	actinomycosis is an infectious disease caused by certain actinomyces species. actinomyces are gram - positive, non - spore forming organisms characterized by obligate or facultative anaerobic rods that normally inhabit anaerobic niches of the human oral cavity. cervicofacial, abdominal, pelvic and thoracic infections of actinomyces are not uncommon, but endobronchial actinomycosis is rarely reported. endobronchial actinomycosis can be misdiagnosed as unresolving pneumonia, endobronchial lipoma or malignancies. endobronchial actinomycosis should be included in the differential diagnosis of any endobronchial mass.we report a case of a 43-year - old man who presented with a productive cough and pulmonary consolidation at the right lower lobe on chest radiograph. fiberoptic bronchoscopy revealed obstruction of the right superior segment of the lower bronchus with an exophytic endobronchial mass. endobronchial actinomycosis was confirmed by demonstration of sulfur granules in the bronchoscopic biopsy of the mass. intravenous administration of penicillin g followed by oral amoxacillin / clavulanic acid therapy for 3 months resulted in improving symptoms. infiltrative consolidation on the chest x - ray was markedly decreased.
the success of any dental restoration is based on the high adhesive property of the material. various materials are available which utilizes this adhesive property such as, glass ionomer cement restoration, composite restorations, and pit and fissure sealants. among these composite resins have been developed since few years in order to provide the best esthetics to the anterior restorations as well as for posterior restorations. dental adhesive systems are agents used to promote adhesion in between dental structure and composite resin, and should give a similar performance on enamel and dentine. it differs from enamel, as it has smear layer, organic contents, and presence of fluid inside the dentinal tubules.1 dentin is a biologic composite of a collagen matrix filled with sub - micron to nanometer - sized carbonate - rich apatite crystallites dispersed between hyper mineralized collagen poor hollow cylinders. it is very well understood that the density of dentinal tubules varies with dentinal depth and as well as the water content of dentin is lowest in superficial dentin and highest in deep dentin. in superficial dentin, which contains fewer tubules and the permeation of resin into intertubular dentin will be responsible for most of the bond strength. in dentin, dentinal tubules are more in number and hence, the intratubular permeability of resins will be responsible for higher bond strength. total - etch technique involves the simultaneously removal of the smear layer from both enamel and dentin surface followed by the utilization of single bottle agent that incorporates the primer and adhesive in one solution. self - etching technique - their bonding mechanism is based on the simultaneous etching, priming and adhesive of the dentin surface in a single bottle.2 bonding to enamel was achieved earlier and easier (buonocore, 1955) because enamel is mostly composed of hydroxyapatite crystals. although it is possible to obtain predictable and reliable adhesion to enamel, adhesion to dentin, which is the largest part of the tooth, has proved to be more challenging because of its heterogeneous nature. the mechanism of dentin adhesion, enhanced by hybrid layer formation between the resin and dentin, was proposed by nakabayashi (1982). the adequate hybrid layer formation is believed to be essential to create a strong and durable bond between resin and dentin. adhesive restorations have been widely accepted for both anterior and posterior use in restorative dentistry. patient s demands for esthetic restorations have caused a recent increase in the use of tooth - colored restorative materials. to achieve clinical success with such restorations good adhesion between restorative materials and tooth substrates is of crucial importance in order to ensure good marginal sealing, reinforcement of the tooth structure, and longer life of the restoration. during the last two decades, a variety of adhesive systems has been continuously developed in order to produce good adhesion to dental substrates. these great advances in the adhesive dentistry have changed the concepts of cavity preparation based on the principals proposed by black (1955) into more conservative and minimally invasive ones. the current self - etching adhesives provide monomer formulation for synchronic conditioning and priming of enamel and dentin. as of today, less research is available to indicate the effectiveness of new generation self - etching primers against superficial and deep dentin. the aim of this study was to evaluate the shear bond strength of the newer bonding systems on superficial dentine and deep dentin. the present in vitro study was conducted in the department of conservative dentistry and endodontic, mr ambedkar dental college and hospital, bengaluru. sixty intact human maxillary premolar teeth extracted for orthodontic reasons were collected from oral and maxillo - facial department at mr ambedkar dental college and hospital. the teeth were stored, disinfected and handled as per rules given by osha and cdc. group a and b were further subdivided into subgroup a and subgroup b, of ten each. all teeth were sectioned at various levels using a carborundum disc under copious water and embedded in an acrylic block of a specific size. group i : superficial dentin 20 specimens subgroup a : superficial dentin (tetric n bond) 10 specimenssubgroup b : superficial dentin (single bond universal) 10 specimens group ii : deep dentin 20 specimens subgroup a : deep dentin (tetric n bond) 10 specimenssubgroup b : deep dentin (single bond universal) 10 specimens group iii : control group 20 specimens subgroup a : superficial dentin 10 specimenssubgroup b : deep dentin 10 specimens group i : superficial dentin 20 specimens subgroup a : superficial dentin (tetric n bond) 10 specimenssubgroup b : superficial dentin (single bond universal) 10 specimens subgroup a : superficial dentin (tetric n bond) 10 specimens subgroup b : superficial dentin (single bond universal) 10 specimens group ii : deep dentin 20 specimens subgroup a : deep dentin (tetric n bond) 10 specimenssubgroup b : deep dentin (single bond universal) 10 specimens subgroup a : deep dentin (tetric n bond) 10 specimens subgroup b : deep dentin (single bond universal) 10 specimens group iii : control group 20 specimens subgroup a : superficial dentin 10 specimenssubgroup b : deep dentin 10 specimens subgroup a : superficial dentin 10 specimens subgroup b : deep dentin 10 specimens the occlusal surfaces of teeth were ground on a water - cooled trimming wheel to produce flat dentin surfaces. all the specimens were etched the prepared dentinal flat surface with (n etch) and washed. the surface was smudged with gauze to yield a visible damp dentin surface. the total - etching adhesive (tetric n bond) was applied on the prepared dentinal flat surface left undisturbed for 20 s and the excess solvent was with a gentle stream of air. after curing the bonding agent, resin (tetric n ceram) was placed on the prepared dentinal surface using teflon mold and cured according to manufacturer s instructions. the self - etching adhesive (single bond universal) was applied on the prepared dentinal flat surface left undisturbed for 20 s and the excess solvent was removed with a gentle stream of air. after curing the bonding agent, nanocomposite resin was placed on the prepared dentin using teflon mold and cured according to manufacturer s instructions. the same procedure was carried out on the 10 specimens in this group. the same procedure as carried out in the group i, subgroup a is carried out on all specimens in this group. the same procedure as carried out in the group a, subgroup b is carried out on all specimens in this group. no bonding agent was applied. specimens were then stored at room temperature for 48 h. the specimens were then thermocycled for 500 cycles between 50c and 55c water bath. all the 60 specimens were transferred to the instron testing machine and subjected to shear bond strength test. the statistical data derived from the four subgroups were analyzed using anova test, bonferroni test, and paired t - test. all the specimens were etched the prepared dentinal flat surface with (n etch) and washed. the surface was smudged with gauze to yield a visible damp dentin surface. the total - etching adhesive (tetric n bond) was applied on the prepared dentinal flat surface left undisturbed for 20 s and the excess solvent was with a gentle stream of air. light curing was done for 40 s with a visible light curing unit. after curing the bonding agent, resin (tetric n ceram) was placed on the prepared dentinal surface using teflon mold and cured according to manufacturer s instructions. the self - etching adhesive (single bond universal) was applied on the prepared dentinal flat surface left undisturbed for 20 s and the excess solvent was removed with a gentle stream of air. after curing the bonding agent, nanocomposite resin was placed on the prepared dentin using teflon mold and cured according to manufacturer s instructions. all the specimens were etched the prepared dentinal flat surface with (n etch) and washed. the surface was smudged with gauze to yield a visible damp dentin surface. the total - etching adhesive (tetric n bond) was applied on the prepared dentinal flat surface left undisturbed for 20 s and the excess solvent was with a gentle stream of air. light curing was done for 40 s with a visible light curing unit. after curing the bonding agent, resin (tetric n ceram) was placed on the prepared dentinal surface using teflon mold and cured according to manufacturer s instructions. the self - etching adhesive (single bond universal) was applied on the prepared dentinal flat surface left undisturbed for 20 s and the excess solvent was removed with a gentle stream of air. light curing was for 40 s with a visible light curing unit. after curing the bonding agent, nanocomposite resin was placed on the prepared dentin using teflon mold and cured according to manufacturer s instructions. the same procedure as carried out in the group i, subgroup a is carried out on all specimens in this group. the same procedure as carried out in the group a the same procedure as carried out in the group i, subgroup a is carried out on all specimens in this group. the same procedure as carried out in the group a, subgroup b is carried out on all specimens in this group. specimens were then stored at room temperature for 48 h. the specimens were then thermocycled for 500 cycles between 50c and 55c water bath. all the 60 specimens were transferred to the instron testing machine and subjected to shear bond strength test. the statistical data derived from the four subgroups were analyzed using anova test, bonferroni test, and paired t - test. for superficial dentin - higher mean shear bond strength was recorded in fifth generation bonding system followed by seventh generation bonding system and control respectively as seen in graph 1. the difference in mean shear bond strength between the groups was not statistically significant (p > 0.05). deep dentin : higher mean shear bond strength was recorded in fifth generation bonding agent followed by seventh generation bonding agent and control respectively as seen in graph 2. the difference in mean shear bond strength between the groups was found to be statistically significant (p 0.05) as seen in graph 3. mean shear bond strength using fifth generation bonding agent at superficial dentin and deep dentin. the difference in bond strength using seventh generation bonding agent in superficial and deep dentin was statically significant (p 0.05). at deep dentin level, higher mean shear bond strength was recorded in fifth generation bonding system followed by seventh generation bonding system and control group, respectively. the difference in mean shear bond strength between the groups was found to be statistically significant (p < 0.001). at deep dentin level, statistically significant results were obtained with the fifth generation (tetric n bond) bonding system which had higher mean shear bond strength values compared to the seventh generation self - etch bonding system (single bond universal). there was a statistically significant difference in shear bond strength values with fifth generation bonding system and control group (without bonding system) at deep dentin. there was a significant fall in bond strength values as one reaches deeper levels from the superficial dentin to deep dentin.	background : the purpose of this study was to compare the shear bond strength of nanocomposite resin to superficial dentin and deep dentin using two different dentin bonding systems.materials and methods : all teeth were sectioned at various levels (superficial dentin : dentin within 0.5 - 1 mm of dentinoenamel junction ; deep dentin : dentin within 0.5 mm of the highest pulp horn) using a carborundum disc and embedded in acrylic block of specific size. selected specimens (60 premolar teeth) were grouped randomly into three groups, the groups were differentiated into superficial dentin, deep dentin, and control group which were further divided into sub group a and subgroup b containing 10 teeth each, depending on the bonding agents used. in subgroup a, tetric n bond, and in subgroup b single bond universal were used. in the control group no bonding agent was used. the specimens were thermocycled for 500 cycles between 5c and 55c water bath for 40 s. finally, the specimens were subjected to shear bond strength study under instron machine (universal testing machine). the maximum shear bond strengths were noted at the time of fracture (de - bonding) of the restorative material. results were analyzed using anova test, bonferroni test, and paired t-test.results:bond strength values of fifth generation bonding system (tetric n bond) showed higher mean shear bond strength compared to seventh generation bonding system (single bond universal). there was a significant fall in bond strength values as one reaches deeper levels of dentin from superficial to deep dentin.conclusion:there was a significant difference between the bond strength of fifth generation bonding system (tetric n bond) and seventh generation bonding system (single bond universal). decrease in the bond strength values is seen for the deeper level of dentin as compared to superficial dentin.
subdural hemorrhage (sdh) occurs from tearing of the bridging and cortical veins, often in the elderly after mild trauma. chronic sdh usually occurs after 3 weeks to 4 months after head trauma, but sdh developed 9 months after the head trauma has been reported5). sdh can manifest with various neurologic symptoms such as altered mental status, focal neurologic deficit, headache, and seizures. common neurological deficits include hemiparesis, bilateral oculomotor nerve palsies, nystagmus, ataxia, and aphasia4). however, the evaluation of sdh in hand weakness has rarely been reported. he did sustain an injury on his left shoulder without direct head trauma 2 months prior to the visit. a neurologic examination showed motor weakness in the left abductor digiti quinti muscle and first dorsal interosseous muscles. there were no sensory deficits to light touch, pain, position, and vibration sense. a nerve conduction study and needle electromyography was done in a local physical medicine and rehabilitation clinic. the left ulnar sensory responses were of low amplitude and increased proportion of polyphasic motor unit potentials were observed in the left biceps brachii, brachioradialis, anconeus, triceps, flexor digiti indicis, abductor digiti quinti, and left c5, 6, and 8 paravertebral muscles, suggestive of left c5, 6, and 8 radiculopathy. a brain mri was done to the patient due to his old age and a history of hypertension and slipping down. sdh was found in the right frontal and parietal convexity and along the falx (fig. a 83-year - old female visited with a tingling sensation in her right hand and weakness for 3 days. she had hypertension and cervical multiple spondylosis and had fallen backwards, hitting the occiput with no loss of consciousness, 3 days before the symptom started. there were no sensory deficits to light touch, pain, position, and vibration sense. two days later, the patient revisited the hospital with aggravated hand weakness and newly developed motor aphasia. he did sustain an injury on his left shoulder without direct head trauma 2 months prior to the visit. a neurologic examination showed motor weakness in the left abductor digiti quinti muscle and first dorsal interosseous muscles. there were no sensory deficits to light touch, pain, position, and vibration sense. a nerve conduction study and needle electromyography was done in a local physical medicine and rehabilitation clinic. the left ulnar sensory responses were of low amplitude and increased proportion of polyphasic motor unit potentials were observed in the left biceps brachii, brachioradialis, anconeus, triceps, flexor digiti indicis, abductor digiti quinti, and left c5, 6, and 8 paravertebral muscles, suggestive of left c5, 6, and 8 radiculopathy. a brain mri was done to the patient due to his old age and a history of hypertension and slipping down. sdh was found in the right frontal and parietal convexity and along the falx (fig. a 83-year - old female visited with a tingling sensation in her right hand and weakness for 3 days. she had hypertension and cervical multiple spondylosis and had fallen backwards, hitting the occiput with no loss of consciousness, 3 days before the symptom started. there were no sensory deficits to light touch, pain, position, and vibration sense. two days later, the patient revisited the hospital with aggravated hand weakness and newly developed motor aphasia. in the first patient, the motor deficit was limited to the left abductor quinti and first dorsal interosseous muscles, both innervated by the ulnar nerve. despite the absence of sensory change, the nerve conduction study revealed low amplitude sensory nerve action potential suggestive of left ulnar neuropathy. also, in addition to the shoulder trauma history, the needle electromyography showed abnormalities in various other muscles, suggestive of left brachial plexopathy. however, neither of these diagnoses was compatible with the focal hand weakness without any sensory change. in the second patient, the history of multiple cervical spondylosis and hand weakness with mild hypoactive deep tendon reflex were suggestive findings of cervical radiculopathy. however, the diagnosis of cervical radiculopathy with no sensory symptoms was insufficient. however, since the patient was initially discharged against the advice of the hospital, we did not have time for further studies. as mentioned previously, when a possible diagnosis does not exactly fit the neurologic examination in elderly patients with the recent history of trauma, subdural hemorrhage should always be suspected. there have been previous reports on interhemispheric subdural hematoma presented with one leg paresis or a hemiparesis worse in the leg than the arm, also known as ' falx syndrome'3). the cases with only hand weakness mimicking peripheral nerve lesion were reported in ischemic stroke. castaldo.2) reported a series of 35 stroke patients with isolated distal arm or hand weakness with small cortical infarction involving precentral and/or postcentral gyrus and often the parietal lobe. timsit.6) reported of six patients showing evolving isolated hand palsy with infarction of the angular gyrus of the inferior parietal lobe. in our cases, although the parietal lobe is known as sensory cortex, motor fibers and subcortical association fibers are known to be interconnected in the parietal lobe. thus, the cause of weakness in our cases may be due to the compression of the contralateral parietal lobe. since subdural hemorrhage can present with only hand weakness and mimic peripheral neuropathy, we suggest that the clinicians be more careful in recording patients ' history and recommend a ct or mr scan in the elderly patients.	subdural hemorrhage (sdh) can manifest various neurologic symptoms. however, sdh presenting with only hand weakness has rarely been reported. we report two sdh cases with only hand weakness mimicking peripheral neuropathy. since sdh can present with hand weakness only, we suggest the clinicians to do a careful history taking and recommend a ct scan in the elderly patients.
syphilis is a sexually transmitted infection (sti) caused by the bacterium treponema pallidum (t. pallidum). the inability to culture the causative organism in vitro and the limited availability of nucleic acid amplification techniques makes the diagnosis of this infection difficult. moreover, direct visualization of the organism does not seem to be feasible since it mandates the presence of lesions and of facilities with either dark field or fluorescent microscopy. serodiagnosis of syphilis relies on detection of two types of antibodies - antibodies against the cardiolipin antigen, and the treponema - specific antibodies. a major diagnostic limitation encountered with the use of anticardiolipin antibody - based tests (nontreponemal tests) is the occurrence of biological false positive (bfp) reactions. it is, therefore, recommended to use nontreponemal tests such as venereal disease research laboratory (vdrl) and rapid plasma reagin (rpr) test as screening assays followed by confirmation of the nontreponemal reactivity by the more specific treponemal tests like t. pallidum hemagglutination assay (tpha) and fluorescent treponemal antibody absorption test. moreover, the tests lack sensitivity in the late latent stage of infection. a major drawback of the laboratory procedures currently in use for syphilis serodiagnosis is that they require laboratory facilities (refrigeration, water bath, centrifuge, rotator, etc.) ; stringent quality control measures and skilled persons to perform the tests, as well as trained health professionals to read and interpret the results. in resource constraint settings, laboratory infrastructure and facilities for syphilis diagnosis might not be widely available, and the delay encountered in getting the samples tested from referral laboratories may preclude timely initiation of treatment. the current situation mandates the need for rapid and reliable tests to serve as screening and confirming assays in all stages of syphilis. rapid serological procedures offer a potential option with assured rapid availability of results usually in < 15 min and ease of use by health professionals allowing on - site testing. the world health organization sexually transmitted diseases diagnostic initiative has laid down the assured criteria that define the ideal characteristics of a rapid and point - of - care test : affordable, sensitive, specific, user - friendly, rapid and robust, equipment free, and deliverable to those who need them. several rapid, point - of - care assays based on recombinant t. pallidum antigens are now commercially available. despite the benefits that the rapid tests offer over traditional laboratory methods for syphilis serodiagnosis, their diagnostic performance remains a matter of concern and is still not widely documented. in this study, the authors have evaluated the performance of sd bioline syphilis 3.0 (sd biostandard diagnostics private limited, gurgaon, haryana, india), a rapid immunochromatographic assay that qualitatively detects antibodies against t. pallidum. to the best of our knowledge, there is no published peer - reviewed article in medical literature regarding the evaluation of this test kit in an indian scenario. this prospective study was conducted at the department of microbiology of a tertiary care health facility in new delhi. the serology laboratory of the department receives on an average 15,000 serum samples annually for syphilis serology from both the inpatient and outpatient departments of the linked hospitals. consecutive blood samples received at the serology laboratory from october 2013 to february 2014 with request for syphilis screening were included in the analysis. on arrival in the laboratory, the samples were allowed to coagulate and centrifuged at 2500 rpm (rotations per minute) for 10 min. the sera were separated and stored at 28c till vdrl (employing antigen from serologist to government of india, kolkata) was put up (generally every 2 or 3 day). after being tested as per the above protocol, the sera were aliquoted and stored in eppendorfs at 20c until a panel of 100 sera comprising of 50 vdrl reactive and 50 nonreactive samples were reached. a different technician who did not have access to results of the screening assay, performed the immutrep tpha (omega diagnostics limited, scotland, united kingdom) as per the manufacturer 's instructions in the kit insert. the third laboratory technician, who was again blinded to the vdrl and immutrep tpha results performed sd bioline syphilis 3.0 in accordance with the manufacturer 's guidelines. the procedural details of the tests employed in this study along with their interpretative criteria are provided in table 1 while their characteristics are summarized in table 2. methodology and interpretation of tests used for serological diagnosis of syphilis characteristics of tests used for serological diagnosis of syphilis the results of vdrl test, immutrep tpha, and sd bioline syphilis 3.0 were entered in microsoft excel sheet and any discordant results by immutrep tpha and sd bioline syphilis 3.0 were tested again by both the procedures before they were finally recorded as positive or negative. the sensitivity and specificity calculations and estimation of negative and positive predictive values of sd bioline syphilis 3.0 were done by comparing its performance with immutrep tpha (reference technique). these values were calculated with their respective confidence interval of 95% (95% ci). sensitivity was calculated as true positives/(true positives + false negatives) 100 ; specificity as true negatives/(true negatives + false positives) 100 ; negative predictive value as true negatives/(true negatives + false negatives) 100, and positive predictive value as true positives/(true positives + false positives) 100. of the 50 vdrl reactive samples, 42 (84%) were tpha positive, and 40 (80%) were reactive with sd bioline syphilis 3.0. considering tpha as the gold standard, the 8 vdrl reactive and tpha negative samples can be considered as bfps. all but one of these 8 bfp samples was correctly identified as negative by sd bioline syphilis 3.0. in addition, of the 42 true positives picked up by immutrep tpha, 3 were wrongly identified as negative by sd bioline syphilis 3.0 and remained so on repeat testing. two of the samples with false - negative rapid test results had a vdrl titre of 1:2, and one sample had a vdrl titre of 1:16. all the vdrl nonreactive sera (n = 50) were negative by both immutrep tpha and the rapid test. the performance of sd bioline syphilis 3.0 and vdrl as against immutrep tpha is presented in tables 3 and 4, respectively. performance of sd bioline syphilis 3.0 in comparison with immutrep tpha (reference standard) and vdrl performance of vdrl in comparison with immutrep tpha (reference standard) compared to immutrep tpha as the gold standard, the sensitivity, specificity, and positive and negative predictive values of sd bioline syphilis 3.0 were 92.86% (95% ci : 80.5298.50%), 98.28% (90.7699.96%), 97.50% (86.8499.94%), and 95.00% (86.0898.96%), respectively. the kappa value was 0.917, showing a very good strength of agreement between the two tests. the performance characteristics of the currently employed vdrl test in comparison to the gold standard (immutrep tpha) are as follows : sensitivity = 100.00% (91.59100.00%), specificity = 86.21% (74.6293.85%), positive predictive value = 84.00% (70.8992.83%), and negative predictive value = 100.00% (92.89100.00%). thus, while the sensitivity of sd bioline syphilis 3.0 was lower than that of vdrl (92.86% vs. 100.00%), the specificity was much higher (98.28% vs. 86.21%). we report sensitivity and specificity values of 92.86% and 98.28%, respectively, for sd bioline syphilis 3.0, compared to tpha as the reference standard. a study conducted in tanzania found the test to be 79% sensitive and 96% specific. in another study conducted in china, the reported sensitivity and specificity values were 95.5% and 97.9%, respectively. thus, keeping in view the high sensitivity and specificity of sd bioline syphilis 3.0, we conclude that the test can be used both as a screening assay and as an alternative to tpha for detection of antibodies to t. pallidum. we found sd bioline syphilis 3.0 to be less sensitive but more specific than vdrl. lower sensitivity of immunochromatographic tests has also been reported in previous studies, and the reason cited has been primarily the low levels of antibodies found in sera of patients with early primary syphilis. the discrepant results between immutrep tpha and sd bioline syphilis 3.0 (immutrep tpha positive and sd bioline syphilis 3.0 negative sera) can be explained by the fact that a large number of treponemal antigens are exposed during the tpha test. therefore, some cross reaction with other organisms may occur. moreover, as has been hypothesized by some researchers, though the recombinant antigens employed in the rapid tests are specific to t. pallidum, posttranslational processing of these protein in vivo or genetically determined unresponsiveness or restricted responsiveness to these antigens could lead to lack of reactivity even in the presence of disease. sd bioline syphilis 3.0 is a rapid, one - step test that is easy to execute, read, and interpret. apart from serum samples, the test is also compatible with whole blood and plasma. therefore, neither does the test require any preprocessing of specimens nor does it require any laboratory infrastructure or trained technical personnel for performing the test procedure. this makes the test an ideal on - site screening option in resource - constraint health - care settings without laboratory facilities. in addition, since the test detects treponema - specific antibodies, bfps do not occur and the test can also be used as an alternative to tpha for confirming the reactivity in nontreponemal assays. the rapid availability of results with the use of this test would also ensure prompt treatment of reactive patients in their very first visit to the health care services.. an important constraint to its application as a screening tool is that the treponema - specific antibodies detected by the test are retained for years and thus it can not distinguish past treated infections from recent or active ones. this implies that a large number of previously treated infections would also be picked up by sd bioline syphilis 3.0, particularly in high - prevalence settings, thereby leading to unnecessary treatment of patients. the test can also give a positive result in various nonvenereal treponematoses such as yaws and pinta. however, some researchers argue that false positive results are preferable to false negative ones. while with a false negative result, a syphilitic patient may go untreated and transmit the infection to others ; a false positive serology would at least trigger repeat testing with alternative methods before a definitive diagnosis is made. in this context, we also suggest adopting a reverse algorithm, whereby a nontreponemal assay such as vdrl or rpr could be used to document active disease in patients with a reactive point - of - care treponemal test. novel dual rapid point - of - care tests have also been devised where both treponemal and nontreponemal tests are combined into one device and can thus simultaneously serve as screening, as well as confirmatory assays. however these tests are still under evaluation and not available for routine use. our study has a great relevance from public health perspective and based on our findings we recommend the use of rapid point - of - care procedures such as sd bioline syphilis 3.0 as primary screening assays for serodiagnosis of syphilis where laboratory facilities are not available and as tests to confirm nontreponemal reactivity where tpha can not be done. the study was conducted at a regional sti reference laboratory located at a tertiary care center in north india. the laboratory screens a large number of samples from in - patient and out - patient sections of various departments of the linked hospitals and thus caters to a diverse population base. first, the number of sera in the panel used for kit evaluation was small. in addition, ours was a laboratory - based study and the evaluation was not done under field or clinical conditions for which the test is primarily intended. moreover, the test kit was exclusively evaluated using serum samples and its performance characteristics with whole blood and plasma specimens were not determined. also, we did not have clinical information of the cases from which the reactive sera were obtained and, therefore, performance of the test according to the stage of syphilis could not be assessed. to summarize, we strongly recommend the use of sd bioline syphilis 3.0 for rapid on - site diagnosis of syphilis. further field studies are required before the test can be routinely implemented as a screening assay.	background : serology is considered the mainstay of syphilis diagnosis. the limitations of the traditional serological methods and the advent and availability of novel immunochromatographic assays have led to the widespread application of rapid point - of - care procedures as screening tools for syphilis. however, these tests have not been extensively evaluated. this study was designed to evaluate the performance of a rapid syphilis diagnostic test known as sd bioline syphilis 3.0 (sd biostandard diagnostics private limited, gurgaon, haryana, india).materials and methods : a panel comprising of 50 venereal disease research laboratory reactive and 50 nonreactive sera was tested using sd bioline syphilis 3.0. the performance of the test was evaluated using immutrep treponema pallidum hemagglutination assay (tpha) (omega diagnostics limited, scotland, united kingdom) as the reference standard and sensitivity, specificity, and negative and positive predictive values were calculated.results:the sensitivity, specificity, and positive and negative predictive values of sd bioline syphilis 3.0 were 92.86% (confidence interval of 95% : 80.5298.50%), 98.28% (90.76 - 99.96%), 97.50% (86.8499.94%), and 95.00% (86.0898.96%), respectively, compared to tpha as the gold standard.conclusion:keeping in view the high sensitivity and specificity of sd bioline syphilis 3.0, we conclude that the test can be used as a tool for rapid on - site diagnosis of syphilis and as an alternative to tpha for detection of antibodies to treponema pallidum.
since the first studies on the prognosis of root canal treatments, the quality of the filling material was considered essential to their success. the presence of voids in the filling mass was associated with bacterial proliferation and the development of periapical lesions. among the most important factors influencing the quality of the treatment these materials should be biocompatible and they must have the adequate physicochemical and antimicrobial properties. in this context, it is important to study the properties of root canal filling materials in order to determine the optimum parameters for the development of new ones and to evaluate those that are already on the market. among other physicochemical properties, the ideal root canal sealer should present sufficient radiopacity to be distinguished from adjacent anatomical structures, such as dental tissue or jaw bone. higginbotham was the first researcher who published a comparative study on the radiopacity of different root canal fillings and gutta - percha cones used in root canal treatment. eliasson and haasken have established a comparison standard for radiopacity studies using optical density values for impression materials and calculating the equivalent thickness of aluminum required to result in similar radiographic density. beyer - olsen and orstavik introduced a comparison standard using an aluminum step wedge with 2-mm increments in thickness to evaluate the radiopacity of root canal sealers. in 1983, the american national institute for standards and technology (nist) and the american dental association (ada) issued a series of rules and tests - called specification no. 57 - to assess the physicochemical properties of root canal sealers in an attempt to standardize their testing and promote quality in dental materials research. this specification was revised in 2000 and includes the following tests : film thickness, setting time, flow, radiopacity, solubility and dimensional change following setting. due to the diversity of composition of the available sealers and considering the ansi / ada standards, the purpose of this study was to evaluate the radiopacity of two bioceramic - based root canal sealers in this study we evaluated the radiopacity of two bioceramic root canal sealers, one that is already on the market - total fill bc sealer (fkg dentaire switzerland) and a new experimental filling material developed in collaboration with raluca ripan institute for research in chemistry, cluj - napoca (table i). five disc - shaped samples were prepared from each material (10 mm diameter 1 mm thickness) according to manufacturers instructions. pure aluminum step wedges with a thickness ranging from 1 to 12 mm were used as standards. the purity of the graded standard used in this study, as measured by optical spectroscopy, was 99.52% al, 0.22% fe and 0.001% cu, in accordance with the recommendations in the literature. the discs were placed together with standard aluminum on an in vitro intraoral sensor (figure 1). digital radiographic images (figure 2) were obtained using an xios plus (sirona) intraoral sensor system and a soredex (minray) x - ray unit at 70 kv, 7 ma, 0.32 seconds exposure time at a focal length of 30 cm. radiopacity was determined by digitally processing the radiographed discs using a computer application (figure 3). four areas have been selected for each sample, corresponding to a different quadrant (quadrants i, ii, iii, iv) of the prepared disc. the area of choice involved an area where no air bubbles are present and the homogeneity - determined using the same applications - meets the standard (minimum 30% homogeneity) (figure 4). statistical interpretation of the results was performed using one - way anova, and the significance level was set at p0.05. table ii shows the radiopacity of the two materials in the four quadrants (i, ii, iii, iv) of the disc to be sealed. radiopacity is expressed in terms of equivalent aluminum thicknesses (in millimeters), higher values accounting for higher radiopacity. the average radiopacity of total fill bc sealer is 40.15, and that of the experimental material is 3.770.27. although there were differences between the values determined for the two materials, they were not statistically significant (p>0.05). the tightness of root fillings is imperative to maintain the sterile environment obtained from the mechanical and antiseptic treatment of the root canal. radiopacity is a desirable quality of root canal sealers as it allows the estimation of the length, width and the shortcomings that may occur during treatment. using a material with a very high radiopacity may give the false impression of compact filling, despite the presence of voids in its mass. conversely, a low radiopacity of the material can be interpreted as its absence in areas where it is found in a very small amount. conventional radiography and optical densitometry have been usually used in the literature to evaluate the radiopacity of filling materials. the indirect method has also been used in some studies, converting existing radiographs into digital images. meanwhile, the use of digital radiographs has become more popular, as they save time and increase image quality. in this study, digital radiographic images were evaluated using computer software for analyzing and comparing the images obtained. by using digital radiography, it was not necessary to convert conventional film radiographic images or to perform image calibration, since both samples and standards were positioned simultaneously on the intraoral sensor. iso standards do not mention the use of the indirect or direct method for assessing the radiopacity of dental materials, so this should be taken into account in the future by the international organization for standardization. there can also be differences in aluminum step wedge alloy, shutter speed, focal length, kvp, mas, all of which influence radiopacity measurements. according to international standards, the radiopacity of root canal sealing materials should be 3 mm equivalent aluminium thickness, although a few filling materials on the market do not comply with this requirement. some authors have suggested that a value of at least 4 mm equivalent aluminum thickness would be more appropriate. based on the results obtained in this study, total fill presented a radiopacity close in value to that of the experimental material and similar to another bioceramic sealer - endosequence bc which, according to a study by candeiro., showed a radiopacity of 3.84 mm equivalent aluminium thickness. considering that the experimental material studied in this paper is a resin - based material, the incorporation of radiopaque agents can easily modify radiopacity, but an increased radiopacity might hide filling mass imperfections, especially when the sealer is used in combination with gutta - percha. aoyagi. have reported that radiopacity increased with the increase in opaque agent content, as well as with the increase in the atomic number of the element. chang - kyu kim. have assumed that the increase in radiopacity of a root canal filling material entails the increase in cytotoxicity, but they havent detected any correlation between these two factors. this means that the radiopaque agent is not the only one that is cytotoxic, but other components of the sealers also contribute to its cytotoxicity. the radiopacity of the materials in this study varied, but the differences were not statistically validated. all values determined were higher than 3 mm aluminium thickness and therefore they have all complied with iso standards.	background and aimsthe aim of this study was to assess the radiopacity of two bioceramic - based root canal sealers, the conventional totalfill bc sealer (fkg dentaire switzerland) and a new experimental filling material developed in collaboration with raluca ripan institute for research in chemistry, cluj-napoca.methodsfive disc samples were prepared using both materials (10 mm diameter 1 mm thickness), being subjected to digital radiography together with aluminum step wedges (1 to 12 mm in thickness), in accordance with iso 6876 : 2012. radiopacity was determined by the computer analysis of the images obtained. four different areas were selected for each sample, corresponding to a disk - sample quadrant. statistical analysis was performed using anova.resultsboth materials showed a radiopacity that was 3 mm greater than the equivalent thickness of aluminum. total fill bc showed greater radiopacity than the experimental material, but the differences were not statistically significant.conclusionsboth materials comply with iso 6876 : 2012 recommendations on minimum radiopacity.
both primary and secondary types of tuberculosis can cause lesions in the oral cavity. in secondary tuberculosis, lesions of the oral cavity may accompany lesions in the pharynx, lungs, lymph nodes, or skin. when oral lesions of tuberculosis are the sole manifestations of the disease, the clinician may face a diagnostic challenge. during the past decades, primary bovine tuberculosis of the oral cavity became extremely rare and occurs mainly through consumption of raw milk from infected cattle in developing countries. the purpose of this report is to present an unusual case of primary oral tuberculosis due to mycobacterium bovis in a 6-year - old boy residing in an urban area. a 6-year - old, previously healthy boy, presented to the pediatric department because of a strongly positive tuberculin skin test (34 mm) and mild chronic swelling with a skin fistula in the left submandibular area, which developed 3 months prior to admission. he was initially treated with oral and then intravenous broad spectrum antibiotics for 3 weeks with the presumptive diagnosis of cervical adenitis with no response at another hospital. after evaluation by an oral and maxillofacial surgeon, the diagnosis of a submandibular abscess originating from the decayed primary second lower left molar was made (fig. the patient underwent incision and drainage of the abscess extraorally and extraction of the involved tooth. the following 2 months, he had persistent moderate swelling, and a fistula developed at the incision site. fig. 1radiological imaging. a panoramic x - ray showing the decayed primary second lower left molar and (b) computed tomography showing chronic osteomyelitis with osteoblastic subperiosteal activity and thickening around the lower mandibular border radiological imaging. a panoramic x - ray showing the decayed primary second lower left molar and (b) computed tomography showing chronic osteomyelitis with osteoblastic subperiosteal activity and thickening around the lower mandibular border the patient had no history of contact with a case of active tuberculosis, and investigation of family contacts was negative. he lived in an urban area and had no contact with cattle or history of consumption of unpasteurized dairy products. both grandparents and parents were born in the former soviet union and had immigrated to greece 20 years ago. a left submandibular firm, erythematous swelling measuring 3 3 cm, was palpated with a draining sinus tract. blood tests revealed a white blood cell count of 15,200/mm with 74% polymorphonuclears, 18% lymphocytes, and 8% monocytes ; erythrocyte sedimentation rate was 50 mm / h, and c - reactive protein was 77 measurement of serum immunoglobulins, determination of lymphocyte subpopulations by flow cytometry, and nitroblue tetrazolium test were performed and were normal. serologic testing ruled out human immunodeficiency virus (hiv) 1 and 2 (enzyme - linked immunosorbent assay method), toxoplasmosis, cytomegalovirus, epstein furthermore, chest radiography and abdominal ultrasound were normal, while culture of gastric aspirate was negative. quantiferon - tb gold in - tube (qft - it) test was positive. a computed tomography of the region revealed the presence of chronic osteomyelitis of the mandibular body adjacent to the extraction site with osteoblastic subperiosteal activity and thickening around the lower mandibular border (fig. antituberculous treatment was initiated with isoniazid, rifampicin, and pyrazinamide (pza), and the patient underwent surgical excision of the submandibular mass and the draining sinus. the histopathological examination revealed no lymphoid but only fatty and fibrous tissue with granulomatous caseating infection. bovis by means of the genotype mycobacterium tuberculosis complex (mtbc) assay (hain lifescience, germany). this assay is based on the dna - strip technology and permits the genetic differentiation of species belonging to the mycobacterium complex on the basis of gyrase b polymorphisms. based on these results, pza was discontinued, and ethambutol was added to the therapeutic regimen. by the end of a 6-month therapy, the lesion had healed and showed complete resolution. the global prevalence of human tb due to m. bovis has been estimated at 3.1% of all human tb cases. according to the report of the european food safety authority, greece is not officially bovine tuberculosis - free in cattle, but no cases of human tuberculosis were confirmed, to our knowledge, during the last 10 years. as stated by the above report, several human cases of bovine tb have been reported from other european countries which have been approved as being free from bovine tb in cattle like germany or austria. it is possible that human bovine tb is under - diagnosed in our settings due to difficulties in obtaining samples or differentiating the organism from m. tuberculosis in culture. people can be infected with m. bovis, by inhalation of tubercle bacilli or by consumption of infected products. pulmonary tuberculosis due to m. bovis is more common in rural dwellers or people with occupational exposure, as a result of inhalation of dust particles or bacteria - containing aerosols shed by infected animals. those who live in urban areas usually acquire the infection via the gastrointestinal route due to consumption of poorly cooked meat, unpasteurized milk, or milk products and develop extrapulmonary tuberculosis [2, 3 ]. although, human - to - human transmission of m. bovis has been reported [2, 4 ], this mode of transmission is rare and is likely to occur in populations particularly susceptible to tuberculosis such as the hiv - infected people. the intact oral mucous membrane presents a natural resistance barrier to mycobacterial invasion, and any break or loss of this barrier may provide a route of entry. it is most likely that, in the case presented, the patient had primary oral tuberculosis since there was no pulmonary involvement. it is assumed that the mycobacterium was directly inoculated into the dental socket and the mandible through the decayed primary tooth, causing infection and abscess formation with subsequent spread to the submandibular space. following surgical treatment, there was some improvement initially but since the cause of the mandibular infection was not treated, symptoms persisted, and soon, mandibular osteomyelitis was obvious radiographically. the isolation of m. bovis from an immunocompetent child residing in an urban area raises the question of how the infection was acquired. there was no history of unpasteurized dairy products consumption, but this possibility can not be ruled out since most cases with primary oral tuberculosis in the literature were due to consumption of infected milk or milk products. by analogy, history of consumption of unpasteurized dairy products is negative in about 20% of cases with brucellosis, another zoonosis. however, the likelihood of reactivation of an old bovine tuberculosis infection in family members was considered unlikely after thorough investigation. finally, transmission of m. bovis infection after dental manipulations has been reported, but our patient did not have such a history. the identification of mtbc isolates depends on biochemical testing, drug susceptibility testing, and dna techniques. bovis and must raise suspicion in the appropriate epidemiological setting. the definite diagnosis and identification was facilitated in our case by the application of a molecular genetic approach which is a useful tool for the differentiation at species / strain level of the closely related members of mtbc. this test measures interferon gamma production from a person 's white blood cells in response to stimulation with antigens specific to mtbc complex organisms including m. bovis, but absent from all m. bovis bacillus calmette gurin strains and most nontuberculous mycobacterium species. this method can detect mtbc infection but can neither differentiate between species nor between infection and active disease. in conclusion, we report a case of dental abscess due to m. bovis in an immunocompetent child who lacked epidemiological risk factors for this infection. the inclusion of tuberculosis in the differential diagnosis of unclear pulmonary and extrapulmonary disease remains important, and tuberculosis has to be managed according to international standards. although, childhood tuberculosis represents an important part of the disease burden, yet its diagnosis remains challenging. the application of molecular genetic techniques that can easily differentiate tubercle bacilli belonging to mtbc could also specify the true prevalence of m. bovis infections that are currently underestimated.	bovine tuberculosis is a zoonotic disease, and although its incidence has dramatically decreased in developed countries where effective control measures are applied, it still remains a potential health hazard in the developing world. tuberculosis of the oral cavity is extremely rare and is usually secondary to pulmonary involvement. we present the unusual case of an immunocompetent 6-year - old child residing in an urban area with primary oral tuberculosis due to mycobacterium bovis, which was confirmed by the application of a molecular genetic approach. m. bovis belongs to mycobacterium tuberculosis complex which comprises species with close genetic relationship, and for this reason, the use of new molecular techniques is a useful tool for the differentiation at species level of the closely related members of this complex.
pulmonary thromboembolism (pte) and deep vein thrombosis (dvt) are part of the spectrum of venous thromboembolism. the annual incidence of pte is 100 - 200 cases/100,000 population, 1 2 and the overall 30-day mortality rate ranges from 6.7% to 11.0%, 3 5 reaching 30.0% in the absence of treatment. 7 the underdiagnosis of pte might be due, at least in part, to the wide variability in the clinical presentation of pte and to the fact that the findings are often nonspecific. the clinical findings consistent with suspected pte are dyspnea, chest pain, hemoptysis, and tachypnea that have an acute onset. in situations that are more dramatic, in which much of the pulmonary circulation is affected or there is a history of significant cardiopulmonary disease, there can be signs of clinical instability, such as hypotension, signs of low cardiac output, and significant hypoxemia. 8 the presence of risk factors for pte in combination with a suggestive clinical picture reinforces the clinical suspicion of pte. the clinical suspicion of pte should be investigated by specific tests that vary according to the clinical status of the patient. the use of chest ct angiography (cta) has increased sharply in recent years, and this imaging test has been used as the first - line tool for suspected cases of pte in various institutions. 9 11 a safe, noninvasive imaging test that allows direct detection of the intra - arterial pulmonary thrombus, cta is rapidly performed, results generally being available within 24 h. reported rates of cta sensitivity range from 64% to 100%, whereas those of cta specificity range from 89% to 100%. 12 14 chest cta results are positive for pte in 6.6 - 60.0%, depending on the criteria used for cta referral. 9 15 21 in parallel with the recognition of the importance of chest cta in the diagnosis of pte, its excessive and sometimes unnecessary use raises concerns about cost - effectiveness issues and about adverse effects related to intravenous contrast use and high radiation rates. 22 a potential advantage of cta is identification of an alternative diagnosis when no findings of pte are detected. alternative diagnoses, such as pneumonia, cancer, pleural effusion, heart failure, copd exacerbation, etc., or incidental findings, such as benign nodules, adenopathy, and granulomatous disease scarring, have been reported in 25.4% to 70.0% of chest cta results. 9 15 21 the risks, benefits, and costs associated with the investigation of such findings need to be further elucidated, given that the investigation has therapeutic consequences in less than 5% of cases. 20 in addition, the role played by chest cta in the absence of pte in making alternative diagnoses that were missed by simple tests, such as chest x - ray, has not been fully investigated. 18 20 the objective of the present study was to investigate the contribution of chest cta findings to identifying alternative diagnoses to pte that would explain the clinical picture of the patient and that were missed on chest x - ray. this was a cross - sectional, retrospective study carried out in the department of radiology of the hospital de clnicas de porto alegre (hcpa), a general university hospital located in the city of porto alegre, brazil. the hcpa has an operational capacity of 845 beds, of which 87 are icu beds and 47 are er beds ; there are also 35 operating rooms. in 2012, the hcpa uses an online medical record system (known as the hospital management applications web) that was developed in - house and, in september of 2009, was integrated into the radiology information system and into a system for the management and storage of medical images (picture archiving and communication system). in parallel, an image and information management system (impax ; agfa healthcare, mortsel, belgium) was employed as a technological option for performing the tasks of transmission, storage, and retrieval of medical images. the study was approved by the hcpa research ethics committee. given the retrospective nature of the study, informed consent was not required. the authors signed a confidentiality agreement for the use of data. between september of 2009 and may of 2012 we included all cases involving adult patients (18 years of age) referred to the computed tomography division of the hcpa for chest cta for suspected pte. we excluded cases referred from the outpatient clinic for investigation of chronic pulmonary hypertension, as well as cases in which chest cta was ordered for reasons other than the investigation of pte (figure 1). patient demographic and clinical data, such as age, gender, race, smoking history, symptoms at the time of suspicion of pte, presence of risk factors for pte, and comorbidities, as well as the health care setting from which the patient was referred at the time of suspicion, length of hospital stay, and clinical outcome (discharge or death), were obtained from the electronic medical records of the hcpa. the medical records were reviewed for the presence of clinical pretest probability scores for pte. the geneva score, in its original version, 8 was calculated retrospectively. 8 chest cta images were obtained with a 16-detector helical ct scanner (brilliance ct ; philips healthcare, best, the netherlands). all images were viewed on a workstation, with the image storage and transmission systems mentioned above, by two independent radiologists, trained to interpret ctas. scans were considered positive for pte if they revealed filling defects within the pulmonary artery or any of its branches. for positive scans, pte was classified as central (up to the first branch of the segmental artery), peripheral (beyond the first branch of the segmental artery), or diffuse (central and peripheral). in the presence of adequate opacification of the pulmonary vascular bed, with no filling defects alternative diagnoses were reviewed on the basis of clinical and imaging findings, by two pulmonologists. findings suggestive of an alternative diagnosis were defined as cta abnormalities that could explain the symptoms (such as dyspnea, chest pain, hypoxemia, and tachycardia). the following criteria were used to establish the alternative diagnoses : 1) pneumonia - presence of cough, expectoration, and systemic findings (tachycardia, leukocytosis or leukopenia, and fever) associated with new pulmonary infiltrate ; 2) decompensated heart failure - a clinical diagnosis of heart failure and radiological signs of heart enlargement and pulmonary edema or pleural effusion ; 3) pleural effusion due to other causes - signs of at least moderate pleural effusion that was not associated with heart failure ; 4) lymphangitic carcinomatosis or progression of cancer - a histological diagnosis of lung cancer with radiological signs of interstitial involvement (with or without pleural effusion or enlarged mediastinal lymph nodes) or an increase in the lung tumor relative to the previous scan ; 5) noncardiogenic pulmonary edema - radiological signs of pulmonary edema, including interstitial infiltrate, confluent consolidation, ground - glass attenuation associated with septal thickening, and/or unilateral or bilateral pleural effusion, in the absence of heart failure and in the presence of one or more conditions that explained the edema ; 6) connective tissue disease - related lung disease - a previous diagnosis of connective tissue disease (rheumatoid arthritis, systemic lupus erythematosus, or systemic sclerosis) with pleural and pulmonary involvement attributed to the connective tissue disease itself, including interstitial pulmonary infiltrate, consolidation, pulmonary nodules, pleural effusion, and pulmonary hypertension ; and 7) atelectasis - signs of atelectasis (opacity and reduced lung volume) of at least one lung lobe, with significant ventilatory impairment. in addition, we reviewed the respective chest x - rays, together with the chest x - ray reports and the related notes contained in the medical records, and we determined whether the same alterations had been previously detected on chest x - rays. taking into account the prevalence of alternative diagnoses made on the basis of chest cta findings in previous studies 9 15 21 and the high prevalence of tuberculosis in brazil, we calculated that, in order to obtain an expected proportion of alternative diagnoses of 60% and achieve a power of 80%, at a level of significance of 5% (two - tailed test), the sample size would have to be 188 patients. data were entered into a microsoft excel spreadsheet and were analyzed with the statistical package for the social sciences, version 18.0 (spss inc., quantitative data were presented as mean and standard deviation or as median and interquartile range. the prevalence of pte, abnormal cta findings, and alternative diagnoses was expressed as absolute numbers or as absolute numbers and proportions. the groups of patients with and without pte were compared with the independent sample t - test or the mann - whitney test, depending on data distribution. the mean age was 59.3 17.1 years, and women predominated. of those, 113 patients (59.2%) were referred from the emergency room at the time of suspicion, whereas 71 (37.2%) were referred from the ward and 7 (3.7%) were referred from the icu. characteristic result age, years59.3 17.1gender male128 (67.0)female63 (33.0)white race169 (88.5)smoking history89 (46.6)smoking history, pack - years 59.3 17.1health care setting where the suspicion was raised emergency room113 (59.2)ward71 (37.2)icu7 (3.7) values expressed as n (%) or mean sd. n = 78 values expressed as n (%) or mean sd. n = 78 the demographics, symptoms, risk factors, comorbidities, length of hospital stay, and survival of the 191 patients are shown in table 2, stratified by the presence (n = 47) or absence (n = 144) of pte. the most common clinical complaints leading to referral for cta in the 191 patients were sudden dyspnea, in 75.4% ; chest pain, in 33.0% ; and cough, in 25.1%. less common complaints were anxiety (in 9.4%), syncope (in 6.3%), and hemoptysis (in 3.1%), there being no difference between the groups with and without pte (6.4% vs. 2.1% ; p > 0.05). the most common comorbidities were systemic arterial hypertension, diabetes, copd, and previous stroke. there were no significant differences in demographic variables, symptoms, or risk factors between the patients with and without pte (p > 0.05). there was a trend toward a higher prevalence of pte in patients in whom suspicion was raised in the ward than in those in whom suspicion was raised in the emergency room (31.0% vs. 21.6% ; p = 0.09). patients with pte had longer hospital stays than did those without (median : 18 days vs. 9.5 days ; p = 0.0001). among the 191 patients, mortality was 13.6%, being 12.8% and 13.9% in the groups with and without pte, respectively (p > 0.05). table 2.comparison of characteristics between patients with positive and those with negative ct angiography results for pulmonary thromboembolism. characteristic all patients patients with positive cta results for pte patients with negative cta results for pte (n = 191) (n = 47) (n = 144) age, years 59.3 17.160.3 17.258.9 17.2female gender128 (67.0)28 (59.6)100 (69.4)smoking89 (46.6)18 (38.3)71 (49.3)symptoms sudden dyspnea144(75.4)36 (76.6)108 (75)chest pain63 (33)17 (36.2)46 (31.9)cough48 (25.1)11 (23.4)37 (25.6)expectoration27 (14.1)7 (14.9)20 (13.9)anxiety18 (9.4)3 (6.4)15 (10.4)syncope12 (6.3)5 (10.6)7 (4.9)hemoptysis6 (3.1)3 (6.4)3 (2.1)risk factors cancer49 (25.7)9 (19.1)40 (27.8)hospital admission in the last 3 months48 (25.1)10 (21.3)38 (26.4)being bedridden for more than 3 days31 (16.2) 6 (12.8)25 (17.4)obesity31 (16.2)9 (19.1)22 (15.3)decompensated heart failure28 (14.7)5 (10.6)23 (16.0)copd exacerbation16 (8.4)3 (6.4)13 (9.0)recent surgery25 (13.1)9 (19.1)16 (11.1)previous dvt13 (6.8)4 (8.5)9 (6.3)previous pte11 (5.8)2 (4.3)9 (6.3)intravenous catheter9 (4.7)1 (2.1)8 (5.6)paralysis of the lower limbs4 (2.1)2 (4.3)2 (1.4)fracture4 (2.1)1 (2.1)3 (2.1)comorbidities systemic arterial hypertension89 (46.6)24 (51.1)65 (45.1)diabetes mellitus39 (20.1)12 (25.5)27 (18.8)copd35 (18.3)6 (12.8)29 (20.1)stroke28 (14.7)9 (19.1)19 (13.2)ischemic heart disease19 (9.9)4 (8.5)15 (10.4)thyroid disease17 (8.9)4 (8.5)13 (9.0)renal failure14 (7.3)3 (6.4)11 (7.6)diffuse connective tissue disease13 (6.8)2 (4.3)11 (7.6)asthma9 (4.7)0 (0.0)9 (6.3)outcomes length of hospital stay, days 11.0 (4 - 22)18.0 (8 - 35)9.5 (3 - 19)death26 (13.6)6 (12.8)20 (13.9)cta : ct angiography ; pte : pulmonary thromboembolism ; and dvt : deep vein thrombosis. an objective, systematic evaluation of pretest probability of pte, using a clinical score, was found in 14 of the 191 medical records (7.3%). the geneva score, calculated retrospectively, was 5.9 3.2 points in the patients with pte and 5.0 2.6 in the patients without pte (p = 0.06). in a three - level stratification of clinical risk probability, 26.2% of the patients were classified as having low risk, 70.2% were classified as having intermediate risk, and 3.7% were classified as having high risk. there were no significant differences in risk stratification between the patients with and without pte (p = 0.07). a review of the chest ctas identified six scans of poor technical quality. however, those six allowed a conclusive interpretation. a diagnosis of pte was made in 47 patients (24.6%), and, in most cases, the thrombi were peripheral, being located either on the right side or bilaterally (table 3). among the 47 patients with pte, 31 had other abnormal findings on cta. the most common findings were atelectasis, in 31.9% ; pleural effusion, in 25.5% ; consolidation, in 17.0% ; enlarged mediastinal lymph nodes, in 14.9% ; pulmonary nodules, in 12.8% ; and enlarged cardiac silhouette, in 6.4%. table 3.chest ct angiography results in 191 patients with suspected pulmonary thromboembolism. finding patients confirmed pulmonary thromboembolism47type of involvement peripheral 31central13mixed 3location right - sided22bilateral20left - sided5unconfirmed pulmonary thromboembolism144normal results24abnormal findings120findings unrelated to the alternative diagnosis45findings suggestive of an alternative diagnosis75findings also present on chest x - ray36findings on ct angiography only39the findings explain the patient 's symptoms. the findings explain the patient 's symptoms. among the 144 cta scans that were negative for pte, 24 were considered completely normal, 21 revealed one abnormality, and 99 revealed multiple findings. the most common findings were atelectasis, in 48.6% of the cases ; pulmonary nodules, in 30.6% ; pleural effusion, in 29.9% ; consolidation, in 21.5% ; and emphysema, in 21.5%. table 4.abnormal findings in 144 patients with negative ct angiography results for pulmonary thromboembolism. variable n % findings in the chest atelectasis7048.6pulmonary nodules4430.6pleural effusion4329.9consolidation3121.5emphysema3121.5enlarged mediastinal lymph nodes2920.1enlarged cardiac silhouette2920.1calcified nodules2215.3ground - glass infiltrate128.3micronodules96.3pericardial effusion96.3interstitial infiltrate85.6vertebral or rib fracture85.6pleural plaques64.2elevated hemidiaphragm32.1extrathoracic findings hiatal hernia42.8thyroid nodule32.1enlarged axillary lymph nodes32.1findings suggestive of pancreatitis21.4 among the 120 cta scans that were negative for pte and revealed abnormal findings, there were 75 that were consistent with an alternative diagnosis that explained the clinical picture of the patient. however, among those cases, there were only 39 (20.4%) in which the same alterations had not been previously detected on chest x - rays (table 3). the major diagnoses made on the basis of cta, in the absence of chest x - ray abnormalities, are shown in table 5. table 5.alternative diagnoses made solely on the basis of the chest ct angiography findings. % pneumonia2051.2decompensated heart failure820.5pleural effusion due to other causes410.3lymphangitic carcinomatosis / progression of cancer37.7noncardiogenic pulmonary edema25.1connective tissue disease - related lung disease12.6atelectasis12.6total39100.0 our study assessed 191 consecutive ctas that were performed for suspected pte, and the major findings were as follows : 1) the prevalence of pte was 24.6% ; 2) clinical complaints, risk factors, comorbidities, and the proportion of deaths were similar between the groups with positive and negative cta results for pte ; however, length of hospital stay was longer for the former group ; and 3) findings consistent with an alternative diagnosis that explained the patient 's symptoms were detected on the cta scans that were negative for pte in 39.3% of the cases, although that proportion dropped to 20.4% when only the findings that had not been previously detected on chest x - rays were taken into account. the prevalence of pte in our study was comparable to that seen in other studies (19.0 - 24.3%), 11 20 21 23 but it was higher than that reported by other authors (8.6 - 9.5%). 16 18 24 factors such as level of clinical suspicion and compliance with guidelines for the investigation of pte can affect the prevalence rate of the disease. an objective, systematic evaluation of pretest probability of pte was described in only 7.3% of the medical records in the present study. in a recent study reviewing 641 chest ctas, the prevalence of pte diagnosed by chest cta was relatively low (9.5%). a careful review conducted by those authors showed that, in 90 cases in their study, the patients had a low probability of having pte (d - dimers 0,05). os fatores de risco para tep mais frequentemente observados foram cncer e hospitalizao prvia, e as comorbidades foram hipertenso arterial sistmica, diabetes, dpoc e acidente vascular cerebral prvio. no houve diferenas significativas nas variveis demogrficas, nos sintomas e nos fatores de risco quando foram comparados os pacientes com e sem tep (p > 0,05). houve uma tendncia a maior prevalncia de tep em pacientes cuja suspeita foi levantada na enfermaria quando comparada quela na emergncia (31,0% vs. 21,6% ; p = 0,09). pacientes com tep tiveram um tempo de internao maior que os pacientes sem tep (mediana : 18 dias vs. 9,5 dias ; p = 0,0001). a mortalidade nos 191 pacientes foi de 13,6%, sendo de 12,8% e 13,9% nos grupos com tep e sem tep, respectivamente (p > 0,05). tabela 2.comparao das caractersticas dos pacientes com angiotomografia computadorizada de trax positiva ou negativa para tromboembolia pulmonar. caractersticas total de pacientes pacientes com angio - tc positiva para tep pacientes com angio - tc negativa para tep (n = 191) (n = 47) (n = 144) idade, anos 59,3 17,160,3 17,258,9 17,2sexo feminino128 (67,0)28 (59,6)100 (69,4)tabagismo89 (46,6)18 (38,3)71 (49,3)sintomas dispneia sbita144(75,4)36 (76,6)108 (75)dor torcica63 (33)17 (36,2)46 (31,9)tosse48 (25,1)11 (23,4)37 (25,6)expectorao27 (14,1)7 (14,9)20 (13,9)ansiedade18 (9,4)3 (6,4)15 (10,4)sncope12 (6,3)5 (10,6)7 (4,9)hemoptise6 (3,1)3 (6,4)3 (2,1)fatores de risco cncer49 (25,7)9 (19,1)40 (27,8)hospitalizao nos ltimos 3 meses48 (25,1)10 (21,3)38 (26,4)imobilizao por mais de 3 dias31 (16,2) 6 (12,8)25 (17,4)obesidade31 (16,2)9 (19,1)22 (15,3)insuficincia cardaca descompensada28 (14,7)5 (10,6)23 (16,0)exacerbao da dpoc16 (8,4)3 (6,4)13 (9,0)cirurgia recente25 (13,1)9 (19,1)16 (11,1)tvp prvia13 (6,8)4 (8,5)9 (6,3)tep prvia11 (5,8)2 (4,3)9 (6,3)cateter endovenoso9 (4,7)1 (2,1)8 (5,6)paralisia de membros inferiores4 (2,1)2 (4,3)2 (1,4)fratura4 (2,1)1 (2,1)3 (2,1)comorbidades hipertenso arterial sistmica89 (46,6)24 (51,1)65 (45,1)diabetes mellitus39 (20,1)12 (25,5)27 (18,8)dpoc35 (18,3)6 (12,8)29 (20,1)acidente vascular cerebral28 (14,7)9 (19,1)19 (13,2)cardiopatia isqumica19 (9,9)4 (8,5)15 (10,4)tireoidopatia17 (8,9)4 (8,5)13 (9,0)insuficincia renal14 (7,3)3 (6,4)11 (7,6)doena difusa do tecido conjuntivo13 (6,8)2 (4,3)11 (7,6)asma9 (4,7)0 (0,0)9 (6,3)desfechos tempo de internao em dias 11,0 (4 - 22)18,0 (8 - 35)9,5 (3 - 19)bito26 (13,6)6 (12,8)20 (13,9)angio - tc : angiotomografia computadorizada ; tep : tromboembolia pulmonar ; e tvp : trombose venosa profunda. angio - tc : angiotomografia computadorizada ; tep : tromboembolia pulmonar ; e tvp : trombose venosa profunda. uma avaliao objetiva e sistemtica de probabilidade pr - teste de tep, usando o escore clnico, foi encontrada em 14 dos 191 pronturios (7,3%). o escore de genebra, calculado retrospectivamente, foi de 5,9 3,2 pontos nos pacientes com tep e de 5,0 2,6 pontos nos pacientes na estratificao de probabilidade clnica de risco em trs nveis, 26,2% dos pacientes apresentavam risco baixo, 70,2% apresentavam risco intermedirio, e 3,7%, risco alto. no houve diferenas significativas na estratificao de risco nos pacientes com tep e sem tep (p = 0,07). a reviso das angio - tcs de trax identificou seis exames com m qualidade tcnica, mas que permitiram uma interpretao conclusiva. o diagnstico de tep foi realizado em 47 pacientes (24,6%) e, na maior parte dos casos, os trombos eram perifricos, com localizao direita ou bilaterais (tabela 3). dos 47 pacientes com tep, 31 apresentaram outros achados na tc de trax. os achados mais frequentes foram atelectasia, em 31,9% ; derrame pleural, em 25,5% ; consolidao, em 17,0% ; adenomegalias mediastinais, em 14,9% ; ndulo pulmonar, em 12,8% ; e aumento de rea cardaca, em 6,4%. tabela 3.resultados da angiotomografia computadorizada de trax em 191 pacientes com suspeita de tromboembolia pulmonar. achados pacientes tromboembolia pulmonar confirmada47tipo de acometimento perifrico 31central13misto 3localizao lado direito 22bilateral20lado esquerdo5tromboembolia pulmonar no confirmada144resultado normal24achados anormais120achados no relacionados ao diagnstico alternativo45achado sugestivo de diagnstico alternativo75achado tambm presente na radiografia de trax36achado somente na angiotomografia computadorizada39os achados explicam os sintomas do paciente. das 144 angio - tcs entre as quais no se pde visualizar tep, o exame foi considerado completamente normal, em 24 casos ; apresentou uma alterao, em 21 ; ou apresentou mltiplos achados, em 99. os achados mais frequentes foram atelectasia, em 48,6% ; ndulo pulmonar, em 30,6% ; derrame pleural, em 29,9% ; consolidao, em 21,5% ; e enfisema, em 21,5%. tabela 4.achados anormais em 144 pacientes com angiotomografia computadorizada de trax negativa para tromboembolia pulmonar. variveis n % achados no trax atelectasia7048,6ndulo pulmonar4430,6derrame pleural4329,9consolidao3121,5enfisema3121,5adenomegalias mediastinais2920,1aumento de rea cardaca2920,1ndulos calcificados2215,3infiltrado em vidro fosco128,3microndulos96,3derrame pericrdico96,3infiltrado intersticial85,6fratura de vrtebra ou costela85,6placas pleurais64,2elevao da hemicpula diafragmtica32,1achados extratorcicos hrnia de hiato42,8ndulo de tireoide32,1adenomegalias axilares32,1achados sugestivos de pancreatite21,4 das 120 angio - tcs negativas para tep que mostraram anormalidades, 75 foram compatveis com um diagnstico alternativo que explicava o quadro clnico dos pacientes. entretanto, em apenas 39 exames (20,4%), os achados no haviam sido visualizados previamente na radiografia de trax (tabela 3). os principais diagnsticos corroborados pela angio - tc, na ausncia de alteraes na radiografia de trax, so mostrados na tabela 5. em 31 casos, a anormalidade foi pulmonar e, em 8, essa foi cardaca ; o diagnstico mais prevalente foi pneumonia, em 20 casos. tabela 5.diagnsticos alternativos cujos achados radiolgicos foram identificados somente na angiotomografia computadorizada de trax. diagnsticos n % pneumonia2051,2insuficincia cardaca descompensada820,5derrame pleural por outra causa410,3linfangite carcinomatosa / progresso de neoplasia37,7congesto pulmonar no cardiognica25,1pneumopatia relacionada colagenose12,6atelectasia12,6total39100,0 nosso estudo avaliou 191 angio - tcs realizadas consecutivamente por suspeita de tep e os principais achados foram os seguintes : 1) a prevalncia de tep foi de 24,6% ; 2) as queixas clnicas, os fatores de risco, as comorbidades e a proporo de bitos foram semelhantes nos grupos com angio - tc positiva e negativa para tep ; porm, o tempo de internao foi maior no primeiro grupo ; 3) nas angio - tcs negativas para tep foram identificados achados compatveis com um diagnstico alternativo que explicava os sintomas dos pacientes em 39,3% dos casos ; entretanto, essa proporo caiu para 20,4% quando foram considerados apenas os achados no visualizados na radiografia de trax. a prevalncia de tep observada em nosso estudo foi comparvel de outras sries (19.0 - 24,3%), 11 20 21 23 mas foi superior descrita por outros autores (8,6 - 9,5%). 16 18 24 fatores como o grau de suspeita clnica e o seguimento de diretrizes para a investigao de tep podem influenciar na taxa de prevalncia da doena. uma avaliao objetiva e sistemtica de probabilidade pr - teste de tep estava descrita em apenas 7,3% dos pronturios no nosso estudo. num estudo recente que revisou 641 angio - tcs de trax, a prevalncia de tep diagnosticada por angio - tc de trax foi relativamente baixa (9,5%). uma reviso criteriosa feita pelos autores mostrou que em 90 casos da srie os pacientes tinham baixa probabilidade de apresentar tep (dmeros - d < 500 ng / ml, e escore de wells 4), no havendo indicao formal de investigao com angio - tc. apenas 2 daqueles casos apresentavam tep. 18 paralelamente, uma alta positividade de tep na angio - tc pode refletir uma baixa suspeita clnica da doena. portanto, alm da prevalncia de tep, importante avaliar simultaneamente o grau de adeso ao algoritmo diagnstico proposto por diretrizes de manejo da tep. 8 25 embora no nosso estudo no tenha sido avaliado o grau de adeso ao protocolo institucional para tep, nossos dados de prevalncia so semelhantes ao de outro estudo (23,3%). dispneia, dor torcica e tosse foram os principais sintomas relatados por pacientes com suspeita de tep no nosso estudo, sem diferena entre os pacientes com angio - tc positiva ou negativa para tep, sugerindo que os sintomas no so especficos para a doena. os mesmos sintomas despontam como os mais frequentes em outras sries 9 27 ; porm, com uma frequncia maior de dor pleurtica (76%) e tosse (44%) numa delas 27 e frequncia menor de tosse na outra (10,8%). 9 em outro estudo, a dor torcica foi o sintoma mais frequente (41,4%), e a dispneia foi observada em apenas 10% dos pacientes. 24 a grande discrepncia entre os dados das diferentes sries pode, ao menos em parte, estar associada ao registro dos sintomas, uma vez que em estudos retrospectivos, como nesse ltimo, 24 as queixas podem ter sido subestimadas. a hemoptise observada nos pacientes no nosso estudo est dentro da margem de prevalncia em outros estudos prvios em pacientes com tep (1,9%-6,0%). 4 9 18 23 27 histria de cncer, hospitalizao prvia e imobilizao foram os principais fatores de risco para tep identificados na nossa srie e tambm relatados por outros autores. 9 cirurgia recente foi o stimo fator de risco em frequncia na nossa casustica (13,1%), embora essa proporo seja comparvel reportada em dois estudos (11,8% e 14,4%)mas inferior reportada em outro estudo. 18 a menor proporo de pacientes em situao ps - operatria no nosso estudo pode estar associada origem dos casos, uma vez que a maioria era oriunda do setor de emergncia. embora tanto os fatores de risco como as comorbidades tenham sido altamente prevalentes na nossa srie, no se observaram diferenas significativas entre os grupos de pacientes com angio - tc positiva e negativa para tep. do mesmo modo, no foi identificada diferena na mortalidade entre os dois grupos. a mortalidade intra - hospitalar dos pacientes com angio - tc positiva para tep na nossa srie 3 5 por outro lado, pacientes com angio - tc positiva para tep tiveram um tempo de internao significativamente maior que os pacientes com investigao negativa para tep. a tep por si s pode prolongar o tempo de internao, mas tambm pode ser um marcador de gravidade do paciente, que se associa permanncia hospitalar. a proporo de angio - tcs normais foi 12,6% no nosso estudo ; em outras sries, essa variou de 12,5% a 29,3%. 18 21 23 os nossos principais achados anormais nos exames negativos para tep foram atelectasia, ndulo pulmonar, derrame pleural, consolidao e enfisema. chama a ateno a significativa proporo de ndulos e microndulos na nossa srie em relao s demais, 16 18 20 23 24 o que pode estar relacionado com a alta prevalncia de doenas granulomatosas no nosso meio. 29 esses achados incidentais, que no estavam associados ao quadro agudo dos pacientes, com frequncia podem necessitar de investigao adicional ou seguimento em longo prazo. 18 20 avaliamos a relevncia clnica dos achados tomogrficos para estabelecer um diagnstico alternativo nos casos com angio - tc negativa para tep. os achados tomogrficos corroboraram um diagnstico alternativo, que explicava os sintomas do paciente, em 39,3% dos nossos pacientes (14,3 - 33,0% dos casos em outros estudos). 16 18 21 entretanto, os achados foram identificados somente na angio - tc, isto, no estavam evidentes simultaneamente na radiografia de trax, em 20,4% dos casos. no nosso conhecimento, apenas trs estudos consideraram os achados concomitantes da radiografia de trax na anlise de diagnsticos alternativos. num estudo, um diagnstico alternativo foi estabelecido com base nos achados na angio - tc em 33% dos casos ; porm, em cerca da metade desses, as mesmas alteraes j haviam sido identificadas na radiografia de trax.outros autores identificaram um diagnstico alternativo em 14,3% dos pacientes, sendo que a radiografia havia mostrado os mesmos achados em 9,8% dos casos. 18 ainda, em outra srie, a angio - tc mostrou achados que suportavam um diagnstico alternativo em 28% de 203 pacientes, sendo o achado no suspeitado antes da angio - tc em apenas 8,8% dos casos.pneumonia foi o diagnstico alternativo mais frequente no nosso e em outros estudos, 18 20 21 enquanto derrame pleural predominou em outras duas sries. o estudo foi realizado em um nico centro, o que limita a generalizao dos resultados, e a coleta dos dados foi retrospectiva. estudos retrospectivos correm risco de vieses de seleo (por perda de casos), bem como de aferio (dados obtidos de pronturios mdicos). entretanto, a forma sequencial de seleo dos nossos casos, a partir do registro de todas as angio - tcs realizadas na instituio, minimizou o risco de vis de seleo. uma limitao adicional que o seguimento dos pacientes foi realizado apenas durante a internao, fornecendo apenas dados de mortalidade intra - hospitalar, sem dados de sobrevida em mdio e longo prazo. por outro lado, um aspecto positivo do estudo foi a interpretao dos exames de imagem, de forma independente, por dois radiologistas experientes na interpretao de angio - tc. ainda, todos os casos com angio - tc negativa para tep foram revisados por dois pneumologistas, do ponto de vista clnico e radiolgico, obtendo - se uma opinio de consenso no final, o que permitiu um maior rigor diagnstico. em concluso, a angio - tc de trax foi positiva para tep em 24,6% dos casos. no houve diferenas nos achados clnicos e na mortalidade intra - hospitalar quando comparados os grupos de pacientes com e sem tep, exceto pelo maior tempo de internao nos pacientes com angio - tc positiva para tep. observamos um grande nmero de angio - tcs com achados anormais, sendo os mesmos compatveis com um diagnstico alternativo que explicasse o quadro clnico dos pacientes em 39,3% dos casos. entretanto, em cerca de metade desses casos, os mesmos achados j haviam sido identificados na radiografia de trax. resumindo, nossos resultados demonstram que a angio - tc de trax til na em casos com suspeita de tep, pois pode confirmar o diagnstico e evidenciar achados sugestivos de um diagnstico alternativo em um significativo nmero de pacientes.	objective : to determine the prevalence of alternative diagnoses based on chest ct angiography (cta) in patients with suspected pulmonary thromboembolism (pte) who tested negative for pte, as well as whether those alternative diagnoses had been considered prior to the cta. methods : this was a cross - sectional, retrospective study involving 191 adult patients undergoing cta for suspected pte between september of 2009 and may of 2012. chest x - rays and ctas were reviewed to determine whether the findings suggested an alternative diagnosis in the cases not diagnosed as pte. data on symptoms, risk factors, comorbidities, length of hospital stay, and mortality were collected. results : on the basis of the cta findings, pte was diagnosed in 47 cases (24.6%). among the 144 patients not diagnosed with pte via cta, the findings were abnormal in 120 (83.3%). such findings were consistent with an alternative diagnosis that explained the symptoms in 75 patients (39.3%). among those 75 cases, there were only 39 (20.4%) in which the same alterations had not been previously detected on chest x - rays. the most common alternative diagnosis, made solely on the basis of the cta findings, was pneumonia (identified in 20 cases). symptoms, risk factors, comorbidities, and the in - hospital mortality rate did not differ significantly between the patients with and without pte. however, the median hospital stay was significantly longer in the patients with pte than in those without (18.0 and 9.5 days, respectively ; p = 0.001). conclusions : our results indicate that chest cta is useful in cases of suspected pte, because it can confirm the diagnosis and reveal findings consistent with an alternative diagnosis in a significant number of patients.
performed the first robotic cholecystectomy using a prototype system with remote control and a three - dimensional viewing through the use of specially developed glasses. more sophisticated robotic systems were later developed which provided or improved upon a stable camera platform, three - dimensional imaging, excellent ergonomics, instruments with a wide - range degree of freedom and modulation of motion amplitude with tremor filtering. the current da vinci robotic system (intuitive surgical, sunnyvale, ca) is the most highly used system worldwide. the first da vinci system was installed in geneva in 2003, and to the best of our knowledge, our team has had the most experience among swiss clinicians in gastrointestinal cases using this system. the aim of the study reported here was to determine surgical feasibility, evaluate functional outcome and assess patients satisfaction using this system. to date, 94 consecutive patients have been treated by our team. from january 2003 to december 2006, the three - arm da vinci system was used (73 cases), and from january 2007 to september 2007, our team used the four - arm da vinci system (21 cases). during the entire study period, 409 robotic procedures were performed at our institution, consisting of 300 urologic cases (73%), 94 gastrointestinal cases (23%) and 15 gynecologic cases (4%). each patient s data were prospectively collected using a robotic logbook ; a clinical information sheet for each individual was filled up by the surgeon and anesthesiologist and later stored in our computerized database system. the follow - up was done during a patient 's visit to the attending physician and/or telephone interview. they were 27 men (29%) and 67 women (71%) in our patient cohort, with a mean age of 53 years (range 1984 years). overall, mean hospital stay was 7 days (range 224 days), and mean body mass index (bmi) was 25 (range 1647). a blood transfusion was needed in one case (1%). benign cases accounted for 88 patients (94%). only six patients (6%) the surgical procedures consisted of 40 colorectal cases (43%), 31 anti - reflux cases (33%), 14 morbid obesity cases (15%) and nine miscellaneous cases (10%). colorectal procedures consisted of sigmoidectomy (n = 20), sigmoidectomy and rectopexy (n = 8), right colectomy (n = 3), rectopexy (n = 3), low anterior resection (n = 2), caecectomy (n = 1), anterior resection (n = 1), abdomino - perineal resection (n = 1) and resection of recto - vaginal endometriosis nodule (n = 1). diagnoses prior to the surgical procedure were : diverticular disease (n = 20, 50%), rectal prolapse (n = 11, 27.5%), adenocarcinoma (n = 6, 15%), benign multiple polyp or endoscopically non - resectable polyp (n = 2, 5%) and recto - vaginal endometriosis (n = 1, 2.5%). the mean age of patients undergoing colorectal procedures was 60 years (range 3284 years). the mean hospital stay was 9 days (range 324), and the mean bmi was 22 (range 1940). two patients were re - operated (one for a left ureter injury in which a robotic suture was performed, another for pelvic bleeding by conventional laparoscopy). there were three conversions to laparoscopy (two cases for difficult dissection, one for a robotic technical problem) and two conversions to laparotomy (one robotic technical problem, and one difficult dissection). the mean age of patients undergoing this procedure was 49 years (range 1968 years). the mean hospital stay was 6 days (range 413 days), while the mean bmi was 27.3 (range 1936). two patients required a nissen redo procedure, and there was one conversion to laparotomy during a nissen redo because of an esophageal tear. gastric banding was the third most common surgical procedure in our series (14 cases). the mean age of patients undergoing this procedure was 41 years (range 2058 years). the mean hospital stay was 4 days (range 37 days), and mean bmi was 41 (range 3446). there was one conversion to laparotomy. among the miscellaneous procedures, there were four intra - abdominal tumor resections (three benign cysts and one teratoma), two cholecystectomies, two partial gastric resections and one case of hepatic cyst resection. a patient 's satisfaction was evaluated using a visual analog score system : the mean value of functional outcome of the surgical procedure was 8 (range 310), and of satisfaction with regard to the entire procedure was 7 (range 210). mean follow - up time for all patients was 11 months (range 15 days to 34 months). several studies have reported surgical results using the da vinci robotic system in which this technique has been shown to be applicable and safe [39 ]. no morbidity related to the system has yet been observed. overall, the reported mortality rate has ranged between 0 and 1.8%, while the rate of complication has been less than 10% ; conversion to conventional laparoscopy and/or laparotomy was less than 5%. almost all studies reported a longer operative time using the robotic system as compared to conventional laparoscopy. increased overall costs due to longer operative time and the use of more expensive instruments was reported for cases of nissen fundoplication and colectomy [1015 ]. our team has not experienced the death of a patient during the robotic procedure, and we have observed a morbidity of 3.1%. conversion to laparoscopy / laparotomy was 5.3%. at the beginning of our learning experience, operative time was long, and although it has diminished with time and experience, it is still longer than conventional laparoscopy. to date, no proven patient - specific advantage of robotic surgery over laparoscopic techniques has been demonstrated. the robotic system allows the surgeon to perform more sophisticated procedures, whereas classical gastrointestinal procedures can be performed safely. the development of mini robots and augmented reality techniques will boost robotic surgery in the future [9, 16 ]. we have found that colon and rectum resections, anti - reflux surgery, and obesity surgery were safe and efficacious for the patients. our experience in terms of operative time, conversion rate, mortality, complications rate and length of stay are similar to those reported in the literature (table 1). it is our impression that the use of the da vinci system is from a technical point of view particularly useful for proctectomy with total mesorectum excision (tme) in cases of rectal cancer, as lymph node harvesting, nerve sparing and anatomical tme is enhanced by the three - dimensional vision of the pelvis. as reported by others, we prefer to do a hybrid procedure (conventional laparoscopic inferior mesenteric high vessel ligature and splenic flexure take down) and robotic proctectomy with tme. right colectomy for cancer with primary vessels ligation can be performed robotically with great ease ; at the end of the procedure, the anastomosis is performed outside the abdomen. sigmoidectomy for benign disease can also be performed easily as no high vascular ligature is necessary and the splenic flexure is left in place. the operative time for a right colectomy is shorter than that for a left colectomy. table 1literature survey of robotic colon and rectum surgeryauthoryearnumber of casesmean operative time, minutes (range)conversion (%) mortality (%) complications (%) mean length of hospital stay, days (range)delaney.. 200630rc 177 (103306)lc 225 (147283)6.6020rc 5.2 (227)lc 6 (330)pigazzi. 20066280 (200330)0016.64.5 (311)current series200740162 (60330)12.5059 (324)na, not available literature survey of robotic colon and rectum surgery given the limited space for dissection and the need to mobilize the great gastric curvature, robotic nissen fundoplication and other anti - reflux techniques have been often performed [34, 6, 1113, 2632 ]. the surgical outcome of our series is in line with other reports as shown in table 2. randomized controlled trials have demonstrated that conventional nissen fundoplication give similar results to the robotic approach, but it is less expensive and less time consuming [1113, 32 ]. cost analysis should be performed in each country as health systems are differ significantly. table 2literature survey of robotic nissen fundoplicationauthoryearnumber of casesmean operative time, minutes (range)conversion (%) mortality (%) complications (%) mean length of stay, hours (range)cadire. 2007112200094current series200731166 (60270)3.303.36 (413)na, not available literature survey of robotic nissen fundoplication it has recently been suggested that the use of the da vinci robotic system is valuable for obesity surgery, specifically in cases of gastric banding [3, 7, 29, 3335 ] and gastric bypass surgery [36, 37 ]. the totally robotic roux - en - y gastric bypass is feasible and safe, but this technique is still being evaluated and compared to conventional laparoscopy, although it seems promising in terms of surgical outcome and bmi reduction [36, 37 ]. table 3literature survey of robotic gastric bandingauthoryearnumber of casesmean operative time, minutes (range)conversion (%) mortality (%) complications (%) mean length of stay, days (range)cadire. 200510167000nacurrent series200714141 (90240)12.5054 (37)na, not available literature survey of robotic gastric banding da vinci robotic use is being developed in foregut surgery, i.e. heller myotomy and gastric cancer resections [3840 ]. future developments will include technologies of information systems, virtual reality, and micro - robotics. in conclusion, future developments in gastrointestinal robotic surgery should include cost analysis studies and evaluations of quality of life outcomes.	the continuing development of robotic surgery supports its use in laparoscopic gastrointestinal surgery. our study retrospectively reviewed the surgical outcome and patient s satisfaction of gastrointestinal laparoscopic robotic procedures. from january 2003 to september 2007, 94 patients (27 women, 67 men) with a mean age of 53 years (range 1984 years) underwent laparoscopic surgery with a da vinci robotic system. there were 40 colorectal cases (43%), 31 anti - reflux surgery cases (33%) and 14 obesity surgery cases (15%) ; the remaining cases consisted of gastric and gallbladder surgery, intra - abdominal tumour excisions, and hepatic cyst resections. the majority of the cases (88, 94%) were performed for benign disease. the mean operative time was 153 min (range 60330 min). one patient needed a blood transfusion. the mean body mass index was 25 (range 1647). no death occurred. five cases (5.3%) were converted to conventional laparoscopic surgery (n = 3) or to laparotomy (n = 2). morbidity consisted of one nissen redo surgery to loosen a tight anti - reflux valve 6 days after robotic surgery, a robotic left ureter repair and pelvic haemorrhage following proctectomy requiring re - operation to control haemostasis and to remove pelvic haematoma. mean follow - up time was 11 months (range 15 days to 34 months). one case of incisional trocar hernia needed re - operation. overall patient s satisfaction was high : few scars were chelod, while functional surgical outcome was rated high by most of the patients. our preliminary experience was encouraging, with minimal morbidity and very high acceptance by patients.
cystic fibrosis (cf ; mim 219700) is a lethal disease, which is induced by mutations in the cystic fibrosis transmembrane conductance regulator (cftr) gene on chromosome 7q31. cf is one of the most frequently - occurring autosomal recessive disorders in caucasians, with an incidence of 1 in every 2,000 to 3,000 (1). however, cf is quite rare in the asian population that an epidemiological study in the japanese population revealed the incidence of cf being about 1 in 350,000 (2,3). cf is also extremely rare in the korean population and there have been only a few reports of this condition thus far (4 - 6). here, we report a korean patient with cf, who was diagnosed by both a sweat chloride test and genetic analysis of the cftr gene. a 15-yr - old boy was admitted to the samsung medical center, complaining of chronic productive cough and dyspnea without any gastrointestinal symptoms. during the first decades of life, the patient had been treated for several episodes of bronchiolitis and pneumonia, necessitating frequent hospital admission. six years prior to the current hospitalization, the patient 's respiratory symptoms became aggravated. he was admitted to a civil hospital for the treatment of pulmonary tuberculosis, and received antituberculosis drug therapy for 12 months. he developed frequent rhinorrhea and nasal stuffiness, and underwent sinus surgery at the age of twelve. his respiratory symptoms progressively worsened, and he was diagnosed with bronchiectasis three years ago. he had a 25-yr - old brother and a 24-yr - old sister, with an unremarkable past and a current medical history. on admission, the patient 's blood pressure was 128/69 mmhg, heart rate 80 beats per minute, respiratory rate 20 breaths per minute, body temperature 36.0, height 174 cm, and he weighed 53.6 kg. he was also determined to exhibit 96 percent oxygen saturation when breathing room air. upon physical examination, diffuse crackles and rhonchi chest radiographs and computed tomographic (ct) scans of the chest, performed without intravenous contrast material administration, revealed bilateral multifocal bronchiectasis and multiple centrilobular nodules (fig. pulmonary function studies showed a forced vital capacity of 2.62 l (63% of predicted), with a forced expiratory volume of 1.36 l per second (36% of predicted). the patient 's total lung capacity was measured to be 6.39 l (121% of predicted). a ct scan of his paranasal sinuses evidenced severe inflammatory changes, associated with pansinusitis. conditions such as immunodeficiency and ciliary dyskinesia were ruled out by the normal results of serum immunoglobulin tests, and by the finding of normal bronchial mucosal biopsy. the sweat chloride concentration was measured by a quantitative pilocarpine iontophoresis sweat test, in accordance with the guidelines of the national committee for clinical laboratory standards (7). l, a finding that exceeded the reference limit (g ; q98r) and a heterozygous c to t transition in exon 6a (c.658c > t ; q220x). a family study demonstrated that the patient 's father and elder brother both also possessed the q98r mutation, whereas his mother harbored the q220x mutation. the patient 's sister, however, harbored no such mutations. based on our search of the cystic fibrosis mutation database (http://www.genet.sickkids.on.ca/cftr/), q98r and q220x mutations have been reported in cf patients from southern france and southern england (8,9). cf is the most frequently encountered life - limiting autosomal recessive genetic disorder in caucasians, with an incidence of 1 in every 3,200 newborns in the united states (1). however, it is less common in african americans (1 in 15,000) (10) and quite rare in asians (1 in 350,000 in the japanese population) (2,3). cf is normally suspected upon the finding of one or more typical cf phenotypical features, including chronic sinopulmonary disease, gastrointestinal / nutritional abnormalities, salt loss syndrome, and obstructive azoospermia in males (7). cf is caused by mutations in the cftr gene, which is located on the long arm of chromosome 7. this gene consists of 27 exons, and encodes for an integral membrane protein composed of 1,480 amino acids. functionally, cftr regulates the expression of the chloride (cl) channel, which is expressed in the apical membrane of exocrine epithelial cells (1). currently, approximately 1,366 mutations and polymorphisms are listed in the cystic fibrosis mutation database (http://www.genet.sickkids.on.ca/cftr/). however, only 11 mutations have been reported in the korean population thus far (table 1). in the present study, we report two additional mutations in the list of cftr gene mutations (q98r and q220x), which have been identified in koreans. it is of note that these two mutations have previously been reported in cf patients from southern france (8) and southern england (9), respectively, but are not frequently encountered mutations even in the caucasian population. as shown in the table, no mutation except ivs8 t5 variant has repeatedly been observed in korean population. furthermore, only two mutations (r117h and delf508) are found among 25 mutations in the screening panel for caucasian cf patients recommended by the american college of medical genetics (11). (12), a possible common mutation might not exist in the korean population. although cf tends to be discovered early in life, the diagnosis of cf is being made in adults with increased frequency (13,14)., they exhibit a milder variant of lung disease, a lesser degree of pseudomonas infection, and are more likely to be pancreatic - sufficient than are patients in whom cf is diagnosed at earlier ages (13,14). physicians who are unfamiliar with the spectrum of cf phenotypic manifestations may not consider cf as a component of their differential diagnoses, thus delaying accurate diagnosis. in summary, we report here a korean cf patient, who was diagnosed via a sweat chloride test, as well as a molecular genetic analysis of the patient 's cftr gene. although cf continues to be quite a rare condition in koreans, cf should still be suspected in patients exhibiting recurrent and unexplained chronic respiratory symptoms.	although cystic fibrosis (cf) is one of the most frequently seen autosomal - recessive disorders in caucasians, it is extremely rare in the korean population. recently, a 15-yr - old korean boy was admitted to our hospital complaining of coughing, sputum, and exertional dyspnea. chest radiographs and computed tomographic chest and paranasal sinus scans revealed diffuse bronchiectasis and pansinusitis. pulmonary function tests revealed severe obstructive impairment. the average sweat chloride concentrations on both of the patients ' forearms were 63.0 mm / l (reference limit : < 40 mm / l). upon mutation analysis, two different mutations (q98r and q220x) were identified in the cystic fibrosis transmembrane conductance regulator gene, both of which had been previously detected in cf patients, one from france and the other from england. as cf is quite rare in korea, the diagnosis of cf in this patient might be delayed. therefore, we recommend that a diagnosis of cf should be suspected in patients exhibiting unexplained chronic respiratory symptoms.
a 34-year - old man with fever (38c), tender cervical lymph nodes, and symptoms of pharyngeal infection was admitted to a hospital in istanbul, turkey on february 3, 2010. his blood pressure was 120/70 mm hg and heart rate was 96 beats / min. laboratory findings included thrombocytopenia (63,000 platelets / mm [reference 13045010 platelets / mm ]) and mild elevation of transaminase levels (alanine transaminase 95 iu / l [reference 1540 iu / l ]) (table 1). diarrhea developed during the first day of hospitalization, and conjunctival suffusion was observed on ocular examination. ciprofloxacin was given to treat suspected salmonellosis at a dosage of 400 mg every 12 hours. dobv, dobrava - belgrade virus ; alt, alanine transaminase ; ast, aspartate aminotransferase ; bun, blood urea nitrogen ; aptt, activated partial thromboplastin time ; pt, prothrombin time ; tsh, thyroid stimulating hormone ; acth, adrenocorticotropic hormone ; adh, antidiuretic hormone ; lh, luteinizing hormone ; fsh, follicle - stimulating hormone ; igf, insulin - like growth factor. his platelet count decreased to 20,000/mm, and his serum creatinine level increased to 1.8 mg / dl (reference 0.71.2 mg / dl). results of a transthoracic echocardiogram showed heart chambers of normal size, a left ventricular ejection fraction of 60%, and moderate pericardial effusion. broad - spectrum antimicrobial drugs were initiated as treatment for suspected sepsis, and platelet and albumin transfusions were given as supportive therapy. on day 3 of hospitalization, the patient experienced tachycardia, petechial lesions appeared on his extremities, and his platelet count dropped to 13,000/mm. septic shock developed in the patient, and inotropic therapy was initiated. on the same day, acute respiratory distress syndrome developed, and assisted ventilation was started. results of a thoracic computerized tomographic scan showed focal infiltration on the upper zone of the left lung and minimal pleural effusion on the right lung. results of abdominal computerized tomographic scan revealed ascites and multiple mesenteric lymphadenomegalies. on the basis of these clinical and laboratory findings, blood samples were obtained for hantavirus testing, and oral ribavirin was added at an initial dose of 30 mg / kg, followed by 15 mg / kg every 6 hours for 4 days, then 7.5 mg / kg for 6 days. on the same day, the patient s urinary output gradually increased, pulmonary symptoms regressed, and his ventilation tube was removed on day 12 of hospitalization. the patient stayed in the intensive care unit for 18 days and was then transferred to a standard care unit. no microbial growth was observed in the cultures of samples taken during the first days of hospitalization. serum samples were tested for antibodies against a panel of pathogens, including salmonella typhi, s. paratyphi a and b, brucella spp., hiv, crimean - congo hemorrhagic fever virus, epstein - barr virus, cytomegalovirus, parvovirus b19, adenovirus, dengue virus, rickettsia spp. treponema pallidum, and hepatitis viruses a, b, c, and e. results of these serologic tests were negative or inconspicuous. results of pcr analysis conducted for leptospirosis, respiratory viruses, and cytomegalovirus were below the detection limit of the assays. serologic testing for hantavirus was performed by using the recomline bunyavirus igg / igm immunoassay (mikrogen, neuried, munich, germany) ; results showed strong reactivity for igg and igm antibodies, indicating an acute hantavirus infection. the blot data provided evidence of an infection with dobv ; to confirm the results, we performed serotyping by using focus reduction neutralization tests (table 2). the results confirmed a dobv infection most likely caused by a strain of dobv - af. reverse transcription pcr results for hantaviral rna were positive for the first acute - phase blood sample. subsequent nucleotide sequence determination of parts of the 3 genomic segments and molecular phylogenetic analysis of these small, medium (figure 1), and large gene sequences (data not shown) showed that the isolate was most closely related to dobv - af. dobv, dobrava - belgrade virus ; aa, apodemus agrarius ; af, apodemus flavicollis ; sk, strain slovakia ; slo, strain slovenia. molecular phylogenetic analysis of small (s) and medium (m) gene segments. consensus neighbor - joining phylogenetic tree (tamura - nei 93 evolutionary model) of hantavirus strains was constructed as described (9) based on partial sequences of the s (panel a) and m segment (panel b). bootstrap values > 70%, calculated from 10,000 replicates, are shown at the tree branches. saav est, saaremaa virus from estonia ; dobv anop, dobrava - belgrade virus (lineage dobv - af) from greece ; dobv slo, dobrava - belgrade virus (lineage dobv - af) from slovenia ; dob sk, dobrava - belgrade virus (lineage dobv - aa) from slovakia ; htnv, hantaan virus ; seov, seoul virus ; andv, andes virus ; snv, sin nombre virus ; puuv, puumala virus ; tulv, tula virus ; phv, prospect hill virus. scale bars indicate an evolutionary distance of 0.1 substitutions per position. on day 19 of hospitalization, the results of the basal levels of hormonal studies are shown in table 1. on day 20 of hospitalization, hormone replacement therapy using hydrocortisone, thyroxin, and testosterone was immediately initiated. on day 22 of hospitalization, the patient was discharged on day 33 of hospitalization. at the final outpatient followup visit, the patient had normal urine output and his serum creatinine level was 1.5 mg / dl. his pulmonary examination results and chest radiograph findings were normal, but he continued to require hormonal replacement therapy at the 16th month of followup care. magnetic resonance images showing hemorrhage of the pituitary gland and pituitary atrophy as indicated by arrows. a) t1-weighted coronal image shows high signal intensity on the right side of the adenohypophysis consistent with hemorrhage. b) t2-weighted sagittal image shows decreased pituitary gland height and heterogenous low signal intensity of the central adenohypophysis due to hemorrhagic infarction. the general symptoms and results of the serologic tests and molecular analyses for this patient were consistent with a hantavirus infection. after the acute phase of the illness, characterized by renal and pulmonary failure, panhypopituitarism developed in the patient. case reports have documented that pituitary hemorrhage followed by panhypopituitarism may complicate hfrs (24), and puumala virus was reported as the causative agent in most of these cases. in addition, hormonal deficiencies have been shown to be common features of puumala virus infections, and chronic hormonal deficits develop in some patients (5). our findings demonstrate that development of hypopituitarism can also be associated with infection by dobv. since the patient does not belong to any groups that have high exposure to hantaviruses (such as farmers, forest workers, and military recruits) and did not report any travel in hantavirus - endemic areas, the source of infection remains unclear. in contrast to what is known about hantavirus presence in other balkan states, little is known about the distribution, diversity, or host range of hantaviruses in turkey (6). in 2009, a hantavirus outbreak occurred in turkey ; 3 of the 5 infected persons died (7). serologic assays have been used to detect dobv infection in patients in turkey, but focus reduction neutralization tests have not been used for serotyping. dobv rna was found only in 1 urine sample from a hantavirus - infected patient from turkey ; however, sequence data were not provided (8). prevalence studies of rodents collected in turkey revealed 6% seropositivity to puumala virus in microtus voles and no appearance of hantavirus antibodies in the apodemus species of rodents (9). focus reduction neutralization testing and virus sequencing confirmed that the patient in our study was infected with dobv - af. this strain is associated with a. flavicollis field mice and yet has not been reported in animals in turkey (see 10,11 for the molecular classification of apodemus spp.associated dobv virus lineages). this case should alert physicians that hantavirus infection should be considered in the differential diagnosis of patients who have high fever and thrombocytopenia, including those without known risk factors for hantavirus exposure. since severe hormonal deficiencies are life - threatening, neuroendocrinologic complications should be taken into account and the endocrine status should be investigated to prevent panhypopituitarism even after recovery from hfrs.	we identified dobrava - belgrade virus infection in turkey (from a strain related to hantavirus strains from nearby countries) in a patient who had severe symptoms leading to panhypopituitarism, but no known risk for hantavirus. our findings emphasize the need for increased awareness of hantaviruses in the region and assessment of symptomatic persons without known risk factors for infection.
stroke is the second leading cause of death in korea and is associated with severe disability and mortality.17) the identification and treatment of stroke risk factors has important consequences on the prognosis of stroke.4)7) well - known definite risk factors for ischemic stroke include dyslipidemia, hypertension, diabetes mellitus, ischemic heart disease, atrial fibrillation, valvular heart disease, carotid stenosis, cigarette smoking, and obesity. we have frequently seen ischemic stroke patients with left atrial enlargement (lae) in clinical practice. this condition indicates increased left ventricle (lv) filling pressure and the subsequent remodeling process in hypertensive heart disease.3) in the past few years, lae has been found to be a risk factor for cardiovascular disease, atrial fibrillation, systemic thromboembolism, congestive heart failure (chf) and stroke.5)6)8)12) the framingham study noted that lae may be a risk factor for ischemic stroke in men partially mediated by lv mass.2) however, the role and basic characteristics of lae in acute cerebral infarction patients have not been sufficiently described in the literature. especially, the relation between stroke subtypes and lae is not clear in patients with acute ischemic stroke. in this study, we investigated the relation of lae at the time of acute ischemic stroke with conventional stroke risk factors and stroke subtypes. the enrolled study population included 182 patients with acute ischemic stroke (mean age : 68.01 12.53 years and 106 males), who were admitted from september 2010 to june 2011 and examined with brain magnetic resonance imaging (mri). routine laboratory tests included complete blood counts, glucose, serum electrolytes, coagulation studies, liver function tests, and lipid profile. systemic hypertension was defined as systolic blood pressure values > 140 mm hg or diastolic blood pressure > 90 mm hg during the interview, or the presence of a medical history or antihypertensive treatment. diabetes mellitus was identified based on of elevated fasting blood glucose level, positive medical history, or current antidiabetic treatment. hyperlipidemia was determined by a fasting total cholesterol level higher than 220 mg / dl, or antilipid agent treatment. coronary artery disease included the presence of an abnormal diagnostic test (stress test or coronary angiography), a history of typical angina or myocardial infarction, or appropriate drug treatment. chf was defined based on symptoms, clinical signs, and/or anti - failure treatment. stroke subtypes were sorted in to 5 classes based on the national institute of neurological disorders and stroke and trial of org 10171 in acute stroke treatment (toast) criteria as follows:1) 1) large artery atherosclerosis included patients with clinical and brain imaging findings of either significant (> 50%) stenosis or occlusion of a major brain artery or cortical artery branch, probably due to atherosclerosis. 2) cardioembolism was defined as patients with cerebral infarction probably due to an embolus originated in the heart. 3) small artery occlusion (lacuna) included patients with classical lacunar syndromes and no evidence of cardiac embolism and significant large artery stenosis. the patient should also have a significant posterior fossa level or hemispheric subcortical lesion with a diameter of less than 1.5 cm on neuroimaging. 4) patients with rare causes of stroke such as nonatherosclerotic vasculopathies, hypercoagulable states, or hematologic disorders were classified as the category of other cause. 5) patients whose cause of a stroke can not be identified with any degree of evidence were labeled as unknown cause. diagnostic evaluations included laboratory assessments, neuroimaging (mri, etc.), electrocardiogram, cardiac imaging (echocardiography, etc.), and duplex ultrasound imaging of extracranial arteries. transthoracic echocardiography was performed for all patients within 30 days of diagnosis of cerebral infarction. studies and measurements were taken based on the guidelines of the american society of echocardiography.15) measurement of left atrial anteroposterior diameter especially was performed according to a leading edge - to - leading edge convention at the aortic valve level. indexed left atrial volume (lavi) was calculated from the left atrial anteroposterior diameter indexed to body surface area. the data collected included continuous variables (clinical and echocardiographic data) and categorical variables (stroke risk factors). differences in baseline characteristics between stroke patients with normal and increased lavi were analyzed by using an unpaired, 2-tailed student 's t - test. differences of stroke risk factors between patients with and without lae in the categorical variable values were evaluated using the chi - square test. null hypotheses of no difference were rejected if p values were less than 0.05, or, equivalently, if the 95% confidence intervals (cis) of risk point estimates excluded 1. logistic regression analysis was employed to determine the correlation of stroke subtypes and increased lavi. all statistical analysis has been performed using spss for windows, version 17.0 (spss inc., the enrolled study population included 182 patients with acute ischemic stroke (mean age : 68.01 12.53 years and 106 males), who were admitted from september 2010 to june 2011 and examined with brain magnetic resonance imaging (mri). routine laboratory tests included complete blood counts, glucose, serum electrolytes, coagulation studies, liver function tests, and lipid profile. systemic hypertension was defined as systolic blood pressure values > 140 mm hg or diastolic blood pressure > 90 mm hg during the interview, or the presence of a medical history or antihypertensive treatment. diabetes mellitus was identified based on of elevated fasting blood glucose level, positive medical history, or current antidiabetic treatment. hyperlipidemia was determined by a fasting total cholesterol level higher than 220 mg / dl, or antilipid agent treatment. coronary artery disease included the presence of an abnormal diagnostic test (stress test or coronary angiography), a history of typical angina or myocardial infarction, or appropriate drug treatment. chf was defined based on symptoms, clinical signs, and/or anti - failure treatment. stroke subtypes were sorted in to 5 classes based on the national institute of neurological disorders and stroke and trial of org 10171 in acute stroke treatment (toast) criteria as follows:1) 1) large artery atherosclerosis included patients with clinical and brain imaging findings of either significant (> 50%) stenosis or occlusion of a major brain artery or cortical artery branch, probably due to atherosclerosis. 2) cardioembolism was defined as patients with cerebral infarction probably due to an embolus originated in the heart. 3) small artery occlusion (lacuna) included patients with classical lacunar syndromes and no evidence of cardiac embolism and significant large artery stenosis. the patient should also have a significant posterior fossa level or hemispheric subcortical lesion with a diameter of less than 1.5 cm on neuroimaging. 4) patients with rare causes of stroke such as nonatherosclerotic vasculopathies, hypercoagulable states, or hematologic disorders were classified as the category of other cause. 5) patients whose cause of a stroke can not be identified with any degree of evidence were labeled as unknown cause. diagnostic evaluations included laboratory assessments, neuroimaging (mri, etc.), electrocardiogram, cardiac imaging (echocardiography, etc.), and duplex ultrasound imaging of extracranial arteries. transthoracic echocardiography was performed for all patients within 30 days of diagnosis of cerebral infarction. studies and measurements were taken based on the guidelines of the american society of echocardiography.15) measurement of left atrial anteroposterior diameter especially was performed according to a leading edge - to - leading edge convention at the aortic valve level. indexed left atrial volume (lavi) was calculated from the left atrial anteroposterior diameter indexed to body surface area. the data collected included continuous variables (clinical and echocardiographic data) and categorical variables (stroke risk factors). differences in baseline characteristics between stroke patients with normal and increased lavi were analyzed by using an unpaired, 2-tailed student 's t - test. differences of stroke risk factors between patients with and without lae in the categorical variable values were evaluated using the chi - square test. null hypotheses of no difference were rejected if p values were less than 0.05, or, equivalently, if the 95% confidence intervals (cis) of risk point estimates excluded 1. logistic regression analysis was employed to determine the correlation of stroke subtypes and increased lavi. all statistical analysis has been performed using spss for windows, version 17.0 (spss inc., one hundred thirty eight (75.8 %) of patients had increased lavi (42.58 17.40 ml / m). patients with normal lavi were 44 (24.2 %) whose mean lavi was 22.09 3.39 ml / m. age and sex were not significantly different between those with increased lavi and normal lavi. patients with increased lavi tend to have more stroke risk factors than patients with normal lavi. patients with increased lavi were significantly different from patients with normal lavi in the prevalence of hypertension (81.2% vs 52.3% ; p = 0.000), history of previous stroke (21% vs 6.8% ; p = 0.031), atrial fibrillation (29.7% vs 4.5% ; p = 0.001), and valvular heart disease including mitral stenosis (17.4% vs 2.3% ; p = 0.011) but not in the prevalence of coronary artery disease, chf and diabetes mellitus. patients with increased lavi commonly had cardioembolic stroke subtype (41, 29.7%) compared with patients with small vessel occlusion (38, 27.5%), large vessel atherosclerosis (36, 26.0%), and unknown (20, 14.4%) subtype (table 2). the proportion of lae in patients with cardioembolic stroke subtype was significantly high, namely, 41 patients had increased lavi while only 4 patients had normal lavi. an increased lavi was associated with a cardioembolic subtype compared with small vessel disease (table 3). an increased lavi risk was present after adjustment for age and sex, and the adjusted odds ratio was 6.749 (p = 0.002) compared with small vessel disease. the major findings of this study were as follows : first, increased lavi is more common in patients who had cardiovascular risk factors, such as hypertension, valvular heart disease, atrial fibrillation, and previous stroke. second, increased lavi influences most patients in all ischemic stroke subtypes, and is especially highly prevalent in the cardioembolic stroke subtype. therefore, increased lavi is more likely a risk factor of ischemic stroke, especially cardioembolic stroke, than normal lavi. there are suggestions that lae is associated with the risk of stroke.5)6)9)11) a recent study in a hypertensive population revealed that a first - ever ischemic stroke was associated with lae, and lavi was the best predictor of stroke in left atrial assessment.11) they enrolled a hypertensive population with or without first ischemic stroke as study population and there were no differences in ischemic heart disease, paroxysmal atrial fibrillation and heart failure between groups. they showed, however, that paroxysmal atrial fibrillation and other stroke risk factors are associated with lae in stroke patients with hypertension. one study reported that for patients with first - ever ischemic stroke in an ethnically diverse population, lae was associated with an elevated risk of cerebral infarction after adjustment for other stroke risk factors in acute ischemic stroke patients with significantly increased mean left atrial anteroposterior diameter and lavi compared with control group.5) another study found that 75% of first ischemic stroke patients have increased lavi.6) ischemic stroke patients with increased lavi were older and had more cardiovascular risk factors than those with normal lavi. the current study has similar results in that patients with increased lavi have more cardiovascular risk factors and previous stroke history. left atrium (la) volume is related to severity and chronicity of diastolic dysfunction in the absence of severe valvular disease and atrial fibrillation.. continuous exposure to abnormal lv filling pressure may result in la remodeling and lae. the cardiovascular health study observed an independent relation of la volume in the patient group with a higher incidence and prevalence of chf compared with control group.8) in a study of elderly subjects with preserved lv systolic function, lavi larger than 32 ml / m was seen to independently predict first chf.14) contrary to those results, this study found no significant difference in the prevalence of chf between patients with and without lae. the subject population of this study was younger than previous studies, in which subjects 65 years of age were enrolled. the mechanisms of the relation between ischemic stroke and an lae are not completely understood. a potential explanation is that the increased hemostasis and thrombus formation might arise as the left atrial size increases. the thrombogenicity of an enlarged la is suggested by the results of several transesophageal echocardiographic studies.10)13) research to determine possible intracardiac sources of cerebral emboli report that lae is the best predictor for either clot in left atrial appendage or spontaneous echo contrast.10) an enlarged atrial size may be the consequence of a raised intra - atrial pressure, which diminishes the flow velocity in the left atrial appendage and therefore increases the thrombogenicity and the embolic risk.13) lae has been reported to be a potent risk factor for the development of atrial fibrillation. the framingham heart study showed that every 5 mm increment in la size raised the risk of atrial fibrillation by 39%.16) the cardiovascular health study noted that lae was an independent risk factor of new onset atrial fibrillation in older adults and la with larger than 0.5 mm diameter had a 4-fold risk of new atrial fibrillation.12) these observations may explain part of the association between stroke and lae. there is potential for intrinsic selection bias in the sample because this study enrolled patients having echocardiography at the time of stroke. the study population has relatively small sample size to represent the general characteristics of ischemic stroke patients fully and there is no proper control group to strengthen the results. further study remains to be performed on a large population and with a normal control group. however, there was a lack of investigations about clinical characteristics and considerations as a stroke risk factor. from the results of this study, lae was found to be a significant risk factor of ischemic stroke, particularly in case of cardioembolic stroke subtype. it may be useful in the estimation of stroke risk and treatment of ischemic stroke in the clinical field.	objectiveincreased atrial size is frequently seen in ischemic stroke patients in clinical practice. there is controversy about whether left atrial enlargement (lae) should be regarded as a risk factor for cerebral infarction. we investigated the association between indexed left atrial volume (lavi) and conventional stroke risk factors as well as stroke subtypes in acute ischemic stroke patients.methodsone hundred eighty two acute cerebral infarction patients were included in this study. brain magnetic resonance imaging and transthoracic echocardiography were done for all patients within 30 days of diagnosis of acute cerebral infarction. echocardiographic lae was identified when lavi was more than 27 ml / m2. stroke subtypes were classified by the trial of org 10171 in acute stroke treatment classification.resultsthere were significant differences between subjects with normal and increased lavi in prevalence of stroke risk factors including atrial fibrillation (p = 0.001), hypertension (p = 0.000), valvular heart disease (p = 0.011) and previous stroke (p = 0.031). an increased lavi was associated with cardioembolic subtype with an adjusted odds ratio was 6.749 (p = 0.002) compared with small vessel disease.conclusionincreased lavi was more prevalent in those who had cardiovascular risk factors, such as atrial fibrillation, hypertension, valvular heart disease and history of previous stroke. lae influenced most patients in all subtypes of ischemic stroke but was most prevalent in the cardioembolic stroke subtype. increased lavi might be a risk factor of cerebral infarction, especially in patients with cardioembolic stroke subtype.
as telehealth services expand nationwide, the department of defense (dod), veterans health administration (vha), and the defense and veterans brain injury center (dvbic) are working to keep pace. through the telehealth initiatives of the dod, vha, and dvbic office of telemedicine, the development and implementation of services two such services are described below, with both providing treatment for military members with a history of traumatic brain injury (tbi) or concussion. through the support of these clinics, we are able to serve active duty service members, as well as veterans who require specialized care. the army s northern regional medical command (nrmc), with clinician support from dvbic, created a tele - tbi clinic to provide specialty support to remote and troop intensive sites. the tele - tbi clinic is comprised of an interdisciplinary team that includes occupational therapy, speech - language pathology, physical therapy, social work, psychology, neuropsychology, neurology, and psychiatry. the nrmc tele - tbi clinic s network with military medical commands allows clients across the northeast united states to receive specialty care closer to home, with improved access. this model is particularly beneficial in areas with both increased traffic congestion and service members residing in geographically distant locations, wherein a simple appointment typically requires an all - day commitment or even an overnight stay. the tele - tbi clinic s network helps to bring care closer to the service members and their families, no matter their location. the james a. haley va hospital (jahvah) in tampa, florida is currently in the collaborative phase to determine protocols for future telerehabilitation programs. jahvah is one of four major polytrauma hospitals serving our active duty military members and veterans. dvbic based its hub for telehealth services at the jahvah due to its ability to provide comprehensive care with state - of - the art equipment and research efforts. for our clients with tbi and concussion, we have programs set up within the hospital to address their needs and assist with re - integration into their units, the community, and the civilian work force. due to the large number of clients seeking services, we hope to better transition and assist them via the use of clinical video telehealth, as well as mobile and web - based applications. in order to provide services within the dod and the vha systems the validation process ensures that providers have the proper education and training to practice and provide care. this credentialing process recognizes licensure from all 50 us states, the district of columbia, and us territories. the dod and vha also have a credentials transfer process for ease of clinician transfer from facility to facility. the existing credentials transfer process also allows for telehealth services to occur from one medical facility to another, as long as the provider is credentialed in both locations (i.e., the provider and the client locations).(1) (2) as we attempt to reach clients closer to home, one challenge we face is that often their home is several hours from the nearest military treatment facility or veterans hospital. the servicemembers telemedicine and e - health portability (step) act was drafted and approved by congress in may, 2011, and is currently awaiting approval from the committee on armed services and the committee on veterans affairs. the original verbiage of the step act is comprised of a mere 387 words which authorizes the secretary of defense (dod) to allow certain licensed health - care professionals to provide care to members of the armed forces at any location and regardless of where the professional of client is located, so long as the practice is within the scope of authorized federal duties.(3) the final language for the step act has not yet been determined. however, we hope that the final version will allow providers to see service members in their homes via telehealth technology, using the home as an extension of the military facility. this would greatly increase access to specialty care for our service members, decrease family burden, and increase family involvement in the service member s recovery process. from our understanding, the step act represents progress toward true inter - state practice, specifically for the treatment of military personnel without the licensure barrier. the step act could allow for improved access to care to a large population of service members who may not be close to specialty care and demonstrate the need for an improved system, country wide. furthermore, the dod s telehealth credentialing model is a hub and spoke model which could be duplicated by state licensure boards. the james a. haley va hospital (jahvah) in tampa, florida is currently in the collaborative phase to determine protocols for future telerehabilitation programs. jahvah is one of four major polytrauma hospitals serving our active duty military members and veterans. dvbic based its hub for telehealth services at the jahvah due to its ability to provide comprehensive care with state - of - the art equipment and research efforts. for our clients with tbi and concussion, we have programs set up within the hospital to address their needs and assist with re - integration into their units, the community, and the civilian work force. due to the large number of clients seeking services, we hope to better transition and assist them via the use of clinical video telehealth, as well as mobile and web - based applications. in order to provide services within the dod and the vha systems, each provider must complete a credentialing and privileging appointment process. the validation process ensures that providers have the proper education and training to practice and provide care. this credentialing process recognizes licensure from all 50 us states, the district of columbia, and us territories. the dod and vha also have a credentials transfer process for ease of clinician transfer from facility to facility. the existing credentials transfer process also allows for telehealth services to occur from one medical facility to another, as long as the provider is credentialed in both locations (i.e., the provider and the client locations).(1) (2) as we attempt to reach clients closer to home, one challenge we face is that often their home is several hours from the nearest military treatment facility or veterans hospital. the servicemembers telemedicine and e - health portability (step) act was drafted and approved by congress in may, 2011, and is currently awaiting approval from the committee on armed services and the committee on veterans affairs. the original verbiage of the step act is comprised of a mere 387 words which authorizes the secretary of defense (dod) to allow certain licensed health - care professionals to provide care to members of the armed forces at any location and regardless of where the professional of client is located, so long as the practice is within the scope of authorized federal duties.(3) the final language for the step act has not yet been determined. however, we hope that the final version will allow providers to see service members in their homes via telehealth technology, using the home as an extension of the military facility. this would greatly increase access to specialty care for our service members, decrease family burden, and increase family involvement in the service member s recovery process. from our understanding, the step act represents progress toward true inter - state practice, specifically for the treatment of military personnel without the licensure barrier. the step act could allow for improved access to care to a large population of service members who may not be close to specialty care and demonstrate the need for an improved system, country wide. furthermore, the dod s telehealth credentialing model is a hub and spoke model which could be duplicated by state licensure boards. while tele - practitioners throughout the united states face the challenges of inter - state licensure barriers, the dod and vha, in collaboration with dvbic, continue to forge ahead with the creation of new telehealth services. their mission is to serve our military members and veterans, first and foremost, through the most comprehensive of services. the views expressed in this article are those solely of the authors and do not express the views of the department of the army, department of defense, department of veterans affairs or the u.s. government.	the telehealth initiatives of the department of defense (dod) and veterans health administration (vha) continue to test the limits of technology to provide the best care to our service members, veterans and their families. the dod and vha have credentialing systems in place to allow clinical practice between facilities. new legislation in the form of the servicemembers telemedicine and e - health portability (step) act will potentially expand telehealth clinical services across state lines into the homes of our service members and veterans.
hepatitis c virus (hcv) is one of the primary causes of chronic liver disease across the world.1 as per the world health organization, this bloodborne pathogen is estimated to infect ~3% of the population.2 the high - level of incidence of hcv makes it one of the few diseases with great economic implications. this infection is recognized as a globally important disease requiring active interventions and extensive measures for treatment and prevention. numerous long - term studies have showed that ~80% of acute infections progress to chronic phase, with 10%20% of these further ending up with life - threatening conditions, such as hepatocellular carcinoma and cirrhosis.35 the prevalence of hcv is the highest in asian and african subcontinents, whereas european, north american, and australian regions have the lowest incidence. middle eastern regions have a low (2.9%) incidence of hcv, with bahrain estimated to have a low incidence.6 hcv (genus : hepacivirus ; family : flaviviridae) is a single strand positive sense rna virus made of 9400 base pairs genome,7,8 encoding ~3000 amino acids. six genotypes (16) of hcv have been identified, which9 further have large number of subtypes. epidemiological studies of hcv are largely based on genotyping as there is a wide geographical variation in the genotypic distribution.10,11 genotypes 13 frequently occur in japan, north america, and europe ; genotype 4 in middle east, egypt, and central africa ; genotype 5 in south africa ; and genotype 6 in asia. these genotypes harbor ~31%34% sequence variation, resulting in ~30% variation in their amino acid products. hence, it is essential to characterize the genotype specific incidence and understand the interrelationship of these genotypes with patient - related factors to employ appropriate and effective preventive and treatment measures. for instance, compared to genotypes 2 and 3, genotypes 1 and 4 are less susceptible to ribavirin and interferon therapy.12 in our previous study, which was a first attempt from bahrain, we showed that genotype 1 was predominant (36.6%) in the patients with hepatitis c, followed by type 3 (18.6%), type 4 (9.8%), and type 2 (5.5%).13 interestingly, contrast to the established predominance of genotype 4 in middle eastern regions including bahrain,14 genotype 1 was most common in our study cohort. hence, in continuation of our previous work, this study was aimed at finding interrelationship of the genotypes with patient characteristics in the cohorts attending our medical center. this study was conducted at medical department of salmaniya medical complex in the kingdom of bahrain. medical records of 122 patients positive for hcv rna test collected between 2013 and 2014 were included. in these subjects, serum was obtained from blood collected by venipuncture and stored at 20c as part of routine diagnosis. for this study, chronic hcv infection was defined as inflammation of the liver caused by hcv lasting 6. this study was reviewed and approved by the salmaniya medical complex institutional review board. due to the retrospective nature of the study, written informed consent only adult patients with positive hcv rna and available medical records were included in the study ; children were excluded. real - time ultrasound was used to evaluate for cirrhosis by demonstrating typical morphological characteristics in contour, texture, and portal collaterals of the liver. fully automated abbott m2000 machine was used along with the manufacturer supplied reagent kits for hcv genotyping and consolidated hcv viral load estimation (abbott molecular, abbott park, il, usa). this assay quantifies hcv rna using in vitro reverse transcription - polymerase chain reaction (pcr) method. this assay has a sensitivity of 12 iu / ml for 0.5 ml and 30 iu / ml for 0.2 ml sample volume with a detection range of 12 iu / ml (log 1.08 iu / ml) to 100 million iu / ml (log 8.0 iu / ml). genotyping was done using standard oligonucleotide specific primers through pcr as described in our earlier report.13 the statistical tests were chosen to understand the interrelationship of the hcv genotypes with patient characteristics. association of continuous variables with genotypes was analyzed through analysis of variance with post hoc evaluation for significant results. all the tests were performed through statistical package for social sciences, version 16 (spss inc., this study was conducted at medical department of salmaniya medical complex in the kingdom of bahrain. medical records of 122 patients positive for hcv rna test collected between 2013 and 2014 were included. in these subjects, serum was obtained from blood collected by venipuncture and stored at 20c as part of routine diagnosis. for this study, chronic hcv infection was defined as inflammation of the liver caused by hcv lasting 6. this study was reviewed and approved by the salmaniya medical complex institutional review board. due to the retrospective nature of the study, written informed consent only adult patients with positive hcv rna and available medical records were included in the study ; children were excluded. real - time ultrasound was used to evaluate for cirrhosis by demonstrating typical morphological characteristics in contour, texture, and portal collaterals of the liver. fully automated abbott m2000 machine was used along with the manufacturer supplied reagent kits for hcv genotyping and consolidated hcv viral load estimation (abbott molecular, abbott park, il, usa). this assay quantifies hcv rna using in vitro reverse transcription - polymerase chain reaction (pcr) method. this assay has a sensitivity of 12 iu / ml for 0.5 ml and 30 iu / ml for 0.2 ml sample volume with a detection range of 12 iu / ml (log 1.08 iu / ml) to 100 million iu / ml (log 8.0 iu / ml). genotyping was done using standard oligonucleotide specific primers through pcr as described in our earlier report.13 the statistical tests were chosen to understand the interrelationship of the hcv genotypes with patient characteristics. association of continuous variables with genotypes was analyzed through analysis of variance with post hoc evaluation for significant results. all the tests were performed through statistical package for social sciences, version 16 (spss inc., of the 122 patients selected, 103 were bahrainis, 18 non - bahrainis, and 1 was unregistered. just more than half of the subjects (57%) were males (table 1). genotype 1 was most common (53%), followed by type 3 (23%) and type 4 (20%). only 1 patient each had genotypes 2 and 5. subtyping, further, showed that type 1a was the predominant (30.33%) one, followed by 1b (22.13%), 4 (18.85%), and 3 (18.03%), with other types detected in very low frequency (table 2). the patients recorded 6 primary symptoms at the time of clinical history taking. abdominal pain (4.1%) was the most common symptom, followed by bleeding (3.4%), fatigue (3.3%), and jaundice (2.5%). none of these symptoms were dependent on specific genotypes or subtypes (all p>0.05). on clinical examination, hepatomegaly was the most common sign (4.9%), followed by splenomegaly (4%), ascites (2.5%), and jaundice (0.8%). both hepatomegaly and splenomegaly were present more frequently in patients with genotype 3 (14.28% ; p=0.032 and 10.71% ; p=0.028, respectively). evaluation for most concomitant and risk factors revealed that 12.2% of the subjects were alcohol users, with 4% positive for human immunodeficiency virus infection and 2.4% were positive for hepatitis b virus infection. there was, however, no correlation between the above risk factor with any of the genotypes. ultrasonography of the hepatic and portal structures demonstrated changes suggestive of cirrhosis in 25.2% of the patients. hepatomegaly was reported in 23.5%, splenomegaly in 12.5%, portal hypertensive changes in 6.7%, and ascites in 3.8%. patients with genotypes 1a and 5a had a higher rate of ascites and cirrhotic changes when compared to those with other genotypes (x=38.29 ; p=0.000 and 23.83 ; p=0.005, respectively). hepatomegaly (x=9.20 ; p=0.419), splenomegaly (x=14.10 ; p=0.119), and portal hypertension (x=4.74 ; p=0.856) did not show statistical significance across genotypes. the mean alanine amino transferase (114.2641.45 u / l ; n=116), alkaline phosphatase (114.2445.86 u / l ; n=116), and gamma - glutamyl - transpeptidase concentration (134.44120.15 u / l ; n=116) were above the normal range ; however, the mean of both albumin (37.385.22 g / l ; n=116) and total bilirubin (14.792.05 mg / dl ; n=116) were normal. the average hcv rna load in the circulating blood of the study cohorts was 1.210 (n=122). the hcv rna data were not normally distributed (anderson darling test ; p<0.01). the hcv rna viral load showed a statistically significant variation across the genotypes (h=12.89 ; p=0.002 ; figure 1a), with a mean rank of 67 for genotype 1, 63 for genotype 3, and 38 for genotype 4. similarly, further analysis also showed that the hcv rna load varied significantly across the subtypes (h=26.50 ; p=0.000 ; figure 1b), with a mean rank of 58 for genotype 3, 36 for type 4, 76 for type 1a, 46 for type 1b, and 76 for type 3a. there have been limited studies on the prevalence of hcv in the kingdom of bahrain. an earlier study had showed that the overall prevalence of hcv was 0.3% in normal population;13 however, this was contradicted by a more recent study that reported a much higher prevalence at a rate of 3.6%.16 the ministry of health in bahrain has recently produced a report that put the prevalence of hcv at 0.99%.17 the genotype distribution in earlier studies was similar to our findings,13 and those reported by janahi.18 the risk factor for transmission remains unknown in 40% of patients ; however, blood transfusion was a major risk factor in the cohorts that we reported in an earlier study.13 one - third of the study cohort were infected with genotype 1a, followed by 1b (22%), 4 (19%), and 3 (18%). while the prevalence pattern for genotypes 1a and 1b is concurrent with previous reports for this region,13,14 the genotypes 3 and 4 showed a difference. our previous report had recorded 26% and 15% each of genotypes 3 and 4. however, in this study these genotypes were detected to have similar occurrence. the symptoms, clinical signs, concomitant risk factors, and the liver function changes in the study cohort were found to follow the established pattern reported by numerous earlier studies.19 no variation was recorded across genotypes. a very important and interesting finding in this study was the significant variation of cirrhosis and ascites rates across the genotypes, unlike the other recorded changes, such as hepatomegaly or splenomegaly. although this is a very interesting finding, the small sample size of this study provides low - level of confidence for generalization of this finding. the main changes in the liver function tests noted in this cohort of patients were of the alanine transaminase (alt), total bilirubin, and gamma - glutamyltransferase (ggt) raised levels. the bilirubin and albumin levels, however, were within the normal range. the findings are not dissimilar to those reported in other studies.20 there were no significant statistical differences in the liver function abnormalities among the different genotypes. it is not uncommon to find disproportionate changes in liver function parameters even though the hcv infection and hepatitis are established. earlier studies have also encountered this important laboratory picture where not all the patients, even after chronic infection with hcv, showed abnormal levels of alt.21,22 silini showed that in a cohort of 341 subjects chronically infected with hcv, 70% of patients had normal alt.21 furthermore, these subjects also did not show alt derangement although chronic hepatitis was confirmed histologically. these findings are important and suggest that the severity or presence of hcv infection can not be solely based on the liver function tests.22 the circulating hcv rna load showed highly significant variation across genotypes. those with genotypes 1 and 4 had on average higher levels of hcv rna load, unlike the patients of genotypes 2 and 3b where significant lower levels were detected in the blood. the significance of both viral genotype and load in relation to the severity of liver disease is unclear. studies have demonstrated normality of both alt and liver histology in a background of high viral load.2325 earlier studies found hcv genotype to be associated with a more severe liver disease, including a higher frequency of cirrhosis and hepatocellular carcinoma. however, many of these studies did not control for important confounding factors, such as age, source, and duration of infection. in studies with adjustment for these variables, the association between genotype 1 b and a more severe liver disease has not been found.23 further studies are warranted in order to better delineate the relationship of the genotype on the outcome of hcv - related liver disease. one interesting application to hcv rna quantification is demonstrated in a recent study that looked at shortening hcv treatment duration. the combination of lidepasvir / sofosbuvir was found to be effective if given for 8 weeks rather than 12 weeks to hcv genotype 1 patients who had a viral load of < 6,000,000 iu / ml.26 the study has also delineated the relationship of hcv rna viral load with the severity of liver disease. this study was conducted to understand the clinical interrelationship of hcv genotypes with patient characteristics in 122 bahraini adult patients positive for hcv rna. genotype 1 was the predominant (53%) one, followed by types 3 (23%) and 4 (20%). although alt, total bilirubin, and albumin levels were increased, ggt and alp levels were normal. about 12% of the subjects were alcohol users, 4% were positive for hiv infection, and 2.4% were positive for hepatitis b virus infection. the circulating hcv rna load was at medium - level in the study cohort and showed significant association with the hcv genotypes and subtypes. patients with genotype 1a had 6 times more load than patients with type 4 (p<0.05). this study reconfirmed the incidence and distribution of different genotypes in bahrain population, and delineated the relationship of hcv rna viral load with the severity of liver disease in our cohort.	aimhepatitis c virus (hcv) shows genotype - based variation in prevalence across geographical regions. this study was conducted to understand the clinical interrelationship of hcv genotypes with patient characteristics.methodsmedical records of 122 patients positive for hcv rna test collected during 2013 and 2014 were included for analysis. only adults were included in the study. hcv rna extraction and genotyping was done as part of the routine diagnostic requirements. the association of continuous and categorical variables with genotypes was analyzed through analysis of variance and chi - square tests, respectively.resultsof the 122 patients selected, 103 were bahrainis, 18 non - bahrainis, and 1 was unregistered. genotype 1 was the predominant (53%) one, followed by types 3 (23%) and 4 (20%). classical symptoms, clinical signs, liver function test, and ultrasonographic results were recorded. cirrhosis and ascites showed significant variation across genotypes. although alanine transaminase, total bilirubin, and albumin levels were increased, gamma - glutamyltransferase and alkaline phosphatase levels were normal. about 12% of the subjects were alcohol users, 4% were positive for hiv infection and 2.4% were positive for hepatitis b virus infection. the circulating hcv rna load was at medium - level in the study cohort and showed significant association with the hcv genotypes and subtypes. patients with genotype 1a had 6 times more load than patients with type 4 (p<0.05).conclusionthis study reconfirmed the incidence and distribution of different genotypes in bahrain population, and delineated the relationship of hcv rna viral load with the severity of liver disease in our cohort.
in various species, including human beings, spermatozoa must undergo a series of biochemical, molecular and functional changes before fertilization. these changes enable spermatozoa to gain hyperactive motility and undergo acrosome reaction to initiate oocyte plasma membrane fusion. capacitation occurs in female genital tract but it can be induced in vitro by media containing a source of metabolic energy such as glucose (2), electrolytes such as ca, nahco3 (3, 4), and serum albumin serving as the cholesterol receptor (5). capacitation includes cholesterol efflux, ion fluxes and increase in membrane fluidity, which causes an increase in tyrosine phosphorylation, induction of hyperactive motility, interaction with zona pellucida and acrosome reaction (6, 7). increase in the level of tyrosine phosphorylation is an important aspect of sperm capacitation during which signal transduction pathways are activated. capacitation is associated with increases in adenyl cyclase activity (8), intracellular cyclic amp (camp) concentration (9) and protein kinase a (pka) activity (10). activation of signal transduction cascade causing induction of tyrosine kinase activity and protein tyrosine phosphorylation occurs uniquely in male germ line (11). protein tyrosine phosphorylation in sperm tail during capacitation has been demonstrated in a number of species (1214). protein tyrosine phosphorylation in sperm tail has been shown to have an important role in sperm hyperactive motility (1215) which is one of the important aspects of capacitation. spermatozoa from asthenospermic males may be compromised in their ability to phosphorylate proteins (5, 15). tyrosine phosphorylation of plasma membrane proteins during capacitation has been reported in boar spermatozoa (16). it has been shown that during capacitation sperm plasma membrane proteins become phosphorylated and play a role in the interaction with zona pellucida (11). teratospermia is a condition in which more than 70% of the sperm in the ejaculates have abnormal morphologies. such abnormal spermatozoa from teratospermic ejaculates have diminished capacitation and acrosomal reaction (18). in addition, protein tyrosine phosphorylation is compromised (19) even in normal appearing spermatozoa from teratospermic ejaculates in comparison with spermatozoa from normospermic cats. thus, the etiology of dysfunctional spermatozoa from teratospermic subjects involves an array of biochemical, molecular and regulatory factors at cellular levels. no reports are available in humans regarding the tyrosine phosphorylation pattern in spermatozoa from teratospermic male. in the present study, we investigated the etiology of compromised sperm capacitation in teratospermic and normospermic groups by analyzing tyrosine phosphorylated proteins before and after capacitation. this study is the first to examine the status of tyrosine phosphorylated proteins in the sperm of terato - spermic individuals. the research project was approved by the bioethics committee of avicenna research institute and informed consents were obtained from the patients attending avicenna infertility clinic in tehran, iran. the staff at andrology department of avicenna iinfertility clinic assessed semen parameters such as sperm count, motility and morphology according to the world health organization (who) manual. after preliminary semen analysis, the remnants of the samples were used for the rest of the study. 2010 spermatozoa / ml, percentage of motile cells > 50%, percentage of viable spermatozoa > 80%, and percentage of spermatozoa with normal morphology > 30%) and 20 teratospermic sperm samples (with sperm concentrations > 2010 spermatozoa / ml, percentage of motile cells > 50%, percentage of viable spermatozoa > 80%, and percentage of spermatozoa with normal morphology 50%, percentage of viable spermatozoa > 80%, and percentage of spermatozoa with normal morphology > 30%) and 20 teratospermic sperm samples (with sperm concentrations > 2010 spermatozoa / ml, percentage of motile cells > 50%, percentage of viable spermatozoa > 80%, and percentage of spermatozoa with normal morphology 70% of spermatozoa are morphologically abnormal). it has been reported that capacitated sperm from teratospermic ejaculates are less able to complete acrosome reaction at the presence of calcium ionophore (27) or progesterone (28) compared to sperm from normospermic ejaculates. sperm capacitation and acrosome reaction have been shown to be compromised in the spermatozoa from teratospermic cats (18). additionally, malformed sperm are not able to penetrate the zona pellucida (29). the etiology of dysfunctional spermatozoa from teratospermic ejaculates involves an array of biochemical, molecular and regulatory factors at cellular levels. tyrosine phosphorylation is one of the key biochemical and molecular factors which is less active in the spermatozoa from teratospermic ejaculates even under capacitating conditions (19). in the present study, the status of tyrosine - phosphorylated proteins in spermatozoa from infertile men with teratospermic condition was evaluated compared with that of the normo - spermic men before and after capacitation. this should advance our knowledge and contribute to a deeper understanding of the basic molecular and cellular mechanisms regulating sperm functions in vitro. to do so, the presence of at least six tyrosine phosphorylated proteins (170, 130, 95, 70, 43 and 25 kilo daltons) was determined in human spermatozoa. second, it was demonstrated that spermatozoa from teratospermic males had a compromised ability to undergo tyrosine phosphorylation following capacitation when compareed with normospermic counterparts. phosphotyrosine containing proteins have been reported to be present in the spermatozoa of numerous species and they are believed to have an important role in sperm functions such as hyper - active motility, interaction with zona pellucida and acrosome reaction. in mouse sperm, anti - phosphotyrosine antibody was used to study capacitation and three proteins with molecular weights of 52kda, 75kda and 95kda were identified. the 95kda protein was reported to have enhanced immunoreactivity after sperm capacitation (30). tyrosine phosphorylation has been demonstrated in the sperm of several mammalian species including humans, rats, rabbit, and mice. in human sperm, four sets of tyrosine - phosphorylated protein with molecular weights ranging form 95/943kda, 463kda, 257kda to 122kda have been reported (26). some of these phosphorylated proteins have been shown to bear an important role in sperm motility (26). in another study two plasma membrane proteins (35kda and 46kda), isolated from capacitated boar sperm cells, were reported to have high binding affinity with zona pellucida (16). in the present study we have demonstrated that tyrosine phosphorylation of several proteins from human spermatozoa are similar to that of mice (30) and boars (16) after capacitation. specifically, tyrosine phosphorylation of six sets of sperm proteins (170kda, 130kda, 95kda, 70kda, 43kda and 25kda) appeared to be compromised in teratospermic samples when compared with normospermic ones. thus, it is proposed that diminished protein tyrosine phos - phorylation may constitute one of the factors responsible for compromised sperm functions in teratospermic males. furthermore, it was demonstrated that tyrosine phosphorylation of the 95kda sperm protein from teratospermic males was compromised which is similar to the findings reported in wild felids (31) and domestic cats (19). this protein is localized to the acrosomal region in the spermatozoa from domestic cats and it is known to interact with zona pellucida protein(s) (19, 31). similar findings in humans and cats indicate that the 95kda protein may play a role in one or more steps associated with sperm - oocyte interaction, which enables zona penetration (19, 31). in human sperm, a 463kda protein is phos - phorylated at tyrosine residues during capacitation (26). more recently, a 46kda protein was reported in boar sperm (16), which was phosphorylated in tyrosine residues during capacitation. this 46kda protein is located in plasma membrane of sperm head and it has been demonstrated to have an important role in interacting with zona pellucid. in this study, it was shown that tyrosine phos - phorylation of a 43kda protein is compromised during capacitation in teratospermic males. the size of this protein is very similar to the protein previously reported in human (26) and boar sperm (16). these observations suggest that this 43kda plasma membrane protein may be involved in the interaction with zona pellucida. for a better understanding of capacitation process in human sperm, it is important to continue studying post - translational modifications of proteins because of their role in sperm functions. to accomplish this goal, studies are underway in our laboratories to identify the tyrosine - phosphorylated proteins that might play a role in the capacitation of sperm. these findings can help the characterization of molecular mechanisms of sperm functions and can further explain the causes of male infertility. tyrosine phosphorylation of sperm proteins may have an important role in the function of sperm and defects in the level of tyrosine - phosphorylated proteins may contribute to low fertilization rates in teratospermic men.	introductionin mammalian system, spermatozoa are not able to fertilize the oocyte immediately upon ejaculation, thus they undergo a series of biochemical and molecular changes which is termed capacitation. during sperm capacitation, signal transduction pathways are activated which lead to protein tyrosine phosphorylation. tyrosine phosphorylated proteins have an important role in sperm capacitation such as hyperactive motility, interaction with zona pellucida and acrosome reaction. evaluation of tyrosine phosphorylation pattern is important for further understanding of molecular mechanisms of fertilization and the etiology of sperm dysfunctions and abnormalities such as teratospermia. the goal of this study is to characterize tyrosine phosphorylation pattern in sperm proteins isolated from normospermic and teratospermic infertile men attending avicenna infertility clinic in tehran.materials and methodssemen samples were collected and the spermatozoa were isolated using percoll gradient centrifugation. then the spermatozoa were incubated up to 6h at 37c with 5% co2 in 3% bovine serum albumin - supplemented ham 's f-10 for capacitation to take place. the total proteins from spermatozoa were extracted and were subjected to sds - page before and after capacitation. to evaluate protein tyrosine phosphorylation pattern, western blotting with specific antibody against phosphorylated tyrosines was performed.resultsthe results upon western blotting showed : 1) at least six protein bands were detected before capacitation in the spermatozoa from normospermic samples. however, comparable levels of tyrosine phosphorylation was not observed in the spermatozoa from teratospermic samples. 2) the intensity of protein tyrosine phosphorylation appears to have been increased during capacitation in the normospermic relative to the teratospermic group.conclusionfor the first time, these findings demonstrate and suggest that the differences in the types of proteins and diminished tyrosine phosphorylation efficiency in sperm from teratospermic men may be responsible for their compromised capacitation and low fertilization success rates.
human papilloma viruses (hpvs) are the most important risk factor of cervical cancers, which is the second most prevalent cancer in females (1). the prevalence of invasive cervix cancer is low in some areas and its low incidence could be attributed to pap smear screening test and hpv dna test (2). human papilloma viruses belong to the large and diverse papillomaviradea family with more than 200 different subtypes, although only 15 types (including 16, 18, 31, 33, 35, 39, 45, 51,52, 56, 58,59, 68, 73 and 82) have been suggested as oncogenes (3). among these types, high - risk hpv strains such as hpv 16 and 18 cause about 70% of cervical cancers. several studies have shown that about 1.3% of 75000 women that died from cervix cancer had a negative pap smear test (4). thus identification of hpv and treatment of this infection could prevent precancerous lesions progressing toward cancer (5 - 7). in vaccination programs it should be documented that immunization against hpv 16 and 18 would be a valuable way to reduce the incidence of cervical cancer, supporting the application of hpv dna testing (3). different methods have been used to detect hpv, such as reverse hybridization and direct sequencing, however application of specific polymerase chain reaction (pcr) seems to be a relatively simple, easy and economical method for the identification of hpv (8 - 13). on the other hand, there are a few studies on the prevalence of papilloma cases and abundance of high - risk types of hpv in squamous cell carcinoma (scc) and dysplasia in iran. however, cervical cancer is still a leading cause of cancer - related death for women in developing countries, including iran, which is mainly because of the lack of hpv program and hpv dna test as a part of regular screening. therefore, the present study sought to determine the abundance of high - risk types of papilloma virus, i.e. subtype 16 and 18, in biopsy samples of cervical cancer patients. overall, 117 paraffin - embedded cervical tissue samples with dysplastic and cancerous lesions of cervix were collected from mirza - kochakkhan - jangali hospital, tehran. tumors were stained for confirmation of cancer tissue and for dna extraction using haematoxylin and eosin staining. informed written consent was obtained from all participates, and the research protocol was approved by the ethics committee of kashan university of medical sciences (no. dna was extracted from all formalin - fix and paraffin - embedded (ffpe) tissue, as described previously (14) and stored at -20c. positive hpv samples were identified by my09 and my11 primers (table 1) (15). genome of hpv was determined using the bio pap kit (biotools b & m labs sa, madrid, spain). presence of 450 bp band indicated that it is generic and its association with the 250 bp band makes it an oncogene (5, 6). moreover, hpv genotyping was performed using the biotypap kit (biotools, b & m labs, sa, spain) for detection of hpv- 67, 68, 6, 11, 13, 16, 17, 30, 69, 39, 40, 42, 64, 66 and 51 to 59. the reaction was done by the enzymatic digestion method (table 2) and the tests were performed using the rflp technique. the positive control was a plasmid dna consisting of some hpv sequences in tris - hcl - edta solution (5, 7). these results showed that 78 samples were positive and 39 samples did nt have any virus (figure 1). the generuler 100 bp dna ladder plus weight (thermo scientific, waltham, ma, usa), was used as the standard for detection of fragments (in base pairs). a, beta globin gene (band size : 468 bp) ; b, 450 bp and 250 bp were detected, suggesting oncogene or generic of the candidate gene ; and c, polymerase chain reaction results from biotaypap kit restriction analysis of the patient infected with hpv 16. lane a, digestion of a pattern ; lane b, digestion of b pattern ; lane c, digestion of c pattern ; lane e, digestion of e pattern ; lane d, digestion of d pattern ; lane nc, negative control ; lane m, molecular marker. overall, 117 paraffin - embedded cervical tissue samples with dysplastic and cancerous lesions of cervix were collected from mirza - kochakkhan - jangali hospital, tehran. tumors were stained for confirmation of cancer tissue and for dna extraction using haematoxylin and eosin staining. informed written consent was obtained from all participates, and the research protocol was approved by the ethics committee of kashan university of medical sciences (no. dna was extracted from all formalin - fix and paraffin - embedded (ffpe) tissue, as described previously (14) and stored at -20c. positive hpv samples were identified by my09 and my11 primers (table 1) (15). genome of hpv was determined using the bio pap kit (biotools b & m labs sa, madrid, spain). presence of 450 bp band indicated that it is generic and its association with the 250 bp band makes it an oncogene (5, 6). moreover, hpv genotyping was performed using the biotypap kit (biotools, b & m labs, sa, spain) for detection of hpv- 67, 68, 6, 11, 13, 16, 17, 30, 69, 39, 40, 42, 64, 66 and 51 to 59. the reaction was done by the enzymatic digestion method (table 2) and the tests were performed using the rflp technique. the positive control was a plasmid dna consisting of some hpv sequences in tris - hcl - edta solution (5, 7). these results showed that 78 samples were positive and 39 samples did nt have any virus (figure 1). the generuler 100 bp dna ladder plus weight (thermo scientific, waltham, ma, usa), was used as the standard for detection of fragments (in base pairs). a, beta globin gene (band size : 468 bp) ; b, 450 bp and 250 bp were detected, suggesting oncogene or generic of the candidate gene ; and c, polymerase chain reaction results from biotaypap kit restriction analysis of the patient infected with hpv 16. lane a, digestion of a pattern ; lane b, digestion of b pattern ; lane c, digestion of c pattern ; lane e, digestion of e pattern ; lane d, digestion of d pattern ; lane nc, negative control ; lane m, molecular marker. the risk factors and demographic characteristics associated with cervical cancer in patients are summarized in table 3. the results of the pap smear showed the presence of malignancy in 71 cases with relative frequency of 0.87 ; 11 cases had an absence of malignancy in their report. among the patients, 26 cases had sexual transmitted diseases with relative frequency of 0.58 (table 3). in order to explore the frequency of high risk hpvs in cervical cancer patients, we determined the presence of more than 20 phenotypes of hpv in patients with squamous cell dysplasia or carcinoma. this analysis showed that 66.66% of samples had oncogene and non - oncogene types of this virus (figure 1 c and table 4), while no hpv was detected in 33.33% of cases. moreover, 23.07% of cases had non - oncogene virus, 31.62% of the subjects had type 18. moreover, 27.35% of cases had type 16, 15.38 had type 33, 4.27% had type 31, 7.69% had type 58, 5.98% had type 52 and 5.12% of cases had type 35 (table 4). the risk factors and demographic characteristics associated with cervical cancer in patients are summarized in table 3. the results of the pap smear showed the presence of malignancy in 71 cases with relative frequency of 0.87 ; 11 cases had an absence of malignancy in their report. among the patients, 26 cases had sexual transmitted diseases with relative frequency of 0.58 (table 3). in order to explore the frequency of high risk hpvs in cervical cancer patients, we determined the presence of more than 20 phenotypes of hpv in patients with squamous cell dysplasia or carcinoma. this analysis showed that 66.66% of samples had oncogene and non - oncogene types of this virus (figure 1 c and table 4), while no hpv was detected in 33.33% of cases. moreover, 23.07% of cases had non - oncogene virus, 31.62% of the subjects had type 18. moreover, 27.35% of cases had type 16, 15.38 had type 33, 4.27% had type 31, 7.69% had type 58, 5.98% had type 52 and 5.12% of cases had type 35 (table 4). to the best of our knowledge this is the first study evaluating the association of 23 different hpvs (including hpv- 67, 68, 6, 11, 13, 16, 17, 30, 69, 39, 40, 42, 64, 66 and 51 to 59) in iranian patients with cervical cancer. 45% of the dysplasia group had an infection with hpv. amongst the different genotypes, the relative frequencies of hpv of 16 and 31 were ranked as the highest and lowest, supporting the hpv dna test for screening and vaccination programs of cancerous and precancerous lesions of cervical cancer. cervix cancer in many countries is considered as an important health issue (16). there is a growing body of evidence showing the impact of particular types of hpv in pathogenesis of cervix dysplasia (17). human papilloma virus infection has been known as a risk factor of cervical cancer (16), although there are a few data about the frequency of specific types and their correlation with dysplasia in iran. therefore, in the present study we investigated the frequency of infection with hpv types in individuals diagnosed with dysplasia and cancer of the cervix. our data showed that more than 63% of patients with squamous - cell carcinoma were under the age of 18 years old. human papilloma virus 18, 32, 26 and 33 were found in 37, 32 and 18 cases, respectively. among the types, the frequency of hpv 16 was high, compared to the other types. in agreement with our observation, farjadian. (18) showed that more than 87% of samples were positive for hpv-16. several other studies have also shown that 94% of cervix cancers and 88% of dysplasia were positive for hpv16 (19). cytological limitations in prediction of cervix cancers have led to the development of more sensitive methods for hpv detection such as pcr (20). in addition, considering the problems of general tests, e.g. pap smear, for detecting wide spectrum of hpv in lower limited detection (19) and also carcinogenesis of dangerous types of virus, differentiation of oncogene types of virus seems to be necessary (9). our results reinforce the rationale for hpv testing in combination with, or even instead of, cytology in a population - based screening program (21, 22). a major strength of the study was that it was performed on a large number of hpv types at different stages of the disease ; however the main limitation of this study was the modest sample size. in conclusion, we demonstrated the significant association of high risk hpvs in cervical cancer patients and showed that patients carrying hpv 16/18 had the highest relative risk, compared to 51, 31 and 33 genotypes, supporting further studies evaluating the role of these types in a larger multi center setting for early detection of high risk types, by using molecular methods at earlier stages. evaluation of the results showed that quality of obtained products of extracted dna from paraffin block cuts was sufficient for the pcr test. considering the efficacy of this protocol, we can routinely perform this test in pathology labs using simple and cheap facilities. in cervix cancer, due to the availability of the tissue, the effects of prevention, early diagnosis and immediate treatment on reduction of mortality rate is evident more than any other cancer. this test, in addition to be used for vaccination after hpv determination can also help with the screening of the population with subclinical infection of the virus. we concluded that the use of a sensitive pcr method in the detection of cervix cancer and determination of genome presence is helpful, plus with this technique we can detect different types of virus in patients and even apparently healthy individuals and this system can also make a major contribution to epidemiology studies of hpv.	backgroundcervical cancer is one of the leading causes of cancer - related death in females. human papilloma virus (hpv) is the major risk factor of cervical cancer.objectivesthe aim of the current study was to explore the frequency and role of 23 different hpvs in patients with cervical cancer.materials and methodsoverall, 117 formalin - fix and paraffin - embedded (ffpe) tissues from cervical cancer patients with squamous cell carcinoma (scc) or dysplasia were collected from mirza - kochakkhan - jangali hospital, tehran, iran during year 2013, to investigate the presence of hpv- hpv- 67, 68, 6, 11, 13, 16, 17, 30, 69, 39, 40, 42, 64, 66 and 51 to 59 genotypes.resultsthe pap smear report illustrated the presence of malignancy in 71 cases, while 11 cases had no evidence of malignancy. among the patients, 26 cases had sexually transmitted disease with relative frequency of 0.58. infection with papilloma virus was observed in 83.6% of scc patients and 45% of the dysplasia group. the most prevalent hpv genotypes were 18 with 31.62% and 16 with 27.35% of cases. moreover the relative frequencies of hpv-33, -6, -58, -52, -35 and -51, genotypes were 15.38, 7.69, 5.98, 5.12 and 3.41%, respectively. among the different genotypes of hpv, 31 had the lowest and 16 had the highest relative frequency.conclusionsour findings demonstrate that hpv-16 and -18 have a higher prevalence in our population than 31 and 51. further investigations are required to evaluate the role of these genotypes in a larger multicenter setting for establishing their values for early detection of patients, which is useful for screening and vaccination programs of cancerous and precancerous lesions of cervical cancer.
cystic lucent lung lesion on chest radiograph is a common finding in children presenting with acute respiratory distress. the prominent differentials for cystic lucent chest lesion on chest radiograph in infants and young children (under 3 years of age) with respiratory distress are post - infective pneumatocele, pneumothorax, congenital diaphragmatic hernia (cdh), congenital cystic adenomatoid malformation (ccam), congenital lobar emphysema (cle), pulmonary sequestration, bronchogenic cyst, and bullous lung disease. congenital cystic lung lesions are relatively rare in incidence and have a varied clinical presentation. majority of the patients usually present in infancy or within the first 3 years of life. on chest radiograph, these patients usually present as cystic lucent lung lesions with or without mass effect. in developing countries, the incidence of chest infection and infection - related complications (pneumothorax and pneumatoceles) is high, and they are considered to be the more common causes of lucent cystic lesions on chest radiograph in patients with respiratory distress. chest tube insertion into congenital cystic lung lesions, mistaking them for an infection - related complication, is a recurring clinical problem which has been reported in the literature as isolated case reports / series and is associated with increased rate of complications in the patient [table 1 ]. the major errors in diagnosis leading to chest tube insertion are due to the inability to differentiate ccam from tension pneumothorax / pneumatocele and cle from a tension pneumothorax. the purpose of the study was to evaluate the imaging findings and complications associated with chest tube insertion in congenital cystic lung lesions and review the role of imaging for appropriate management of these patients. we reviewed the case records of children with congenital cystic lung malformations who were operated at our institution from 1998 to 2011. patients with congenital cystic lung malformation confirmed on imaging (chest radiograph and ct), surgery, and histopathology, and who had an inadvertent chest tube insertion preoperatively were included in the study. we collected information related to clinical presentation, imaging findings on chest radiograph and ct, and operative and histopathology findings. an investigator studied the imaging findings on plain chest radiograph (pa or ap) before and after tube insertion. ct scan was performed with a 40-slice multi - detector computerized tomography (mdct) scanner (philips brilliance) using a routine chest ct protocol for children, with 80-kvp tube voltage and 80-ma tube current. intravenous iodinated contrast medium (low osmolar, non - ionic, 300 mg / ml iodine content) was used routinely at a dose of 2 ml / kg body weight, administered by hand injection. the mean ct dose index volume in our study was found to be 3.0 0.8 mgy. we also studied the ct images for changes in imaging appearance and complications due to chest tube insertion. out of 54 children operated for congenital cystic lung lesion, 15 children (27%) with an inadvertent chest tube insertion preoperatively based on plain radiograph findings were identified. the children were categorized into two groups based on the final diagnosis as ccam and cle. there were 10 cases (66%) of ccam in the series [table 2 ]. respiratory distress was present in all 10 patients at presentation, fever in 3 patients, cough in 3 patients, and cyanosis in 1 patient. a prenatal ultrasound (us) multiseptated cystic hyperlucent lesion was seen in nine cases, while one case showed a single cystic lucency with no septations. the cyst size was uniformly the same in two cases, while in seven cases there were variable - sized cystic lucencies with a single large dominant cystic lucency. in seven cases, the cystic hyperlucent lesion was seen localized to a lung segment, while in three cases it was seen to involve the whole lung field. mass effect with resultant mediastinal shift was seen in seven cases, while three cases showed cystic lucent lesion with no mass effect. following chest tube insertion, respiratory distress increased in all 10 patients and a ct scan was performed to rule out complications. worsening of respiratory distress was determined based on clinical notes ; in some cases, objective measures (increased oxygen requirements) were available. summary of cases with complications on ct, multiple thin - walled cystic lesions involving an entire lobe of lung were seen. multiseptated cystic lesions with single dominant cyst were seen in nine cases, while single air - filled cystic lesion was seen in one case. the diagnosis was type 1 ccam in nine cases and type 2 ccam in one case. the affected lung segment showed no normal intervening lung parenchyma between the cysts in any of the cases. of the three cases in which the cystic lucency was seen involving the whole lung field on plain radiograph, ct showed the lesion to lie in the left lower lobe in two cases and in the right lower lobe in one case. one of the cases who did not have mass effect on initial chest radiograph showed mediastinal shift on ct following chest tube insertion. due to chest tube insertion, moderate hydropneumothorax and pneumothorax were seen in two cases each, resulting in some volume loss of the surrounding lung. pleural effusion was noted in one case and subcutaneous emphysema in two cases. in four cases, air fluid levels and consolidation developed following chest tube insertion, which was not present on the initial radiograph, which suggested the development of secondary infection. all 10 patients underwent lobectomy and diagnosis was confirmed at histopathology. as a result of the complications due to chest tube insertion, adhesions there were five cases of cle misdiagnosed as pneumothorax on initial plain radiograph with resultant chest tube insertion in the study. a prenatal us study had not been performed in any of the patients in the study. all patients had dyspnea at presentation, but only three patients were in severe respiratory distress. chest radiograph was diagnostic in four cases, showing hyperinflated lung field herniating across the midline with associated mediastinal shift. faint vessel markings were seen in all cases within the hyperlucent lung field. in one case, however, the hyperinflated lung field was not obvious on the initial radiograph because of prior chest tube insertion. due to non - improvement of patient 's condition, in all five cases, the diagnosis was mistaken as tension pneumothorax on initial chest radiograph with resultant chest tube insertion. due to worsening of the patient 's dyspnea following tube insertion, ct scan was performed. ct was diagnostic in four cases of cle, showing hyperinflated affected lobe with attenuated vascular markings and herniation of lung across the midline with mediastinal shift. in one case of cle, multiple chest tube insertions resulted in consolidation, pulmonary hemorrhage, pneumothorax, and led to loss of typical diagnostic hyperinflated lung segment on ct. this made it difficult to make a definite diagnosis of cle on the initial ct. however, despite the chest tube insertion, the affected lung segment looked mildly hyperlucent compared to the surrounding lung and a possibility of cle was suggested. a repeat ct of the patient 3 months later showed the classical hyperinflated lobe suggestive of cle. due to chest tube insertion, subcutaneous emphysema was seen in three patients and moderate pneumothorax in three patients resulting in minimal volume loss of the adjacent lung. there were 10 cases (66%) of ccam in the series [table 2 ]. respiratory distress was present in all 10 patients at presentation, fever in 3 patients, cough in 3 patients, and cyanosis in 1 patient. a prenatal ultrasound (us) multiseptated cystic hyperlucent lesion was seen in nine cases, while one case showed a single cystic lucency with no septations. the cyst size was uniformly the same in two cases, while in seven cases there were variable - sized cystic lucencies with a single large dominant cystic lucency. in seven cases, the cystic hyperlucent lesion was seen localized to a lung segment, while in three cases it was seen to involve the whole lung field. mass effect with resultant mediastinal shift was seen in seven cases, while three cases showed cystic lucent lesion with no mass effect. following chest tube insertion, respiratory distress increased in all 10 patients and a ct scan was performed to rule out complications. worsening of respiratory distress was determined based on clinical notes ; in some cases, objective measures (increased oxygen requirements) were available. summary of cases with complications on ct, multiple thin - walled cystic lesions involving an entire lobe of lung were seen. multiseptated cystic lesions with single dominant cyst were seen in nine cases, while single air - filled cystic lesion was seen in one case. the diagnosis was type 1 ccam in nine cases and type 2 ccam in one case. the affected lung segment showed no normal intervening lung parenchyma between the cysts in any of the cases. of the three cases in which the cystic lucency was seen involving the whole lung field on plain radiograph, ct showed the lesion to lie in the left lower lobe in two cases and in the right lower lobe in one case. one of the cases who did not have mass effect on initial chest radiograph showed mediastinal shift on ct following chest tube insertion. due to chest tube insertion, moderate hydropneumothorax and pneumothorax were seen in two cases each, resulting in some volume loss of the surrounding lung. pleural effusion was noted in one case and subcutaneous emphysema in two cases. in four cases, air fluid levels and consolidation developed following chest tube insertion, which was not present on the initial radiograph, which suggested the development of secondary infection. all 10 patients underwent lobectomy and diagnosis was confirmed at histopathology. as a result of the complications due to chest tube insertion, adhesions there were five cases of cle misdiagnosed as pneumothorax on initial plain radiograph with resultant chest tube insertion in the study. a prenatal us study had not been performed in any of the patients in the study. all patients had dyspnea at presentation, but only three patients were in severe respiratory distress. chest radiograph was diagnostic in four cases, showing hyperinflated lung field herniating across the midline with associated mediastinal shift. faint vessel markings were seen in all cases within the hyperlucent lung field. in one case, however, the hyperinflated lung field was not obvious on the initial radiograph because of prior chest tube insertion. due to non - improvement of patient 's condition, in all five cases, the diagnosis was mistaken as tension pneumothorax on initial chest radiograph with resultant chest tube insertion. due to worsening of the patient 's dyspnea following tube insertion ct was diagnostic in four cases of cle, showing hyperinflated affected lobe with attenuated vascular markings and herniation of lung across the midline with mediastinal shift. in one case of cle, multiple chest tube insertions resulted in consolidation, pulmonary hemorrhage, pneumothorax, and led to loss of typical diagnostic hyperinflated lung segment on ct. this made it difficult to make a definite diagnosis of cle on the initial ct. however, despite the chest tube insertion, the affected lung segment looked mildly hyperlucent compared to the surrounding lung and a possibility of cle was suggested. a repeat ct of the patient 3 months later showed the classical hyperinflated lobe suggestive of cle. due to chest tube insertion, subcutaneous emphysema was seen in three patients and moderate pneumothorax in three patients resulting in minimal volume loss of the adjacent lung. ccam is a developmental abnormality of lung in which there is abnormal adenomatoid proliferation of bronchial structures as cysts rather than normal alveoli and is limited to a single lobe in the majority of cases. these abnormalities usually present in neonates and infants, but are also known to occur in adults. classified ccam into three types depending upon the size of cysts contained in the lesion. the appearance of the lesion on chest radiograph and ct depends upon the number, size, and amount of fluid within the lesion. on plain radiograph, there are multiple isolated reports of chest tube insertion in ccam, after mistaking it for complicated pneumatocele and tension pneumothorax. in our study, ccam was mistaken as pneumatocele in five cases and pneumothorax in five cases on initial chest radiograph, resulting in chest tube insertion. in seven cases, cystic lucency was localized to a part of the lung field either in the upper or lower lobe, while in three cases of ccam involving the lower lobes, it was seen to extend over the entire lung field [figure 1 ]. thus, ccam involving the lower lobes might resemble a pneumothorax by showing lucency across the entire lung field ; however, the presence of septations within the cystic lucent lesion, presence of vascular markings, and relative lack of lucency in the uninvolved apical region point toward a diagnosis of ccam. a multiseptated cystic lucent lesion was seen in nine cases, while single cystic lucency was noted in only one case. thus, the presence of septations within a cystic lucent lesion should suggest a possibility of ccam. mass effect and the resultant mediastinal shift were seen in the majority of our cases. a 3-month - old infant with respiratory distress. (a) chest radiograph shows cystic hyperlucent left lung field with septations (arrow) and mass effect. (b) ct chest axial section with lung window shows a thin - walled cyst in left lower lobe with mass effect. diagnosis was confirmed after surgery as type 1 ccam as noted in our study, ct was diagnostic in all cases, and helped in correctly localizing the lesion, detecting the position of chest tube, and demonstration of complications due to chest tube insertion. the complications noted in our series were pneumothorax, hydropneumothorax, pleural effusion, infection, and subcutaneous emphysema [figures 2 - 4 ] [table 2 ]. in addition, tube insertion resulted in the formation of adhesions around the cyst due to infection which caused difficulty during surgical excision. (a) chest radiograph shows a multiseptated cystic lucency (black arrow) in the right lung field with mediastinal shift. a chest tube (white arrow) (b) ct chest axial section with lung window shows multiple thin - walled cysts in the right upper lobe. diagnosis was confirmed after surgery as type 1 ccam ct chest lung window axial sections show (a) chest tube insertion (black arrow) in a multiseptated cystic lesion in the right upper lobe, (b) resulting in the formation of hydropneumothorax (white arrow). (c) coronal image shows the chest tube (black arrow) in the multicystic lesion and the resultant pneumothorax (white arrow). diagnosis was confirmed after surgery as type 1 ccam (a) chest radiograph shows cystic lucency involving the whole of the right lung field with chest tube insertion and subcutaneous emphysema. (b) chest ct axial section with lung window shows multiple thin - walled cystic lesions showing air fluid level (black arrow) and pneumothorax (white arrow). diagnosis was confirmed after surgery as type 1 ccam in our series, 50% cases of ccam were misdiagnosed for complicated pneumatoceles. pneumatoceles are post - infectious thin - walled collections of air within the lung parenchyma, commonly seen in infants and children up to 3 years of age, and are generally known to appear during the resolving stages of staphylococcal and streptococcal pneumonitis. noted that pneumatoceles may be seen on the first chest radiograph in 80% of the cases and that up to 90% of the pneumatoceles show spontaneous resolution by 3 months. the imaging findings of ccam may be indistinguishable from those of pneumatoceles in some cases on chest radiograph and ct. the presence of normal lung parenchyma between the cysts may be an important ct criterion to differentiate a pneumatocele from a ccam in which the entire affected part of lung parenchyma is abnormal. although in developing countries pneumothorax / pneumatocele may be more common causes of cystic chest lucency, a possibility of a congenital cystic malformation must be always be kept in mind, especially in children less than 3 years of age, when multiple cystic lucencies are seen in a localized area with no surrounding consolidation. there is paucity of literature regarding the ability of plain radiograph and ct to correctly differentiate ccam from pneumatoceles, and further studies may be required. chest tube insertion should be avoided in congenital cystic lung lesions, and a prior ct is advocated if atypical findings are found on the chest radiograph. direct chest tube insertion is recommended only in extreme cases with severe respiratory distress and significant mediastinal shift which may decompress a potential pneumothorax, tension pneumatocele, or a large ccam. also, as noted in our study, no increased mortality was seen due to chest tube insertion. cle is a rare congenital lung anomaly characterized by postnatal overexpansion of one or more lobes of a histologically normal lung. the most common lobes affected are left upper lobe followed by right middle and right upper lobes. the left upper lobe (four cases) was the most commonly affected lobe in our study. majority of the patients present in the immediate neonatal period ; however, presentation in late infancy and early childhood is well known. on chest radiograph, a hyperlucent, hyperexpanded affected lung field is seen that shows herniation across the midline and mediastinal shift. this radiographic appearance can be mistaken for a pneumothorax / tension pneumothorax with resultant chest tube insertion, and this problem has been widely reported in the literature. chest tube insertion in cle worsens the patient 's condition by producing pneumothorax, subcutaneous emphysema, and increase in mediastinal shift, and may result in delay in diagnosis as was noted in our study. careful observation of the chest radiographs in cle will demonstrate scant lung markings within the hyperlucent area, which will not be seen in case of a pneumothorax [figure 5 ]. this finding is, however, subject to interpretive error as evidenced by multiple case reports reporting this problem. in addition, in cle compression of the adjacent lobes pushes them cephalad or caudal toward the diaphragm, while in cases of pneumothorax, the lung collapses toward the hilum. ct is diagnostic in cle, and is recommended for confirmation for diagnosis and for delineation of the affected lobe preoperatively. on ct, hyperinflated lobe with attenuated and displaced pulmonary vessels is a typical appearance of cle. however, as seen in one of our cases, with multiple chest tube insertions this typical imaging appearance may be lost making a definitive diagnosis difficult even at ct [figure 6 ]. (a) chest radiograph showing hyperlucent left lung field herniating across the midline with preserved vascular markings. (b) ct axial section lung window shows a hyperinflated left lung segment with attenuated lung markings. (c) peroperative image shows the hyperinflated left upper lobe (black arrow) in comparison to the normally aerated left lower lobe (white arrow). diagnosis after surgery confirmed this as a left upper lobe cle a 7-month - old child with multiple chest tube insertions due to clinical suspicion of pneumothorax. chest ct images lung window axial and coronal views (a - c) show a slightly hyperinflated left upper lobe with a pneumothorax due to multiple chest tube insertions (black and white arrows). however, on comparison with adjacent lung segments, the hyperinflation is not obvious for definitive diagnosis of cle. (d) follow - up ct 3 months later shows hyperinflated left upper lobe with attenuated vascular markings, diagnostic for cle. diagnosis was confirmed after surgery chest radiograph is an important initial investigation in the evaluation of cystic lucent lung lesions. appropriate differentials may be considered by noting the size, position, location, number, and evolution of cysts, the age of patient, and by comparison with previous chest radiographs. detailed chest radiograph evaluation of the lung borders, bronchovascular markings, mediastinal shift, septations within cysts, consolidation surrounding the cystic lucency, position of collapsed lobes, diaphragmatic margins, and review of previous x - rays in an emergency setting is vital for correct diagnosis. however, the imaging findings of ccam may be indistinguishable from those of pneumatoceles in some cases on chest radiograph. us is a sensitive modality for the detection of pneumothorax. the absence of comet tail artifacts and sliding lung sign is indicative of air in the pleural cavity, and us may be used as an initial screening modality in differentiating a cle from a tension pneumothorax. ccam may be demonstrated on us as multiple cystic anechoic to homogenously solid hyperechoic lesions within the lung parenchyma. however, differentiation of large cystic ccam from pneumatocele and pneumothorax may be difficult on us and further studies are required. chest ct has a definite role in the management of children with respiratory distress and cystic lucent lung lesion on chest radiograph, as was noted in our study. ct will help in confirming the diagnosis and provides vital information for operative and image - guided intervention. it is only recommended when the imaging findings are atypical and respiratory distress does not improve or worsens following chest tube insertion, which would not be the case in a simple pneumothorax. role of ct in such cases would be to diagnose alternate conditions such as large ccam / cle, which may mimic pneumothorax / pneumatocele on chest radiograph. in addition, it may help in detecting complications associated with chest tube insertion, which can help explain patient 's symptoms. due to the retrospective nature and criteria for inclusion for the study, we could not opine in how many cases chest ct would have prevented a chest tube insertion. however, in all cases ct provided a diagnosis of congenital lung abnormality and this resulted in removal of the chest tube. thus, this made us conclude that a prior ct would have prevented a chest tube insertion. a potential limitation of our study was that we did not include cases of complicated pneumatocele for imaging comparison. also, we did not have ct scans of patients before chest tube insertion and it was assumed that the complications were a result of the chest tube insertion based on clinical and imaging findings on initial chest radiograph. due to the retrospective nature of our study over a long duration, the exact details of the training level of the primary reader of the chest radiograph were not available. in addition, the criteria for inclusion in our study (chest tube insertion in patients with congenital lung lesions) probably resulted in inclusion of cases with relatively atypical findings on chest radiograph. chest tube insertion in congenital cystic lung lesions worsens the respiratory distress and increases the rate of complications like pneumothorax, hydropneumothorax, infection, and subcutaneous emphysema. it may also result in change in imaging findings, resulting in delay in diagnosis. chest ct has an important role in confirming the diagnosis, detecting the complications, and deciding appropriate therapy.	background : chest tube insertion in congenital cystic lung lesions is an important problem in children with acute respiratory distress having a cystic lucent lesion on chest radiograph.objective:to evaluate the imaging findings and complications in cases of congenital cystic lung lesions with chest tube insertion and suggest the role of appropriate imaging for management of these patients.materials and methods : chest radiographs and ct scans of children with congenital cystic lung lesions who had inadvertent chest tube insertion preoperatively were retrospectively reviewed for imaging appearances and complications.results:fifteen patients comprising 10 cases of congenital cystic adenomatoid malformation (ccam) and 5 cases of congenital lobar emphysema (cle) were included. majority of the cases were infants. ccam was misdiagnosed as complicated pneumatocele (n = 5) and pneumothorax (n = 5), while cle was misdiagnosed as tension pneumothorax (n = 5) on the chest radiograph findings. final diagnosis was made on ct and operative findings with histopathology. complications noted were pneumothorax, hydropneumothorax, and infection in cases of ccam, and change in imaging appearance and pneumothorax in cases of cle.conclusion:chest tube insertion in congenital cystic lesions increases the rate of associated complications. chest ct has a definite role in early diagnosis and deciding appropriate management in these cases.
	predicting the molecular complexity of a genomic sequencing library has emerged as a critical but difficult problem in modern applications of genome sequencing. available methods to determine either how deeply to sequence, or predict the benefits of additional sequencing, are almost completely lacking. we introduce an empirical bayesian method to implicitly model any source of bias and accurately characterize the molecular complexity of a dna sample or library in almost any sequencing application.
genome - wide linkage studies, the traditional approach to identifying risk loci, provide chromosomal locations for risk - influencing loci based on the observation of coinheritance of marker alleles and the disease trait in families. to be comprehensive, this approach has been used for cocaine, opioid, and nicotine dependence, and for related traits. we are aware of only one linkage study of cocaine dependence (cd) ; we studied a sample of small families each with at least one subject affected with cd, which included 528 full and 155 half sibpairs and was 45.5% european - american (ea) and 54.5% africanamerican (aa). we completed an autosomal genomewide linkage scan for the cd diagnosis, cocaine - induced paranoia, and cocaine - related subphenotypes derived using cluster analytic methods. the subtyping procedure was used to identify more genetically homogeneous subgroups of subjects in which the effects of individual risk loci might be more prominent. for cd, we found suggestive linkage signals on chromosome 10, in the full sample, and on chromosome 3, in the ea part of the sample. much stronger results were obtained for the cluster - derived subtypes, including genome - widesignificant lod scores for membership in the heavy use, cocaine predominant cluster on chromosome 12 and for membership in the moderate cocaine and opioid abuse only, we observed a genome -wide - significant lod score on chromosome 9 for the trait of cocaine - induced paranoia. genome -wide significance was defined on the basis of lander and kruglyak 's 1995 criteria. there have been three independent genome -wide linkage studies of opioid dependence (od). we completed a genome -wide linkage scan for dsm - iv od, and, as for the cd study, for clusterdefined phenotypes, a heavy - opioid - use cluster, and a non - opioid - using cluster. the strongest results were, again, seen with the cluster - defined traits : for the heavy opioid users cluster there was a genome - widesignificant linkage for ea and aa subjects combined, on chromosome 17. for the nonopioid users cluster, there was a genome -wide - significant linkage elsewhere on chromosome 17, for ea subjects only. lachman studied a mixed us sample of 305 od - affected sibling pairs, and identified evidence for linkage on a region of chromosome 14 overlying the neurexin 3 gene (nrxn3). finally, glatt studied a sample of nearly 400 independent affected sibling pairs ascertained in china near the golden triangle, one of asia 's largest illicit opium - producing areas, but did not identify any strongly -supported linkage signals, despite the presumed genetic homogeneity of the sample. the strongest signal they observed was on chromosome 4q. there have been numerous genome -wide linkage scans for smoking and related phenotypes, reviewed in ref 20. completed genome scan meta - analysis (gsma) of genome -wide linkage scans for nicotine dependence (nd) and related traits, pooling all available independent genome scan results on smoking behavior. to minimize locus heterogeneity, subgroup analyses of the smoking behavior assessed by the fagerstrom test for nicotine dependence (ftnd) and maximum number of cigarettes smoked in a 24-hour period (maxcigs24) were also carried out. fifteen genome scan results were available for analysis, including 10 253 subjects in 3 404 families. the primary gsma across all smoking behavior identified a genome -wide suggestive linkage in chromosome 17q24.3-q25.3. but the strongest result derived from the subgroup analysis of maxcigs24 (including 966 families with 3 273 subjects), which identified a genome - wide significant linkage in 20q13.12-q13.32. chrna4, a strongly supported nd candidate gene, is located in this interval ; li previously reported on association of chrna4 variants to nd. a high level of statistical support for a genetic linkage is very valuable, but the ultimate proof that a disease - influencing locus underlies a statistical linkage peak is the identification of a risk gene in the peak that accounts for the linkage signal. the next step is typically genetic association analysis, ie, evaluation of a set of markers that map under the linkage peak for association with the trait. genetic association provides another degree of statistical evidence, but eventually, proof of a disease - gene relationship must rely on demonstration of a functional effect of a variant or variants at the risk locus. nd is the furthest of all drug - dependence (dd) traits along this pathway, with numerous loci supported on the basis of statistical genetic association evidence, and some of these loci have received the higher level of support of functional data. strategy for single - nucleotide polymorphism (snp) selection plays a key role in association study outcome. in general, variants predicted to have functional consequences, eg, because they alter predicted amino acid sequence, have been favored for study ; alternatively, researchers often try to capture most of the genetic variation at a locus via selection of haplotype tagging snps followed by haplotype reconstruction. it is important to recognize some of the limitations of these strategies at the outset. although most common putatively functional snps are known, rarer snps may have large phenotypic effects, and there are many such variants yet to be discovered. snps vary by population, and populations differ in the extent to which common genetic variation has been identified. the same population variation is reflected in differences in haplotype structure. finally, haplotype reconstruction is almost always accomplished via computer algorithms, and the results are estimated. with these limitations in mind, we discuss several examples of genetic associations with dd phenotypes, focusing on interesting physiological candidates and on replicated findings. association of variants that map at or near the d2 dopamine receptor (drd2 locus) with drug or alcohol dependence was proposed many years ago and has been widely debated. we identified a suggestive linkage peak for nd at the region of chromosome 11 that includes the ncam1-ttc12-ankk1-drd2 gene cluster. the inconsistent results with drd2 may be attributable to an indirect effect - observed association could actually be mediated through variation at a nearby locus in linkage disequilibrium with drd2. to test this hypothesis, we genotyped 43 snp markers in a region including drd2 and the three adjacent genes, in an sd linkage sample of > 1600 subjects. we found very strong evidence of association of multiple snps at ttc12 and ankk1 in two different populations, eas and aas (minimal p=0.0007 in aas and minimal p=0.00009 in eas), and highly significant association of a single haplotype (set of markers) spanning ttc12 and ankk1 to nd in the pooled sample (p=0. thus, a risk locus for nd maps to a region that spans ttc12 and ankk1. the exact localization of the risk haplotype depends on the disease definition, and whether and which co - occurring diagnoses are present in the study sample. these results support the hypothesis that the drd2 findings could be attributable to variants in nearby loci. such variants could reflect either functional variation that affect those loci (and not drd2), or relatively distant regulatory regions important for drd2 function. another set of risk loci that are of interest in relation to the risk of drug dependence are those encoding proteins that regulate or mediate opioidergic function. all of the opioid receptor genes have been reported to be associated with substance dependence liability. a functional polymorphism in oprm1 (asn40 asp), which encodes the mu - opioid receptor, has been the most extensively studied in this regard, though the association is controversial. although multiple studies have shown a significant allelic association with dd, they are nearly evenly divided between those showing a significant excess of the asp40 allele among cases and those showing a significant excess of the asp40 allele among controls. consequently, meta - analyses of that literature failed to show a reliable association of the snp with either od or any sd disorder. however, zhang examined 13 snps spanning the coding region of oprm1 in a sample of eas with ad and/or dd and 338 ea healthy controls. there were significant differences between cases and controls in allele and/or genotype frequencies for snps in block i and in block ii, after correction for multiple testing. haplotypes constructed from five tag snps differed significantly in frequency between both ad and dd subjects and controls. logistic regression analyses in which the sex and age of subjects and alleles, genotypes, haplotypes, or diplotypes of the five tag snps were considered confirmed the association between oprm1 variants and sd. zhang also examined the genes encoding the other two opioidergic receptors : oprd1 (which encodes the delta receptor) and oprk1 (which encodes the kappa receptor). eleven snps spanning oprd1 were examined in eas with ad, cd, and/or od, and control subjects. although nominally significant associations were observed for five snps with sd, only the association of the nonsynonymous variant g80 t with od remained significant after correction for multiple testing. haplotype analyses with six tag snps indicated that a specific haplotype was significantly associated with ad and od (p 30 000 snps selected from the first stage were screened individually in ~1000 each cases and controls. numerous genes were identified as possibly associated to nd, including both novel genes and genes that were previously considered candidates based on known physiology (eg, cholinergic receptor, nicotinic, beta 3, chrnb3). the latter finding has been confirmed in larger studies : subsequent gwass have demonstrated highly significant associations between variation in the nicotinic receptor gene cluster chrna5-chrna3-chrnb4 and nd and related traits and with lung cancer. in a hypothesis - generating study, we studied a set of 5633 snp markers in 1699 subjects from 339 aa families and 334 ea families ascertained through a sib pair meeting dsm - iv criteria for either cd or od. it is expected to interrogate < 10% of the genome, thus, can not be considered to be a study of truly genome -wide depth. associations between these markers and five substance dependence traits (cd, od, ad, nd, and cocaine - induced paranoia) were assessed by family -based association tests (fbat). the top - ranked result was an association of a specific snp in the manea gene with cocaine - induced paranoia. this study provided an initial sd trait - specific blueprint of associated regions for future candidate gene studies. there are, at the time of this writing, no published gwas studies for several of these traits. we identify two main ways to account for the relatively consistent results seen in this field. second, the phenotypes are relatively straightforward because they are, in their essence, pharmacogenetic. that is, sd phenotypes reflect genetic moderation of the subjective response to drugs of abuse. while results in this research field have been relatively consistent, most of the genetic risk for dd has yet to be attributed to specific alleles. initially, it was thought that the gwas was the answer to the problem. but application in other complex traits (eg, schizophrenia, bipolar affective disorder, autism) has revealed a more complex picture, such that even clinical samples that should have been adequately powered have fallen short of providing definitive and significant results. the explanation for this situation may reside in the fundamental genetic architecture of some complex traits. gwas is based on a common - disease - influenced - by - common - allele model. however, we are now learning that many phenotypes are influenced instead by sets of variants, in sets of loci, each of which is rare on a population level. such variants are likely to be uncovered only by extensive sequencing of affected and unaffected individuals. copy number variation (cnv) is another mechanism that is proving to be important in modulating disease risk. such variation is important for at least some behavioral traits ; for example, sebat have reported on the relationship of cnv to autism, and several groups have reported association of rare structural variants with schizophrenia. we have seen several successful examples of genetic association identified following a linkage finding, a sequence that demonstrates the main utility of genetic linkage. but there have also been surprisingly many instances when strong genetic association has not been identified readily. there are many ways to account for such a circumstance - genetic heterogeneity, random variation, and population variation, to name a few. the linkage - to - association - to - gene model is premised basically on the common disease - common variant model discussed above. this model may not be as applicable as was thought ; there is increasing evidence that heritability may be accounted for by many rare variants in either a single locus, or a set of related loci. since linkage depends on the identification of coinheritance of trait and marker within families, it stands to reason that a set of different rare variants could be detected by linkage (even if the responsible variants differed greatly between families in the discovery set). similarly, such variants would be expected to be refractory to discovery by gwas methodology. for example nejentsev found a set of individually rare variants at the ifih1 locus that affect risk for type 1 diabetes, following up on a gwas study. ji started with a set of genes known to have large effects on blood pressure in a small number of severely affected families, and sequenced them in a large number of unrelated individuals. these findings are likely to be relevant for sd genetics research as well, inasmuch as deep sequencing of candidate loci in many unrelated individuals may be necessary to account for a greater proportion of the genetic risk than is presently known. whole - genome sequencing is becoming progressively less expensive, and will surely ultimately be feasible for locating genetic variants that increase risk for complex genetic traits, albeit at the risk of daunting statistical problems. sequencing of expressed sequences only (' whole exome) may be a valuable interim step. new developments in a variety of genetic methods and in the accumulating molecular evidence of the genetic risk for sd promise to yield greater insights into the etiology of these disorders, bringing into relief the environmental contributions and creating opportunities for prevention and new therapeutic options.	drug - dependence disorders (we focus here on cocaine, opioid, and nicotine dependence) are genetically influenced. risk genes have been located based primarily on genetic linkage studies, and identified primarily based on genetic association studies. in this article we review salient results from linkage, association, and genome - wide association study methodologies, and discuss future prospects for risk allele identification based on these, and on newer, methodologies. although considerable progress has been made, it is likely that the application of more extensive sequencing than has previously been practical will be required to identify a fuller range of risk variants.
our society is entering an era of aging with increasing life expectancies owing to the remarkable advancement of medical science. an aging society means the ratio of elderly people over 65 years old comprises more than 7% of the overall population. aging is an inevitable and natural process of life, but with increases in the aged population, social concerns of aging and anti - aging are greater than ever. all body organs show dysfunction and reduced capacity with aging (kosmadaki and gilchrest, 2004). in skin, aged phenotypes which are commonly characterized as increased wrinkling, sagging, and laxity are more apparent than in other internal organs. since skin has a physiological function as a barrier for protection against environmental damages but also serves as a social interface between an individual and society (robert, 2009), the interests and efforts for preventing skin aging are growing rapidly in the dermatology field and cosmetic industry. another feature of skin aging is that the skin suffers both intrinsic and extrinsic aging (fisher, 2002) whereas internal body organs only undergo intrinsic aging. intrinsic skin aging is an innate aging process resulted from the passage of time and is thus called chronological aging. it is generally determined genetically and has been thought to begin upon the onset of switch genes at a particular time according to a biologic clock programmed in the body (uitto, 1989 ; kosmadaki and gilchrest, 2004). the tissue function of the skin deteriorates by intrinsic aging similar to internal body organs but the changes of cutaneous aging are easily recognized by clinical manifestations including fine wrinkling, loss of elasticity, and increased fragility (gilchrest, 1982). on the other hand, it can arise from external factors such as sun exposure, smoking (turchin, 2009), nutrition, etc., of which uv irradiation from the sun damages human skin more severely than do other factors. it has been proposed that up to 90% of visible skin aging is due to cumulative exposure to sunlight (sudel, 2005). thus, extrinsic skin aging is termed photoaging (kligman, 1989). photoaged skin also reveals distinct deleterious changes such as coarse and deep wrinkling, thickened and leathery appearance, and irregular pigmentation (kligman and kligman, 1986). in general, photoaging is seen with intrinsic aging, especially in sun - exposed skin (fisher, 2002). whilst these intrinsic and extrinsic aging processes of skin have different etiologies and changes, they both exert harmful effects on dermal connective tissue (chung, 2001 ; fisher, 2002 ; sardy, 2009). in this article, we summarize the molecular mechanisms of skin aging that have prominent effects on dermal matrix components and studies in in vivo mouse models. skin is made up with three cutaneous layers of epidermidis, dermis, and subcutaneous tissue. the epidermis is the most superficial layer and its main function is to protect the body from harmful environmental stimuli and diminish fluid loss. it is a multilayered stratified squamous epithelium which is constantly renewed throughout life by the keratinization, a process of shedding of cornified cells. keratinocytes are the principal cells of the epidermis and maintain epidermal homeostasis (blumenberg and tomic - canic, 1997). the dermis is located under the epidermis and is attached to it by the dermo - epidermal junction. dermal connective tissue forms the dermis and contains extracellular matrix proteins such as collagen, elastic fibers, fibronectin, glycosaminoglycans, and proteoglycans which are produced and secreted into the extracelluar space by fibroblasts, the main cell type of the dermis (makrantonaki and zouboulis, 2007). collagen is the most abundant protein in dermal connective tissue, making up to 70% of the dry skin mass (gniadecka, 1998), and has a characteristic triple - helix configuration through an enzymatic process. the triple - helix complexes associate with small proteoglycans to form regularly arranged fibrillar structures called collagen bundles (bateman and chothia, 1996) that are responsible for conferring strength and support to human skin (oxlund and andreassen, 1980 ; uitto, 1986). elastic fibers are mainly composed of elastin and fibrillin, and are formed in a way in which cross - linked elastin forms the central fiber core and fibrillin - rich microfibrils surround the core. these account for less than 1 - 2% of the dermal weight but play a functional role in resisting deformational forces and providing elasticity by forming a fine network (makrantonaki and zouboulis, 2007). proteoglycans are also non - collageneous proteins similar to elastic fibers comprising only 0.1 - 0.3% of the dry weight of skin, and fill the majority of extracellular interstitial spaces within the tissue (frantz, 2010). they are responsible for the assembly of the extracellular matrix by affecting collagen fibrillogenesis and regulating the diameter and structure of collagen fibrils, thus preventing abnormal fibril assembly (okamoto and fujiwara, 2006). decorin and dermatopontin are the main molecules of proteoglycans (okamoto and fujiwara, 2006). these various extracellular matrix proteins in the dermal connective tissue mainly contribute to the maintenance of skin integrity and architecture. therefore, damages to the dermal connective tissue by the aging process can be closely related to structural changes of the skin like wrinkling, loss of elasticity, and sagging. cellular senescence of dermis - derived cells that are maintaining homeostasis of the dermal connective tissue is a fundamental cause of skin aging and brings about growth retardation due to a decreased proliferative capacity of cells mostly by the intrinsic aging process. as in all organs, dermal fibroblasts responsible for producing matrix proteins also show replicative senescence with age (praeger, 1986 ; cristofalo and pignolo, 1993). in the estimation of cellular senescence of human dermal fibroblasts, the population doubling - time of older cells (passage 28) was three times longer than of younger cells (passage 4) (yoon, 2004). from the cell cycle profile by flow cytometry in this study, it was also found that older cells have increased g0/g1 phase cell populations and a decrease in the s and g2/m fractions. growth reduction and cell cycle arrest at the g0/g1 phase can be ascribed to the repression of several regulatory genes that are important for cell cycle progression and cell proliferation (yaar, 2002). these genes include c - fos that encodes a component of the ap-1 transcription factor (seshadri and campisi, 1990 ; sheerin, 2001), id1h and id2h encoding helix - loop - helix proteins (hara, 1994), the e2f family of transcription factors that induce the expression of the genes required for cell proliferation (dimri, 1994), and cenp - f (mitosin) and kif4fa which are related with the onset of mitosis and mitotic chromosomal positioning (rattner, 1993 ; lee and kim, 2003) ; all of these are downregulated in senescent fibroblasts. on the contrary, the p21 and p16 inhibitors of cyclin - dependent protein kinases are up - regulated to interrupt the cell cycle progression (noda, 1994 ; hara, 1996). many specific genes associated with cellular senescence have been identified but the existence of a master switch that induces the activity of these genes at a particular time remains to be determined (jenkins, 2002). another mechanism for explaining cellular senescence is that age - associated changes in gene expression patterns and cellular proliferative capacity are likely to be under the control of telomeres (kosmadaki and gilchrest, 2004).. the length of telomeres decreases by up to 150 base pairs with cell division (harley, 1990) and is inversely related to an individual 's age (allsopp, 1992). when telomeres become critically short, cells enter proliferative senescence and thus telomeres may serve as a biologic clock indicating whether cells are young or old (wright and shay, 1992). therefore, telomere shortening can account for cellular senescence being mainly induced by intrinsic aging because it arises from serial cell division. however, it has also been determined that photoaging appears to influence telomere attrition (li, 2003). as a result of cellular senescence, non - dividing aged dermal fibroblasts accumulate in the dermal connective tissue and the synthesis of various extracellular matrix proteins by senescent fibroblasts is undoubtedly reduced. it is well known that the synthesis of fibrillar collagen, which is the major extracellular matrix protein and provides a supportive extracellular framework, is reduced in aged skin (uitto, 1989). in a study of collagen production in chronologically aged skin, the content of type i collagen, the major collagen in the skin and a marker of collagen synthesis, is deceased by 68% in old skin versus young skin, and cultured young fibroblasts synthesize more type i collagen than old cells (varani, 2006). the main reason for decreased production of collagen was found to be due to decreased synthesis of mrna for type i collagen, and there was a three - fold reduction in the steady - state level of type i collagen mrna in senescent fibroblasts (furth, 1991). more fundamental mechanisms for age - related collagen synthesis involve the transforming growth factor- (tgf-)-induced signaling pathway of collagen synthesis (figure 1). the ability of tgf- to stimulate the synthesis of extracelluar matrix components in cultured fibroblasts is well - documented (ignotz and massague, 1986 ; raghow, 1987 ; bitzer, 2000). tgf--induced smad2 phosphorylation is an initial molecular event mediated by tgf- receptor i kinase. phosphorylated smad2 dissociates from the receptor and then forms a complex with smad4. the smad complex translocates to the nucleus where it activates the transcription of target genes including col1a2, a gene for type i procollagen. in the early step of this process, smad2 phosphorylation may also occur as the result of activation of a kinase located downstream of mek-1 and upstream of the mapk / erk pathway (brown, 1999). other intermediate factors may be involved in this signaling pathway ; one protein that induces the gene expression of extracellular matrix protein is the connective tissue growth factor (ctgf), a cystein - rich peptide (grotendorst, 1997). ctgf is secreted by fibroblast cells after activation by tgf- and acts as a downstream mediator of tgf--induced collagen synthesis (duncan, 1999). it has been recently reported that ctgf is potently induced by tgf- and stimulates type i procollagen expression through col1a2 promotor activation (holmes, 2001 ; gore - hyer, 2002). accordingly, the tgf-/smad / ctgf / procollagen axis is the main signaling pathway for collagen synthesis in dermal fibroblasts and it is also found that tgf-, ctgf, and type i procollagen genes are all downregulated in aged human skin in vivo (quan, 2010). in this study, expression levels of tgf-, ctgf, and type i procollagen genes were reduced to 70, 50, and 75%, respectively, in aged dermis compared to young dermis. therefore, reduced synthesis of collagen in aged skin can be clearly explained by downregulation of signaling proteins in tgf--mediated collagen synthesis. the loss of collagen synthesis is recognized as a characteristic of chronologically aged skin by the cellular senescence of fibroblasts. however, there are some reports providing evidence that collagen synthesis is decreased by uv irradiation, the major factor for extrinsic skin aging (fisher, 2000 ; quan, 2004). the mechanism of reduced collagen production by uv irradiation is also related to the tgf-/smad pathway (figure 1). in human skin, uv irradiation impairs the tgf-/smad pathway by downregulating tgf- type ii receptor, and leads to reduced tgf- responsiveness and repression of tgf- target genes including type i procollagen (quan, 2001 ; quan, 2006). uv irradiation can also reduce collagen production in dermal fibroblasts by a different mechanism associated with mechanical tension. according to previous studies, fibroblasts in healthy cells have normal mechanical tension by attaching to intact collagen fibrils and containing abundant actin in their cytoplasm (grinnell, 2000, 2003). in contrast, cells in photoaged skin are in a mechanically relaxed state from contacting fragmented or amorphous collagen and having lower amounts of actin. with reduced mechanical tension, signaling through mapk or tgf- is not effectively transduced to the nucleus and subsequent transcription of collagens genes is inhibited (grinnell, 1999 ; varani, 2004). another key condition of skin aging associated with atrophy of the dermal connective tissue is the destruction of extracellular matrix (ecm) components, in particular collagen fibers. whereas both intrinsic and extrinsic factors are related with this destructive phenomenon, photoaging by uv irradiation has a much more substantial effect than other factors and the underlying mechanisms have been extensively investigated in many studies. in brief, the first step is the generation of reactive oxygen species (ros) by uv irradiation (black, 1987). ros, including superoxide anions, hydrogen peroxide, hydroxyl radicals, and singlet oxygen (masaki, 2010) can activate growth factor and cytokine receptors in fibroblasts (sardy, 2009). activated receptors stimulate members of very complex mapk signaling pathways such as akt, jnk, erk, and p38. next, the activation of nf-b, c - jun, and c - fos successively follows in the nucleus and the latter two proteins combine together to form ap- 1, the transcription factor which stimulates the transcription of matrix metalloproteinase (mmp) genes (pimienta and pascual, 2007 ; yaar and gilchrest, 2007). mmps are ubiquitous endopeptidases responsible for the turnover and degradation of ecm, the main target molecules being ecm proteins including all types collagens, elastins, and proteoglycans (nagase, 2006). ap-1 also inhibits procollagen gene expression by blocking tgf- type ii receptor / smad signaling (quan, 2004). consequentially, uv irradiation gives rise to dermal damage from collagen degradation by increased expression of mmps and inhibition of procollagen synthesis (figure 1). therefore, the accumulation of dermal damage by repeated sun exposure may result in characteristic wrinkling of photoaged skin. intrinsic or chronological aging can also contribute to the collagen damage even though its influence is less potent than that of photoaging. the main causative agent in this situation is also ros that are produced as a consequence of oxidative metabolism (sohal and weindruch, 1996). ros are inevitably created during the process of mitochondrial oxidative energy generation and are considered to be the cause of intrinsic aging by the destruction of important cellular constituents including proteins, lipids, and dna (muscari, 1996 ; sohal and weindruch, 1996 ; masaki, 2010). ros also accumulate due to the reduced antioxidant capacity of aged cells as a result of cellular senescence (kohen and gati, 2000) and act as a common molecular mediator for both over - expression of mmps and reduction of type i procollagen seen in photoaging - induced dermal damage. various phenomena and mechanisms of skin aging have been verified by in vivo experimental data from mouse models. mice are useful models for studying cutaneous aging as they are genetically similar to humans, affordable for experiments, and easy to manage. they are particularly favorable for investigating intrinsic skin aging because of problems associated with reliable sampling and effects of the environment in human studies (bhattacharyya and thomas, 2004). therefore, the changes of connective tissue by intrinsic or chronological aging have been confirmed in many mouse models (fornieri, 1989). in one study of changes in collagen synthesis and degradation in male lewis rat models between the ages of 1 and 24 months, collagen synthesis rates in skin at 24 months of age had decreased by at least 10-fold compared to rates in 1-month old animals and the proportion of newly synthesized collagen degraded in the skin was increased from 6.4% at 1 month of age to 56% at 15 months of age (mays, 1991). in another report, total collagen synthesis in mouse skin expressed as the amount of hydroxyproline according to the wet weight was decreased by about 30% between 2 and 22 months of age (boyer, 1991). different results are available from histomorphological observations in the cba mouse model in which a notable attrition of epidermal thickness and the number of epidermal cells could be correlated with age, but no conspicuous changes were noted in the depth of the dermis and the percentage area of collagen in aged mice (bhattacharyya and thomas, 2004). in another study with rats, dermal thickness remained constant from ages 2 to 22 months in fisher rats while the dermis width increased until 1 year of age and thereafter remained constant in wistar rats (sundberg and king, 2001). these morphological observations rather than biochemical data do not always corroborate with in vivo mouse model experiments. that is because there are inherent differences in genetic constitution among different strains of colony - raised rats and mice, and technical factors that influence morphometric microscopic evaluation (bhattacharyya and thomas, 2004). mouse models can be also effectively used for elucidating the roles of ecm components in the dermis because of the possibility of transgenic modification for examining a target gene. as an example, a study of collagen - associated protein dermatopontin knockout mice represented the involvement of dermapontin in the formation of collagen fibrils (takeda, 2002). in this study, the dermatopontin - null mice showed that the skin collagen content was 40% lower and the initial elastic modulus was 57% lower compared to wild - type mice. these data indicate that dermatopontin plays a critical role in skin elasticity and collagen fibrillogenesis in vivo. phenotypic abnormalities have also been observed in various transgenic models for other ecm proteins such as decorin (danielson, 1997), fibromodulin-(svensson, 1999) and thrombospondin-2 (kyriakides, 1998) null mutant mice that show skin laxity and fragility, and collagen fibril abnormalities. these transgenic mouse models are helpful to understand not only the roles of these proteins in dermal connective tissue but also the skin aging process because expression of these proteins is down - regulated with age. studies of uv radiation - induced connective tissue damage in mouse models were first attempted in the 1960s. in the early stage, normal mice were used but hairless mice were adopted in the 1980s because normal mice with fur were not an appropriate model for human responses to uv irradiation. currently, the most commonly used hairless mouse for studying photoaging is the albino skh - hairless-1 (kligman, 1996). the common feature of various alterations found in the dermal connective tissue following uv irradiation is elastosis, an accumulation of amorphous elastin material replacing the normal dermis (baumann, 2007). elastotic changes mainly appear in photoaged human and mouse skin, and are considered to be an important influence on the mechanical property of skin as well as the putative reason for the development of wrinkles (carneiro, 2007). other dermal changes, such as type i / iii collagen and total collagen contents, induced by uv irradiation have been reported differently in several previous studies. in the report by kligman, there were increases in total collagen in uv - irradiated hairless mice (kligman, 1989). additionally, in an experiment with photoaged c3h / hen, c3h / hej, and balb / c mice, uvb irradiation led to an increase in collagen, elastin, and glycosaminoglycans in all three strains (kochevar, 1994). however, in another study the intact ultrastructural appearance of dermal collagen fiber bundles (dcfb) was gradually lost with increasing uv dosage in uv - irradiated hairless mouse skin and changes of the percent area of wrinkles was significantly correlated with degenerative changes of dcfb (nishimori, 2001). this result concurs with the histologic and quantitative study of human facial skin by warren (1991) in which a group with higher sun exposure had more wrinkles, more severe elastosis, and less collagen than a group with less sun exposure. because of the complexity of skin organization and the genetic diversity among various strains of rats and mice, different histomorphologic and biochemical studies of the aging process in skin may only give partial pictures of overall cutaneous senescence. notwithstanding, mouse models have been adopted and developed effectively for studying the mechanisms of skin aging and testing the effects of pharmacological tools to retard this process. cellular senescence of dermis - derived cells that are maintaining homeostasis of the dermal connective tissue is a fundamental cause of skin aging and brings about growth retardation due to a decreased proliferative capacity of cells mostly by the intrinsic aging process. as in all organs, dermal fibroblasts responsible for producing matrix proteins also show replicative senescence with age (praeger, 1986 ; cristofalo and pignolo, 1993). in the estimation of cellular senescence of human dermal fibroblasts, the population doubling - time of older cells (passage 28) was three times longer than of younger cells (passage 4) (yoon, 2004). from the cell cycle profile by flow cytometry in this study, it was also found that older cells have increased g0/g1 phase cell populations and a decrease in the s and g2/m fractions. growth reduction and cell cycle arrest at the g0/g1 phase can be ascribed to the repression of several regulatory genes that are important for cell cycle progression and cell proliferation (yaar, 2002). these genes include c - fos that encodes a component of the ap-1 transcription factor (seshadri and campisi, 1990 ; sheerin, 2001), id1h and id2h encoding helix - loop - helix proteins (hara, 1994), the e2f family of transcription factors that induce the expression of the genes required for cell proliferation (dimri, 1994), and cenp - f (mitosin) and kif4fa which are related with the onset of mitosis and mitotic chromosomal positioning (rattner, 1993 ; lee and kim, 2003) ; all of these are downregulated in senescent fibroblasts. on the contrary, the p21 and p16 inhibitors of cyclin - dependent protein kinases are up - regulated to interrupt the cell cycle progression (noda, 1994 ; hara, 1996). many specific genes associated with cellular senescence have been identified but the existence of a master switch that induces the activity of these genes at a particular time remains to be determined (jenkins, 2002). another mechanism for explaining cellular senescence is that age - associated changes in gene expression patterns and cellular proliferative capacity are likely to be under the control of telomeres (kosmadaki and gilchrest, 2004).. the length of telomeres decreases by up to 150 base pairs with cell division (harley, 1990) and is inversely related to an individual 's age (allsopp, 1992). when telomeres become critically short, cells enter proliferative senescence and thus telomeres may serve as a biologic clock indicating whether cells are young or old (wright and shay, 1992). therefore, telomere shortening can account for cellular senescence being mainly induced by intrinsic aging because it arises from serial cell division. however, it has also been determined that photoaging appears to influence telomere attrition (li, 2003). as a result of cellular senescence, non - dividing aged dermal fibroblasts accumulate in the dermal connective tissue and the synthesis of various extracellular matrix proteins by senescent fibroblasts is undoubtedly reduced. it is well known that the synthesis of fibrillar collagen, which is the major extracellular matrix protein and provides a supportive extracellular framework, is reduced in aged skin (uitto, 1989). in a study of collagen production in chronologically aged skin, the content of type i collagen, the major collagen in the skin and a marker of collagen synthesis, is deceased by 68% in old skin versus young skin, and cultured young fibroblasts synthesize more type i collagen than old cells (varani, 2006). the main reason for decreased production of collagen was found to be due to decreased synthesis of mrna for type i collagen, and there was a three - fold reduction in the steady - state level of type i collagen mrna in senescent fibroblasts (furth, 1991). more fundamental mechanisms for age - related collagen synthesis involve the transforming growth factor- (tgf-)-induced signaling pathway of collagen synthesis (figure 1). the ability of tgf- to stimulate the synthesis of extracelluar matrix components in cultured fibroblasts is well - documented (ignotz and massague, 1986 ; raghow, 1987 ; bitzer, 2000). tgf--induced smad2 phosphorylation is an initial molecular event mediated by tgf- receptor i kinase. phosphorylated smad2 dissociates from the receptor and then forms a complex with smad4. the smad complex translocates to the nucleus where it activates the transcription of target genes including col1a2, a gene for type i procollagen. in the early step of this process, smad2 phosphorylation may also occur as the result of activation of a kinase located downstream of mek-1 and upstream of the mapk / erk pathway (brown, 1999). other intermediate factors may be involved in this signaling pathway ; one protein that induces the gene expression of extracellular matrix protein is the connective tissue growth factor (ctgf), a cystein - rich peptide (grotendorst, 1997). ctgf is secreted by fibroblast cells after activation by tgf- and acts as a downstream mediator of tgf--induced collagen synthesis (duncan, 1999). it has been recently reported that ctgf is potently induced by tgf- and stimulates type i procollagen expression through col1a2 promotor activation (holmes, 2001 ; gore - hyer, 2002). accordingly, the tgf-/smad / ctgf / procollagen axis is the main signaling pathway for collagen synthesis in dermal fibroblasts and it is also found that tgf-, ctgf, and type i procollagen genes are all downregulated in aged human skin in vivo (quan, 2010). in this study, expression levels of tgf-, ctgf, and type i procollagen genes were reduced to 70, 50, and 75%, respectively, in aged dermis compared to young dermis. therefore, reduced synthesis of collagen in aged skin can be clearly explained by downregulation of signaling proteins in tgf--mediated collagen synthesis. the loss of collagen synthesis is recognized as a characteristic of chronologically aged skin by the cellular senescence of fibroblasts. however, there are some reports providing evidence that collagen synthesis is decreased by uv irradiation, the major factor for extrinsic skin aging (fisher, 2000 ; quan, 2004). the mechanism of reduced collagen production by uv irradiation is also related to the tgf-/smad pathway (figure 1). in human skin, uv irradiation impairs the tgf-/smad pathway by downregulating tgf- type ii receptor, and leads to reduced tgf- responsiveness and repression of tgf- target genes including type i procollagen (quan, 2001 ; quan, 2006). uv irradiation can also reduce collagen production in dermal fibroblasts by a different mechanism associated with mechanical tension. according to previous studies, fibroblasts in healthy cells have normal mechanical tension by attaching to intact collagen fibrils and containing abundant actin in their cytoplasm (grinnell, 2000, 2003). in contrast, cells in photoaged skin are in a mechanically relaxed state from contacting fragmented or amorphous collagen and having lower amounts of actin. with reduced mechanical tension, signaling through mapk or tgf- is not effectively transduced to the nucleus and subsequent transcription of collagens genes is inhibited (grinnell, 1999 ; varani, 2004). another key condition of skin aging associated with atrophy of the dermal connective tissue is the destruction of extracellular matrix (ecm) components, in particular collagen fibers. whereas both intrinsic and extrinsic factors are related with this destructive phenomenon, photoaging by uv irradiation has a much more substantial effect than other factors and the underlying mechanisms have been extensively investigated in many studies. in brief, the underlying molecular mechanism of photoaging includes the following processes. the first step is the generation of reactive oxygen species (ros) by uv irradiation (black, 1987). ros, including superoxide anions, hydrogen peroxide, hydroxyl radicals, and singlet oxygen (masaki, 2010) can activate growth factor and cytokine receptors in fibroblasts (sardy, 2009). activated receptors stimulate members of very complex mapk signaling pathways such as akt, jnk, erk, and p38. next, the activation of nf-b, c - jun, and c - fos successively follows in the nucleus and the latter two proteins combine together to form ap- 1, the transcription factor which stimulates the transcription of matrix metalloproteinase (mmp) genes (pimienta and pascual, 2007 ; yaar and gilchrest, 2007). mmps are ubiquitous endopeptidases responsible for the turnover and degradation of ecm, the main target molecules being ecm proteins including all types collagens, elastins, and proteoglycans (nagase, 2006). ap-1 also inhibits procollagen gene expression by blocking tgf- type ii receptor / smad signaling (quan, 2004). consequentially, uv irradiation gives rise to dermal damage from collagen degradation by increased expression of mmps and inhibition of procollagen synthesis (figure 1). therefore, the accumulation of dermal damage by repeated sun exposure may result in characteristic wrinkling of photoaged skin. intrinsic or chronological aging can also contribute to the collagen damage even though its influence is less potent than that of photoaging. the main causative agent in this situation is also ros that are produced as a consequence of oxidative metabolism (sohal and weindruch, 1996). ros are inevitably created during the process of mitochondrial oxidative energy generation and are considered to be the cause of intrinsic aging by the destruction of important cellular constituents including proteins, lipids, and dna (muscari, 1996 ; sohal and weindruch, 1996 ; masaki, 2010). ros also accumulate due to the reduced antioxidant capacity of aged cells as a result of cellular senescence (kohen and gati, 2000) and act as a common molecular mediator for both over - expression of mmps and reduction of type i procollagen seen in photoaging - induced dermal damage. various phenomena and mechanisms of skin aging have been verified by in vivo experimental data from mouse models. mice are useful models for studying cutaneous aging as they are genetically similar to humans, affordable for experiments, and easy to manage. they are particularly favorable for investigating intrinsic skin aging because of problems associated with reliable sampling and effects of the environment in human studies (bhattacharyya and thomas, 2004). therefore, the changes of connective tissue by intrinsic or chronological aging have been confirmed in many mouse models (fornieri, 1989). in one study of changes in collagen synthesis and degradation in male lewis rat models between the ages of 1 and 24 months, collagen synthesis rates in skin at 24 months of age had decreased by at least 10-fold compared to rates in 1-month old animals and the proportion of newly synthesized collagen degraded in the skin was increased from 6.4% at 1 month of age to 56% at 15 months of age (mays, 1991). in another report, total collagen synthesis in mouse skin expressed as the amount of hydroxyproline according to the wet weight was decreased by about 30% between 2 and 22 months of age (boyer, 1991). different results are available from histomorphological observations in the cba mouse model in which a notable attrition of epidermal thickness and the number of epidermal cells could be correlated with age, but no conspicuous changes were noted in the depth of the dermis and the percentage area of collagen in aged mice (bhattacharyya and thomas, 2004). in another study with rats, dermal thickness remained constant from ages 2 to 22 months in fisher rats while the dermis width increased until 1 year of age and thereafter remained constant in wistar rats (sundberg and king, 2001). these morphological observations rather than biochemical data do not always corroborate with in vivo mouse model experiments. that is because there are inherent differences in genetic constitution among different strains of colony - raised rats and mice, and technical factors that influence morphometric microscopic evaluation (bhattacharyya and thomas, 2004). mouse models can be also effectively used for elucidating the roles of ecm components in the dermis because of the possibility of transgenic modification for examining a target gene. as an example, a study of collagen - associated protein dermatopontin knockout mice represented the involvement of dermapontin in the formation of collagen fibrils (takeda, 2002). in this study, the dermatopontin - null mice showed that the skin collagen content was 40% lower and the initial elastic modulus was 57% lower compared to wild - type mice. these data indicate that dermatopontin plays a critical role in skin elasticity and collagen fibrillogenesis in vivo. phenotypic abnormalities have also been observed in various transgenic models for other ecm proteins such as decorin (danielson, 1997), fibromodulin-(svensson, 1999) and thrombospondin-2 (kyriakides, 1998) null mutant mice that show skin laxity and fragility, and collagen fibril abnormalities. these transgenic mouse models are helpful to understand not only the roles of these proteins in dermal connective tissue but also the skin aging process because expression of these proteins is down - regulated with age.. studies of uv radiation - induced connective tissue damage in mouse models were first attempted in the 1960s. in the early stage, normal mice were used but hairless mice were adopted in the 1980s because normal mice with fur were not an appropriate model for human responses to uv irradiation. currently, the most commonly used hairless mouse for studying photoaging is the albino skh - hairless-1 (kligman, 1996). the common feature of various alterations found in the dermal connective tissue following uv irradiation is elastosis, an accumulation of amorphous elastin material replacing the normal dermis (baumann, 2007). elastotic changes mainly appear in photoaged human and mouse skin, and are considered to be an important influence on the mechanical property of skin as well as the putative reason for the development of wrinkles (carneiro, 2007). other dermal changes, such as type i / iii collagen and total collagen contents, induced by uv irradiation have been reported differently in several previous studies. in the report by kligman, there were increases in total collagen in uv - irradiated hairless mice (kligman, 1989). additionally, in an experiment with photoaged c3h / hen, c3h / hej, and balb / c mice, uvb irradiation led to an increase in collagen, elastin, and glycosaminoglycans in all three strains (kochevar, 1994). however, in another study the intact ultrastructural appearance of dermal collagen fiber bundles (dcfb) was gradually lost with increasing uv dosage in uv - irradiated hairless mouse skin and changes of the percent area of wrinkles was significantly correlated with degenerative changes of dcfb (nishimori, 2001). this result concurs with the histologic and quantitative study of human facial skin by warren (1991) in which a group with higher sun exposure had more wrinkles, more severe elastosis, and less collagen than a group with less sun exposure. because of the complexity of skin organization and the genetic diversity among various strains of rats and mice, different histomorphologic and biochemical studies of the aging process in skin may only give partial pictures of overall cutaneous senescence. notwithstanding, mouse models have been adopted and developed effectively for studying the mechanisms of skin aging and testing the effects of pharmacological tools to retard this process. the phenotypes of aged skin such as wrinkling, laxity, sagging, and fragility have become a target for anti - aging studies from a standpoint of the skin 's role as a social interface as well as its physiological function. the aging process in skin is a more complex process influenced by intrinsic and extrinsic factors than that of any other body organs, and the effects of these two types of factors overlap for the most part. dermal matrix alterations are also affected by the combined effects of these two aging processes. studies of skin aging have been extensively performed to elucidate the underlying mechanisms and to develop anti - aging measures. for this, various mouse models have been used to establish the processes of skin aging through experimental modifications. although mouse models do not always corroborate the biochemical data and produce consistent results, they are the utmost useful tools to examine the fundamental etiology of skin aging and anti - aging effects.	skin is the most superficial body organ and plays an important role in protecting the body from environmental damage and in forming social relations. with the increase of the aging population in our society, dermatological and cosmetic concerns of skin aging are rapidly increasing. skin aging is a complex process combined with intrinsic and extrinsic factors. intrinsic or chronological skin aging results from the passage of time and is influenced by genetic factors. extrinsic skin aging is mainly determined by uv irradiation, also called photoaging. these two types of aging processes are superimposed on sun - exposed skin, and have a common feature of causing dermal matrix alterations that mostly contribute to the formation of wrinkles, laxity, and fragility of aged skin. the dermal matrix contains extracellular matrix proteins such as collagen, elastin, and proteoglycans that confer the strength and resiliency of skin. skin aging associated with dermal matrix alterations and atrophy can be caused by cellular senescence of dermal cells like fibroblasts, and decreased synthesis and accelerated degradation of dermal matrix components, especially collagen fibers. both intrinsic aging and photoaging exert influence during each step of dermal matrix alteration via different mechanisms. mouse models of skin aging have been extensively developed to elucidate intrinsic aging and photoaging processes, to validate in vitro biochemical data, and to test the effects of pharmacological tools for retarding skin aging because they have the advantages of being genetically similar to humans and are easily available.
chest x - ray (cxr) is a key diagnostic tool in the health care system. it is used in the evaluation of acute patients with respiratory problems and chronic disorders and in the diagnostic process of serious diseases. in denmark, more than 600,000 cxrs are performed each year, and it is thus the most requested radiologic procedure.1 we know that about 20% of lung cancer patients have had a false - negative cxr prior to diagnosis.2,3 therefore, it is essential to obtain more knowledge about the use of cxr in order to optimize the diagnostic process for different diseases, including cancer. registry - based epidemiologic research, based on secondary data, has the advantages of large sample sizes, high statistical precision, and limited risk of recall and nonresponse bias. on the other hand, this type of research greatly depends on the validity and completeness of the data. studies using registry data are vulnerable (eg, to missing data and selection bias), often with no possibility to discover such problems. the danish national patient registry (dnpr) was established in 1977 as a nationwide registry for patient contacts to hospitals. the recorded data is based on the unique civil registration number assigned to all danish citizens at birth.4 registrations were adapted to the international classification of diseases, version 10 (icd-10) in 1994. dnpr has formed the basis for remuneration of services provided by public hospitals since 2000. it has been mandatory since 2002 for all danish hospitals to report all performed radiological procedures to the dnpr ; this also includes registration of all cxrs performed. all danish radiology departments use a version of an electronic registration system called the radiology information system (ris). the version may vary between hospitals, but all systems ensure that a valid civil registration number is registered before an x - ray can be conducted. therefore, this system is considered to contain complete and valid information on all radiology procedures performed. data from the ris are not readily accessible to researchers, but the ris provides the basis for the information in the dnpr.5 the dnpr was hosted by the national board of health until march 2012 after which the state serum institute (ssi), the danish national institute for health and disease control, was given the responsibility. data are accessible for researchers either directly from the ssi or through statistics denmark which receives copies from ssi on an annual basis. statistics denmark receives the data for national statistical figures, but the organization also hosts data for researchers. only two previous studies have investigated the completeness of the registrations in dnpr of performed diagnostic procedures (coronary computed tomographic [ct ] angiography and fetal umbilical artery flow velocity) ; both of these studies showed low completeness.6,7 the aim of this study was to investigate the completeness of the cxr registrations in the dnpr by comparing the two versions from the ssi and statistics denmark, respectively, using ris as the reference standard. we conducted a validation study comparing dnpr data from two different sources (the ssi and statistics denmark) and used the ris data to examine the completeness of the performed cxr coding. we invited the administrators of ten different randomly selected radiology departments to provide data for the study. data from ris were collected from these nine radiology departments that were located across the nation and covered 20 different hospitals. we established a cohort of all persons with a valid civil registration number who had a cxr performed in one of the included radiology departments between january 1, 2004, and december 31, 2011, and were aged between 40 and 99 years at the date of investigation. from each department, we requested data for three different weeks during the entire study period. the weeks were randomly distributed in the period 20042011 using the rand function in excel. aarhus university hospital did not have data before 2009 and could therefore only provide information on two of the randomly assigned weeks, which gave a total of 29 different weeks of cxr. using the civil registration number provided to all danish citizens, we were able to link data at the individual level. we did not have information on the exact time of the day for each cxr. therefore, we allowed inclusion of only one cxr per person per day. from the ssi, we had information on all cxrs performed at danish hospitals in the period 20042011 according to the dnpr for persons aged 4099 years. these data were searched for cxrs performed on our study population at the relevant hospitals. from statistics we extracted data on our study population regarding performed cxrs at the relevant hospitals (procedure code : uxrc00). in all registrations of diagnostic procedures in both versions of the dnpr, the patient was assigned a main department that was responsible for their procedure and treatment. this is generally the department referring for the procedure. for outpatients with cxr referral from elsewhere, the classification of cxr (uxrc00) and other procedures in the dnpr are based on the web - based health care classification (sks) system.8 the sks system is a collection of international, nordic, and danish classifications that contain up to ten alphanumeric characters. the first is a letter representing a primary group, and the records follow a mono - hierarchical classification system. the sks system covers diagnoses, surgery, other treatments, anesthesia, and examinations.5 the code uxrc00 encompasses both frontal and lateral cxr. however, lateral cxr is performed only in patients who can mobilize from the bed. completeness was calculated as the proportion of performed cxr procedures from ris registered with the ssi or statistics denmark, respectively. we also compared the registrations of the main department between the two versions of the dnpr. analyses were stratified according to age at investigation (4054, 5569, 7084, and 8599 years) and year of investigation. mean age was calculated on the basis of the integer of age at the date of the cxr. all analyses and data management were carried out using the statistical software stata 14.1 (statacorp lp, lakeway drive, college station, tx, usa). according to danish law, approval from the national committee on biomedical research ethics was not required as no biomedical intervention was performed. patient written informed consent was deemed not required for this study according to danish law, due to the use of de - identified register data. we conducted a validation study comparing dnpr data from two different sources (the ssi and statistics denmark) and used the ris data to examine the completeness of the performed cxr coding. we invited the administrators of ten different randomly selected radiology departments to provide data for the study. nine departments agreed to participate. data from ris were collected from these nine radiology departments that were located across the nation and covered 20 different hospitals. we established a cohort of all persons with a valid civil registration number who had a cxr performed in one of the included radiology departments between january 1, 2004, and december 31, 2011, and were aged between 40 and 99 years at the date of investigation. from each department, we requested data for three different weeks during the entire study period. the weeks were randomly distributed in the period 20042011 using the rand function in excel. aarhus university hospital did not have data before 2009 and could therefore only provide information on two of the randomly assigned weeks, which gave a total of 29 different weeks of cxr. using the civil registration number provided to all danish citizens, we were able to link data at the individual level. we did not have information on the exact time of the day for each cxr. we invited the administrators of ten different randomly selected radiology departments to provide data for the study. nine departments agreed to participate. data from ris were collected from these nine radiology departments that were located across the nation and covered 20 different hospitals. we established a cohort of all persons with a valid civil registration number who had a cxr performed in one of the included radiology departments between january 1, 2004, and december 31, 2011, and were aged between 40 and 99 years at the date of investigation. from each department, we requested data for three different weeks during the entire study period. the weeks were randomly distributed in the period 20042011 using the rand function in excel. aarhus university hospital did not have data before 2009 and could therefore only provide information on two of the randomly assigned weeks, which gave a total of 29 different weeks of cxr. using the civil registration number provided to all danish citizens, we were able to link data at the individual level. we did not have information on the exact time of the day for each cxr. from the ssi, we had information on all cxrs performed at danish hospitals in the period 20042011 according to the dnpr for persons aged 4099 years. these data were searched for cxrs performed on our study population at the relevant hospitals. from statistics we extracted data on our study population regarding performed cxrs at the relevant hospitals (procedure code : uxrc00). in all registrations of diagnostic procedures in both versions of the dnpr, the patient was assigned a main department that was responsible for their procedure and treatment. this is generally the department referring for the procedure. for outpatients with cxr referral from elsewhere, the classification of cxr (uxrc00) and other procedures in the dnpr are based on the web - based health care classification (sks) system.8 the sks system is a collection of international, nordic, and danish classifications that contain up to ten alphanumeric characters. the first is a letter representing a primary group, and the records follow a mono - hierarchical classification system. the sks system covers diagnoses, surgery, other treatments, anesthesia, and examinations.5 the code uxrc00 encompasses both frontal and lateral cxr. however, completeness was calculated as the proportion of performed cxr procedures from ris registered with the ssi or statistics denmark, respectively. we also compared the registrations of the main department between the two versions of the dnpr. analyses were stratified according to age at investigation (4054, 5569, 7084, and 8599 years) and year of investigation. mean age was calculated on the basis of the integer of age at the date of the cxr. all analyses and data management were carried out using the statistical software stata 14.1 (statacorp lp, lakeway drive, college station, tx, usa). according to danish law, approval from the national committee on biomedical research ethics was not required as no biomedical intervention was performed. patient written informed consent was deemed not required for this study according to danish law, due to the use of de - identified register data. the cohort from ris consisted of 11,235 persons who had a total of 12,513 cxrs performed during the 29 investigated weeks. mean age of patients was 66.3 years (table 1). in the data from the ssi, we were able to link 12,265 (98.0%) performed cxrs (table 2) with a mean patient age of 66.3 years (table 1). the completeness of data was comparable across age groups and throughout the years (table 2). the proportion of missing data was almost equally distributed across the radiology departments (table 2). only small differences existed across radiology departments when stratified according to age (table 4) in the version of the dnpr from statistics denmark, we were able to link 9,151 cxr (73.1%) to the reference standard. the proportion of linked cxr varied between the years from 50.9% in 2005 to 95.8% in 2011. most of the patients had internal medicine as their main department (table 3). only 6.9% had a radiology department as their main department (table 3) ; all of these 629 cxr were registered in 2011 (data not shown). therefore, the main reason for the higher proportion of missing data in statistics denmark was that no cxrs were registered with radiology as the main department before 2011. some differences in the proportion of missing data existed across radiology departments (table 2). there was a tendency for a higher completeness among the elderly, both overall (table 2) and for each hospital (table 4). the cohort from ris consisted of 11,235 persons who had a total of 12,513 cxrs performed during the 29 investigated weeks. in the data from the ssi, we were able to link 12,265 (98.0%) performed cxrs (table 2) with a mean patient age of 66.3 years (table 1). the completeness of data was comparable across age groups and throughout the years (table 2). the proportion of missing data was almost equally distributed across the radiology departments (table 2). only small differences existed across radiology departments when stratified according to age (table 4) in the version of the dnpr from statistics denmark, we were able to link 9,151 cxr (73.1%) to the reference standard. the proportion of linked cxr varied between the years from 50.9% in 2005 to 95.8% in 2011. the mean age of the patients was 67.5 years (table 1). most of the patients had internal medicine as their main department (table 3). only 6.9% had a radiology department as their main department (table 3) ; all of these 629 cxr were registered in 2011 (data not shown). therefore, the main reason for the higher proportion of missing data in statistics denmark was that no cxrs were registered with radiology as the main department before 2011. some differences in the proportion of missing data existed across radiology departments (table 2). there was a tendency for a higher completeness among the elderly, both overall (table 2) and for each hospital (table 4). we found that the completeness of the dnpr varied according to the data source. compared to the data obtained directly from the radiology departments, the version from the ssi had a high completeness ; 98% of all cxrs the version from statistics denmark had a higher proportion of missing data ; this was mainly because no cxr was registered with radiology as the main department before 2011. all patients registered with a diagnostic procedure in the dnpr are assigned a main department. an algorithm identifies this department according to whether a patient is hospitalized or receives ongoing outpatient care at a department at the time of the procedure. in the former cases, patients referred from general practice for a diagnostic investigation at a radiology department will be assigned the radiology department as the main department. the data from statistics denmark generally lacked information on these investigations from 2004 to 2010. the completeness increases with age, which may be because a lower proportion of older patients have their cxr taken after referral from general practice. furthermore, the completeness varies between hospitals ; this indicates differences in the proportion of cxrs requested from general practice at the different hospitals. after personal correspondence with both the ssi and statistics denmark, it has been confirmed that, in this period of time, all data on visits at a number of laboratory specialties, including radiology, have been deleted before the yearly copies were sent from the ssi to statistics denmark. therefore, researchers are recommended to ensure that full copies of the registry are retrieved and to use the ssi to explore the use of cxr. there may, for example, be small errors in the coding of the civil registration numbers, or different choices may have been made in the algorithms used before the data were sent to the ssi or other reasons. however, no previous studies have investigated the completeness and validity of radiology codes in dnpr. previous studies exploring the validity of the dnpr have mainly focused on specific diagnoses and have shown high completeness and validity often over 90%, with some variation between studies.917 one study, however, found a positive predictive value of 32% when looking at vitamin b12 deficiency anemia.13 only two studies have looked at diagnostic investigations. nielsen found a completeness of registrations of coronary ct angiographies of 72% in the dnpr. in a report from the danish health and medicines authority from 2003, the completeness of the records of fetal umbilical artery flow velocity in the dnpr was 47% compared to the medical records.6 a recent review of all validation studies by schmidt also confirmed considerable variation in the validity and completeness across diagnoses and treatments. together with our study, this emphasizes the importance in checking the data thoroughly and critically ; it also stresses the necessity of validity studies when looking at data from the dnpr. furthermore, our findings underscore the significance of good algorithms and reliable data management at the responsible authorities generating the data for the registries. the unique civil registration number assigned to all danish citizens at birth enables linkage of big data sets, but it also ensures complete data from the radiology departments (the reference standard), as the radiologic services can not be initiated without registration of a valid civil registration number. inclusion of data from nine administrative departments covering 20 hospitals ensured that data are representative of the entire population. during the study period however, we could not find any differences in the completeness in the ssi version over time for the different hospitals (data not shown). throughout the study period, the coding of cxr has formed the basis for reimbursement for services provided, which ensures a high completeness of registrations in the reference standard. the random selection of weeks, including weekends and holidays, was also considered a strength. a potential limitation of the current study is that we only had information on patients aged between 40 and 99 years. yet, no major variation in the completeness was found for different age groups in the included patients in the ssi version, and it seems unlikely that this would be different among the younger patients. furthermore, the studied age group receives more than 85% of the cxrs performed in denmark. we only included persons with a valid civil registration number and thus excluded tourists ; this is because differences exist in the registration of these patients. it would have been a strength if we had been able to calculate the positive predictive value and the validity of a cxr registration in the dnpr, but this was not possible because valid identification of the relevant cxr in the dnpr was uncertain because of numerous administrative changes at radiology departments and hospitals during the study period. it is also a limitation that we were not able to investigate the validity of other information in the records such as the referring body or whether the investigation was performed as inpatient or outpatient. the completeness of registrations of cxrs in the dnpr differs according to the source of the data. a high completeness was found in the version obtained directly from the ssi, whereas the completeness was under 75% in the data obtained from statistics denmark. this calls for meticulous assessment of both data and data sources in future studies investigating radiologic procedures on the basis of records in the dnpr.	objectivethe aim of this validation study was to assess the completeness of the registrations of chest x - rays (cxr) in two different versions of the danish national patient registry (dnpr).material and methodswe included electronic record data on cxr performed on patients aged 40 to 99 years from nine radiology departments covering 20 danish hospitals. from each department, we included data from three randomly selected weeks between 2004 and 2011 (reference standard). in two versions of the dnpr from the state serum institute (ssi) and statistics denmark, respectively, we investigated the proportion of registered cxr compared to the reference standard. furthermore, we compared the completeness of the recorded data according to the responsible department (main department).resultswe identified 11,235 patients and 12,513 cxr in the reference standard. the data from the ssi contained 12,265 (98%) cxr, whereas the data from statistics denmark comprised 9,151 (73.1%) cxr. the completeness of the ssi data was fairly constant across years, radiology departments, medical specialties, and age groups. the data from statistics denmark was almost complete in 2011 (95.8%). however, for the remaining study period, the data with radiology departments registered as the main department were lacking in the version from statistics denmark, and so the overall completeness was 73.1%.conclusionthe completeness of cxr registrations varied between 98% and 73% depending on the information source, and this should be considered when investigating radiology services in the basis of dnpr.
the incidence and clinical features of several types of vasculitides differ between japan, europe and north america, unlike those of rheumatoid arthritis, systemic lupus erythematosus, and other rheumatic diseases in these geographical regions [1, 2 ]. these vasculitides are more rare and heterogeneous in terms of clinical features, types of anti - neutrophil cytoplasmic antibody (anca) and response to treatment. because geographical differences in the incidence of anca - associated vasculitis (aav) have been demonstrated in europe, we extended our research to determine the incidence, clinical phenotype and the associated genetic factors of vasculitides between japan, europe, and north america. in this review, tak and gca are two types of vasculitis characterized by inflammation of the large vessels. fewer patients with gca have been reported in the japanese literature than in the european and north american literatures. in contrast, more patients with tak have been reported in japan than in europe or the usa. the point prevalence of gca in japan was 690 patients in 1997 (95 % confidence interval [ci ] 400980). the prevalence of patients 50 years of age was 1.47 cases (95 % ci 0.862.10) per 10 million people in japan compared with 200 and 60 cases per 10 million people in the usa and spain, respectively [6, 7 ]. the reason for the low incidence of gca in japan remains unclear ; however, genetic factors affecting the incidence of these diseases are unique and important. the hla - drb1 0401 and hla - drb1 0404 haplotypes are predominantly (60 %) detected in patients with gca in america. these haplotypes were less frequently detected in 493 japanese healthy controls (2.9 and 0.7 %, respectively) than in 60 american healthy controls (15.9 and 3.2 %, respectively). moreover, our study found no significant differences in the clinical features of gca between japan and other countries, although gca cases are less common in japan than in the usa or europe. tak, which predominantly affects young females in japan, affects the aortic arch (type i), as determined by angiography. the incidence of hla - b52 (56 %) and hla - b39 (17 %) was significantly higher in patients with tak than in healthy controls (25 and 6 %, respectively) in a japanese study. however, several patients with tak in india and other east asian countries are reportedly middle - aged males who express hla - b39 and exhibit abdominal aorta involvement (type iii). asahikawa city (43.5n) on hokkaido island is close to the latitude of lugo, spain (42n). on this island, there are more patients with microscopic polyangiitis (mpa) ; a higher number of patients with aav are mpo - anca - positive than granulomatosis with polyangiitis (gpa)- or pronase 3 (pr3)-positive. gca and gpa occur more frequently in north europe and north america whereas takayasu arteritis and mpa occur more frequently in japan geographical differences in the incidences of vasculitides. gca and gpa occur more frequently in north europe and north america whereas takayasu arteritis and mpa occur more frequently in japan on the other hand, it is interesting to note that a study from beijing (39.5n), china, demonstrated that 60.7 % (54/89) of patients with gpa were mpo - anca - positive and 38.2 % (34/89) were pr3-anca - positive. patients with mpo - anca had multiorgan involvement with higher serum creatinine levels than pr3-anca - positive patients with gpa. differences in renal involvement in gpa and mpa between patients in the uk and japan were reported by watts.. supporting data indicated that patients with localized gpa were more frequent than gpa patients with renal involvement in japan, which was reported by harabuchi. from asahikawa medical university and confirmed in our investigation. moreover, two studies demonstrated renal involvement in 1240 % of 21 patients with gpa [13, 14 ]. in another hospital - based, nationwide, retrospective study conducted in japan from 1988 to 1998 by the japanese ministry of health, labour and welfare, renal involvement was diagnosed in 3963 % of 172 patients. in two studies by gross. in germany and hoffman. in the usa, renal involvement was diagnosed in 77 % of 155 patients and 77 % of 70 patients with gpa, respectively [15, 16 ]. a genetic analysis of patients with mpa was initiated in 1997 by the research committee of intractable vasculitis of the japanese ministry of health and welfare (chief investigator prof. hiroshi hashimoto). a significant association between hla - drb1 0901 and mpa (p = 0.037 ; odds ratio [or ] 2.44 ; 95 % ci 1.334.46) as well as mpo - anca positivity (p = 0.014 ; or 2.44 ; 95 % ci 1.414.22) was demonstrated by tsuchiya. [17, 18 ]. another report published in 1996 demonstrated an association between hla - dr9 in 62.5 % patients and canca - positive gpa (10/16) compared with 26 % in healthy controls (p 80 % patients in japan were panca- and/or mpo - positive, whereas two - thirds of patients in the uk were canca- and/or pr3-positive. renal involvement in patients with mpa was common in both countries but it was significantly less common in gpa patients in japan than in gpa patients in the uk. there was no major difference in the incidence of aav between japan and the uk, but this prospective study found that mpa and mpo - anca were more common in japan whereas gpa and pr3-anca were more common in the uk. asahikawa city (43.5n) on hokkaido island is close to the latitude of lugo, spain (42n). on this island, there are more patients with microscopic polyangiitis (mpa) ; a higher number of patients with aav are mpo - anca - positive than granulomatosis with polyangiitis (gpa)- or pronase 3 (pr3)-positive. gca and gpa occur more frequently in north europe and north america whereas takayasu arteritis and mpa occur more frequently in japan geographical differences in the incidences of vasculitides. gca and gpa occur more frequently in north europe and north america whereas takayasu arteritis and mpa occur more frequently in japan on the other hand, it is interesting to note that a study from beijing (39.5n), china, demonstrated that 60.7 % (54/89) of patients with gpa were mpo - anca - positive and 38.2 % (34/89) were pr3-anca - positive. patients with mpo - anca had multiorgan involvement with higher serum creatinine levels than pr3-anca - positive patients with gpa. differences in renal involvement in gpa and mpa between patients in the uk and japan were reported by watts.. supporting data indicated that patients with localized gpa were more frequent than gpa patients with renal involvement in japan, which was reported by harabuchi. from asahikawa medical university and confirmed in our investigation. moreover, two studies demonstrated renal involvement in 1240 % of 21 patients with gpa [13, 14 ]. in another hospital - based, nationwide, retrospective study conducted in japan from 1988 to 1998 by the japanese ministry of health, labour and welfare, renal involvement was diagnosed in 3963 % of 172 patients. in two studies by gross. in germany and hoffman. in the usa, renal involvement was diagnosed in 77 % of 155 patients and 77 % of 70 patients with gpa, respectively [15, 16 ]. a genetic analysis of patients with mpa was initiated in 1997 by the research committee of intractable vasculitis of the japanese ministry of health and welfare (chief investigator prof. a significant association between hla - drb1 0901 and mpa (p = 0.037 ; odds ratio [or ] 2.44 ; 95 % ci 1.334.46) as well as mpo - anca positivity (p = 0.014 ; or 2.44 ; 95 % ci 1.414.22) was demonstrated by tsuchiya. [17, 18 ]. another report published in 1996 demonstrated an association between hla - dr9 in 62.5 % patients and canca - positive gpa (10/16) compared with 26 % in healthy controls (p 80 % patients in japan were panca- and/or mpo - positive, whereas two - thirds of patients in the uk were canca- and/or pr3-positive. renal involvement in patients with mpa was common in both countries but it was significantly less common in gpa patients in japan than in gpa patients in the uk. there was no major difference in the incidence of aav between japan and the uk, but this prospective study found that mpa and mpo - anca were more common in japan whereas gpa and pr3-anca were more common in the uk. these findings provide useful information on the aetiology and pathogenesis [22, 23 ] of primary systemic vasculitides in various geographical regions.	the epidemiology of systemic vasculitides differs between japan, europe and north america. takayasu s arteritis occurs frequently in japan, unlike giant cell arteritis. a collaborative international study comparing the epidemiology of anti - neutrophil cytoplasmic antibody (anca)-associated vasculitis between japan and the united kingdom (uk) demonstrated that microscopic polyangiitis and myeloperoxidase - anca were more common in japan whereas granulomatosis with polyangiitis and pronase 3-anca were more common in the uk. these differences may be attributed to differences in latitude and genetic backgounds. these findings provide useful information on the aetiology and pathogenesis of primary systemic vasculitides in various geographical regions.
it accounts for 12% of all sarcomas [1, 2 ]. while it can occur in any part of the body, skin, soft tissue, breast and liver it has a predilection for skin and soft tissues in head and neck region, given the vascular density and exposure to ultraviolet radiation. cutaneous angiosarcomas commonly occur in the face and scalp region ; they account for about 60% of all angiosarcomas [3, 4 ]. soft tissue angiosarcomas and breast angiosarcomas roughly account for about 25 and 8% of angiosarcomas, respectively [3, 4 ]. in the us, the incidence of angiosarcoma is reported to be higher amongst caucasians compared to african americans. risk factors for angiosarcoma include exposure to agents such as thorotrast, vinyl chloride, insecticides containing arsenic, long - term anabolic steroid or estrogen therapy ; morbid obesity ; chronic venous ulceration ; chronic lymphedema (stewart - treves syndrome) ; prior radiotherapy ; renal transplantation ; familial syndromes such as klippel - trenaunay syndrome, maffucci syndrome, retinoblastoma, xeroderma pigmentosum, neurofibromatosis ; pre - existing cancers such as germ cell tumors, vestibular schwannomas, leiomyomas, nerve sheath tumors ; foreign bodies such as vascular graft material, surgical sponges, dacron, plastic, steel. in general, angiosarcomas have a poor prognosis. prognosis depends upon factors such as depth of tumor invasion, tumor diameter, local regional spread, distant metastasis, positive margins on surgical tumor resection and tumor recurrence. in the us, the overall 5-year survival is reported to be in the range of 1045%. given the rarity of this cancer, no standard treatment has been established. in addition, the multifocal nature of the disease, as well as different combinations of disease location and subtypes, makes the treatment challenging. in spite of limited retrospective and prospective nonrandomized data on the treatment of angiosarcoma, radical surgical resection followed by wide - field postoperative radiotherapy chemotherapy may be especially helpful for short - term palliation. in regard to definitive treatment, the role of adjuvant chemotherapy remains unclear. doxorubicin, paclitaxel, and subcutaneous interferon alpha-2a with oral 13-cis - retinoic acid have been reported to be used. the patient is an 81-year - old caucasian woman with a 15-pack year smoking history and no family or past medical history of cancer who presented to our multidisciplinary head and neck tumor board at the university of wisconsin, madison, wisc., usa, with a large erythematous lesion on her nose with bilateral malar extension. the lesion also extended superiorly to the infraorbital region and into the lower eyelids ; it further extended inferiorly into the nasal vestibule and into the nasal cavity. the lesion was characterized by multiple papules and eschars, most prominently over the bridge over her nose (fig. ten months prior to presentation, the patient developed red papules on her bilateral nasal ala. the initial biopsy was read as benign. however, the lesion on the right side of her nose continued to grow and bled intermittently over a 10-month period. she eventually underwent excision of a portion of the lesion elsewhere. a subsequent pathology report revealed an epithelioid hemangioma. the patient was managed with continued observation. however, the lesion progressed and this prompted her to undergo reevaluation of the lesion by a dermatologist. she underwent a repeat biopsy of the lesion and the pathology returned positive for angiosarcoma. the patient was referred to the university of wisconsin for recommendations concerning her treatment options. the pathology was reviewed and the diagnosis of angiosarcoma was confirmed. at presentation, the patient 's review of systems was essentially negative for any constitutional symptoms or those referable to head and neck. she reported bilateral orbital swelling that developed over 2 months prior to her presentation, excessive lacrimation and intermittent bleeding from the lesion. her past medical history was unremarkable for prior radiotherapy, chronic venous ulceration, lymphedema, or other causative factors of angiosarcoma. of note, she donated one of her kidneys to her son in the past. she had a history of bilateral cataract removal, hypothyroidism, and diabetes mellitus type ii at presentation. head ct demonstrated that the lesion extended into the nasal cavity, and a chest ct demonstrated a 1-cm pleural based pulmonary nodule in the posteromedial aspect of the left upper lobe. the patient underwent a pet / ct scan that demonstrated a mildly diffuse fdg avidity associated with her known angiosarcoma without any evidence of local regional lymph nodes or distant metastasis. it did not reveal any abnormal fdg avidity associated with the patient 's previously appreciated left upper lobe subpleural pulmonary nodule. therefore, this abnormality was deemed to be nonspecific and most likely represented a prior granulomatous infection, given the presence of densely calcified mediastinal and right hilar lymph nodes. an mri of the orbit, face, and neck was performed which demonstrated the angiosarcoma of the nose with bilateral malar extension and without evidence of perineural spread. the tumor measured 7.0 cm cephalad to caudad, 12.0 cm in the lateral dimension and there was 3.0 cm extension along the floor of the nasal cavity, as measured from the nasal vestibule. considering the patient 's age, medical condition, only one functional kidney and refusal to receive chemotherapy, systemic agents were not given. a total dose of 66 gy in 33 fractions, utilizing 12 mev electrons (custom bolus for uniform dosing) was delivered to the central face. a dose of 57.2 gy in 29 fractions was delivered to the bilateral cheeks using 6 mv photons. the patient tolerated radiotherapy well with the expected side effects. at nearly 2.0 years following treatment, she remains free of disease recurrence with the only late complication consisting of bilateral trichiasis (fig. the diagnosis and treatment of angiosarcoma presents unique challenges. given the poor overall survival (os) for this tumor, it is crucial to perform a thorough history and physical examination with a high index of clinical suspicion when evaluating skin and vascular lesions in the head and neck region. mortality typically results from either extensive local disease or distant metastasis to organs such as lungs. her tumor was well - differentiated ; however, in contrast to other sarcomas, grade is not helpful in predicting survival. furthermore, no correlation has been shown between local recurrence or survival and tumor characteristics such as ulcerated, diffuse, or nodular. nevertheless, multifocal disease, positive surgical margins, size of the tumor (> 5 cm of external diameter of the tumor), mitotic rate (> 3 hpf), depth of invasion (> 3 mm), local regional recurrence and distant metastases have been shown to correlate with poor outcomes. in our patient, difficulty in making the diagnosis placed her at significant risk for reduced survival, especially given the size of her tumor. often, cutaneous angiosarcomas will initially be perceived as benign. according to one study, clinical signs of disease exist for an average of 5.1 months prior to diagnosis of scalp angiosarcomas. in some cases, diagnosis may be delayed for as long as 1 year despite continued signs and symptoms of disease. expanding nodular or papule type lesions that bruise or bleed for a prolonged period of time should raise concerns about an underlying malignancy and should be promptly investigated. a recent retrospective study reported on survival outcomes of 48 patients who were treated for angiosarcoma of face and scalp between 1987 and 2009 with either a single modality or combination of surgery, radiotherapy, chemotherapy, and immunotherapy. the median follow - up for all 48 patients was 13.7 months (range 2.5105.9). surgery and radiotherapy were found to be significant favorable and independent prognostic factors for os. patients who underwent both surgery and radiotherapy (2-year os : 45.8%) had a significantly more favorable os (p < 0.0001) compared with patients treated with either surgery or radiotherapy (2-year os : 11.1%) alone and patients who received no surgery or radiotherapy (2-year os : 0%). these findings corroborate data from a literature review which suggests that the optimal treatment for angiosarcoma of head and neck is surgery followed by wide - field radiotherapy. however, the tumor is often so extensive at diagnosis that complete surgical resection of the tumor may not be feasible. even with optimal local regional treatment, the likelihood of a local recurrence in the radiation field or distant metastases through hematogenous spread is quite high. mendenhall. reported 5-year local regional control from 40 to 50%, 5-year distant metastasis - free survival from 20 to 40%, and 5-year os from 10 to 30%. available data suggests usefulness for palliation with progression - free survival rates ranging from 1 to 5 months. nevertheless, there are a few case reports that have reported complete or partial response of tumor to chemotherapy when delivered either as a single modality or in combination with surgery and/or radiation. koontz. reported two cases of nasal angiosarcoma that responded with complete remission to treatment with bevacizumab, radiotherapy, and surgical resection with response duration of 26 and 8.5 months for the two cases. a retrospective study by schlemmer. reported on treatment outcomes for 8 patients with angiosarcoma of scalp and face who were treated with paclitaxel with or without other modalities such as surgery and radiotherapy. the authors reported one case (1/8) of complete remission with response duration of 42 months and five cases of partial response (5/8) with mean response duration of 5.8 months. a retrospective study by nagano. reported treatment responses for 9 patients with cutaneous angiosarcoma treated with docetaxel with or without previous treatment by surgery and radiation. the authors reported two cases of complete remission (2/9) and four cases of partial remission (4/9) ; of these, only one case had a single modality treatment with paclitaxel, resulting in a partial response. there are few other case reports that report complete remission of cutaneous angiosarcoma of scalp and face treated with combined liposomal doxorubicin and radiotherapy ; response duration ranged from 15 months to 4 years [2, 13, 14 ]. in summary, data on the role of chemotherapy in the definitive treatment of cutaneous angiosarcoma of face are limited and varied. to the best of our knowledge, there is only one documented case in the past literature that reports radiotherapy as a single modality treatment for angiosarcoma of the face. reports durable complete remission of a well - differentiated exophytic angiosarcoma of the face that responded well to radiotherapy alone. given the size and location of the tumor, our patient was deemed ineligible for surgery. aggressive surgical resection would have resulted in significant life - long morbidity with compromised cosmesis. from a definitive standpoint, combined chemotherapy and radiotherapy was the next available option. therefore, the patient underwent successful treatment with definitive radiotherapy, using electrons and photons. definitive radiotherapy may be an effective treatment in a select group of patients with head and neck angiosarcoma in whom chemotherapy and surgery may not be practically feasible. a delay in the diagnosis of angiosarcoma in the head and neck region could present with significant treatment challenges due to increased tumor size, especially since surgery and postoperative radiotherapy is the mainstay therapy in many patients. hence, it is very important to diagnose patients with angiosarcoma in a timely fashion. given the propensity of angiosarcoma to develop metastases, a high index of clinical suspicion early in the clinical course is crucial in order to maximize patient survival.	angiosarcoma is a rare, aggressive malignancy of endothelial cells lining blood vessels. it poses therapeutic challenges since there is no standard established treatment. it is typically treated with resection and wide - field postoperative radiation therapy. chemotherapy and radiation therapy have also been reported as initial therapies. regardless of the treatment rendered, the risk of local regional failure and distant relapse remains high for this disease. we present the case of a patient who developed a well - differentiated angiosarcoma of the nose with bilateral malar extension. no commonly associated risk factors such as lymphedema, prior radiotherapy or chronic venous ulceration were present. given her age, pre - existing renal condition and preference not to receive chemotherapy, systemic therapy was not utilized. surgery was also refused by the patient due to the projected cosmetic deficit. the patient was ultimately treated with definitive radiotherapy, utilizing electrons to the central face, differential thickness bolus, an intraoral stent, eye shields, an aquaplast mask for immobilization and a wax - coated lead shield over the face in order to limit penumbra of the radiation beam. right and left anterior 6-mv photons were used to tangentially treat the bilateral malar region in order to extend the field edges. at the time of this report, the patient remains disease free at nearly 2.0 years after radiotherapy. to the best of our knowledge, this represents only the second case in the literature reporting radiotherapy as a single modality treatment that resulted in complete remission of an angiosarcoma of the face.
the online version of this article (doi:10.1007/s00439 - 010 - 0939 - 8) contains supplementary material, which is available to authorized users. the concept of personalized medicine assumes that prediction of phenotypes based on genome information can enable better prognosis, prevention and medical care which can be tailored individually (brand. however, practical application of genome - based information to medicine requires the disease risk to be predicted with high accuracy, while knowledge on genetics of common complex diseases is still insufficient to allow their accurate prediction solely from dna data (alaerts and del - favero 2009 ; chung. knowledge gained on externally visible characteristics (evc) from genotype data obtained by examination of crime scene samples may be used for investigative intelligence purposes, especially in suspect - less cases (kayser and schneider 2009). the idea is based on using dna - predicted evc information to encircle a perpetrator in a larger population of unknown suspects. such approach could also be useful in cases pertaining identification of human remains by extending anthropological findings on physical appearance of an identified individual. one notable exception is eye (iris) color, where previous candidate gene studies and especially recent genome - wide association studies (gwas) revealed 15 genes involved (eiberg. 2002 ; kayser. 2008 ; liu. 2010 ; rebbeck. one of them, herc2, harbors genetic variation most strongly associated with human eye color variation (eiberg. 2008 ; kayser. moreover, a recent systematic study investigating the predictive value of eye color associated single nucleotide polymorphisms (snps) (liu. 2009) found that a model with 15 snps from 8 genes predicts categorized blue and brown eye color with high accuracies with a subset of only 6 snps covering most of the predictive information. the irisplex system employing these six snps and a prediction model based on data from thousands of europeans was recently developed and validated for dna prediction of human eye color in forensic applications (walsh. 2010a, b). furthermore, a recent gwas on quantitative eye color explained about 50% of continuous eye color variation by genetic factors (liu. the recent progress on dna prediction of human eye color raises expectations for the dna prediction of other human pigmentation traits, such as hair color. inheritance of one particular hair color in humans i.e., red hair, has already been explained to a significant degree. (1995) have found that red hair color is mainly associated with polymorphisms in the mc1r gene. this information has been confirmed since in many other studies performed on various population samples (box. mc1r snps are fairly indicative for red hair and thus have already been implemented in forensic science (branicki. 2007 ; grimes. 2001), but the practical application of red hair color prediction (without the ability to predict other hair colors) strongly depends on the population it is applied to, given the strong differences in red hair color frequency between populations. additional data on red hair color inheritance came from a recent gwas in icelanders, which revealed two snps in the asip gene, representing a mc1r antagonist, as significantly associated with red hair color (sulem. moreover, a position in the 3-utr of the asip gene was previously associated with dark hair color in european populations (kanetsky. the candidate gene approach also delivered two non - synonymous snps in slc45a2 (matp) with association to dark hair color in another study with several confirmation studies (branicki. 2005). via several large gwass various snps in / nearby genes in addition to mc1r, asip and slc45a2 have been found with association to human hair color variation, such as oca2, herc2, slc24a4, kitlg, tyr, tpcn2, tyrp1, irf4, exoc2, kif26a, and obscn (han. 2007, 2008). furthermore, two recently published studies tested for hair color association of snps from a large number of candidate genes, not only confirmed some previously known hair color genes, such as kitlg, oca2, mc1r, tyrp1, tyr, slc45a2, herc2, asip, but additionally reported association with quantitative measures of hair color of snps in additional genes, such as slc24a5, myo5a, myo7a, mlph, gpr143, dct, hps3, gnas, prkaria, ercc6, and dtnbp1 in one or both studies (mengel - from. 2010). in the present study, we tested in polish europeans with single - observer hair color phenotype data the predictive power of 45 snps from 12 genes previously implicated with replicated evidence in human hair color variation the study was approved by the ethics committee of the jagiellonian university, number kbet/17/b/2005 and the commission on bioethics of the regional board of medical doctors in krakow number 48 kbl / oil/2008. samples were collected from patients attending dermatological consultations at the department of dermatology of the jagiellonian university hospital. hair color phenotypes were collected by a combination of self - assessment and professional single observer grading. a single dermatologist interviewed and assessed all the subjects. the questionnaire included basic information, such as gender and age as well as data concerning pigmentation phenotype. the study included 385 individuals (39.0% male) after genetic and phenotypic quality control. categorical hair color was assessed by examination of the scalp in majority of cases. in a rare number of elderly volunteers and volunteers with dyed hair at the time of inspection we used i hair color was classified into 7 categories : blond (16.4%), dark - blond (37.7%), brown (9.4%), auburn (3.1%), blond - red (11.2%), red (10.6%), and black (11.7%). for some analyses, we grouped blond and dark - blond into one blond group (54.1%) and auburn, blond - red, and red into one red group (24.9%) resulting into 4 categories. notably, the frequency of red hair color in our study population is higher than expected in the general polish population because of an enrichment of red hair colored individuals in the sampling process. this was done to demonstrate prediction accuracy of red hair, similar to other hair color, as red hair normally is relatively rare in the polish population. hence, the color distribution of the selected samples in our study does not represent that of the general polish population (a point not relevant for the purpose of our study). this study was based on 45 snps from 12 genes (table 1), including slc45a2, irf4, exoc2, tyrp1, tpcn2, tyr, kitlg, slc24a4, oca2, herc2, mc1r, and asip that were associated with human hair color variation in several previous studies (duffy. 2007 ; graf. 2005 ; han. 2008 ; kanetsky. 2002, 2004 ; valverde. 1995 ; sulem. a subset of 25 snps were genotyped via mass spectrometry using sequenom multiplexing (see supplementary table s1 for additional details about markers and methods). multiplex assay design was performed with the software massarray assay design version 3.1.2.2 (sequenom inc. the results were two 7-plexes and one 11-plex (see supplementary table 1). a 2 ng of dried genomic dna in 384-well plates (applied biosystems) was amplified in a reaction volume of 5 l containing 1 pcr buffer, 1.625 mm mgcl2, 500 m dntps, 100 nm each pcr primer, 0.5 u pcr enzyme (sequenom). the reaction was incubated in a geneamp pcr system 9700 (applied biosystems) at 94c for 4 min followed by 45 cycles of 94c for 20 s, 56c for 30 s, 72c for 1 min, followed by 3 min at 72c. to remove the excess dntps, 2 l sap mix containing 1 sap buffer and 0.5 u shrimp alkaline phosphatase (sequenom) this was incubated in a geneamp pcr system 9700 (applied biosystems) at 37c for 40 min followed by 5 min at 85c for deactivation of the enzyme. then 2 l of extension mix is added containing a concentration of adjusted extend primers varying between 3.5 and 7 m for each primer, 1 iplex buffer (sequenom), iplex termination mix (sequenom) and iplex enzyme (sequenom). the extension reaction was incubated in a geneamp pcr system 9700 (applied biosystems) at 94c for 30 s followed by 40 cycles of 94c for 5 s, 5 cycles of 52c for 5 s, and 80c for 5 s, then 72c for 3 min. after the extension reaction it is desalted by the addition of 6 mg clean resin (sequenom) and 16 l water, rotating the plate for 15 min and centrifuging. the extension product was spotted onto a g384 + 10 spectrochip (sequenom) with the massarray nanodispenser model rs1000 (sequenom). the chip is then transferred into the massarray compact system (sequenom) where the data are collected, using typeranalyzer version 4.0.3.18 (sequenom), spectroacquire version 3.3.1.3 (sequenom), genoflex version 1.1.79.0 (sequenom), and massarraycaller version 3.4.0.41 (sequenom). the data were checked manually after the data collection. vaguely positioned dots produced by typeranalyzer 3.4 (sequenom) and all the wells that 50% or more failed snps were excluded from analysis. blanks (2%), controls (2%), and duplicates (9%) were checked for any inconsistencies and false positives. furthermore, snps in the mc1r gene were analyzed by amplification and cycle sequencing of the complete mc1r exon using the procedure described in branicki. selected polymorphisms within genes asip (rs6058017), oca2 (rs1800407, rs1800401, rs7495174, rs4778241, rs4778138), slc45a2 (rs16891982, rs26722), and herc2 (rs916977) were analyzed using minisequencing procedure and snapshot multiplex kit. briefly, the pcr reaction was composed of 5 l of qiagen multiplex pcr kit (qiagen, hilden, germany), 1 l of primer premix, 2 l of q solution, and 2 l (approximately 5 ng) of template dna. the temperature profile was as follows : { 94 for 15 min (94 for 30 s, 5860c for 90 s, and 72 for 90 s) 32, 72/10 min } 4c/. the pcr products were purified with a mixture of exoi and sap enzymes (fermentas, vilnius, lithuania) and subjected to multiplex minisequencing reactions with a snapshot multiplex kit (applied biosystems, foster city, ca, usa). a 2 l of snapshot kit was combined with 1 l of extension primers premix, 1 l of the purified pcr product, and nuclease - free water up to 10 l. the products of extension reactions were purified with sap enzyme and analyzed on abi 3100 avant genetic analyzer (applied biosystems, foster city, ca, usa).table 1single snp hair color association in a polish samplevariantchrpositiongeneabmafcoloror95% lower ci95% upper cip valotherrs16891982533987450slc45a2gc0.02black5.111.7914.550.002yesrs28777533994716slc45a2ac0.02black7.052.2322.250.001yesrs26722533999627slc45a2ga0.02black5.531.6418.680.006yesrs122035926341321irf4ct0.08black2.351.224.540.011yesrs93788056362727irf4ac0.450.103rs49592706402748exoc2ca0.46black0.560.350.910.020yesrs1408799912662097tyrp1ct0.290.097rs2733832912694725tyrp1tc0.400.177rs683912699305tyrp1ac0.340.099rs352648751168602975tpcn2at0.230.158rs38292411168611939tpcn2ga0.370.183rs23054981168623490tpcn2ga0.270.230rs10111761168690473tpcn2tc0.360.096rs10426021188551344tyrca0.280.255rs13933501188650694tyrga0.25brown1.701.022.820.041yesrs128212561287852466kitlgtc0.090.052rs128963991491843416slc24a4gt0.440.064rs49048681491850754slc24a4ct0.46blond0.640.430.970.037rs24021301491870956slc24a4ag0.16d - blond0.620.390.960.033rs18004071525903913oca2ct0.07red3.231.079.760.038rs18004011525933648oca2ct0.060.105rs169508211525957102oca2ct0.120.170rs71740271526002360oca2ct0.120.146rs47781381526009415oca2tc0.16brown1.801.023.190.043yesrs47782411526012308oca2gt0.180.078rs74951741526017833oca2tc0.050.069rs129138321526039213herc2ct0.22black3.331.995.574.3e06yesrs71838771526039328herc2ca0.070.124rs116358841526042564herc2tc0.010.135rs9169771526186959herc2ct0.15red0.340.180.650.001yesrs80391951526189679herc2tc0.11red0.300.140.640.002yesmc1r_r16mc1rwtr0.31red12.647.0322.742.5e17yesmc1r_r16mc1rwtr0.20red2.501.354.310.003yesrs18050051689985844mc1rgt0.08blond2.991.525.860.001yesy152och1689986122mc1rac0.000.982n29insa1689985753mc1r insa0.01red53.601.292221.720.036rs18050061689985918mc1rca0.000.476rs22284791689985940mc1rga0.09red0.430.190.970.043rs115474641689986091mc1rga0.02red3.351.0410.760.042rs18050071689986117mc1rct0.11red6.693.5012.799.3e09yesrs11104001689986130mc1rtc0.020.314rs18050081689986144mc1rct0.16red5.693.319.783.2e10yesrs8854791689986154mc1rga0.03blond2.901.216.960.017rs18050091689986546mc1rgc0.01red31.852.61388.280.007yesrs10153622032202273asipct0.30b - red1.671.022.750.043rs60580172032320659asipag0.130.211rs23782492032681751asipag0.18red2.341.144.820.021yesmaf minor allele frequency, color the most significantly associated color, or the allelic odds ratio for the minor b allele, shown only if p 0.05) did provide independent information toward hair color prediction (such as rs1042602 in tyr, rs683 in tyrp1, and rs12821256 in kitlg). only the non - synonymous snps from the tpcn2 gene tested did not contribute to the prediction model and did not show a statistically significant association with any hair color category. rs35264875 and rs3829241 in tpcn2 had been discovered recently as significantly associated with blond versus brown hair color in icelandic and replicated in icelandic and dutch people (sulem. predicting each color type separately using binary logistic regression yield slightly lower accuracy compared to the multinomial model (supplementary table s3).table 2parameters of the prediction model based on multinomial logistic regression in a polish samplesnpgeneeffect4 hair color categories7 hair color categoriesallelerankb1b2b3rankb1b2b3b4b5b6constant1.701.262.620.501.291.261.934.316.96mc1r_rmc1rr11.110.554.0911.700.980.541.845.416.02rs12913832herc2t21.750.102.4922.581.550.100.894.683.21rs12203592irf4t31.291.151.1341.431.241.141.031.161.19rs1042602tyra40.390.301.2030.530.350.301.071.081.67rs4959270exoc2a50.770.851.1550.560.810.841.240.921.11rs28777slc45a2c61.6913.890.101213.781.3111.097.842.022.91rs683tyrp1c70.100.580.02100.210.210.570.490.090.24rs1800407oca2t80.491.140.1981.010.441.1210.321.021.26mc1r_rmc1rr90.460.550.6160.740.400.550.531.221.69rs2402130slc24a4g100.480.090.5490.220.570.090.610.610.73rs12821256kitlgc110.690.010.87110.450.720.020.710.300.94rs16891982slc45a2c120.8211.783.48130.620.849.099.556.770.97rs2378249asipg130.180.160.4070.170.290.160.540.431.03b1, b2, b3 in the 4 categories are the betas for blond, brown, and red, all versus black ; b1 to b6 in the 7 categories are the betas for blond, d - blond, brown, auburn, b - red, and red, all versus black ; rank, prediction rank with 1 having the highest and 13 having the lowest rank in the prediction analysisfig. auc was plotted against the number of snps included in the multinomial logistic model for predicting 4 (a) and 7 (b) hair color categories. snp annotation and prediction ranks are provided in table 2 parameters of the prediction model based on multinomial logistic regression in a polish sample b1, b2, b3 in the 4 categories are the betas for blond, brown, and red, all versus black ; b1 to b6 in the 7 categories are the betas for blond, d - blond, brown, auburn, b - red, and red, all versus black ; rank, prediction rank with 1 having the highest and 13 having the lowest rank in the prediction analysis accuracy of hair color prediction using dna variants in a polish sample. auc was plotted against the number of snps included in the multinomial logistic model for predicting 4 (a) and 7 (b) hair color categories. snp annotation and prediction ranks are provided in table 2 overall, hair color prediction with 13 dna components from 11 genes showed very good accuracy without cross - validation, such as auc for blond = 0.81, brown = 0.82, red = 0.93, black = 0.87 in the 4 category model (table 3 ; fig. 1a), and auc for blond = 0.78, d - blond = 0.73, brown = 0.82, auburn = 0.82, b - red = 0.92, red = 0.94, black = 0.88 (table 3 ; fig. the mean accuracies derived from 1,000 cross - validations are somewhat lower for all hair color categories (least so for red), likely because of sample size effects as the rare alleles with large effects are not well captured in the training sets (table 3).table 3hair color prediction accuracy using 13 genetic markers in a polish sampleaccuracy4 hair color categories7 hair color categoriesblondbrownredblackblondd - blondbrownauburnb - redredblackusing all sample auc0.810.820.930.870.780.730.820.820.920.940.88 sensitivity0.880.080.780.310.170.800.140.000.530.660.38 specificity0.550.990.950.970.960.530.981.000.950.940.95 ppv0.700.380.840.580.480.510.450.000.560.590.49 npv0.800.910.930.910.860.820.920.970.940.960.92average of 1,000 cross - validations auc0.750.720.900.780.700.660.730.640.850.900.81 sensitivity0.830.050.740.240.150.710.080.000.410.440.29 specificity0.520.980.930.960.930.510.971.000.920.930.94 ppv0.670.210.770.450.330.460.200.000.420.430.43 npv0.720.910.910.900.850.750.910.970.920.930.91auc the area under the roc curves, ppv positive predictive value, npv negative predictive value hair color prediction accuracy using 13 genetic markers in a polish sample auc the area under the roc curves, ppv positive predictive value, npv negative predictive value in general, the sensitivities for predicting brown, red, and black colors were considerably lower than the respective specificities, except for blond in the 4 categories and dark blond in the 7 categories (table 3). the very low sensitivities for brown may reflect uncertainties in distinguishing between the dark - blond and brown colors on one side, and between the auburn, red and blond - red colors on the other side during phenotyping, as well as an additional sample size effect for auburn representing the smallest hair color group in our study (n = 12) however, the final model showed a good power to discriminate highly similar hair color categories, such as red and blond - red, as well as between blond and dark - blond (table 3), underlining the value of the genetic markers involved in our hair color prediction model. the roc curves from the final model (fig. 2) provide practical guides for the choices between desired false positive thresholds (1-specificity) and expected true positive rates (sensitivity) for predicting all color categories. for example, if the desired false positive threshold is 0.2 (in other words, if we use the predicted probability of p > 0.8 as the threshold for prediction, thus we know that we have at least 80% chance to be correct), then the expected true positive rates (or sensitivities) are 0.61 for blond (meaning that if a person has blond hair, our model provides a 61% chance to predict him / her as blond), 0.69 for brown, 0.78 for black, and 0.88 for red. notably, incorrect predictions fall more frequently in the neighboring category than in a more distant category, so the predictive information can still provide useful information.fig. 2roc curves for the final model including 13 dna predictors for 4 (a) and 7 (b) hair color categories in a polish sample roc curves for the final model including 13 dna predictors for 4 (a) and 7 (b) hair color categories in a polish sample we noticed that the prediction accuracies for the blond and brown colors were somewhat lower than those for black and red colors. one reason for this difference may be in the environmental rather than genetic contribution to hair color variation. hair color changes in some individuals during adolescence and such change is most often from blond to brown (rees 2003). since in our study we used adult individuals, those volunteers who had experienced such specific hair color change when being younger were grouped most likely in the brown hair category, although they may have blond associated genotypes. consequently, these individuals would have lowered the prediction accuracy for brown relative to the brown - haired individuals who have not changed from blond. our study design did not allow recording age - dependent hair color change, but this factor may be considered and tested in future studies. although, volunteers in the red hair color group of our study was significantly younger at time of sampling than people in any other hair color category groups (p 50% probability to have red hair, about 70% indeed had red hair. to make these previous findings more comparable with ours, we performed red hair prediction in our data by using only rs1805007 and rs1805008 as used by sulem. notably, this value is considerably lower than the one we received for red hair using all markers analyzed in the present study (0.93 or 0.94). hence, we can conclude that the additional snps we used in our full model, in particular the additional mc1r snps, improved red hair color prediction accuracy in our study. in a second step, (2007) used associated snps from all 6 loci to predict blond, dark blond / light brown, and brown / black hair color categories. they found in their icelandic replication set that among the individuals for whom brown hair color was predicted with > 50% probability, about 60% indeed had brown / black hair. however, their prediction results were much less convincing for blond since, from the individuals predicted to be blond with only > 40% probability (the highest threshold reported for blond), less than 50% were indeed blond, but about 50% were dark blond / brown and a few percentage were dark or red. performing auc prediction in our samples only with the snps used by sulem. (2007) resulted in auc values of 0.69 for blond, 0.71 for brown, and 0.75 for black. again, aucs for all non - red hair color categories as achieved in the present study considerably exceed those estimated from the markers used by sulem. (2007), which demonstrates the extra value of the additional markers we included in our model for accurate prediction also of non - red hair colors. a recently published candidate gene study employed linear regression modeling using snps from hair color candidate genes and found that three snps in herc2, slc45a2 and slc24a5 together explain 76% of total hair melanin in the study population (valenzuela. 2010). it has been shown that the least absolute shrinkage and selection operator (lasso) approach (tibshirani 1996) can be used to estimate marker effects of thousands of snps in linkage disequilibrium (ld) (usai. were in ld, we additionally performed the multinomial lasso regression and compared the prediction results with those from our multinomial logistic regression model. the auc estimates from lasso using all samples (auc blond = 0.88, brown = 0.89, red = 0.96, black = 0.96) are slightly higher than the ones from the multinomial logistic regression (table 3). however, the average auc values from the 1,000 cross - validations of the lasso approach (auc blond = 0.66, brown = 0.62, red = 0.86, and black = 0.76) are considerably lower than the ones obtained from all samples with the same approach, and are also lower than the results from the multinomial logistic regression (table 3). this may indicate that there is a potential over - fitting problem in the lasso method and our data. because the sample size used in this study is relatively small (n = 385), we estimated the effect of the total sample size on the accuracy of pigmentation prediction using a bootstrap analysis of the eye color data published previously (liu. 2009), in which a auc value of 0.91 was obtained for predicting blue eye color based on a large population sample (n = 6,168). as evident from supplementary figure s1, if the total sample size is smaller than 300 individuals, the auc value for blue eye color tends to be under - estimated with large confidence intervals. for example, with only 100 samples the mean auc value from 1,000 bootstrap analyses was considerably lower (auc = 0.85, 95% ci : [0.61.0 ] ; figure s1) than the value of 0.91 as achieved with thousands of samples (liu. 2009). however, this effect quickly diminishes when the sample size increases, and with about 350 samples the mean auc value was close (auc = 0.90, 95% ci : [0.800.97 ] ; figure s1) to the value obtained from thousands of samples, and only increased marginally until 800 samples. from this example of blue eye color we may extrapolate that the aucs for hair color obtained from the 385 samples included in the present study (which are similar to the auc obtained for blue eye color) are unlikely to change drastically when more individuals are added to the hair color model. many genetic studies on hair color (as well as eye and skin color) use phenotypic information provided by self - assessment, i.e. questionnaires filled out by the individual participants (e.g. sulem. 2007, 2008 ; han. 2008), which may be expected not to be completely reliable. to avoid hair color phenotype uncertainties potentially generated by such multiple - observer approach, we performed single - observer hair color grading in the present study. 2008). however, it is not clear how these methods as well as self - assessment and single - observer hair color categorization compare to each other and what the impact on dna - based prediction accuracies is. on the one hand vaughn. (2008) in a phenotypic study found some differences between single - observer hair color grading and spectrophotometric measurement, but the sample size was low (with about 100 individuals). on the other hand shekar (2008) in a genetic study could not confirm the utility of spectrophotometric estimation in relation to hair color rating. the single - observer grading approach we applied in the present study was found to be more accurate than using self - assessed hair color grading (vaughn., we demonstrated that human hair color categories can be accurately predicted from a relatively small number of dna variants. the prediction accuracies achieved here for red and black hair color were in the similarly high precision range as previously obtained for blue and brown eye color, for which practical applications has already been implemented (walsh. 2010a, b). slightly lower prediction accuracies obtained here for blond and brown hair color, which were still higher than previously observed for non - blue / non - brown eye color (liu. 2009), may be influenced by age - dependent hair color change during adolescence, which shall be investigated in more detail in future studies. although our example of using eye color to monitor the effect of sample size to the auc - based prediction accuracy of pigmentation traits indicate that the sample size used here for hair color prediction is large enough to obtain a reasonably accurate prediction model, our results may be further replicated in a larger study. furthermore, it shall be tested in future studies if and to what extent snps from other genes with recently reported hair color association not used here add to the hair color prediction accuracy as presented. overall, we evidently present hair color as the third externally visible characteristic that can be reliably predicted from dna data after iris color (liu. 2009 ; walsh. 2010), and human age, the latter demonstrated recently using quantification of t - cell dna rearrangement (zubakov. we therefore expect dna - based hair color prediction, e.g. using the markers suggested here, to be used in future practical applications, such as in the forensic context. furthermore, our study demonstrates that markers not statistically significantly associated with a trait in a study population can still independently contribute to the trait prediction in the same population, a notion that shall be considered in the design of future genetic prediction studies, including for diseases risks. below is the link to the electronic supplementary material. supplementary material 1 (doc 519 kb)	predicting complex human phenotypes from genotypes is the central concept of widely advocated personalized medicine, but so far has rarely led to high accuracies limiting practical applications. one notable exception, although less relevant for medical but important for forensic purposes, is human eye color, for which it has been recently demonstrated that highly accurate prediction is feasible from a small number of dna variants. here, we demonstrate that human hair color is predictable from dna variants with similarly high accuracies. we analyzed in polish europeans with single - observer hair color grading 45 single nucleotide polymorphisms (snps) from 12 genes previously associated with human hair color variation. we found that a model based on a subset of 13 single or compound genetic markers from 11 genes predicted red hair color with over 0.9, black hair color with almost 0.9, as well as blond, and brown hair color with over 0.8 prevalence - adjusted accuracy expressed by the area under the receiver characteristic operating curves (auc). the identified genetic predictors also differentiate reasonably well between similar hair colors, such as between red and blond - red, as well as between blond and dark - blond, highlighting the value of the identified dna variants for accurate hair color prediction.electronic supplementary materialthe online version of this article (doi:10.1007/s00439 - 010 - 0939 - 8) contains supplementary material, which is available to authorized users.
legg - calv - perthes ' disease (lcpd) is an idiopathic osteonecrosis of the proximal capital femoral epiphysis in children. the healed hip may range from an essentially normal contoured femoral head (stulberg i) to one with incongruous incongruity (stulberg v). as with any pathologic process, lcpd goes through a course of disease denoted by the waldenstrm stages, which are synovitic, avascular, fragmentation (collapse), reossification (healing), and healed (residual). the magnitude of epiphyseal involvement is determined by the catterall class, salter - thompson group, and/or lateral pillar group. the catterall class is determined on both anteroposterior and frog - lateral radiographs during the stage of maximum fragmentation, the salter - thompson group is determined on the frog - lateral radiograph during the avascular / precollapse stage using the subchondral crescent fracture, and the lateral pillar classification is determined on the anteroposterior radiograph during early fragmentation. lcpd has been known by at least 22 different names since its first description in the late 19th and early 20th centuries. since 1963, the official medical subject heading (mesh) used by the national library of medicine is legg - perthes ' disease, but many other names had been previously used. to ensure capture of all the published literature, therefore, the terms used to search for lcpd were arthritis deformans juvenilis, calve - perthes disease, coxa plana, femoral head necrosis, juvenile chondroepiphysitis, legg - calve - perthes disease, legg - perthes disease, legg 's disease, osteochondritis deformans juvenilis, osteochondritis deformans juvenilis coxae, osteochondritis juvenilis, osteochondrosis of capital epiphysis of femur, perthes disease, and pseudocoxalgia. the databases searched were pubmed (http://www.ncbi.nlm.nih.gov/pubmed/), ovid medline, embase, worldcat (books and theses) (http://firstsearch.oclc.org/), and indexcat (index catalogue of the library of the surgeon - general 's office) (http://www.indexcat.nlm.nih.gov/). exclusion criteria were those manuscripts discussing surgery, therapy, rehabilitation, and any foreign language articles without an english abstract. individual journals were also searched for articles published prior to 1996 that predate electronic medline indexing, including journal of bone and joint surgery (american and british), clinical orthopaedics and related research, and acta orthopaedica scandinavica. the dates for the search were 18801961 for indexcat, 19002009 for worldcat, 19481965 for oldmedline, and 1950february 2010 for ovid medline. these 1124 manuscripts were reviewed to find those that discussed any of the topics regarding etiology, epidemiology, demographics, incidence, prevalence, race, gender, family history, genetics, inheritance, age, bone age, weight (either birth weight or normal weight), height, growth, maturation, other anthropometric characteristics, hormone / endocrine, smoking, coagulation, fibrinolysis, congenital anomalies, collagen, immunoglobulin, opposite hip, behavior / psychology, seasonal variation, and infection. of these 1124 manuscripts, 144 provided ample information and are the contents of this paper. the conventional quotation for the incidence of lcpd is the number per 100,000, usually for age lateral pillar b). a marker of type iii collagen synthesis is the procollagen type iii n - terminal propeptide (p3np). type iii collagen synthesis is reduced at diagnosis in children with lcpd as demonstrated by very low levels of p3np. however, there were no controls, and the differences in children with lcpd compared to otherwise normal children in the same geographic / ethnic / socioeconomic situation are not known. a recurrent mutation in type ii collagen (cartilage collagen) in a japanese family with lcpd has been noted. this mutation amino acid change (p.g1170s) perturbs the gly - x - y triple - helix of type ii collagen. similar findings were noted in a chinese family where a p.gly1170s mutation of col2a1 resulted in premature hip osteoarthritis, avascular necrosis of the femoral head, or lcpd, depending upon the age at onset. in a cohort of nonfamilial children with lcpd, glycosaminoglycans (gags) are chains of repetitive disaccharide units linked with proteins in the cartilaginous extracellular matrix to form proteoglycans. upon cartilage degradation, elevated urinary gag levels indicate increased articular cartilage degradation. decreased levels of urinary gags in children with lcpd compared to normal children or those with transient synovitis have been noted. this can be interpreted as either increased preservation of the gags within the hip or a decrease in the quantity of synovial fluid. increased levels of proteoglycan fragments and stromelysin in the synovial fluid of children with lcpd have been noted, consistent with a synovitis. in a review of the radiographs of 153 children with unilateral lcpd, 48.4% demonstrated irregularity of the epiphyseal surface, flattening, or dimpling of the opposite normal hip. in most instances (37%), they were present in the initial radiograph. similar changes were noted in only 10.4% of a control group of 153 age and gender - matched children using intravenous urograms. this was interpreted as the capital femoral epiphysis in the young child being very vulnerable to stress ; the minimal contour irregularities in the normal hip represent one end of the spectrum and frank lcpd, the other as the stress response of the capital femoral epiphysis. another study confirmed that the unaffected hip in lcpd demonstrates anterior and lateral flattening perhaps indicating a constitutional abnormality. in a third study, 15% of the opposite normal hips demonstrated physeal changes, especially decalcification below the physis. the initial radiographs of 125 japanese children with unilateral lcpd demonstrate delayed ossification of the opposite epiphysis as seen by diminished epiphyseal height. children with lcpd are extremely busy and active. an early study (phd thesis) discovered that children with lcpd demonstrated a motor - expressive personality, an active approach to life and had higher psychosomatic and visceral complaints. a later study reviewed the behavioral characteristics of 24 children with lcpd ; 33% of children with lcpd had abnormally high scores in standard psychological child behavioral questionnaires for profiles associated with attention deficit hyperactivity disorder, greater than the 35% of age matched children. certain epidemiologic characteristics of lcpd (gender, socioeconomic status, geographic location, and associated congenital anomalies) are also similar characteristics of attention deficit hyperactivity disorder. these findings were confirmed in a recent study of 19 children with lcpd ; 8 of 12 school - aged children had negative scores in neuropsychological tests and 5 of the 8 had learning difficulties at school. an increase in both major and minor congenital defects in children with lcpd is known. these include anomalies of the genitourinary tract and inguinal region and spina bifida occulta [10, 141, 142 ]. sacral inclination, decreased lumbar lordosis, and an overall more negative spinal balance with vertebral end plate anomalies have been recently described in the spine of lcpd patients. low blood manganese levels were noted in children with lcpd in liverpool, but refuted by others. an increase in igg and igm, but not iga serum immunoglobulin levels in lcpd, are described, suggesting that immunological mechanisms may mediate certain changes in lcpd. there clearly is disharmony between cartilage and bone and growth in lcpd as evidenced by progressive caudal growth impairment and delays in skeletal maturation, both involving the wrist and the pelvis. the insult on skeletal maturation appears to occur early in life, perhaps even prenatally, since there is an increased frequency of minor congenital malformations in children with lcpd. these delays in maturation (both anthropometric and skeletal age) can be due to a combination of familial and environmental circumstances (lower socioeconomic class with malnutrition, underlying genetic / collagen defects, or some other unknown entity). the delay in skeletal ossification results in a weaker skeleton that is more susceptible to trauma. a highly active child incurs more skeletal injuries ; this microtrauma in a biologically susceptible weaker skeleton creates microfractures in the proximal femoral epiphysis and metaphysis. a hypercoagulable state, due to underlying abnormalities in the clotting mechanisms and/or exposure to passive smoke, results in increased thrombosis in the proximal femur after microfractures with subsequent necrosis of the capital femoral epiphysis and the development of lcpd. as a systematic literature review, institutional review board approval is not applicable. this was the 2nd of three presentations on the epidemiology and demographics of pediatric hip disorders given at the ao north american symposium on surgical preservation of the hip, squaw valley, california, january 2009	the etiology of legg - calv - perthes ' disease (lcpd) is unknown. there are many insights however from epidemiologic / demographic information. a systematic medical literature review regarding lcpd was performed. the incidence ranges from 0.4/100,000 to 29.0/100,000 children < 15 years of age. there is significant variability in incidence within racial groups and is frequently higher in lower socioeconomic classes. the typical age at presentation ranges from 4 to 8 years (average 6.5 years), except for children from the indian subcontinent (average 9.5 years). there is a mild familial component. the children demonstrate impaired growth in height, skeletal age, and birth weight. this impaired growth coincides with an age appropriate reduced somatomedin a activity and decreased levels of igf. lcpd can be associated with abnormalities in the coagulation cascade, including an increase in factor v leiden mutation, low levels of protein c and/or s, and decreased antithrombin activity. there is decreased turnover in type i collagen and synthesis of type iii collagen, as well as reduced levels of urinary glycosaminoglycans in the active phases of the disorder. subtle abnormalities in the opposite hip and other minor / major congenital defects are reported. children with lcpd are active and score abnormally in certain standardized psychological tests.
a 40-year - old woman was referred for electroconvulsive therapy (ect) for a severe depressive illness. she had a background of severe psoriasis for which she received methotrexate for nearly 20 years and adalimumab by subcutaneous injection every 2 weeks. she had been commenced on prednisolone, amoxycillin and ipratropium and her first session was cancelled owing to her dyspnea and widespread wheeze, although she was apyrexial. it had already been agreed with the patient that she would commence unilateral ect owing to concerns about cognitive side effects. on the first session the patient was given a 5% stimulus from a thymatron iv machine using the delia placement, producing a 39-s seizure which was taken to be the seizure threshold. no ill effects were noted at any point and the patient made a full recovery. on the second session however the patient received a stimulus of 30% in the same position and administered by the consultant, in line with recent developments in unilateral application. as she recovered, she complained of pain around her right eye and was dysarthric. on clinical examination she was noted to have an injected right conjunctiva and a right facial palsy affecting her right eye, and mouth. the patient made a full recovery but ect was suspended while she had further investigations advised by the neurologist. this included a c - reactive protein which was normal and mri brain which revealed no abnormality. the patient subsequently continued with bilateral ect for 20 sessions and made a good recovery from the depressive disorder and no further neurological sequelae were observed. todd 's paralysis is described as a temporary weakness usually affecting one or more limbs and which usually occurs after a focal seizure. however, a similar phenomenon resulting in language, somesthetic and visual deficits can occur depending on the focal area involved. the rationale for converting to bilateral ect was to avoid a focal seizure which may have been significant in producing this focal neurology. this condition has not been described in connection with ect as far as we are aware. this may represent a rare indication of the need to convert from unilateral to bilateral ect.	this case describes a woman undergoing unilateral electroconvulsive therapy (ect) who developed a todd 's palsy following the treatment, and which resolved when converted to bilateral ect. we go on to hypothesize that this rare side effect may be an indication of the need to switch laterality during a course of ect.
brain tumors are classified based on their relation to the neuraxis those arising within the substance of the brain or spinal cord are termed intraaxial lesions, whereas those that arise outside are termed extraaxial. several studies have established the occurrence of a systemic inflammatory response to brain tumors, similar to that seen with other malignancies. the degree and type of inflammatory response would obviously depend on the location, antigenicity, and vascularity of the lesion both in the brain and elsewhere in the body. thus, it may reasonably be expected that the degree and direction of derangement of the systemic markers of inflammation vary between malignancies, based both on anatomic location and biologic aggressiveness of the neoplasm. the neutrophil to lymphocyte ratio (nlr) is the ratio of the count (or percentage) of neutrophils to lymphocytes in peripheral blood and is a systemic marker of inflammation. the nlr has been shown to have prognostic significance in pancreatic, liver, prostate, and bladder cancers, and more recently, in glioblastoma multiforme (gbm) as well. however, it has not been clearly established whether the nlr and/or other leukocyte counts and indices are deranged in all patients with malignant intraaxial brain tumors and if so, the magnitude and direction of such derangement. moreover, no data have been published documenting the derangement of peripheral counts and ratios in patients with low grade [world health organization (who) grade 1 and 2 ] intraaxial lesions and benign extraaxial lesions. in this study, peripheral blood counts and some leukocyte indices in a control population were compared with those of patients with low and high grade intraaxial lesions and benign extraaxial brain tumors. this institutional review board (irb)-approved study (jip / iec / sc/2014/10/706) was conducted prospectively over a period of 6 months (october 2014 to march 2015). the tumor group included treatment - nave patients with primary brain tumors at the time of initial diagnosis. this group included patients with low grade and high grade intraaxial tumors and benign extraaxial tumors. patients who were on steroids, those suffering from any endocrinopathy, and individuals with suspected or diagnosed infections or autoimmune disease were excluded. age - matched controls were randomly selected from the hemogram data for those healthy individuals who had undergone tests for either pre - employment screening or as part of other routine check - up protocols. for all subjects who had undergone a hemogram study, 2 ml of blood was drawn in an ethylene diamine tetra - acetic acid tube and a complete blood count was obtained using the sysmex 2000i hematology analyzer (sysmex, japan, sysmex india pvt ltd. the parameters measured included hemoglobin, total leukocyte count (tlc), differential count (including neutrophil, lymphocyte, basophil, eosinophil, and monocyte counts), and platelet count. two ratios were calculated from the differential count the nlr and the monocyte to lymphocyte ratio (mlr). in the tumor group, the linear dimensions of each tumor were measured in three orthogonal planes (axial, sagittal, and coronal) on gadolinium contrast - enhanced t1 sequences of magnetic resonance (mr) images and the volume of the tumor was calculated using the formula v = (abc)/2. the data were analyzed using the statistical package for the social sciences (spss) (version 17, ibm, new york, usa) and graphs were plotted on ms excel 2013. the control group had data pertaining to 216 healthy individuals, out of whom 37 were females. the mean nlr for the control group was 1.97 0.84 and the mean mlr was 0.18 0.08. no mathematically definable relation was noted between age and the nlr or age and the mlr [figure 1a and b ]. an inverse linear relationship was noted between the neutrophil and lymphocyte counts and this was maintained across the tumor groups as well. no definite relationship was found between the monocyte and lymphocyte counts [figures 1c and d ] there was no significant difference in the mean nlr between men (2 1.03) and women (1.97 0.87, t = 0.084, p = 0.933) or in the mean mlr between men (0.17 0.07) and women (0.18 1.12) (t = -0.43, p = 0.665). a total of 115 patients were recruited to the tumor group 70 with intraaxial tumors and 45 with extraaxial lesions. the control group. (a) this graph plotted between age and the nlr shows an absence of any relationship between these two variables (b) this panel shows the plot of age versus mlr. there is no clear relation between the two (c) plot of neutrophil count versus lymphocyte count. a definite negative linear relationship is noted (d) there was no definite relation between the lymphocyte and monocyte counts all patients in the intraaxial lesion group had supratentorial gliomas 36 of these patients were eventually found to have gbm (who grade 4) and 9 had who grade 3 lesions. sixteen were low grade gliomas (who grades 1 and 2) and the tumor grade was not known for nine patients. the male to female distribution was 1.33 : 1 and the mean age was 32.89(16.3) years. there is a relative reduction in the hemoglobin level as well as a relative increase in platelet counts in the tumor group when compared to the control group. in the tumor group the neutrophil and eosinophil counts there was no significant difference in the lymphocyte count between the tumor and control groups. the mlr was significantly reduced in the tumor group compared to the control group (p 4 was an independent prognostic factor in patients with glioblastoma. in this study, we found that the nlr in patients with intraaxial tumors was elevated compared to the control group (trend toward significance, p = 0.05). when patients with gbm were analyzed separately, it was found that the elevation in nlr when compared to the controls was statistically significant (p = 0.02). furthermore, we found that this increase in the nlr in the intraaxial tumor group was due to an increase in the neutrophil count rather than a decrease in the lymphocyte count. it is thus evident that there is an increase in the phagocytic cells of inflammation in patients with intraaxial tumors. thus, it appears that in all patients with gbm, there is an elevation in the nlr. the subset of patients who have an nlr>4 may have aggressive forms of the disease and may be prone to earlier recurrence / progression. the occurrence of alterations of the peripheral blood counts and indices in patients with benign extraaxial tumors and low grade gliomas point to the existence of an immune response to these tumors as well. the major limitation of this study is the lack of follow - up and outcome data. a correlation between the mlr / nlr and disease - free survival would make any analysis of significance more robust and credible. this study provides some baseline data about the hematologic derangements that occur in patients with both extraaxial and intraaxial tumors. the derangement of these counts and indices in patients with extraaxial tumors as well as in patients with low grade intraaxial brain tumors is a curious finding. this project was partly funded by the intramural golden jubilee short - term research award for undergraduate students (gj - straus) scheme awarded by jipmer in the year 2014 - 2015. this project was partly funded by the intramural golden jubilee short - term research award for undergraduate students (gj - straus) scheme awarded by jipmer in the year 2014 - 2015.	background : elevation of the neutrophil to lymphocyte ratio (nlr) has been shown to be an indicator of poor prognosis in many malignancies including recurrent glioblastoma multiforme.objectives:this study was aimed at assessing if the nlr and other leukocyte counts and indices were deranged in treatment - nave patients with primary brain tumors when compared with an age - matched healthy control group.materials and methods : this was a prospective comparative clinical observational study by design. a healthy control population was compared with treatment - nave patients diagnosed with intra- and extraaxial brain tumors. leukocyte counts (neutrophil, lymphocyte, monocyte, eosinophil, and basophil counts) as well as leukocyte ratios such as the nlr and the monocyte to lymphocyte ratio (mlr) were calculated. we also evaluated if the counts and indices were related to the tumor volume.results:in all patients with tumors, the platelet and neutrophil counts were elevated when compared to the controls. in contrast, monocyte counts and the mlr were found to be decreased in patients with tumors when compared to the controls. the subset of patients with glioblastoma showed a significant increase in nlr when compared to the controls.conclusions:significant changes in the neutrophil, monocyte, and platelet counts as well as nlr and mlr were observed. prospective longitudinal studies are required to determine the prognostic and therapeutic implications of these findings.
chronic hypertension leads to concomitant remodeling of the cardiac and vascular systems, and various organs, especially the brain, kidney, and retina [1, 2 ]. early detection of hypertensive target organ damage is important for more - successful prevention of cardiovascular diseases and to improve outcomes [1, 2 ]. the brain is an early target for organ damage due to high blood pressure (bp) [1, 2 ], which is the major modifiable risk factor in men and women for ischemic and hemorrhagic stroke, as well as small vessel disease [1, 2, 4 ] predisposing to lacunar infarction, white matter lesions (wml), and cerebral microbleeds, which are frequently silent [1, 2, 5 ]. stroke is the second leading cause of death and the leading cause of disability worldwide. for each decade of life after the age of 55 years, the stroke rate doubles in both men and women, and > 80% of strokes occur in people aged 65 years. because of the aging population the increased vulnerability of elderly people to stroke is associated with changes in the aging brain and also with a higher prevalence of well - documented risk factors for stroke such as hypertension, atrial fibrillation, carotid stenosis, and cardiovascular disease. it is known that wmls are an important prognostic factor for stroke, cognitive impairment, dementia and death. cerebral wml are more common and extensive in patients with cardiovascular risk factors, such as hypertension and diabetes mellitus, heart disease, and symptomatic cerebrovascular disease [710 ]. however, it is controversial whether bp still is associated with wml in patients manifesting vascular disease.. showed in 1030 patients manifesting vascular disease (including cerebrovascular disease, coronary heart disease, peripheral arterial disease, abdominal aortic aneurysm), that bp was not associated with the presence of wml, irrespective of the presence of diabetes mellitus or the localisation of vascular disease. older age and hypertension are constantly reported to be the main risk factors for cerebral wml [4, 10 ]. hypertensive patients have a higher rate and extension of cerebral wml compared with normotensives [10, 12 ], but treated, controlled hypertensive patients have a lower prevalence of wml than both untreated and treated but uncontrolled hypertensives. a magnetic resonance imaging (mri) substudy of the randomized progress trial of bp lowering with perindopril versus placebo in normotensive and hypertensive subjects with cerebrovascular disease found that the mean total volume of new wml was significantly reduced in the active treatment group compared with placebo. a post hoc analysis found that greatest beneficial effect of antihypertensive therapy on wml progression was observed in patients with severe wml at study entry. godin. have recently shown that, in a prospective population - based cohort of 1319 subjects aged 65 years, bp at baseline and changes in bp over a 4-year follow - up are strong predictors of wml volume progression independently of potential confounders and suggest that correct antihypertensive treatment could slow wml progression. interestingly, the cardiovascular health study has been recently shown that diastolic bp had no effect on ischemic stroke incidence in elderly patients with low wml levels but had a marginally significant j - curve relationship with ischemic stroke in elderly with high wml levels. indeed, in elderly individuals with low - grade wml, low dbp however, in high - grade wml, ischemic stroke risk may increase in diastolic bp less than 69 mmhg but is highest more than 80 mmhg. the main current hypothesis concerning the association between high bp and wml is that long - standing hypertension causes lipohyalinosis of the media and thickening of the vessel walls, with narrowing of the lumen of the small perforating arteries and arterioles that nourish the deep white matter. the perforating vessels, which originate in the cortical and leptomeningeal arteries, have a relatively poor anastomotic system, which makes the white matter particularly vulnerable to cerebral ischemia.. showed in a 20-year follow - up study that risk of late - life wml was related to midlife hypertension, and hypertension from midlife to late life also increased the risk of wml. in addition, an association with wml was seen for decreasing bp (hypertension at midlife but not at late life) even after controlling for antihypertensive treatment. authors speculate that the decline in bp could be secondary to dementia - related processes, when structures involved in bp regulation become affected. it has been shown in elderly people that severe wmls frequently coexist with medial temporal lobe and global brain atrophy. there is strong evidence that cerebral wml in hypertensive patients should be considered a silent early marker of brain damage. early atherosclerotic changes in the cerebrovascular system, ultimately leading to incident stroke and cognitive impairment or dementia [4, 7 ], could be mirrored by hypertensive target organ damage in the cardiac, renal, retinal, and vascular and other systems. therefore, assessment of hypertensive target organ damage in these systems may be indicative of the extent of vascular disease in less easily accessible sites such as the brain. several forms of heart damage, such as heart failure, coronary heart disease, and cardiac arrhythmias have been associated with wml although the underlying pathogenetic mechanisms are not clear. studies have reported that echocardiographically determined left ventricular hypertrophy (lvh) is an independent risk factor for cardiovascular morbidity and mortality in essential hypertensive patients [18, 19 ]. demonstrated that left ventricular mass (lvm) was associated with an increased risk of cerebrovascular events such as stroke and transient ischemic attack in an elderly cohort from the framingham heart study. it is suggested that left ventricular geometric patterns add prognostic information on the development of cardiovascular disease and the presence of extracardiac target organ damage in essential hypertension [22, 23 ]. hypertensive patients with concentric lvh have more - advanced target organ damage, including renal [22, 23 ] and retinal involvement, than patients with other patterns of left ventricular geometry. some studies have found an association between lvh and cerebral wml [2328 ], but others have not. we found a close relationship between silent wml and concentric lvh in middle - aged untreated essential hypertensive patients, with wml being more common among patients with concentric lvh. the mechanisms connecting lvh and cerebrovascular damage are unclear and might reflect long - term exposure to genetic, hormonal, or metabolic factors in addition to bp. it is difficult to differentiate the relative role of high bp from the direct contribution of lvh to the increased risk of cerebrovascular disorders, and longitudinal studies are necessary. however, detection of cardiac hypertrophy may help to identify patients at risk of cerebrovascular injury. the brain and the kidney are highly vascular structures that respond to diseases such as hypertension and diabetes mellitus in similar ways at the microscopic level. in nephrosclerosis, gradual alterations in the renal endothelial cells, glomeruli, and interstitial spaces lead to glomerular leakage of serum proteins into the urine. if a similar process were occurring at the endothelial level in cerebral microvessels, serum proteins would pass into the cerebral extracellular space. neuropathological studies show that white matter hyperintensities may represent enlarged perivascular spaces and perivascular demyelination, among other mechanisms. these changes are what might be expected if the cerebral extracellular spaces were exposed to proinflammatory proteins which, in healthy individuals, should remain within the vascular space. while there is no direct proof that this process occurs it has been suggested that the brain and the kidney have a common, unique way of reacting to fluctuations in bp and flow due to similar low - resistance vascular beds. high - pressure fluctuations in the carotid, vertebral, and renal arteries, together with the turbulent flow, expose the small vessels of these two organs to pressure and flow fluctuations that may explain the microvascular damage and the resulting renal failure and neurological and cognitive alterations. in addition, chronic renal disease (crd) has been linked with proinflammatory and procoagulant states, which may contribute to wml [37, 38 ]. small vessel disease is a systemic condition which is induced by aging and exacerbated by vascular risk factors, especially hypertension, and affects the brain and other systems. in the kidney, the damage markers are albuminuria / proteinuria and the estimated glomerular filtration rate (egfr), which shows the functional reduction. studied 615 stroke - free subjects (mean age : 70 years) and found that the prevalence of silent wml was independently associated with crd (people with egfr between 1560 ml / min per 1.73 m when compared to people with 6090 ml / min, and > 90 ml / min) even after adjustment for age, sex, ethnicity, education, and vascular risk factors. the rotterdam study of 484 subjects (mean age : 73.4 years ; mean systolic bp : 145.7 mmhg ; mean egfr : 54.8 ml / min/1.73 m) found that individuals with a lower egfr had more wml. takahashi. recently studied 2,103 asymptomatic individuals with a younger mean age of 56 years and found that the prevalence of subcortical wml and periventricular wml correlated significantly with lower egfr ; in subgroups with egfr 90, 6089, and 61 years, found that subjects with lower egfr had higher grades of silent wml, that mean grades of wml were greater in subjects with albuminuria than in those without, and that age and the prevalence of hypertension were significantly higher in individuals with higher grades of wml.. also found an independent association between microalbuminuria and wml in 1251 asymptomatic individuals (mean age : 63.8 years, 78% with hypertension). the retina offers a unique, noninvasive, and easily accessible window to study the microvascular etiology of cerebrovascular disease. retinal and cerebral small vessels share similar embryological origins, anatomical features, and physiological properties. retinal microvascular abnormalities, such as microaneurysms, retinal hemorrhages, soft and hard exudates, arteriovenous nicking, and retinal focal arteriolar narrowing, usually result from small - vessel damage due to aging and high bp. arteriolar narrowing may be a sign of hypertension, a history of hypertension in the last three to six years, or a risk factor for the onset of hypertension in normotensive individuals. large prospective studies (the aric, beaver dam, blue mountains eye, and rotterdam studies) have found that reduced arteriolar diameter is an independent risk factor for developing hypertension within 3 to 10 years in normotensive individuals. studies have shown that retinal microvascular flow is reduced in persons with wml and lacunar infarction and that retinal and cerebral arterioles share a similar histopathology in patients dying from stroke. in the aric study, retinal abnormalities were associated with concurrent bp. in a cohort of 1684 asymptomatic people aged 5172 years from the aric study, individuals with wml were more likely to have retinal microvascular abnormalities. in general, in this study, wmls were significantly associated with increasing age, black ethnicity, and after adjusting for age, sex, and ethnicity with higher bp and increased carotid intima - media thickness (imt). retinopathy was significantly associated with black ethnicity and, after similar adjustment, with higher systolic bp, fasting glucose level, diabetes mellitus, and increased carotid imt. after a follow - up of 10 years, retinal microvascular abnormalities measured at baseline were prospectively associated with a long - term risk of subclinical cerebrovascular disease on mri, independent of conventional risk factors in this population - based cohort of middle - aged persons without clinical stroke. the authors suggested that retinal microvascular abnormalities are early and, possibly, more sensitive markers of subclinical cerebral small - vessel disease before radiological and clinical manifestations become apparent. in a cohort of 1717 people from the cardiovascular health study (mean age : 78.3 years ; hypertension : 56.3% ; diabetes mellitus : 13% ; previous cardiovascular disease, including stroke : 23.8%) associations were found between wml grade, prevalent lacunar infarct, and a lower arteriovenous ratio. in 174 patients without a history of transient ischemic attack or stroke before follow - up, wmls worsening on mri 5 years later and incident lacunar infarct were associated with a lower arteriovenous ratio. in light of data reporting that persons with a smaller arteriolar - to - venular ratio tended to have more wmls on mri, the rotterdam study evaluated whether this could be due to arteriolar narrowing or venular dilatation. in a population - based cohort study of 490 people without dementia (age 6090 years ; mean age : 68.4 years ; mean systolic bp : 136.7 19.9 mmhg ; diabetic patients : 6.3%), cerebral small vessel disease was evaluated at baseline. lacunar infarcts and wml at baseline, and incident infarct and changes in periventricular and subcortical wml and progression, were evaluated in the follow - up. neither venular nor arteriolar diameters were related to the severity of cerebral small vessel disease. larger venular diameters were, however, associated with a marked progression of cerebral small vessel disease. it has been hypothesized that retinal venular dilatation occurs in response to retinal hypoxia and venular dilatation has been also described as one of the earliest changes in diabetic retinopathy. retinal microcirculation abnormalities, including retinopathy, reduced arteriolar diameter and, increased venular caliber are widely observed in the general population. the abnormalities observed in retinopathy may reflect disorders of the retinal vascular wall, endothelial dysfunction, and inflammation secondary especially to diabetes mellitus, age, hypertension, obesity, and metabolic disorders. the decreased arteriolar diameter signals the presence of hypertension (current or old) and the risk of hypertension onset. an increased venular diameter has been associated with diabetes mellitus, obesity, and metabolic disorders. ultrasonographic findings of increased atherosclerotic plaques and carotid artery imt are regarded as the subclinical markers of early atherosclerosis and are associated with nonmodifiable and modifiable risk factors and the subsequent risk of new or recurrent stroke. some studies have found that the severity of imt and the presence of plaques in the carotid arteries are also predictive of wml [5356 ], although another study in the elderly did not. this association between large and small vessel disease may well be mediated via common intermediary risk factors such as hypertension. it is known that the arterial system gradually stiffens due to the combined effects of aging, high bp, and other vascular risk factors. increased arterial stiffness results in characteristic increased impedance and pulse wave velocity (pwv) in the aorta, which increases systolic and pulse pressure (pp) centrally. stiff arteries cause high pp and pulsatile flow to be transmitted to distal organs during systole, damaging the cerebral microvasculature. it has been hypothesized that cerebral microvascular disease results from the damaging forces of abnormal flow pulsations extending into small cerebral arteries as a consequence of arterial stiffening. in a homogeneous sample of never - treated hypertensive patients aged 5060 years, after exclusion for known risk factors for cerebrovascular damage, such as diabetes mellitus or significant alcohol intake, sierra. found an association between higher pp (including office, ambulatory 24 hours, daytime and nighttime estimates) as a measure of arterial stiffness and wml. in the elderly, laurent. found that aortic stiffness, assessed by carotid - femoral pwv using applanation tonometry, the gold standard for arterial stiffness measurement, was an independent predictor of fatal stroke in patients with essential hypertension. whether the risk of stroke is mediated by large- and/or small - vessel disease is not clear, but the previously reported increased risk of stroke when preclinical cerebral microvascular disease (wml, silent lacunar infarcts, and/or cerebral microbleeds) is present suggests small - vessel disease involvement [4, 7 ]. studied 167 hypertensive subjects (mean age : 51.8 years) and found that higher pwv was significantly associated with a greater volume of wml and lacunar infarcts, but not with cerebral microbleeds after multivariate analyses adjusted for age, sex, brain volume, mean bp, and heart rate. this suggests that aortic stiffness is independently associated with the manifestations of cerebral small - vessel disease in hypertensive patients and links systemic large- to cerebral small - artery disease. with respect to radial arterial pulse wave analysis, shrestha. studied 179 unselected patients with a mean age of 66 years and found that central systolic bp values, measured by radial applanation tonometry, were more closely associated with wml than brachial systolic bp. in this study, the role of inflammation in atherosclerosis and stroke has received increasing attention as basic and clinical research has provided evidence that inflammatory mechanisms play a central role in the pathogenesis and progression of atherosclerosis, plaque rupture, thrombosis, and stroke. whether inflammatory processes, apart from their involvement in large - vessel disease, are also involved in the development and consequences of cerebral small - vessel disease is still poorly understood. some studies have shown a relationship between wml and markers of inflammation, such as c - reactive protein, while others have not [64, 65 ]. a similar relationship has also been found with plasma homocysteine levels, lipoprotein - associated phospholipase a2 (lp - pla2), and myeloperoxidase (mpo). plasma markers of endothelial dysfunction such as intercellular adhesion molecule-1 (icam-1) and p - selectin have also been associated with wml [68, 69 ]. it has recently been suggested that endothelial activation associated with small vessel disease is accompanied by enhanced levels of tissue plasminogen activator (tpa) and low levels of plasminogen activator inhibitor type 1 (pai-1) when lacunar strokes are associated with wml, suggesting that differences in the activity of components of the fibrinolytic system might contribute to the development of wml. inspite of their small simple size one study has reported that altered vascular function and structure in subcutaneous small arteries of patients with late - life depression are related to cerebral small vessel disease, including wml, and basal ganglia and infratentorial changes. the study was performed in 16 patients with late - life depression (68.8% hypertensives) who were compared with 15 controls (80% hypertensives). small arteries were isolated and studied using pressure myography after subcutaneous gluteal fat biopsy. in summary, there is some evidence to show that inflammation, impaired endothelial function, and abnormal wall growth are involved in the pathogenesis of wml.. found that the small cerebral arteries of patients with essential hypertension had an increased media / lumen ratio compared with normotensive individuals, similar finding to those previously observed in subcutaneous small arteries. structural alterations of cerebral small vessels were assessed in 13 hypertensive patients and 15 normotensive individuals undergoing neurosurgery for benign or malign tumors. a small portion of morphologically normal cerebral tissue was excised from surgical samples and examined, and cerebral small resistance arteries were dissected and mounted on an isometric and isobaric myograph. an increased media - to - lumen ratio of subcutaneous small resistance arteries has also been shown to predict the development of cardio - cerebrovascular events in hypertensive patients. early detection of hypertensive target organ damage is important in order to prevent cardiovascular diseases more successfully and improve patients ' outcomes [1, 2 ]. after age, hypertension is the most - important factor for the development of cerebral wml [4, 10 ], which are an important prognostic factor for stroke, cognitive impairment, dementia, and death. various studies have shown an association between wml and a number of extracranial systems affected by high bp. there are findings that suggest that correct antihypertensive treatment could efficiently slow wml progression [1113 ]. strong evidence suggests that cerebral wml in hypertensive patients should be considered a silent early marker of brain damage. the latest reappraisal of the european guidelines on hypertension management reports that, in a group of 192 untreated hypertensive patients (aged 1890 years) without overt cardiovascular disease, silent cerebrovascular lesions (wml, lacunar infarcts, cerebral microbleeds) were even more prevalent (44%) than cardiac (21%) and renal (26%) subclinical damage and frequently occur in the absence of other signs of organ damage. similarly, 58% of patients with demonstrable cardiac or renal damage or both had silent cerebrovascular lesions. with mri being increasingly used for diagnostic procedures, investigation of silent cerebrovascular disease in the meantime, target organ damage in other systems may aid the evaluation of early hypertensive brain damage.	chronic hypertension leads to concomitant remodeling of the cardiac and vascular systems and various organs, especially the brain, kidney, and retina. the brain is an early target of organ damage due to high blood pressure, which is the major modifiable risk factor for stroke and small vessel disease. stroke is the second leading cause of death and the number one cause of disability worldwide and over 80% of strokes occur in the elderly. preclinical hypertensive lesions in most target organs are clearly identified : left ventricular hypertrophy for the heart, microalbuminuria for the kidney, fundus abnormalities for the eye, and intima - media thickness and pulse wave velocity for the vessels. however, early hypertensive brain damage is not fully studied due to difficulties in access and the expense of techniques. after age, hypertension is the most - important risk factor for cerebral white matter lesions, which are an important prognostic factor for stroke, cognitive impairment, dementia, and death. studies have shown an association between white matter lesions and a number of extracranial systems affected by high bp and also suggest that correct antihypertensive treatment could slow white matter lesions progression. there is strong evidence that cerebral white matter lesions in hypertensive patients should be considered a silent early marker of brain damage.
caused by coxiella burnetii, an obligate intracellular gram - negative bacterium, q fever can be acute or chronic. long - term sequelae are beginning to gain acceptance as a third category of this zoonotic disease. endocarditis usually develops in people with underlying heart disease and accounts for 6070% of chronic q fever cases. onset is generally insidious and manifestations are atypical as fever can be absent, and vegetations can be unapparent or small,. the authors report a case of a prosthetic valve endocarditis caused by coxiella burnetii in a 53 year- old patient with recurrent mechanical valve dehiscence. we consider this case of major importance to highlight that even if endocarditis is the most common manifestation of chronic q fever it is still a rare disease and can be difficult to diagnose as its clinical course is variable. a case of a 53-year old patient with a past history of transfusional hepatitis c and a splenectomy at the age of 23, who was admitted in the infectious diseases department of a portuguese hospital in october 2015 because of a recent onset of fever, dyspnea, fatigue and non productive cough. the patient s previous relevant medical history started in june 2004, at the age of 41 years old, when he presented with heart failure symptoms due to mitral stenosis. in october 2005 mitral- valve replacement with a mechanical medtronic valve five months later (in march, 2006), patient began to have anorexia, fatigue, progressive dyspnea on exertion, paroxysmal nocturnal dyspnoea and peripheral edema. transesophageal echocardiogram revealed a severe mitral insufficiency secondary to dehiscence of the prosthesis and a new intervention was performed in august 2006 to correct the leak without valve replacement. six months later he was again admitted for progressively worsening dyspnoea, asthenia and palpitations with one month duration and fever (3839 c) in the previous week. transesophageal echocardiogram revealed slight dilation of the left cavities and right atrium, with preserved ejection fraction and new partial dehiscence of the prosthesis with elevation of the transprothesic gradient (43/15 mmhg) causing severe mitral insufficiency and severe pulmonary hypertension (> 100 mmhg). a diagnosis of culture negative endocarditis was presumed and empirical antimicrobial therapy was started with rifampin (300 mg po tid), vancomycin (500 mg bid iv adjusted to blood levels) and gentamicin (500 mg / day iv). in march 2007 he underwent a third surgical intervention : mitral valve replacement with st jude 27 prosthesis and tricuspid annuloplasty with a medtronic ring. he completed 2 weeks of gentamicin and 4 weeks of vancomycin and rifampin and was started on hypocoagulation with warfarin for his permanent atrial fibrillation. the outcome was favorable. in october 2015 (8 years later), patient was referred to the emergency room of a peripheral hospital with a history of two weeks duration of persistent non productive cough, myalgia, fever and worsened dyspnea on exertion. he denied orthopnea, paroxysmal nocturnal dyspnea, thoracic pain, vomiting, anorexia or other gastrointestinal or urinary symptoms. concerning the patient epidemiological context, he was retired (used to work in metal processing), lived in a rural area with sheep and goats on the surroundings but had no pets or any farm animals in his house. medications taken on a daily basis included warfarin, carvedilol (9.375 mg / day), ramipril (1.25 mg / day), furosemide (20 mg / day), digoxin (0.125 mg / day except weekends). on physical examination, patient was febrile (temperature of 38,2 c), blood pressure was 91/62 mmhg, pulse 80 beats per minute, room air peripheral o2 saturation of 100%, pulmonary auscultation was normal and cardiac auscultation revealed an irregular rhythm with a grade iii / vi systolic murmur heard at the base and apex. blood workup revealed a haemoglobin 9 g / dl, platelets 293.000, leukocytes 10, 95 10 ^ 4, 59% neutrophils, 15% lymphocytes, c - reactive protein 29,6 mg ui / l, alkaline phosfatase 63 ui / l, gamaglutamil transferase 32u / l, lactate dehydrogenase 2822 ui / l, total bilirrubin 2,63 mg / dl. iron 79 ug / dl, iron binding capacity 232 ug / dl, ferritin 11 ng / ml, haptoglobin < 0.8 g / dl, vitamin b12 502 pg / ml, folic acid 5.4 ng / ml. antinuclear, anti - smooth muscle, antimitochondrial anti- dsdna and anti - histone antibodies were all negative. ceftriaxone 2 g / daily was started and he was admitted on the internal medicine department of another hospital for study. an infectious diseases consultation was called for case discussion and complementary exams were suggested, namely : brucella spp. the transesophageal echocardiogram revealed : dilation of the left cavities, preserved systolic function with an ejection fraction of 54%, mild aortic insufficiency and mitral valve prosthesis with two regurgitant jets with a moderate periprosthetic leak. serologies for coxiella burnetti were positive : igg antibodies to phase i were 1:32 768 and igg antibodies to phase ii was 1:16 384. the diagnosis of chronic q fever endocarditis and haemolytic anemia secondarily to periprosthetic leak were presumed. doxycycline (100 mg orally bid) plus hydroxychloroquine (600 mg / day) were started. surgery was performed in december 2015 with valve replacement by a mechanical prosthesis st jude n 27. the patient was discharged one week after surgery with the indication to complete therapy with doxycycline plus hydroxychloroquine for at least 24 months. during follow up no events were reported and 8 months later he is still asymptomatic (no signs or symptoms of heart failure, serial echocardiography showing a preserved integrity of the valve). q fever is a worldwide zoonosis and the most common sources of human infection are farm animals such as cattle, goats and sheep, but also cats, rabbits and dogs can be responsible for infection in an urban context. these mammals, when infected, can shed the desiccation- resistant form of the organism in urine, faeces, milk and, especially, birth products. contact with animals, even if indirect, is important to suggest the diagnosis of q fever. in europe, the clinical picture is very unspecific but it is known that chronic infection can develop month or years after acute q fever, which can be asymptomatic. the most common manifestation of chronic disease is endocarditis and c. burnetii was the leading cause of negative blood culture prosthetic- valve endocarditis in one study of men less than 65 years old. most cases involve the aortic or mitral valve in patients with pre - existing valvular disease or prosthetic valves. infection can be very indolent without fever and prosthetic valves have shown little or no evidence of infection in the valve ring (3 out of 5 patients had no vegetations found). the diagnosis can be delayed due to the protean manifestations of this entity and some patients have undergone several valve replacements before a diagnosis was made. this patient was submitted to two surgical interventions of the mitral valve due to a mechanical valvular leak, one of which was assumed to be a consequence of a culture - negative endocarditis. nine years later, he developed another dehiscence of the same valve with surgical indication and only at that time a q fever serologic test was performed, leading to the diagnosis of q fever endocarditis. we admit that this diagnosis was possibly delayed for all this time and coxiella burnetii endocarditis of the mitral valve could have been present as early as 2006 with recurring infections of the replacement valves until the diagnosis was made serologically and therapy instituted. described 9 patients with q fever endocarditis during a 19-year period, three (33%) of which were unexpected and diagnosed after elective valve surgery. autoantibodies are also frequent in chronic q fever such as rheumatoid factor, anti- smooth muscle, antinuclear and antimitochondrial antibodies and also positive coomb test but in this case all the autoimmune tests were negative. the gold standard for the diagnosis of q fever is indirect immunofluorescence (if). phase i antibodies are raised in chronic disease and if titres igg to phase i antigen of 1:800 or more is considered diagnostic for endocarditis. some authors say that pcr has low sensitivity in blood and high sensitivity in tissue samples, but there is evidence of high sensitivity and sensibility of pcr in specialized laboratories. in our patient the recommended treatment for q fever endocarditis is doxycycline (100 mg / bid) plus hydroxychloroquine (200 mg tid) until a target of < 1:800 for igg is achieved. generally, a minimum of 18 months is needed. according to million. who published the largest cohort of q fever endocarditis to date, for prosthetic valve infections, the optimum management includes a longer course of therapy (24 months) with doxycycline and hydroxychloroquine. intolerance to the recommended drugs, namely photosensitivity can be a problem, and regular heart and eye examinations are needed. when untreated, the disease is usually fatal and, even with appropriate treatment, is associated with a mortality of 10% at five years. million. identified that coxiella burnetii endocarditis on prosthetic valves (compared with the general population and with patients with other valvular heart diseases) was associated with higher mortality, more frequent stroke, delayed serological cure, need for longer treatment course and higher risk of relapse. our patient is still on treatment and free of disease after 8 months of doxycycline plus hydroxychloroquine. we consider this case of a major importance to highlight the need to consider the diagnosis of chronic q fever not only in the presence of a culture - negative endocarditis but also when unexplained valvular leaks occur in prosthetic valves. the authors recommend that all patients with negative blood culture endocarditis or unexplained dehiscence of prosthetic valves undergo serological testing for q fever. a copy of the written consent is available for review by the editor - in - chief of this journal on request. rf, fs, pa and ja were responsible for the patient s management during hospitalization and follow up after discharge, have collected all significant clinical information and drafted this manuscript. jpt and ma were extremely important in collecting technical surgical data and also reviewed this paper. ls, acc and as have reviewed, redrafted and given significant contribution to the final version.	chronic coxiella burnetii endocarditis usually develops in people with underlying heart disease and accounts for 6070% of chronic q fever. onset is generally insidious and manifestations are atypical. the authors report a case of coxiella burnetii prosthetic valve endocarditis in a 53 years- old patient with recurrent mechanical valve dehiscence on mitral position. he lived in a rural area with sheep and goats on the surroundings. during a 9 year- period, he was submitted to three cardiac mitral valve surgeries two of which with no q fever diagnosis suspicion. diagnosis was based on a positive serology test (indirect imunofluorescence). treatment consisted in a combination of prolonged course of hydroxychloroquine plus doxycycline and surgical replacement of the mitral valve, with a favorable outcome. with this case report, the authors pretend to highlight the not always expected diagnosis of q fever endocarditis. if not considered, coxiella burnetii endocarditis may lead to multiple cardiac surgeries, greater morbidity and potentially death.
under the prisma coordination model, all older people benefit from several model components, but only subjects identified with moderate to severe disabilities are eligible for case management. the prisma model has proven its effectiveness at the population level. however, for the sub - group with case management, what is its effect on services use ? we used data from the prisma study on adults aged 75 or over at risk of functional decline to compare subjects exposed to case management to unexposed ones, matched for functional disabilities, age, and gender. we studied change in annual services use, contrasting the year of assignment to the previous year. matched pairs were created for 49 cases for which data were available before and after assignment. change in the number of annual er visits was similar across groups, but the number of annual hospitalisations tended to increase less for exposed subjects (p=0.08). change in annual hours for home maintenance was similar across groups, but more hours of help for personal care were added for case - managed older people (p=0.01), possibly by increasing access to home care.	introductionunder the prisma coordination model, all older people benefit from several model components, but only subjects identified with moderate to severe disabilities are eligible for case management. the prisma model has proven its effectiveness at the population level. however, for the sub - group with case management, what is its effect on services use?studywe used data from the prisma study on adults aged 75 or over at risk of functional decline to compare subjects exposed to case management to unexposed ones, matched for functional disabilities, age, and gender. we studied change in annual services use, contrasting the year of assignment to the previous year.resultsamong subjects in the prisma experimental group, 18% were assigned a case manager. matched pairs were created for 49 cases for which data were available before and after assignment. change in the number of annual er visits was similar across groups, but the number of annual hospitalisations tended to increase less for exposed subjects (p=0.08). change in annual hours for home maintenance was similar across groups, but more hours of help for personal care were added for case - managed older people (p=0.01), possibly by increasing access to home care. this strengthens the value of case management in an isd network.
indirect aesthetic materials have become very popular in making of inlays, onlays, and crowns. lithium disilicate is one such material which has wonderful translucency and shade variety for the manufacture of inlays, onlays, and single crowns. indirect composite is another material which is proven to have improved fracture toughness because of post - cure treatment reinforcement. full - contour zirconia has become the most preferred material for anterior and posterior indirect restorations because of their high flexural strength, excellent biocompatibility, and good esthetics. limited data is available regarding the selection of the appropriate tooth - colored restorative material and its influence on the fracture resistance of cusp - replacing restorations in endodontically treated posterior teeth. the present study assessed the fracture resistance and failure patterns of endodontically treated maxillary premolars restored with three all - ceramic cad - cam (computer aided design - computer aided manufacturing) onlay materials which are lithium disilicate, indirect composite, and full zirconia. forty sound human maxillary premolars atraumatically extracted for orthodontic reasons were selected and were stored in 0.5% chloramine solution (oxford lab chem, india). the roots were embedded in acrylic resin parallel to the long axis of teeth upto 2 mm below the cemento - enamel junction (cej). ten teeth were randomly selected for the control group (group i) in which neither cavity preparation nor root canal treatment was performed. in the remaining 30 teeth, standardized mesio occluso distal (mod) onlay cavities were prepared using fg 271 and fg 169l carbide burs (ss white, lake wood, usa). occlusal cavity width was kept at half the intercuspal distance, pulpal depth at 4 mm from the buccal cusp tip, and 6 taper on each wall. gingival seat in the proximal boxes were placed 1 mm above the cej and width of the seat in mesio - distal direction was 1.5 mm. the buccal and palatal cusps were reduced occlusally by 2 mm from cusp tips and a 1.5 mm width collar was prepared for both cusps. access openings were done with fg round diamond point iso 001 - 009 (dia burs, mani, tochigi, japan). the coronal and middle thirds of the root canals were enlarged using sizes 1 - 3 gates glidden drills (mani tochigi, japan). subsequently the instrumentation of the root canals was done by step back technique using hand k files till clean shavings of dentin was achieved. irrigation was performed using 1 ml of 2.5% naocl (prime dental products, india) after every change of file size and after cleaning and shaping 3 ml of 17% ethylenediaminetetraacetic acid (edta ; prevest denpro, jammu and kashmir, india) was irrigated for 1 minute to remove smear layer. the canals were obturated with gutta - percha (densply maillefer, ballaigues, switzerland) and grossman 's sealer (pharma dent, montevideo, uruguay) by lateral condensation technique. subsequently, the pulp chambers were temporarily filled with cavit (3 m espe, germany) and stored for 24 h at 37c in 100% humidity. gutta - percha was removed upto the orifice of the canals and a zinc phosphate base of 1 mm was placed over the gutta - percha, and the remaining access cavity space was restored with dual cure flowable composite (rebilda dc dual cure flowable composite, voco, germany) till the pulpal floor. ii- indirect composite (paradigm mz 100,3 m espe, st paul, mn) and group iii- lithium disilicate glass - ceramic (ips e. max cad, ivoclar vivadent, schaan, liechtenstein) were fabricated using cad / cam (cerec ; sirona, bensheim, germany) with the cerec 3d system using the software package provided (version 2.80r2402 ; sirona). in group iv- partially sintered zirconia onlays (sagemax - nexxzr, federal way, usa) were fabricated using cad / cam using delcam dent cad and roland milling machine. cementation surfaces of group iii onlays were etched using 5% hydrofluoric acid for 60 s and rinsed under running water for 60 s and air dried for 30 s. a silane coupling agent was applied and allowed to dry for 3 min. enamel and dentin margins were etched using 35% phosphoric acid for 15 s and rinsed with water for 30 s. all the onlays were cemented using multilink automix (ivoclar vivadent, schaan, lichtenstein). multilink primer a and b were mixed and applied to the cavity preparations according to the manufacturer 's recommendation and the onlays were cemented and light - cured for 40 s on all restored surfaces. the same protocol was followed for cementation of groups ii and iv except 5% hydrofluoric etching step was eliminated, instead was replaced by air borne particle abrasion with 27 m aluminium oxide at 30 psi. the silane coupling agent (metal / zirconia primer) was applied for 180 s. after cementation of the restorations, specimens were stored in water for 21 days at 37c. the fracture resistance of the buccal cusp was tested on a universal testing machine (instron, uk) with load applied on the center of the buccal cusp slope with a steel ball of diameter 3.5 mm at an angle of 30 to the long axis of the tooth with a crosshead speed of 0.5 mm / min. after mechanical failure, all specimens were observed under stereomicroscope (2 magnification) to visualize the fracture lines in the restoration and the tooth. failure patterns were evaluated and classified into five categories : type i : no visible fracture in the restoration and tooth type ii : no visible fracture in the restoration but fracture in the tooth type iii : fracture in the restoration only type iv : fracture in the restoration and the tooth above cej type v : fracture in the restoration and the tooth below cej resistance to fracture was submitted to parametric one - way analysis of variance (anova) test. these were performed using statistical package for social sciences (spss), version # 16. resistance to fracture was submitted to parametric one - way analysis of variance (anova) test. these were performed using statistical package for social sciences (spss), version # 16. the mean fracture resistance and standard deviation of the experimental groups is shown in figure 1. groups ii and iii showed significantly lower values than the group i. however, groups ii and iii presented no significant difference from each other. group iii demonstrated 30% type ii fractures, 10% type iv, and 60% type v. group iv demonstrated 90% type i and 10% type ii fractures. bar graph representing mean fracture resistance of four groups bar graph representing comparison of failure patterns frequency table of failure patterns observed in all groups generally, endodontically treated teeth are considered to be more susceptible to fracture than the sound teeth, because of the internal tooth structure removal during endodontic treatment. according to reeh., endodontic treatment performed in intact teeth reduced fracture resistance by 5%, but when combined with an mod cavity preparation, it is reduced to 69%. many authors preferred the onlay design to the inlay design as it would reinforce the cusps. in the present study, the bucco lingual width of half the intercuspal distance was maintained to simulate the clinical situation where a wide bucco lingual access cavity is prepared. earlier studies had maintained 1.5 - 2 mm of occlusal cavity width for the inlays and onlays. buccal and palatal cusps were reduced by 2 mm occlusally and are in line with bruke., who suggested that 2 mm should be the minimal thickness of the indirect restoration that will provide adequate resistance to occlusal forces. in the present study, the fracture resistance of group i presented a mean fracture resistance of 980 n. similar values were found in other studies, which ranged between mean of 882 and 1,568n. this variance of results of the present study from the other studies may be due to difference in the type and design of the load application of the contact device, specimen preparation, test speed, and tooth storage method. studies were mainly done on indirect composite crowns and veneers assessing the fatigue resistance. in the present study, indirect composite cad onlays could not reinforce the endodontically treated teeth to that of sound teeth. again there are no studies in the literature assessing fracture resistance of e - max press and e - max cad onlays. however in the present study, e - max cad onlays could not reinforce the endodontically treated teeth to that of sound teeth. the mean fracture resistance in group iv was 1,524.4n which is highest of all the groups. this high fracture resistance of zirconia could be related to its phase transformation toughening phenomenon. under stresses, when crack propagates within the zirconia mass, the tetragonal grains are transformed to monoclinic with a volume expansion of 3 - 5%. this expansion of the grains will ultimately lead to compressive stresses at the edge of the induced crack front and so extra energy is required for the crack to propagate. in the present study, zirconia mod onlays had reinforced the endodontically treated to a much higher value than that of the sound teeth. in comparison between groups in the present study, the mean fracture resistance of group i was significantly higher than groups ii and iii. although the fracture resistance of groups ii and iii were lower than that of the group i, their fracture resistance values were much higher than the normal masticatory forces which is between 222 and 447 n. but that is not good enough to withstand the highest masticatory forces of 900n in bruxism and hard objects chewing. while comparing groups ii and iii, there is no significant difference statistically. this could be attributed to the increased thickness of e - max cad of 2 mm in this study which could have withstood the testing loads compared to ultrathin models in other studies. interestingly there is almost double the increase in the fracture resistance as compared to all the other groups. these findings were supported by ma., who reported that flexural strength or fracture load of zirconia is 2.5 times greater than lithium disilicate glass ceramics suggesting that zirconia is much more suitable for free standing stress bearing application. coming to modes of failure ; in contrast to many studies, there was not much difference in the fracture modes of groups ii and iii even though the stress distribution varies with the material properties and cavity design. the occlusal cavity depth of 4 mm in the present study could have altered the stress distribution. this can be supported by goel., where they suggested that deeper the prepared cavity, the greater the changes in the stress gradient in the dentin. the changes in the stress gradient could initiate fracture of the remaining cusps, possibly starting in the dentin immediately adjacent to the deepest portion of the cavity preparation. in group iv, none of the onlays fractured, but one sample fractured only in the root. the failure at a higher load could be due to the bond failure in the cement or fractures in the internal surface of the tooth, where more amount of stresses are usually concentrated. within the limitations of this in vitro study, it is interesting to note that full contour zirconia onlays had showed an excellent fracture resistance with no cracks in the restoration at very high loads, compared to lithium disilicate and indirect composite onlays. therefore, full contoured zirconia could be chosen as an ideal esthetic posterior onlay material and could be a good alternative to unesthetic metal onlays.	aim : to compare the fracture resistance and modes of failures of three different aesthetic mod onlays on endodontically treated premolars.materials and methods : forty sound maxillary premolars were selected of which 10 untreated teeth were taken as control (group i). the other thirty premolars were subjected to standardized mod onlay preparations and root canal treatments and divided into 3 equal groups. onlays were prepared in group ii- indirect composite, group iii- lithium disilicate ceramic and group iv- full zirconia. all onlays were cemented using multilink automix. all the 40 samples were subjected to fracture resistance testing on universal testing machine. also fractured specimens were observed under stereo - microscope for modes of failure.results:group iv presented the highest fracture resistance. groups ii and iii presented no significant difference in fracture resistance from each other (p > 0.05). group ii and group iii showed significantly lower fracture resistance values than group i. coming to modes of failure, only group iv had showed no cracks in any of the restorations.conclusion:full zirconia mod onlays increased the fracture resistance of endodontically treated premolars to a significantly higher level than the sound teeth.
	metal ion binding is exploited by proteins in nature to catalyze reactions, bind molecules, and favor discrete structures, but it has not been demonstrated in -peptides or their assemblies. here we report the design, synthesis, and characterization of a -peptide bundle that uniquely binds two cd(ii) ions in a distinct bicoordinate array. the two cd(ii) ions bind with positive allosteric cooperativity and increase the thermodynamic stability of the bundle by more than 50 c. this system provides a unique, synthetic context to explore allosteric regulation and should pave the way to sophisticated molecular assemblies with catalytic and substrate - sensing functions that have historically not been available to de novo designed synthetic proteomimetics in water.
low grade nasopharyngeal papillary adenocarcinoma (lgnppa) is an extremely rare type of nasopharyngeal cancer. the clinicopathological characteristics of this disease were originally described from a series of nine consecutive cases, and only five cases were reported thereafter [2 - 4 ]. although the clinicopathological characteristics have not been completely determined due to the extreme rarity, the clinical characteristics is known to be distinct from those of the ordinary nasopharyngeal carcinomas. surgical resection is regarded as the principal treatment [2 - 5 ]. until now, resection has been performed employing potentially - morbid transpalatal or transfacial approach in all cases [1 - 3,5 ]. even though endoscopic nasal surgery has been adopted to the treatment of various nasal tumors, there has been no report for lgnppa. here we report a case of lgnppa which was successfully treated with exclusive endoscopic resection via combined transnasal and transoral approach. an otherwise healthy 31-year - old female visited with complaints of nasal obstruction and mild postnasal drip for several weeks. the mass was exophytic, irregularly - surfaced, and very fragile even to endoscope - guided careful palpation and suction. subsequent surface bleeding was only scanty without any problems, which suggested hypovascular feature of this tumor. detailed examination of tumor attachment area and adjacent invasion was difficult in the office due to the excessive intracavitary bulging. dislodged tumor dislodged piece of the tumor was sent for pathologic examination, which revealed the presence of lgnppa. computed tomography showed a 2.82.4 cm - sized, relatively hypovascular tumor without deep invasion or cervical lymphatic metastasis. the tumor exhibited typically exophytic feature. through serial images, the point of stalk was estimated to be the narrow area around the cranial end of the nasal septum and the nasopharyngeal vault (fig. 1b). an 18-fluorodeoxyglucose positron emission tomography / computed tomography scan showed a mildly increased standardized uptake value of 2.55 without uptake elsewhere (fig. free t4, thyroid stimulating hormone, and thyroglobulin were all within the normal range. endoscopic exploration via combined transnasal and transoral approach was tried first, under preparation of further transpalatal or transfacial approach. at first, the tumor, protruded into the choana of both nasal cavities, was carefully probed and mobilized. area of tumor attachment was the narrow area in the nasal septum around choana and nasopharyngeal vault. considering the tumor attachment confined only in narrow portion, we then decided to try exclusive endoscopic resection without transfacial or transpalatal approach. we tried to keep the resection margin to 0.5 cm around the tumor. although en - bloc resection was originally planned, tumor was partially dislodged due to its fragility during the manipulation. but subsequent wider surgical field made the endoscopic resection more feasible. eventually, the mass and surrounding mucosa was successfully separated from the nasopharyngeal vault, and delivered transorally due to its large size. the cells lining these structures had bland, round - to - oval, nuclei without mitotic figures (fig. immunohistochemistry disclosed positivity for thyroid transcription factor-1 (ttf-1) (fig. 2c) and cytokeratin 7 (ck7), but negativity for thyroglobulin (fig. the patient was kept in hospital overnight and the posterior packing was removed the next day without any complications or morbidities. this case shows exclusive endoscopic resection via combined transoral and transnasal approach of lgnppa is feasible and sufficient. although only a few patients with lgnppa have been described, their tumors were all considered ' radioresistant, ' with definitive surgery being an effective and powerful therapy [1 - 3,5 ]. there have been no reports of recurrence after surgical resection, whereas one patient failed radiation treatment, in which surgery was performed successfully as a salvage. however, the stromal invasion, the cytological pleomorphism, and recurrence following inadequate therapy have been regarded as indicatives of a malignant neoplasm, albeit a neoplasm of low grade. to date, a transpalatal or transfacial maxillary swing approach to the nasopharynx has been used for all cases of lngppa [1 - 4 ]. however, these procedures can cause several complications, including pain, bleeding, and oronasal fistulae. in addition, lgnppa is usually pedunculated, with limited extension extramucosally, which can make endoscopic resection feasible like this case. the tumor in this case was attached to the nasopharyngeal vault and the posterior end of the nasal septum, which were effectively visualized exclusively by an endoscope. this case shows resection of lgnppa with only endoscopic guidance can be sufficient for complete resection. lgnppas arise from the surface epithelium. unlike the majority of nasopharyngeal carcinomas, such as keratinizing or nonkeratinizing carcinomas more common glandular neoplasms are those originating from the submucosal seromucous salivary gland. unlike this, lgnppas originate within surface mucosal glandular epithelia, a very rare pattern of pathogenesis among nasopharyngeal tumors. lgnppa is a unique tumor type and much less common than nasopharyngeal salivary gland neoplasms. our case was positive for ttf-1 and ck7 but negative for thyroglobulin and ck20, a pattern identical to those previously reported. this pattern is similar to that of papillary thyroid carcinoma, which is usually positive for ttf-1, ck7, and thyroglobulin, but negative for ck20. in addition, histological characteristics of papillary growth of abundant glandular architecture are also similar in lgnppa and thyroid papillary carcinoma. therefore, thyroid sonography has been recommended for lgnppa patients, to identify any sign of hidden thyroid malignancies. this suggests that lgnppa may be a form of metastatic disease originating from occult thyroid papillary carcinoma. to our knowledge, however, there have been no reports showing metastasis of papillary thyroid carcinoma into only the nasopharyngeal mucosa, with a confined pedunculated pattern and without any metastatic lymphadenopathy. such a pattern was also observed in our patient, and we found no sonographic abnormality of thyroid. the clinicobiological similarity between lgnppa and thyroid papillary carcinoma should be further investigated. in conclusion, this case was successfully treated with exclusive endoscopic resection via combined transnasal and transoral approach. exclusive endoscopic resection was effective to obviate the potential morbidities of transpalatal or transfacial approach if performed appropriately in this rare variant of nasopharyngeal cancer.	low grade nasopharyngeal papillary adenocarcinoma (lgnppa) is an extremely rare variant of nasopharyngeal cancer, which exhibits distinct clinicopathological characteristics. surgical resection has been regarded as the principal treatment. for this, transpalatal or transfacial approach has been classically used for exposure of the field. up for now, there has been no report on applying endoscopic approach for this disease, which could be an effective alternative to minimize possible morbidities of palatotomy or maxillotomy. endoscopic approach can be justified considering narrow extent and indolent behavior of lgnppa. we report a patient with lgnppa, which was successfully resected exclusively by endoscopic visualization. our case exhibited narrow - based exophytic features with compatible immunopathologic profiles of lgnppa. exclusive endoscopic resection can be effective and less - morbid modality for this rare disease as in this case.
diverse disciplines, such as psychology, pathology, molecular biology and even computer science converge their efforts to understand how the nervous system works and produces behavior. the clinical relevance of these studies is captured in neurology and neuropathology (often jointly termed clinical neuroscience), as well as psychiatry. each discipline has particular methods and techniques that are appropriate to study their area of interest. these methods often focus on particular parts of the nervous system or their functions, such as the physiological basis of the withdrawal reflex in sea slugs (i.e., electrophysiology) or the neuronal loss in the nigrostriatal pathway and its importance to parkinson s disease (i.e., histopathology). however, these diverse areas often do not directly relate to each other (cross - disciplinarity) and provide a fragmented viewpoint of the nervous system and its functions. to ensure steady progress in our holistic understanding of the nervous system, it is imperative that new generations of scientists are educated in the interdisciplinary nature of neuroscience, rather than merely within a single discipline (i.e., uni - disciplinary) (ramirez, 1997 ; collins, 2002). although a multidisciplinary approach (i.e., different disciplines working side - by - side to address a common circumscribed topic) is more desirable than a cross - disciplinary study (i.e., engaging different disciplines without focusing on a common issue), it is trans - disciplinary investigations (i.e., transcending traditional boundaries of scientific disciplines without fully integrating with another discipline) that are the starting point to overcome the limitations that individual disciplines face while studying the nervous system. trans - disciplinary scientists are overcoming traditional gaps in terminology, methodology, and analytical approaches. however, they do not fully integrate and adopt the expertise of another discipline (table 1). it is the integration of the analytical and methodological strengths, as well as a common terminology, across multiple disciplines that is required to advance an inter - disciplinary field of study, such as neuroscience (http://neuroscienceblueprint.nih.gov). although neuro - science, in practice and in teaching, today is a mix of these different disciplines, it is the interdisciplinary approach that poses the greatest challenge to teach neuroscience within a coherent framework (ramirez, 1997 ; pellmer and eisenberg, 2000 ; holley, 2009). interdisciplinary teaching and learning is a challenge to both teachers and students (woods, 2007 ; bleakley., 2011). teachers are often specialized in a particular discipline and find it challenging to engage with other teachers, as there is a lack of a common vocabulary and educational vision. however, it is this competence in communication between disciplines that is at the heart of interdisciplinary teaching (woods, 2007). teachers need to share a common vision and ideology to design an appropriate curriculum for students. if there is a lack of coherence in the teaching curriculum due to miscommunication between teachers from different disciplines, it is likely that students will get a fragmented view of neuroscience that promotes superficial learning, rather than interdisciplinary learning (figure 1). there is a need for a consistent framework within which structured learning can occur and allow students to assimilate complex ideas into a cohesive knowledge structure (biggs, 2003). the diversity and different viewpoints from multiple disciplines is also an asset to the curriculum as information is presented multiple times within different contexts potentially fostering a deeper learning (i.e., a robust knowledge and understanding of a discrete topic). additionally, increasing links between apparently disparate and complex ideas will be essential to create an interdisciplinary learning process (lattuca., 2004 ; lawson, 2006). indeed, an interdisciplinary neuroscience curriculum aims to link apparently disparate types of information into a continuous and coherent knowledge structure. therefore the curriculum design requires thorough planning to provide an appropriate learning environment for students. it is important to note that the curriculum is more than just the content to be taught on an academic course (fraser and bosanquet, 2006). curriculum design also includes the overall organization of the learning process (i.e., the structure of the teaching program), the teaching methods (e.g., practicals, presentations, discussions) and the content of what is being learnt, as well as the assessments that are used to determine if indeed learning took place (helsby, 1999). recent initiatives have aimed to develop blueprints for neuroscience teaching, but have mostly focused on the content that should be included within an undergraduate degree (ramirez. defining an appropriate curriculum is crucial to specify what learning outcomes are expected of the students. we therefore here discuss the conceptual framework within which an interdisciplinary curriculum in neuroscience can be designed to fulfill the different requirements of the scientific and professional community. curriculum design for neuroscience courses that lead to academic degrees (i.e., the awarded qualification at the end of a defined learning period / course) is geared towards teaching a solid knowledge base of the nervous system, develop appropriate analytical skills (including laboratory techniques), but also to establish the students ability to control their own learning. although degrees in neuroscience are awarded in a variety of universities and colleges, the learning outcomes of the different courses (i.e., implementation of a degree at a given institution) will depend on the specific focus / interest of the university and department (ramos., 2011). the learning outcome will depend on whether students are being prepared, for instance, to proceed to a phd program or if the course is mainly designed to prepare students to become technicians in a laboratory (austin, 2002 ; estes, 2007). although curriculum design needs to account for these different aims, curriculum design will also be highly dependent on the level of the university degree. the specific learning outcomes between a bsc, msc and phd, irrespective of the focus of the university, will require an increasingly more sophisticated scholastic ability, but also more independence in the students learning (table 2). a curriculum developing a scholastic ability in neuroscience needs, therefore, to also account for the students prior knowledge. in the case of undergraduates, it is unlikely that these students will have any prior knowledge of neuroscience and hence they need to be considered novices. in contrast, at the post - graduate level some students might have taken a prior undergraduate degree in neuroscience, and hence, would have a solid knowledge base to build on. with these students, it should be possible to then further develop their skills to a more proficient level (msc courses) and eventually to an expert level (phd programs). based on the dreyfus model (dreyfus and dreyfus, 1980), we can then revisit the aims of undergraduate and post - graduate courses to define an undergraduate course as a course that develops novices into competent professionals, whereas a master s degree produces proficient scientists that can eventually become experts within a phd program (table 3) (curriculum design for phd programs is very specific and goes beyond the scope of the current discussion). as some neuroscience courses will recruit students from other disciplines, such as computer science or philosophy, to foster inter - disciplinarity ; these courses, therefore, can not presume the same background in neuroscience between all students. these courses will require a different curriculum design compared to those where students had a previous exposure to a more general neuroscience training (see below). nevertheless, at the same time, these courses need to also fulfill the learning outcomes that are required to award a neuroscience degree and ideally aim to provide an inter - disciplinary view of the field. one of the challenges of modern curriculum design is therefore to provide a learning environment that is flexible, provides efficient learning and, at the same time, is economically viable. often flexibility and economical viability are tightly linked, as costs are directly linked to the number of students and courses that can be taught using the same resources (grundy, 1987 ; estes, 2007 ; whittaker and akers, 2009). however, to ensure a continued success of these courses, their focus needs to be on efficient learning, rather than purely on it being economical. although some learning is merely a reflection of how much factual information students recall on a particular subject, a more modern perspective on learning would require that students evolve from novices to experts by demonstrating an ability to manipulate knowledge (kinchin and cabot, 2010). as such, novice students would acquire a solid base of core factual information that reflects the wider aspects of their subject, but as the curriculum progresses they specialize to become experts by developing skills that can be applied to a variety of factual information (carracio., 2008). expertise in this case would not be just recollecting a series of facts, but would additionally involve the students ability to use this knowledge in novel ways and generate a new understanding / knowledge through research, ideally by transcending traditional boundaries of scientific disciplines (bennett., 2000 ; prideaux, 2003). kirkpatrick (kirkpatrick, 1994) identified 10 generic points that should be considered in curriculum design (table 4). these 10 points indicate the importance of planning and that curriculum design goes beyond the content of the taught material. even before designing the curriculum, communication between the different actors (i.e., university administrators, course designer / coordinator, teachers and prospective students) is important, as aims, objectives and learning outcomes will be the most important factors that define the learning process in a top - down fashion (prideaux, 2003). outcome measures that the success of a given curriculum needs to be judged. as the course progresses from one academic year to the next, there is a need for a re - evaluation of the designed / planned and created curriculum against the experienced curriculum (figure 2a) (baerheim., 2007). minor or major refinements are constantly required. ideally, the created curriculum is consistent and constructively aligned with the aims of the planned curriculum (biggs, 2003). constructive alignment of the curriculum implies that students will be able to generate meaning from the different activities they encounter as part of the curriculum. the success of the learning process is measured through assessment of the students knowledge and skills in the subject, but also through their feedback on the curriculum of the course (figure 2b). managing students learning is essential for a constructive alignment of the curriculum with learning outcomes. problem - based learning has been suggested to achieve a good constructive alignment of the curriculum for medical - based subjects (fraser and bosanquet, 2006 ; trappler, 2006). if students are faced with professional problems, such as designing an experiment to determine if a mouse modeling alzheimer s disease has a memory disorder, they would construct meaning out of this situation and learn how to solve a particular problem. however, this is not necessarily so, as the problems have to be very well defined and provide a clear process from which the students can learn (tchudi and lafer, 1996). for this, the students will need to know sufficient information to consider a particular problem, as otherwise they are unlikely to really confront the issues at hand (schmidt, 2009). importantly, students need to generate the outcomes / solutions to the problem to ensure that they truly engage with the problem (so called epistemic curiosity). if solutions are presented by the teacher, the students are unlikely to fully engage. there is, nevertheless, a risk in this approach that students feel lost and disengaged with the learning process. therefore, a strong emphasis will be on the teachers and their guidance through this process (ward and lee, 2002). still, it is this interaction between teachers and students that is most likely to yield the best results, although it poses a greater demand on the teachers time and skills. ideally, problem - based learning would allow students to first experience a superficial learning of the subject area in which they will merely connect how different concepts relate to each other without going into the specific complexities within each particular topic (trappler, 2006). these core or introductory sessions could also involve other aspects that enhance interdisciplinary thinking, such as historical, ethical or philosophical aspects of the field (beck, 1986 ; wiertelak and ramirez, 2008). in advanced or specialized modules, students can engage more directly (e.g., practicals) and diversely (e.g., discussions, practicals, research projects) with the topic that will result in a more meaningful and deeper understanding (kolb, 1984 ; beattie., 1997). if appropriate, these specialized modules could be developed into a curriculum that re - focuses learning into a particular discipline, such as a degree in neuroimaging, rather than a more general degree in neuroscience (estes, 2007). in these specialized modules, researchers can present and discuss their latest research, but students can also be asked to critically appraise a specific paper relevant to the subject or present / defend someone else s work in this area (cleland, 2002). this approach will allow them to critically evaluate work in this area. the designation of the course will then depend on the specific content that is taught in both the core and specialized modules (estes, 2007). these novice - expert modules would allow the university to enlarge the number of courses offered (trappler, 2006). a modular approach to curriculum design is often desirable as teaching spans across several academic departments (ackerman., nevertheless, this can lead to each module equating to a specific discipline (e.g., developmental neurobiology, neuropsychology) and is likely to result in a multi - disciplinary curriculum structure. under this structure, the departments are mostly independent to organize the content and structure of these individual course modules. typically modules evolve from developmental neurobiology and neuroanatomy to behavioral, cognitive and clinical neuroscience. this structure is easy to administer and can draw on existing content (commonly an introductory course for that discipline) that is administered on more discipline - focused teaching sessions. within each module, students are provided with a general overview of the discipline and its contribution to the study of the nervous system. typically, following this general overview the content and teaching methods might not be contiguous and hence could prevent the integration of material from the different modules. this structure mainly leaves integration of information to students and does not provide a structure that is conducive to integrate different disciplines, but rather regards them as parallel to each other, potentially fostering a multi - disciplinary view of neuroscience. despite flexibility and the potential to develop expert learning, there have been concerns that a modularized curriculum can endanger coherence in learning (bennett., 2000). although, it will be obvious to students that there is some overlap between disciplines, too few opportunities are given to students in the delivered curriculum to develop a clearly integrated view of neuroscience as an inter - disciplinary field of study. this multi - disciplinary approach to curriculum design, therefore, might be apposite for undergraduate programs or master - level programs that aim to provide a general overview of neuroscience (table 2), but it is unlikely to provide the level of integration that is required for an inter - disciplinary view of neuroscience (boitano and seyal, 2001). an interdisciplinary curriculum needs to achieve an integration of these disparate disciplines to highlight how they all converge to answer a particular question. the development of problem - based learning can provide a platform that affords students to interact with techniques and theoretical constructs from a variety of disciplines (trappler, 2006). for instance, is it the accumulation of aggregated a proteins in the ca1 subfield of the hippocampus that leads to a cognitive impairment in patients with alzheimer s disease ? this requires knowledge of what distinguishes aggregate forms of a, probes neuroanatomy in terms of defining location as an important factor, but also necessitates an understanding of how one can reliably assess cognitive functions in animals and its translational relevance to patients. confronting students repeatedly with similar issues to solve will allow them to refine their learning and improve outcomes. the specific problem or question here, however, is not of as much importance as the principle that they have to engage to solve a particular biological puzzle that requires multiple tools to provide a satisfactory answer. it is the teacher s responsibility here to ensure that the students draw on multiple disciplines and that integration is required to provide an appropriate answer. to ensure persistent learning of this integrative approach, it is important to not overemphasize specific facts, as these are rapidly changing in modern science, but rather to develop competencies (i.e., finding the appropriate type of information to integrate). it is these competencies that are most likely to be useful if students progress in their future careers, be it in academia, industry or government (wiertelak, 2003). spiral curriculum that repeatedly exposes the students to the same aspects in a different context is likely to enhance the interdisciplinary learning and the connection between different facets of the same problem (bruner, 1960 ; masters and gibbs, 2007). for instance, students can learn the basic principles of a aggregation with a lecture, but this same information can be revisited during a discussion of animal models and their pathology and both of these can then be re - discussed within context while seeing patient s with the disease. this forms a spiral of learning where the same information is placed within a new context that adds complexity to the acquired knowledge. importantly, however, the re - presentation and discussion of relevant information requires planning and hence coordination over the whole curriculum. disciplinarity, using this approach, is de - emphasized and solving the scientific question or problem with appropriate means is accentuated. continued assessments and reiterations of general concepts will be needed to ensure persistent learning and inter - disciplinary thinking. careful planning of each module in the context of the overall curriculum is central to ensure that specific aims are defined and accomplished by teaching methods. one approach to implement this progression in the curriculum are core teaching modules that involve a general overview of the subject allowing students to connect with the new material based on the variety of background information they previously acquired. the advantage of this system is that all students will have sufficient background to engage with more complex ideas and gradually learn to integrate ideas and techniques from other disciplines. although inter - disciplinary teaching is thought to be essential to the progress of neuroscience, it is vital to acknowledge that not all students will be adept at this process and, potentially more importantly, not all students are interested in this type of learning. some students will prefer to specialize in one specific area or technique that is relevant to neuroscience. therefore, courses and curriculum design need to reflect the variety of students background, as well as their interests. it is important to note here that no one curriculum structure is necessarily better than another one, provided that it is based on a rational design that delivers the expected learning outcomes (i.e., the product). rational curriculum design will first specify particular aims and outcomes that should be achieved by the curriculum (i.e., the planned curriculum) (kessels and plomp, 1999). elements that are not relevant to these will be omitted. there is a commitment to provide an efficient learning process to achieve these aims. in general, it is thought that there are three parts to a curriculum (figure 2) : the planned curriculum, the created curriculum and the experienced curriculum (stenhouse, 1975 ; knight, 2001). the created curriculum incorporates the different teaching methods and content to achieve these aims (i.e., the syllabus). however, delivery of this is dependent on the teachers, also sometimes referred to as the delivered curriculum. the experienced curriculum is reflected in the learning of the students (i.e., the process). typically, the learning outcomes are evaluated using various forms of assessment. however, as always, to ensure that these different versions of the curriculum converge, communication between the different actors is key (huber, 2002). the main aim of curriculum design is to engage the student and to refine the learning process (brann and sloop, 2006). to this end, the students need to experience as many different ways as possible to interact with the same topic (i.e., neuroscience) and to assimilate interdisciplinary thinking. often, when given a choice to engage with interdisciplinary modules, students choose to stay within their discipline or to very closely related subjects (huber, 2002). although this might be appropriate for some courses that restrict themselves within a particular discipline or scientific question (e.g., neurodegeneration), it does not provide students with a more comprehensive skill set that we would define as inter - disciplinary. it is paramount for the students to be aware that they need to drive the learning process outside of their comfort zone and that curriculum and faculty are but enablers for this process. apart from curriculum design, specific teaching methods used within the course structure are also important aspects to direct students learning (bourner and fowers, 1997). it is here also relevant to remark that neither teachers, nor students act within a vacuum, but their teaching and learning are part of a collective experience (figure 2a). a teacher s methods of teaching are embedded and offset with the remainder of the faculty. it is, therefore, desirable for different teachers to employ a variety of teaching methods (felder, 1993 ; tanner and allen, 2004). this will enable students with different learning styles to engage more effectively, but will also present students with different experiences of the same content (kinchin, 2011). a student s learning experience is not only influenced by their assimilation of what is presented by the faculty, but it is also dependent on their interaction with other students (hirschy and wilson, 2002). an efficient and rational curriculum design for inter - disciplinary topics is, therefore, a dynamic process that requires careful planning, but also a continued engagement of both faculty and students. future employers should be able to associate particular neuroscience degrees with a student that is appropriate for their needs (whittaker and akers, 2009). courses, therefore, will eventually need to define the main responsibilities associated with the student s occupation (i.e., critical work function), but they will also need to identify and measure competencies (i.e., student s key activity), determine effective performance criteria (i.e., provide performance indicators in key activities), establish an essential knowledge associated with the occupation / profession (i.e., technical / scientific facts) (bennett., 2000). these aspects need to be complemented by more general competencies in key activities (e.g., adaptability of skill and knowledge), as well as desirable attitudes to the activity (i.e., ethics, professional conduct, continued professional development). these standards provide practitioners and employers with a certain quality control that allows them to chose neuroscience courses based on their current and desired aptitude, as well as an on their future employability. the conceptualizations about curriculum design illustrate strengths and weaknesses of each approach, but there is little, if any, evidence for any particular curriculum approach being measurably superior in student learning. although there is some recent evidence that a problem - based learning curriculum improved junior doctors preparedness for coping with uncertainty (bleakley and brennan, 2011), it is important to note though that in this case course design and assessment were focused on this specific issue. in some cases of professional development, one particular curriculum design might therefore be favorable over another as it is geared to this specific skill. yet in other circumstances, another curriculum designed to achieve a different outcome might be superior. consequently, it is essential to follow a rational curriculum design that uses the overall expected learning outcomes to align the delivered curriculum accordingly. in neuroscience education it is, therefore, important that an appropriate curriculum design is chosen for the particular circumstances of any given course. although inter - disciplinary teaching is considered an important aspect in furthering our understanding of the nervous system, this is not necessarily the most important aspect to consider for courses that have other outcome priorities (e.g., training of technical staff). importantly then, the faculty needs to evaluate outcome and constantly aim to improve upon the experienced and planned curriculum (felder and brent, 1999). improving the quality of neuroscience teaching, as well as the inter - disciplinary skill base of neuroscience,	teaching of interdisciplinary fields of study poses a challenge to course organizers. often interdisciplinary courses are taught by different departments, and hence, at best provide a multidisciplinary overview. scientific progress in neuroscience, for instance, is thought to depend heavily on interdisciplinary investigations. if students are only taught to think in particular disciplines without integrating these into a coherent framework to study the nervous system, it is unlikely that they will truly develop interdisciplinary thinking. yet, it is this interdisciplinary thinking that is at the heart of a holistic understanding of the brain. it is, therefore, important to develop a conceptual framework in which students can be taught interdisciplinary, rather than multidisciplinary, thinking. it is also important to recognize that not all teaching needs to be interdisciplinary, but that the type of curriculum design is dependent on the aims of the course, as well as on the background of the students. a rational curriculum design that aligns learning and teaching objectives is, therefore, advocated.
one of the issues of debate in myocardial revascularization has been that whether utilizing extracorporeal circulation has an effect on the immediate & mid - term results of the patient. as the aha scientific statement, in spite of merits ; namely, less blood loss, less need for transfusion, less myocardial enzyme release for up to 24 hours, less early neurocognitive dysfunction and less renal insufficiency, fewer distal grafts tend to be anastomosed with off - pump myocardial revascularization than with standard on - pump myocardial revascularization. in a retrospective study by the cleveland clinic, although there was no difference in the mid - term results, survival, freedom from myocardial infarction, and freedom from percutaneous coronary reintervention, the on - pump group received a great number of bypass grafts than the off - pump group (3.51.1 versus 2.81.0, respectively, p0.1). in almost all patients of both groups, furthermore, between the two groups, there is no difference of statistical results according to the completeness revascularization and the revascularized territories, respectively (table 2, 3). there was no difference in the ejection fraction (ef) between the two groups during the follow - up period. however, the length of stay and follow - up periods were significantly shorter in the off - pump group (p0.1). the main complications in the on - pump group were bleeding (n=4), low cardiac output syndrome (n=3), wound infection (n=3), cva (n=3), pneumonia (n=1), sepsis (n=1) and tamponade (n=1). usually one patient had more than one complication. in the off - pump group, however, there were 2 wound infections and 1 case of pneumonia occurring in 2 patients. the deteriorations of renal function developed in 21 patients in the on - pump group and 6 patients in the off - pump group, who did not need rrt preoperatively. postoperatively, they needed periodic permanent hemodialysis but there was no significant difference (p>0.1). at discharge, 74.6% of patients (n=50) in the on - pump group and 61.1% of patients (n=22) in the off - pump group were dependent on periodic renal replacement therapy. death of 6 patients occurred in the on - pump group during the hospitalization period, resulting in a hospital mortality rate of 5.8% (6 of 103). 3 patients died of low cardiac output syndrome with newly developed atrial fibrillation ; the cause of death for the remaining 3 patients was cerebrovascular accident, respiratory failure with pneumonia and sepsis. the mean follow - up periods of the 97 hospital survivors was 36.69 months (range, 0.9 to 116.8 months). during follow - up, 1 patient in the on - pump group and 1 patient in the off - pump group underwent renal transplantation and are still surviving. in the on - pump group, ten of the 23 late deaths were from cardiac causes, 2 patients from cerebrovascular disease, and the other patients from causes unrelated to the vascular system. meanwhile, three of the five late deaths in the off - pump group were from the cardiovascular disease (table 5). to identify factors influencing the survival rate, univariate analysis showed that only the number of total grafts acted as a determinant of the postoperative survival rate. postoperative periodic rrt trended toward being an independent factor for survival (table 6). however, at multivariate analysis using the cox proportional hazards modeling on variables after exclusion of perioperative deaths, the independent predictors for reduced survival after hospital discharge were the number of total grafts and postoperative periodic renal replacement therapy (table 7). the survival rates of all discharged patients at 1, 3 and 5 years were 92.6%, 85.0% and 64.1% respectively (fig. survival rates of the on - pump group declined more rapidly than those of the off - pump group during the immediate postoperative period due to higher in - hospital mortality, but the on - pump group showed a similar survival rate to the off - pump group after the perioperative period (fig. the survival rates of discharged patients at 1, 3 and 5 years were 90.0%, 86.1% and 62.7% in the on - pump group, and 97.1%, 79.0% and 69.1% in the off - pump group, respectively. in spite of the differences in mortality during the perioperative period, there was no significant difference in the 5 year survival rate between the both groups by log rank statistic (p=0.57). freedom from cardiac related death at 3 and 5 years were 95.7% and 77.7% in the on - pump group, and 81.3% and 71.2% in the off - pump group (p>0.1 by log rank statistic) (fig. whether complete myocardial revascularization or the use of extracorporeal circulation is more beneficial in patients with renal insufficiency is a question of great importance for the cardiac surgeon. patients with renal dysfunction have several obstacles that prevent optimal revascularization and contribute to an unfavorable postoperative outcome. pressure overload as a result of stiffness of large capacity arteries like the aorta, and volume overload as a result of chronic fluid retention lead to left ventricular hypertrophy and increased left ventricular end diastolic diameter, which makes patients with renal dysfunction susceptible to ischemic insult and left ventricular dysfunction. in addition, a significantly greater incidence of plaques has been reported in the common carotid artery, rendering patients more prone to perioperative ischemic cerebral insult. high level exposure to traditional risk factors such as smoking and dyslipidemia, and also endothelial dysfunction, commonly characterized by reduced production of the vasodilator nitric oxide (no), are thought to be a factors leading to a worse postoperative outcome. an overview of the available literature summarizing the perioperative mortality rate for isolated myocardial revascularization for patients with renal insufficiency shows a rate of 8.9%, with increased but acceptable perioperative mortality. this mortality ratio is lower than that of isolated cardiac valve surgery and a combined procedure (19.3 and 39.5% respectively). therefore, cardiac procedures, especially myocardial revascularization, to relieve ischemic insult is the best treatment for patients with renal insufficiency having ischemic heart disease. in particular, when comparing myocardial revascularization with pci in patients receiving rrt, there may be better long - term survival and freedom from angina with myocardial revascularization, compared with balloon angioplasty. however, the inherent side effects of conventional myocardial revascularization utilizing extracorporeal circulation may impose more serious problems on patients with renal insufficiency, especially such as bleeding tendency due to platelet malfunction, fluid overloading, systemic inflammatory reactions and compromise of renal function. a prospective randomized study, by ascione. shows that off - pump myocardial revascularization offers superior renal protection when compared with the on - pump, namely conventional myocardial revascularization in first time coronary revascularied patients. furthermore, glomerular filtration was assessed by creatinine clearance and the urinary microalbumin / creatinine ratio, which was significantly worse in the on - pump group. in addition, compared to the off - pump group, renal tubular function was also impaired in the on - pump group as assessed by an increased n - acetyl glucosaminidase activity. also, osaka. suggest that off - pump myocardial revascularization has several advantages compared to on - pump myocardial revascularization for patients with renal insufficiency, in that there is less bleeding, the systemic inflammatory response syndrome can be avoided and electrolyte imbalance can be prevented. in the case of calcified and narrow native stenotic coronary arteries, distal anastomosis may be difficult to perform, so emphasis is placed upon precise preoperative angiographic evaluation of native coronary arteries. however, the authors concluded that graft anastomosis problems in difficult cases may be improved dramatically as experience with off - pump revascularization increases. moreover, it is well known that off - pump myocardial revascularization reduces postoperative complications and early mortality in patients with renal insufficiency [19 - 21 ]. in our study, although there is no difference in the complications other than a renal problem in the both groups (p>0.1), early mortality showed a trend of being lower in the off - pump group, p<0.064. concluded that off - pump myocardial revascularization had a consistent trend of reducing morbidity and early mortality overall, including all high - risk subsets (80 years of age or older, ef<0.25, concurrent chronic obstructive pulmonary disease, prior renal failure, prior neurologic event and reoperation). but what about the mid- or long - term results of the off - pump myocardial revascularization in patients with renal dysfunction ? in an analysis of current trends that divided the study period into 4 periods of time, bechtel. demonstrated that improvement in perioperative survival after cardiac surgery in dialysis dependent patients in recent years does not continuously improve the long - term prognosis. they also noticed that overall survival was significantly dependent on the type of surgery and was better for patients receiving isolated myocardial revascularization than isolated valve surgery and myocardial revascularization with concomitant valve surgery. although we did n't analyze the trend over time in our study, 5 patients among in - hospital death of a total of 6 patients of on - pump group received surgery before the year 2004 (5 deaths out of a total of 69 patients who were operated on in 1999~2003, and 1 death out of a total of 34 patients who were operated on in 2004~2009). dewey. studied patients with end - stage renal disease undergoing myocardial revascularization, comparing the results of on - pump group to off - pump group. although there were early mortality benefits, the long - term survival rate was significantly worse in patients revascularized using off - pump method (p=0.03). the authors suggest the significantly fewer number of grafts performed in the off - pump group may be related to the cause of lower mid- or long - term survival. however in our study, there is no difference in the mid - term survival of patient with renal insufficiency between the on - pump group and off - pump group (p=0.57). particularly, both groups have a similar total number of targets with grafts, and distal number of targets with arterial grafts are even greater in the off - pump group (p<0.001). in addition, the revascularized territories were not different between the two groups. perhaps increased experience and new devices for the stabilization of the heart make it possible for surgeons to adeptly perform complete myocardial revascularization with off - pump methods. because postoperative angiography was not performed in all patients, we can not guarantee the patency of the bypass grafts ; however off - pump myocardial revascularization is not a difficult surgery for multiple or complete revascularization. not a study dealing exclusively with patients with renal insufficiency, puskas. reported that graft patency was not different between the off - pump group and the on - pump group at 1 month postoperative data. however, in another study by khan, the authors found better patency of the graft was shown in the on - pump group than the off - pump group (98% versus 88% respectively, p=0.002). the interesting finding of this study is that graft patency in both groups show different statistical results according to the revascularized territories and the kind of grafts that was used. in considering the susceptibility to development of atherosclerosis in the coronary arteries of patients with renal insufficiency, graft patency may be one of the other key independent determinants that affect mid- or long - term results in these patients. first, we did n't always assess graft patency postoperatively at regular intervals due to the concern of renal toxicity of radiologic contrast, which could affect the survival rate. second, information on the duration of preoperative dialysis was not obtained from medical records. generally, the longer the history of preoperative hemodialysis, the lower the survival rate. third, the current study is not a prospective randomized study, but retrospective observation study. finally, a relatively short term follow - up duration, a small sample size and a selection bias for the choice of utilization of extracorporeal circulation may affect the results of survival analysis. in conclusion, off - pump myocardial revascularization does not affect the number of bypass grafts and does not affect mid - term survival for patients with renal insufficiency, in spite of having the benefit of early mortality, compared to on - pump myocardial revascularization. especially, for patients who are likely to take periodic renal replacement therapy postoperatively, regardless of the use of arterial or venous grafts, myocardial revascularization with a large number of total grafts has a beneficial effect of increasing mid - term survival, irrespective of utilizing extracorporeal bypass.	backgroundmyocardial revascularization in patients with renal insufficiency is challenging to the cardiac surgeon, irrespective of utilizing extracorporeal circulation. this study aimed to compare the number of bypass grafts and the mid - term results and to evaluate independent survival predictors in patients with renal insufficiency undergoing on - pump or off - pump myocardial revascularization.materials and methodswe retrospectively analyzed the data of 103 patients with renal insufficiency, who had isolated myocardial revascularization between january 1999 and january 2009. the patients were divided into two groups, the on - pump group and the off - pump group.resultsthe off - pump group received a significantly greater number of distal arterial grafts than the on - pump group. however, the mean number of total grafts, the degree of complete revascularization, and survival rate of the patients were not significantly different between the two groups. multivariate analysis showed the independent predictors for reduced mid - term survival were the number of total grafts and postoperative periodic renal replacement therapy. off - pump myocardial revascularization does not decrease the number of bypass grafts or influence on the mid - term results for patients with renal insufficiency, compared to on - pump myocardial revascularization.conclusionmyocardial revascularization with a large number of total grafts has a beneficial effect on survival in patients with renal insufficiency, irrespective of utilizing extracorporeal bypass.
during the past few decades, dengue fever has gradually become one of the leading causes of morbidity and mortality in tropical and subtropical areas throughout the world. the dengue virus (denv), a mosquito - borne member of the family flaviviridae, circulates as four distinct serological types denv 1, denv 2, denv 3, and denv 4. over all, two - fifth of the world population are living in areas, at risk for dengue [24 ]. infection with any of these leads to a mild self - limiting febrile illness (dengue fever, df). a more severe form of the disease, dengue hemorrhagic fever / dengue shock syndrome (dhf / dss), is responsible for high mortality rate, especially in children. it has been estimated that about 50 million cases of df occur annually, with 10,000 infant deaths due to dhf / dss. dhf / dss has been postulated to result from immune enhancement after a second heterologous denv infection. in india, denv was first isolated in 1946 and many outbreaks have been reported [710 ]. dhf was first reported in calcutta (kolkata), west bengal in 1963, again in 1964. since then, there are numerous studies from the indian subcontinent investigating dhf in various parts of the country [1322 ]. but there are no studies investigating the overall prevalence of the dengue serotype circulating in the endemic zone, apart from the epidemic outbreak. the purpose of this paper is to present a comprehensive report on the diagnosis of dengue infection amongst the febrile cases, available from january to december, 2010, in the city of kolkata and also to identify the serotype presently circulating in this region. the incidence of df in the rural west bengal is on the increase and is spreading to geographic regions not previously affected [23, 24 ]. antibodies against group b - arthropod - borne viruses in more than 80% of the kolkata population have been recorded almost fifty years back, and that is too possibly due to the infection by dengue viruses. the present study aimed to identify the serotypes of denv in the population of kolkata as well as to study the sociodemographic status in relation to denv infection. the present population of kolkata is 44,86,679 of which 23,62,662 are males and 21,24,017 are females. the city has an international sea and airport and one rail way station (sealdah) which is busiest in the world. the rail road of this station covers a number of districts, situated at the border of bangladesh. adjacent to the city, there is a thickly populated town, howrah, which has one of the biggest terminating railway stations of the eastern india. cases were mainly referred from outpatient department (opd) and indoor of i.d & b. g hospital, attached to this unit, from different medical colleges as well as other hospitals in kolkata along with a short history of the patients. a good number of cases were referred to us by the private practitioners also. in the matter of selection of dengue fever (df) cases, the following criteria were initially considered : (1) high fever ; (2) head ache ; (3) retro - orbital pain ; (4) nausea / vomiting ; (5) malaise / joint pain ; (6) generalized skin rashes. in the present study two or more of these criteria, apart from fever, were fulfilled. the possibilities of bacterial and prokaryotic etiology in the collected samples were excluded through investigations at the respective hospitals. the case history and the investigations of the patients were compiled. in the case of dhf, the history of illness was revealed by the sudden rise of high fever (38.3c39.4c), headache, retro - orbital pain, conjunctival congestion, and facial flashing. in addition to that, some cases had the history of hemorrhagic manifestation either with petechiae or with gum bleeding or malena. in such cases, intermittent or biphasic course of fever was recorded, where the first phase of fever persisted for 27 days and the second bout of fever persisted for 23 days. no cases with the history of plasma leakage were observed. in the admitted cases, we found that fever was also accompanied by generalized malaise and lumbosacral pain. those patients represented rashes, appeared on 25 days of illness in limbs, sparing palm, and souls. sera were separated from the collected blood samples, were stored at 80c, and were tested within 1 month from the date of collection. a total of 378 samples were thus received from the suspected cases and analyzed for the detection of dengue viruses, if any. to study the sociodemographic status in relation to denv infection, all the 378 samples were screened for the presence of dengue igm antibodies by igm capture enzyme - linked immunosorbent assay (elisa) : using a kit, prepared by the national institute of virology, pune, india, following the prescribed protocol. optical density (od) was measured at 492 nm using an elisa reader (titertek multiskan plus, lab systems finland, type-314). to study the molecular typing of denv, attempts were made to isolate the rna from all the samples as well as from four different denv serotype strains, which were used as positive control. viral rna was isolated by using qiagen viral rna isolation kit (qiagen, gmbh, hilden, germany) according to the manufacturer 's protocol. in this study,, viral rna was converted to a dna copy (cdna) prior to enzymatic dna amplification by the use of reverse transcriptase (rt) and the denv downstream consensus primer d2 - 5-ttgcaccaacagtcaatgtcttcaggttc-3 homologous to the genomic rna of the four serotypes. subsequent taq polymerase amplification was performed on the resulting cdna with the upstream dengue virus consensus primer d1 - 5-tcaatatgctgaaacgcgcgagaaaccg-3. target rna was amplified in 25 l volumes containing the following components : 800 mm deoxynucleotide triphosphates (dntps), 8 mm dithiothreitol, 0.24 m each of primers d1 and d2, 0.5 u of amv rt (promega, madison, wi, usa), and 0.625 u of dreamtaq dna polymerase (fermentas inc., the reactions were allowed to proceed for 1 h at 42c and then to proceed with 95c for 3 minutes for initial denaturation followed by 35 cycles of denaturation (95c for 30 sec), primer annealing (55c for 1 min), and primer extension (72c for 2 min) along with final extension (72c for 5 min). denv serotyping was conducted by second - round amplification (nested pcr) initiated with 10 u of diluted material (1 : 100 in sterile distilled water) from the initial amplification reaction. the total 20 l of reaction mixture was prepared using 2 l of diluted first pcr products, 0.8 mm dntps, 0.5 u of dreamtaq dna polymerase and 0.3 m of primer d1 and 0.3 m of dengue virus type - specific primers : ts1 5-cgtctcagtgatccggggg-3, ts2 5-cgccacaagggccatgaacag-3, ts3 5-taacatcatcatgagacagagc-3, and ts4 5-ctctgttgtcttaaacaagaga-3. dithiothreitol and amv rt were eliminated. the samples were subjected to initial denaturation (95c for 3 min) followed by 20 cycles of denaturation (95c for 30 s), primer annealing (55c for 1 min), and primer extension (72c for 1 min) along with final extension (72c for 5 min). the pcr products were analyzed by running a 1.5% agarose gel stained with ethidium bromide. out of 378 samples collected, only 173 samples were reactive to dengue igm antibody by elisa method. maximum numbers of igm positive cases were observed in the age group of 010 years in both male and female patients (figure 1). females were more affected (46.5%) than the males (45.1%). as regards the seasonal prevalence, it is evident from the result that although sporadic cases obtained throughout the year, dengue cases started from the month of july and attained maximum number of cases in the month of november (figure 2). out of 378 samples, seventy - four samples (68.5%) had the denv infection by single different serotypes, of which 7 samples (9.5%) had the monotypic infection with denv-1, 45 samples (60.8%) had the monotypic infection with denv-2 and 22 samples (29.7%) had the monotypic infections with denv-3 serotype. only one sample had all the three serotypes, that is, denv-1, denv-2, and denv-3. out of 22 dhf cases, 3 were found with single - serotype infection and 18 cases with dual infections of denv 2 and denv 3. only a female patient of 24 years of age had the infection with denv-1, denv-2, and denv-3 serotypes at a time and also suffered from dhf. clinical data regarding the death of the dhf cases was inadequate to reach any conclusion about the severity of illness. the specimen containing denv-1, denv-2, and/or denv-3 is identified by the detection of a dna band of 482, 119, or 290 bp in size, respectively, on 1.5% agarose gel, loaded with nested pcr products along with positive controls, stained with ethidium bromide (figure 3). the monitoring of denv activity is required for public health importance, as the dengue fever and dhf / dss are increasing worldwide and are spreading in the places, where it was previously not reported. the first isolation of denv serotype 1 and 4 was reported from india in 1964 [31, 32 ] and serotype 3 in 1968. although concurrent infection with more than one serotype of denv in the same individual is uncommon, high percentage of concurrent infections with different denv serotypes had been detected at an outbreak in delhi, india, in 2006. kolkata is a dengue endemic zone ; frequent outbreaks of df and dhf have been occurring since last centuries. in kolkata, dengue was first documented in 1824 and several epidemics took place in the city during the years 1836, 1906, 1911, and 1972, affecting 40% of the city people. the last large - scale dengue outbreak has been recorded in the year 2005. in the rural areas of west bengal, dengue is gradually spreading and establishes new reports [23, 24 ]. no continuous monitoring of the molecular detection of the dengue serotype has yet been attempted in the city of kolkata, either in epidemic or in sporadic dengue outbreaks. out of 378 blood samples, agewise distribution revealed that the highest number of dengue cases were detected in the age group of 010 years, followed by 1120 years and above (figure 1). in the highest age group (above 50 years), the number of positive cases were too small and only 16.6% dengue igm positivity was found in the male individuals. in all the age groups, females were more affected than the males (figure 1). are domestic and peridomestics in nature, the females get more exposure than the males, as most of the time they reside inside the house. during the year - round study, although small number of samples from suspected dengue cases were referred to us during the period from january to may, only a few were positive to dengue igm antibody by elisa method. it is evident from our study (figure 2) that the dengue cases actually started from the month of june and attained its peak in the month of november during this year, which is the post - monsoon period. it may be explained by the fact that the stagnant fresh water during the rainy seasons (june to october) favoured the breeding of the vector mosquitoes. for the molecular detection, rt - pcr was performed with all samples, of which 108 samples were positive by that method. a total of 74 samples had the monotypic infections, involving denv 1, denv 2, and denv 3, of which denv 2 was predominated. conversely, 33 samples had the dual infections with denv 2 and denv 3, of which all the samples were igm reactive. only one sample produced prominent band against denv1, denv 2, and denv 3 and also contained igm antibody. the detection of the viral rna in presence of the igm antibody may be explained by the fact that, due to the consecutive infection, the igm detected in those samples possibly appeared due to the initial infection, which is evident by the intensity of the bands in gel electrophoresis (figure 3). the hemorrhagic manifestation was found mainly among the patients, infected with multiple dengue serotypes. dhf cases were observed mainly among the young and young - adult age groups (030 years) which might be due to the absence of immunity against all serotypes of denv in them. although the increasing trend of cocirculation of multiple denv serotypes suggests that kolkata is becoming a hyperendemic state, a large - scale monitoring on the circulating strains of denv is highly required to draw a definite conclusion.	dengue is one of the major public health threats in kolkata. every year, blood samples with dengue - like illness are referred to us from different medical colleges and hospitals in kolkata for the detection of dengue infection in them. in 2010, a total of 378 samples were referred to us for that purpose. all the samples were tested for the detection of igm antibodies by elisa method, followed by rt - pcr test for the detection of serotypes. only 173 samples were elisa positive. out of 378 samples, 108 were rt - pcr positive. out of 108 samples, 74 samples had monotypic infection with different serotypes of denv and 33 samples had dual infections with denv-2 and denv-3. only one sample had the infection with denv-1, denv-2, and denv-3. dhf was found mainly among the patients, infected with multiple dengue serotypes. only 3 dengue monotypic infected patients had suffered from dhf.
transient cerebral ischemia - reperfusion (ir) injury is a major complication in stroke, resuscitation, and perioperative period, in which 5070% survivals suffer from severe disabilities. decrease of atp after anoxia or ischemia has been shown to be one of the original steps to cause the following injuries, and the creatine / phosphocreatine system can to some extent compensate for the injuries of anoxia or ischemia. in normal condition, creatine (cr) is synthesized in the brain and is used to form phosphocreatine (pcr) with a reaction catalyzed by creatine kinase (ck), called lohmann reaction. when the phosphate group is detached from pcr, 45 kj / mol of free energy thus, pcr can donate its phosphate group to adp to resynthesize atp under conditions of atp exhaustion. in this way, pcr allows atp synthesis even in the absence of oxygen and glucose [3, 4 ] (1)1cr+atpckpcr+adp+h+. some studies showed that pcr could improve the outcome after neonatal hypoxic ischemic encephalopathy. to investigate whether pcr has protective effect on focal cerebral ir injuries, we introduce exogenous pcr to rats before cerebral infarction and studied the effect of pcr on intracranial pressure (icp), neural dysfunction score, cerebral infarction volume, cell apoptosis, expression of caspase-3, and level of aquaporin-4 (aqp4) in brain. spf grade wistar rats, weighing 250350 g, were obtained from animal center, shengjing hospital of china medical university. the rats were housed in spf facilities with a 12 h dark - light cycle in animal center of shengjing hospital of china medical university and fed ad libitum in experiment. the animal use protocols used were approved by the institutional animal care and use committee at china medical university. all animal experiments were carried out in accordance with the national research council guide for the care and use of laboratory animals, as adopted by the national institutes of health. focal cerebral ir was achieved using transient middle cerebral artery occlusion (mcao). anesthesia was induced by 10% chloral hydrate (0.3 mg / kg i.p.). the left common carotid artery (cca), external carotid artery (eca), and internal carotid artery (ica) after careful preparation of the vessel, a small incision was made on the common carotid artery and a 0.2 mm monofilament nylon suture was introduced through the common carotid artery into the internal carotid artery (about 18 to 20 mm from the external - internal carotid artery bifurcation). at the end of a 2-hour ischemic period, blood flow was restored by carefully removing the nylon filament. during surgery, rats were maintained at constant temperature using a heating pad. according to zea longa score standard, the model was qualified when it scored 24 (score 0, normal, no neurological sign ; score 1, can not completely stretch contralateral forelimbs ; score 2, contralateral circling when walking ; score 3, contralateral fall over when walking ; score 4, can not walk, and lowering of consciousness.). the left common carotid artery (cca), external carotid artery (eca), and internal carotid artery (ica) the rats were operated by the method of animal model, at the end of a 2-hour ischemic period, blood flow was restored by carefully removing the nylon filament., disodium creatine phosphate was dissolved in saline in the concentration of 50 mg / ml) was administered intravenously 30 min before operation. these rats received the same ir, but, in contrast to the ir group, 200 mg / kg of disodium creatine phosphate was administered intravenously 30 min before operation. these rats received the same ir, but, in contrast to the ir group, 400 mg / kg of disodium creatine phosphate was administered intravenously 30 min before operation. we observed neurological deficits at 24 h and 72 h after occlusion, and intracranial pressure (icp) was determined at 1 h, 2 h, 4 h, and 24 h. apoptosis detection by tunel and caspase-3 immunohistochemical staining, aquaporin-4 (aqp4), and ultrastructure observation were detected at 24 h and 72 h after ischemia. 24 h and 72 h after the onset of ischemia, neurologic evaluation was performed by the same investigator, who was not aware of the group assignment. the specific tests included (a) observation of spontaneous ipsilateral circling, graded from 0 (no circling) to 3 (continuous circling) ; (b) contralateral hindlimb retraction, which measured the ability of the animal to replace the hindlimb after it was displaced laterally by 2 to 3 cm, graded from 0 (immediate replacement) to 3 (replacement after minutes or no replacement) ; (c) beam walking ability, graded 0 for a rat that readily traverses a 2.4 cm wide, 80 cm long beam to 3 for a rat unable to stay on the beam for 10 seconds ; (d) bilateral forepaw grasp, which measures the ability to hold onto a 2 mm diameter steel rod, graded 0 for a rat with normal forepaw grasping behavior to 3 for a rat unable to grasp with the forepaws. icp was monitored continuously from the cisterna magna at 50 hz using acqknowledge software (v3.7.3, biopac, systems, santa barbara, ca). a dorsal midline occipitocervical incision was made, and the muscles were dissected off the occiput, atlas and dura overlying the cisterna magna. a 27-gauge needle, attached using short, noncompliant tubing to a pressure transducer (tsd104a ; biopac systems, santa barbara, ca), was inserted into the cisterna magna. brain was sampled after reperfused with sodium chloride and 4% formaldehyde, regularly embedded in paraffinum, and then sectioned at a thickness of 4 m. the level of cell apoptosis was determined with a roche in situ cell - apoptosis - assay kit with nuclei stained in brown particles (roche diagnostics, indianapolis, in). 5 high - power fields (400) were randomly selected, and the number of apoptotic cells was counted for each field. apoptosis index (ai) = number of positive cells / number of total cells. caspase-3 immunohistochemical procedures were performed strictly according to the manufacturer 's guidelines (nanjing jiancheng bio co., ltd). and the slides with the addition of 0.01 mmol / l pbs (containing 1 : 200 nonimmunity animal serum), instead of primary antibody, showed no response. under the microscope, those with brown granules in cytoplasm were considered as positive cells and were counted in 5 random high - power fields (400). materials of 1 mm thickness were fixed for 2 hours in 4% glutaraldehyde (ph 7.27.4, buffered with phosphate) and postfixed with 1% osmium tetroxide in phosphate buffer (ph 7.4) for 2 hours at 4c. the protein level of aqp4 was determined by western blot analysis using a rabbit monoclonal antibody against rat aqp4 (santa cruz). brain samples were pulverized, incubated in lysis buffer and protease inhibitor cocktail (sigma) for 1 h at 4c. after a 10 min centrifugation at 12000r, the supernatant was collected, and the protein content was quantified. 30 mg protein was loaded per lane, separated by 12% sds - page electrophoresis, and electroblotted onto nitrocellulose membranes for 1.5 h at a constant voltage of 10 v using semidry apparatus. the membrane was incubated with a rabbit monoclonal anti - rat aqp4 antibody (lab vision, fremont, ca) overnight at 4c and then incubated with an anti - rabbit immunoglobulin g horseradish peroxidase - conjugated secondary antibody (pierce, rockford, il). the signals were detected with an enhanced chemiluminescence kit (pierce) and exposed on x - ray film. after the film was scanned with a gs-700 imaging densitometer (bio - rad, hercules, ca), a quantitative analysis was performed using multi - analyst software (bio - rad). data of icp, ai, caspase-3 positive cells, and level of aqp4 were employed by repeated - meausures anova for multiple comparisons. if the result was significant, ranks of arranged score were analyzed by student - newman - keuls test for multiple comparisons. high - dose usage of disodium creatine phosphate can cause low blood pressure. in the p3 group, 5 rats were dead from unconsciousness, which was thought to be related to low blood, pressure. 2 rats from the ir group, 1 from the p2 group and 1 from the p3 group were excluded from this study because of the development of subarachnoid hemorrhage. 1 rat from the ir group, 1 rat from the p2 group, 2 rats from the p3 group were excluded from this study because they were not scored 24 zea longa score. 24 h and 72 h after the onset of ischemia, neurologic evaluation was evaluated. there was significant alleviation in motor function in pcr pretreated group than that in the ir group. icp was lower in the ir, p1, p2, p3 groups than that in the sham - operated group during ischemia stage. but icp was higher in the ir and pcr - pretreated group when compared with the sham - operated group after reperfusion. but there were no statistically significant differences among the five groups at all the time points (figure 1). tunel and caspase-3 immunohistochemical assay were adopted to determine neuronal apoptosis. in the study, ai value and caspase-3 positive cells were significantly decreased after pcr treatment (figures 2, 3, and 4). clear bilayer nuclear membrane and nucleolus, rounded or oval - shaped mitochondria, were seen in neuron cells in sham - operated group. in the ir group, shrunken neucleus and aggregated chromatin toward the nuclear membrane were observed. in obviously swollen mitochondrion, cristae were disordered, cracked, or decreased, and membrane structure became incomplete or partially disappeared. in the p1 group, the nuclear membranes were clear, and the granule - like aggregation of chromatin could be seen. but organelles were also disordered. in the p2 group, slightly pycnosis, complete nucleus membrane, and less degenerated organelles could be observed. in the p3 group, the nucleus and mitochondrion changes levels immunoblot analysis showed aqp4 protein in brain homogenates from rats as bands at about 32 and 34 kda representing the two aqp4 protein isoforms expressed in brain (figure 6). quantitative analysis of brain homogenates indicated increased aqp4 protein in the ir group and pcr group compared with those in the sham - operated group. the levels of brain aqp4 protein were decreased in the pcr group compared with the ir group (p < 0.01, figure 7). rapid decrease of atp has been shown after cerebral ischemia, which leads to secondary injuries. we hypothesized that supplication of an exogenous substrate before ischemia reperfusion might alleviate ir damages. study showed that [5, 10 ], after exogenous injection, the distribution of disodium creatine phosphate is mainly in skeletal muscle and myocardium, brain is the second one, and kidney is the third, but less in lung and liver. investigation showed that pcr can go through the blood - brain barrier, even through the cell membrane [11, 12 ], to supply energy to cells directly. but, in actual practice, we do not use disodium creatine phosphate to protect brain from damage at present, especially in early stage (3 hs after infarction) of cerebral ischemia. so the study was performed to investigate the potential effects of pcr on neurologic behavioral assessments, ultrastructural histopathologic changes, and neuronal apoptosis in an animal model of focal cerebral ischemia - reperfusion. studies have shown that the caspase family is the promoter and implementer of apoptosis in mammalian cells, among which, caspase-3 is the most critical downstream apoptosis protease in the caspase cascade waterfall. a variety of extracellular signals activate caspase-8 through fas receptor pathway and caspase-9 via mitochondrial cytochrome c in cerebral ischemia reperfusion injury. activation of caspase-8 and caspase-9 then promote caspase-3, which in turn hydrolyzes cell - specific proteins, and poly - adp ribose polymerase (parp), thereby inducing apoptosis [13, 14 ]. many investigators found the expression of caspase-3 enhanced with the increased cell apoptosis in brain, both in local and focal ischemia. the number and region of apoptosis were related to ischemia time and sensitivity of neurons. after cerebral ischemia, apoptosis cells mainly located in selective infarction area, preoptic region, corpus striatum, inner margin cortex of infarction boundary, corpus striatum, hippocampus, and olfactory tubercle peak in 2448 h. in the study, we examined cell apoptosis by tunel and caspase-3 immunohistochemical staining, and we observed obviously decrease of ai and caspase-3 positive cells after intervention of pcr. some brain regions, including the hippocampus, are more vulnerable to ischemia than others. in the experiment, we observed swelling mitochondrial, disaggregation of polyribosomes, decreased rough endoplasmic reticulum and golgi apparatus in postischemic hippocampal neurons, which is consistent with the studies before. but, in severe cases, organelles in neurons demonstrate condensation, increased matrix density, and deposits of an electron - dense material followed by the disintegration of mitochondria [17, 18 ]. in the experiment and we also demonstrated slightly shrunken nucleus and complete nuclear membrane in the pcr pretreatment group compared with the significantly aggregated chromatin toward the nuclear membrane in the ir group. recently, the bidirectional water channel aquaporin-4 (aqp4) has been found to play an important role in brain - water homeostasis. aqp4 protein is expressed strongly in astroglia at the bbb and csf - brain interfaces, involved in water movement between fluid compartments (blood and csf) and brain parenchyma. it has been suggested that aqp4 deletion markedly reduced brain swelling of cytotoxic brain edema, including water intoxication and focal cerebral ischemia [21, 22 ]. so detection of expression level of aqp4 can indirectly determine the brain swelling extent in cerebral ischemia. in the study, according to ultrastructure observation, less swelling can be seen in plasma and organelles ; meanwhile, we detected significantly decrease of aqp4 expression, so pcr can alleviated brain swelling in cerebral ir injury, although pcr did not decrease icp significantly. in the study, three doses of pcr have been adopted to prove the effect of pcr on focal cerebral ischemia - reperfusion injury. after pretreatment in three groups, animals ' ir injuries were alleviated in motor behavior, apoptosis, brain swelling, and ultrastructural pathological changes. the effects of 200 mg / kg and 400 mg / kg were not significantly different, but the side effect of pcr increased with higher dosage, especially low blood pressure. so, in the experiment, 200 mg / kg is more appropriate for treatment. in conclusion, the study demonstrates pcr has neuroprotective effect on focal cerebral ir injury, which may relate to apoptosis. after pcr pretreatment, neurobehavioral score, infarction volume, and brain swelling	phosphocreatine (pcr) is a natural compound, which can donate high - energy phosphate group to adp to synthesize atp, even in the absence of oxygen and glucose. at present, it is widely used in cardiac and renal ischemia - reperfusion (ir) disease. in this study, to examine the protective efficacy of pcr against cerebral ir, disodium creatine phosphate was injected intravenously into rats before focal cerebral ir. intracranial pressure (icp), neurological score, cerebral infarction volume, and apoptotic neurons were observed. expression of caspase-3 and aquaporin-4 (aqp4) was analyzed. compared with ir group, rats pretreated with pcr had better neurologic score, less infarction volume, fewer ultrastructural histopathologic changes, reduced apoptosis, and lower aquaporin-4 level. in conclusion, pcr is neuroprotective after transient focal cerebral ir injury. such a protection might be associated with apoptosis regulating proteins.
our institutional review board approved this study ; however, informed consents from the subjects were not required for this retrospective study. a computerized search of the medical records between january 1997 and march 2003 at our institution revealed 164 patients who had undergone surgery for epithelial origin ovarian carcinoma. from those patients, we collected the records of 124 patients (mean age : 52 years) who had available abdomen and pelvic ct images obtained before surgery (1 - 30 days), and who had no past history of other malignant disease before the diagnosis of ovarian carcinoma. none of these patients had undergone chemotherapy for ovarian cancer prior to the ct examination. all the tumors included in this study arose from epithelial cells, and the histologic diagnoses are summarized in table 1. based on the pathologic reports of these patients that were written by various staff pathologists at our institution, each epithelial origin ovarian carcinoma was classified as well, moderately or poorly differentiated according to the degree of differentiation by referring to the modified broder 's grading system (10, 11). according to this system, well differentiated cells were more than 75% of the tumor cells in grade 1 (well differentiated) carcinoma, they were 25%-75% of the tumor cells in grade 2 (moderately differentiated) carcinoma and they were less than 25% of the tumor cells in grade 3 (poorly differentiated) carcinoma (10, 11). consequently, the number of patients with well, moderately and poorly differentiated carcinoma was 29, 31 and 64, respectively. one hundred and eleven patients underwent ct examination at our institution, but 13 patients with one well differentiated carcinoma, eight with moderately differentiated carcinoma and four with poorly differentiated carcinoma had ct images obtained at other institutions. the imaging acquisition parameters of the ct images obtained at other institutions were variable, but all the images included contrast - enhanced scans with a 5 - 10 mm slice thickness. at our institution, the ct examinations were performed using a single - detector row helical ct scanner (somatom plus - s ; siemens medical systems, erlangen, germany) for 88 patients, including 26 with well differentiated carcinoma, 16 with moderately differentiated carcinoma and 46 with poorly differentiated carcinoma. multi - detector row helical ct scanners (lightspeed qx / i, general electric medical system, milwaukee, wi) were used for the remaining 23 patients, including two with well differentiated carcinoma, seven with moderately differentiated carcinoma and 14 with poorly differentiated carcinoma. all patients received 500 - 900 ml of oral contrast material (e - z - cat [2% barium sulfate suspension ] ; e - z - em, westbury, ny) 30 minutes prior to the ct examination. intravenous contrast material (ultravist 300 [iopromide ] ; schering, berlin, germany or iopamiro 300 [iopamidol ] ; bracco, milano, italy) was administered into the antecubital vein with using a power injector at a dose of2 ml / kg to a maximum dose of 160 ml at a rate of 3 ml / sec. the scan delay for contrast - enhanced scanning was100 - 120 seconds for the single - detector row helical ct scanning and 90 - 100 seconds for the multi - detector row helical ct scanning. the scanning parameters for single - detector row helical ct were section collimation : 7 mm, pitch : 1.5, table speed : 7.5 mm per rotation (10 mm / sec), reconstruction interval : 5 mm, 120 kv and 210 ma. for multi - detector row helical ct, a section collimation of 5 mm 4, a beam pitch of 1.5, a reconstruction interval of 5 mm, a x - ray tube voltage of 120 kv and a tube current of 210 - 240 ma were used. two radiologists, who were unaware of the pathologic reports of ovarian carcinoma, but who knew about the presence of ovarian cancer in each patient, evaluated the ct findings in a consensus fashion on the picture archiving and communication system, petavision (asan medical center, seoul, korea) ; this made it possible to measure the tumor diameter and attenuation in a particular region of interest. the reviewers evaluated the presence or absence of bilateral ovarian involvement and the nature of the tumor according to the ratio of the cystic portion in the tumor : this included cystic tumor (approximately 100%), predominantly cystic tumor (> 70%), mixed tumor (30 - 70%), predominantly solid tumor (70%), mixed tumor (30 - 70%), predominantly solid tumor (0.05). a septa greater than 3 mm for the maximum thickness was more frequently noted in the poorly differentiated carcinomas (91%) than in the well (63%) or moderately differentiated carcinomas (64%) (p 0.05). among 124 patients, 46 patients had bilateral ovarian involvement. only two (7%) of the 29 patients with well differentiated carcinoma had bilateral ovarian involvement, whereas 13 (42%) of the 31 patients with moderately differentiated carcinoma and 31 (48%) of the 64 patients with poorly differentiated ovarian carcinoma showed bilateral ovarian involvement. the frequency of bilateral ovarian involvement was greater in the moderately and poorly differentiated carcinomas than in the well differentiated carcinomas (p = 0.002 and p 0.05). a septa greater than 3 mm for the maximum thickness was more frequently noted in the poorly differentiated carcinomas (91%) than in the well (63%) or moderately differentiated carcinomas (64%) (p 0.05). our results show that the ct findings of epithelial origin ovarian carcinoma vary according to the degree of differentiation. the frequency of bilateral ovarian involvement and a solid or predominantly solid nature were greater in the poorly and moderately differentiated carcinomas than in the well differentiated carcinomas. tumor seeding was noted in the following order in the poorly, moderately and well differentiated carcinomas. the poorly differentiated carcinoma group was different from the other groups for the tumor morphology, that is, the frequency of thick septa (> 3 mm), papillary projection and necrosis in the solid portion was greater in the poorly differentiated carcinoma than in the other tumor types. the tumor behavior for the poorly differentiated carcinoma was more aggressive than that of the other groups, that is, lymphadenopathy, tumor seeding and a large amount of ascites were more frequently noted in the poorly differentiated carcinoma than in the other tumor types. the presence of papillary projection and a solid portion in the tumor are known as the most predictive findings for ovarian carcinoma (9). in this study, these findings were noted in less than half of the well differentiated carcinomas, whereas most of the cases of poorly differentiated carcinoma showed these findings. moreover, the frequency of these findings in the moderately differentiated carcinoma was between those frequencies of the well differentiated and poorly differentiated carcinomas. with these results, our study shows that the frequency of the well - known ct findings that are predictive of ovarian carcinoma is heavily related to the degree of differentiation. the fact that many cases of well differentiated carcinoma do not show papillary projection or necrosis in the solid portion means that there may be a high risk for misinterpreting well differentiated carcinoma from benign cystic neoplasms in daily practice. therefore, any decision on distinguishing benign from malignant ovarian neoplasms should be reached not only according to the imaging findings, but also according to the clinical findings such as the ca-125 level and the patient 's signs and symptoms. in our study, nearly half the cases of moderately or poorly differentiated carcinomas showed bilateral ovarian involvement. brown. (12) reported that the frequency of multilocularity was significantly higher in primary ovarian tumors than in the metastatic ovarian cancers in bilateral ovarian tumors. however, many cases of moderately and poorly differentiated carcinomas in this study had a predominantly solid or purely solid nature that seemed to be unrelated to multilocularity. therefore, this differentiation is very difficult when bilateral ovarian masses show a solid nature, and in these cases, more effort must be given in these cases to detect primary tumors in organs other than the ovaries. various imaging modalities such as ultrasonography, ct and mr are used for evaluating ovarian tumors. it has been reported that gadolinium - enhanced mr imaging is superior to ct and us for tissue characterization and that ct may miss a small solid portion ; further, it can not differentiate solid nodules from high - attenuation fluid such as mucin or hemorrhage (6, 9). nevertheless, we believe that ct is still widely used for patients with presumed ovarian tumor because it is superior to mr in terms of the scanning time, cost and range of scan coverage. furthermore, ct is undoubtedly better than mr for detecting a possibly hidden primary tumor in organs other than the ovaries. therefore, radiologists should be mindful of the spectrum of ct imaging of ovarian carcinoma according to the degree of differentiation first, this study evaluated only cases of malignant ovarian epithelial neoplasms and it did not include other malignant tumors such as granulosa cell tumor and germ cell tumors, which usually manifest as predominantly solid or purely solid tumors (13, 14). therefore, the ct findings of solid ovarian masses do not always suggest moderately or poorly differentiated ovarian carcinoma, and it is mandatory to consider the clinical or serologic findings such as ca-125, alphafetoprotein and beta human chorioglobin levels when interpreting ct images. the decision for a solid lesion can be made on the basis of enhancement between the unenhanced and contrast - enhanced scans. unfortunately, as all of our patients did not have both these types of images, we could not accurately estimate the extent of the solid portions of the tumors, although we considered a portion with a ct number greater than 60 hu as a solid portion. therefore, there may have been a potential study drawback that mucin or hemorrhage in the tumor was interpreted as a solid portion. third, as we judged the presence of adjacent organ invasion and peritoneal carcinomatosis only by the ct findings, but not by the pathology reports, there might have been false negative or positive cases when evaluating those findings. last, for determining the grade of differentiation in ovarian carcinoma, several grading systems have been introduced based on various findings such as the percentage of undifferentiated or well differentiated cells, the architectural features and the nuclear features (15 - 18). this study used broder 's grading system, which is mainly based on the percentage of well differentiated cells, because this system is generally used in the pathology department of our institution. therefore, the composition of our study population might have changed if another grading system were applied. in summary, our results demonstrate that epithelial origin ovarian carcinoma has various imaging findings according to the degree of differentiation. we suggest radiologists should keep in mind that well differentiated carcinoma may not show the typical findings of ovarian carcinomas and that moderately or poorly differentiated carcinomas may mimic metastatic ovarian cancers.	objectivewe wanted to evaluate the ct findings of epithelial origin ovarian carcinoma according to the degree of histologic differentiation.materials and methodsthis study enrolled 124 patients with 31 well differentiated, 44 moderately differentiated and 95 poorly differentiated carcinomas with epithelial origin. the ct images were retrospectively evaluated with regard to bilateral ovarian involvement, the tumor 's nature, lymphadenopathy, adjacent organ invasion, peritoneal tumor seeding, a large amount of ascites and distant metastasis. in cystic, predominantly cystic and mixed tumors, the tumor wall, septa, papillary projection and necrosis in the solid portion were assessed.resultsbilateral ovarian involvement was more common in the poorly (48%) and moderately (42%) differentiated carcinomas than in the well differentiated carcinomas (7%) (p < 0.05). the frequency of a predominantly solid or solid nature was greater in the moderately and poorly differentiated carcinomas than in the well differentiated carcinomas (p < 0.0001). in the 87 tumors with a cystic, predominantly cystic or mixed nature, septa greater than 3 mm, papillary projection and necrosis in the solid portion were more common in the poorly differentiated carcinoma (91%, 91% and 77%, respectively) than in the moderately (64%, 68% and 34%, respectively) and well differentiated carcinomas (63%, 47% and 27%, respectively) (p < 0.05). lymphadenopathy, organ invasion, tumor seeding and a large amount of ascites were more common in the poorly differentiated carcinomas (38%, 27%, 73% and 69%, respectively) than in the moderately (13%, 10%, 48% and 45%, respectively) and well differentiated carcinomas (3%, 0%, 10% and 17%, respectively) (p < 0.05).conclusionepithelial origin ovarian carcinoma shows different ct findings according to the degree of histologic differentiation.
hypovitaminosis d is a highly prevalent condition, especially in older adults, with an estimated prevalence of 5090% depending on the definition used and the population studied [1, 2 ]. growing evidence has established that vitamin d exhibits not only skeletal effects but also a number of nonskeletal effects [1, 2 ]. this finding explains why vitamin d plays a much greater role in human health than previously thought and why lower 25-hydroxyvitamin d (25ohd) concentrations contribute to cellular dysfunction in many biological systems, which in turn is manifested by organ disorders and chronic diseases [14 ]. in particular, it has been reported, among older inpatients hospitalized in geriatric acute care units, that lower serum 25ohd concentrations at the time of admission are directly associated with a greater severity of chronic diseases and higher risks of acute decompensation and inhospital mortality. in line with this, hypovitaminosis d has been proposed as a biomarker of longer length of stay (los) in acute care units [79 ]. for instance, lower serum 25ohd concentrations were independently associated with longer intensive care unit stay in cardiac surgical patients. hypovitaminosis d also doubled the risk of being hospitalized more than 14 days in a geriatric acute care unit and, in another study, the combination of hypovitaminosis d, male gender, and delirium predicted a 4.8-fold higher risk of longer los among geriatric inpatients. importantly, the average increase in los in geriatric acute care units attributable to hypovitaminosis d remains unknown. moreover, while existing studies report an association between hypovitaminosis d and prolonged hospitalization, it remains unclear whether an inverse linear association exists over a range of 25ohd concentrations and whether any increase in 25ohd is associated with a decrease in los, regardless of the initial level. we had the opportunity to examine these issues in a historical cohort of older inpatients hospitalized in a geriatric acute care unit. the objectives of this analysis were (i) to estimate the difference in hospitalization days between inpatients with and without hypovitaminosis d and (ii) to determine whether there was a linear association between serum 25ohd concentration and los. the studied sample was a convenience of all inpatients consecutively admitted into the geriatric acute care unit of angers university hospital, france, between june and december 2008. study inclusion criteria included unplanned admissions to hospital for people 75 years and willingness to participate. two hundred fifty - three inpatients met the study inclusion criteria and agreed to participate. power analysis determined that 253 participants were sufficient to find significant correlations between serum 25ohd concentration and los with 77% power at p 50 nmol / l. additionally, we report the first evidence of a linear association between 25ohd concentration and los, suggesting that any increase in 25ohd was associated with a decrease in los whatever the initial vitamin d status. vitamin d is a secosteroid hormone that exhibits multiple biological actions mediated by the vitamin d receptor present in many cells and target tissues [13 ]. since vitamin d is necessary for the regulation of cellular growth, differentiation, and function, lower vitamin d status leads to multiple organ dysfunction, disability, and unstable health status, which are all causes of deconditioning, polypharmacy, and longer los [1416 ]. consistently, previous literature from our group has provided compelling evidence that hypovitaminosis d could be regarded as a biomarker of age - related disease burden [4, 5 ] and consequent longer los [79 ]. for instance, it has been reported that the risk of being in the highest tertile of los was multiplied by approximately 2 in case of hypovitaminosis d at baseline among geriatric inpatients. however, the number of additional hospitalization days in case of hypovitaminosis d has not been examined yet. thus, the results of the current study provide extra information, specifically, that older inpatients with hypovitaminosis d were hospitalized 3 days more in our geriatric acute care unit than those with adequate vitamin d status ; that is, they had a 21 percent longer los (table 1). the clinical relevance is that longer los exposes older inpatients to greater risks of disability, nosocomial infections, deconditioning, and higher healthcare costs [16, 17 ]. consequently, any decrease in los is beneficial to older inpatients. in the current study, we found a 0.14-day decrease in los per 1 nmol / l increase in serum 25ohd concentration (figure 1), that is, a 1-day decrease in los per 7.1 nmol / l increase in 25ohd. it has been previously reported that vitamin d supplementation produces an elevation of serum 25ohd concentration of approximately 2.5 it can thus be inferred that each additional 300 iu vitamin d per day is associated with a 1-day shortening of los among geriatric inpatients. such estimates may help to justify, plan, evaluate, and compare the effectiveness of interventions aiming at shortening the los in geriatric acute care units with vitamin d supplements firstly, the study cohort was restricted to caucasian older inpatients hospitalized in a single geriatric acute care unit who might be unrepresentative of all older inpatients. thirdly, although we were able to control for many important characteristics that could bias the results, residual prehospital health conditions influencing both vitamin d status and los, such as the body mass index, were not considered. finally, it has to be kept in mind that, in some cases, los does not only depend on bioclinical aspects but is influenced by other factors, including number of beds available, human resource employees, and health insurance guidelines, which could not be taken into account here. increased serum 25ohd concentrations were linearly associated with shorter los in this cohort of acute care geriatric inpatients. those with an adequate vitamin d status were hospitalized 3 days less than those with hypovitaminosis d. our findings reinforce the conceptualization of vitamin d as a biomarker of the health status of inpatients in geriatric acute care unit and provide an additional rationale for prescribing vitamin d supplements in this population. multicenter randomized controlled trials are required to investigate the benefits of vitamin d supplementation on los with higher levels of evidence.	background. hypovitaminosis d is linked to unstable health in older adults. our objectives were to determine (i) the difference in length of stay (los) in geriatric acute care unit between inpatients with and without hypovitaminosis d and (ii) whether there was a linear association between serum 25-hydroxyvitamin d (25ohd) concentration and los. methods. 253 inpatients admitted in 2008 to the geriatric acute care unit of angers university hospital, france, (mean agestandard deviation, 86.2 6.0 years ; 66.8% female) were included in this historical cohort study. los was calculated by subtracting day of admission from day of discharge. hypovitaminosis d was defined as 25ohd50 nmol / l at the time of admission. age, gender, place of life, functional independence, reason for admission, number of acute diseases and comorbidities, use of vitamin d supplements, and creatinine clearance were used as confounders. results. participants with hypovitaminosis d had longer los than their counterparts (15.2 8.2 days versus 12.1 7.0 days, p = 0.017), underlining a mean difference of 3 days. 25ohd concentration inversely correlated (r = 0.14, p = 0.028) and was inversely associated with los (adjusted = 0.07 [95%ci : 0.14 ; 0.02 ], p = 0.043). conclusions. we found an inverse linear association between serum 25ohd concentrations and los in a geriatric acute care unit. participants with 25ohd>50 nmol / l were hospitalized on average 3 days less than those with 25ohd50 nmol / l.
the incidence of osteoarthritis is increasing as the world 's population is rapidly aging, and total knee arthroplasty (tka) has become a common surgery in the orthopedic field. it has been known that perioperative blood loss is estimated to be between 1,000 ml and 1,500 ml, and transfusion is required in 18%-95% of the patients1). in the meantime, there are increasing doubts about the efficacy of allogeneic transfusion due to the risk of transfusion - transmitted infectious diseases and the absence of management measures during transfusion 234). curran.5) reported on the growing incidence of transfusion - transmitted infectious diseases such as aids following allogeneic transfusion. there have been numerous attempts in the search for an alternative to allogeneic transfusion ; autologous transfusion is considered as one of the options, including the use of preoperative autologous blood deposit, salvation of bleeding during surgery, postoperative reinfusion of drained blood after sterilization, and the use of hematopoietic agents such as erythropoietin 789). a lot of studies have suggested that allogeneic transfusion can be reduced by autologous transfusion3101112). however, existing reports are based on insufficient study population sizes to elucidate whether autologous transfusion can reduce the necessity of allogeneic transfusion. in this study, we investigated whether allogeneic blood transfusion combined with autologous transfusion using an autologous transfusion device can reduce the use of allogenic transfusion compared with allogenic transfusion alone in a substantial number of unilateral tkas. further, we assessed whether the use of an autologous transfusion device has an influence on the complication rate (e.g., infection, embolism, etc.). of the patients who underwent unilateral tka at our institution with a diagnosis of degenerative arthritis of the knee, 245 patients in whom the surgery was performed by the same surgeon were enrolled in this study. the patients were divided into group a and group b according to the use of an autologous transfusion device which was introduced in may 2009 at our institution. group a consisted of 127 patients who underwent tka and allogeneic transfusion between january 2003 and april 2009 without the use of an autologous transfusion device. group b was composed of 118 patients who underwent allogeneic transfusion and autologous transfusion using an autologous transfusion device after tka between may 2009 and january 2014. in group b, cbc ii (consta - vac ; stryker, kalamazoo, mi, usa), an autologous transfusion device, was applied after surgery. it was designed to drain blood after surgery from the operation site under 50 - 100 mmhg of negative pressure and reinfuse it 6 hours later to the patient via venous access through a filter that removes impurities without any other washing process. we inserted the drainage line into the joint space immediately prior to wound closure through an aseptic procedure and performed autologous transfusion once 6 hours later. three surgical instruments - scorpio nrg (stryker orthopaedics, mahwah, nj, usa), nexgen lps (zimmer inc., warsaw, in, usa), and vanguard (biomet, warsaw, in, usa)-were randomly applied during surgery. in all surgery, pneumatic tourniquet was applied on the proximal femoral region at 300 mmhg of pressure. criteria for allogeneic transfusion were below 9.0 g / dl postoperative hemoglobin level and unstable vital signs irrespective of hemoglobin level. in each patient, the hemoglobin level was measured before operation, immediately after operation, and 1 day after operation, and the amount of drainage was aggregated at 24 hours and 48 hours after surgery. the presence of side effects (pulmonary embolism, deep vein thrombosis, acute infection, etc.) was examined based on medical records including vital signs, collaboration / consults with other departments, etc. statistical analysis was done using spss ver. 21.0 (ibm corp., armonk, ny, usa). a t - test was conducted to determine the significance of differences in the average volume of drainage and transfusion between the groups. fisher 's exact test was done to identify if there is difference in the complication rate between the groups. there was no statically significant difference in demographics between group a and group b. in group a, the mean age was 74.7 years (range, 73.4 to 76.0 years), and there were 99 female and 28 male patients. in group b, the mean age was 69.1 years (range, 67.6 to 70.6 years), and there were 92 females and 26 males (table 1). the total drainage for 24 hours after operation was a mean of 718.5 ml (standard deviation [sd ], 505.0 ml) in group a and a mean of 906.6 ml (sd, 410.2 ml) in group b. the mean value between 24 and 48 hours after surgery was 129.2 ml (sd, 112.3 ml) in group a and 112.7 ml (sd, 103.1 ml) in group b. at more than 48 hours after surgery, the total amount of drainage was measured as a mean of 65.0 ml (sd, 60.0 ml) in group a and a mean of 19.6 ml (sd, 44.3 ml) in group b. the total drainage amount was a mean of 942.4 ml (sd, 611.2 ml) in group a and a mean of 1,048.8 ml (sd, 455.9 ml) in group b. there was no statistically significant difference in the total drainage amount between the two groups (p>0.05) (table 2). in group b, after 6 hours of application of the autologous transfusion device, a mean of 625.2 ml (sd, 119.3 ml) autologous transfusion was done. and allogenic transfusion (packed red blood cells) was done if the patient still needed blood transfusion after autologous transfusion (table 3). the amount of allogeneic transfusion was a mean of 415.7 ml (sd, 471.1 ml) in group a and a mean of 200.0 ml (sd, 317.0 ml) in group b during 24 hours after operation ; the value was a mean of 157.5 ml (sd, 334.9 ml) in group a and a mean of 88.1 ml (sd, 228.8 ml) in group b between 24 and 48 hours after surgery. it was a mean of 97.6 ml (sd, 240.2 ml) in group a and a mean of 88.1 ml (sd, 240.4 ml) in group b at 48 hours after surgery. the total amount of allogeneic transfusion showed statistically significant difference between groups : the mean value was 670.9 ml (sd, 585.3 ml) in group a and 376.3 ml (sd, 456.8 ml) in group b, thus it was 294.6 ml less in group b than group a (p<0.05) (table 4). regarding major postoperative side effects, wound infection occurred in 3 knees in group a and in 2 knees in group b, which healed without re - operation and uncomplicated by superficial infection, showing no statistically significant difference between groups. there were 3 patients who were medicated for skin rash in group a and none in group b (table 5). other postoperative complications, such as pulmonary embolism and deep vein thrombosis, did not develop in both groups. this study demonstrated two major findings among numerous tka patients : first, using an autologous transfusion device can result in statistically significant reduction in the allogeneic blood transfusion volume compared to allogeneic transfusion alone ; and second, there was no significant difference in complication, such as infection or embolism, in spite of the loss of blood from the operation site for autologous transfusion. tka may necessitate postoperative transfusion according to the degree of injury to soft tissue or bone tissue that may occur during surgery 313). there are some methods for autologous transfusion ; however, we could not utilize a short - term preservation method which is performed after dilution of blood during surgery or the intraoperative blood collection method due to tourniquet application during surgery. preoperative blood deposit method was not chosen because of inappropriateness in elderly patients according to adalberth.14). therefore, recollection of blood in the operated joint by an autologous transfusion device and reinfusion via venous access method was chosen. there has been controversy regarding the efficacy of autologous transfusion in reducing the need for allogeneic transfusion. marks.15) reported that autologous transfusion through an autologous transfusion device failed to prove itself as an efficient method to decrease the amount of allogeneic transfusion. lee.16) insisted reinfusing lost blood during tka does not guarantee beneficial outcome. also, abuzakuk.4) reported that autologous transfusion can not replace allogeneic transfusion. ha.10), however, insisted autologous transfusion could decrease allogeneic transfusion based on a comparative study of 71 patients who underwent minimally invasive knee arthroplasty. simpson.11) implied that autologous transfusion via an autologous transfusion device can decrease the amount of allogeneic transfusion based on a prospective study of 24 tka patients. our study also confirmed that autologous transfusion after tka decreases the use of allogeneic transfusion. even though the total amount of drainage had no statistically significant difference between the groups, group b had significantly greater amount of drainage than group a within 24 hours after surgery. we attribute this to the greater negative pressure for the use of autologous transfusion device in group b ; we applied -100 mmhg pressure for the first 6 hours when using the autologous transfusion device. the total amount of drainage in group b was an average of 906.6 ml for 24 hours. the average amount of drainage was 625.2 ml for the first 6 hours, which was equal to the amount of autologous transfusion. considering that the amount of drainage for the rest 18 hours was not more than the half of the original volume, we think that bleeding was induced in early stage due to the greater negative pressure. there are a lot of arguments about the safety of reinfusion of collected blood. according to friedman.13), there were fewer infection events in groups that received autologous transfusion alone than those with allogeneic transfusion. there was no systemic infection in the group with autologous transfusion, whereas 2 infections were observed in the group with allogeneic transfusion in our study, which also supports the study results of friedman.13). furthermore, according to han.8), although there was decreased antithrombin iii in blood collected by an autologous transfusion device, no clinical symptoms including side effects caused by thrombosis were reported after reinfusion. nor were there reported side effects associated with thrombosis in this study. yim.3) suggested using two blood filters can reduce the possibility of thrombosis formation by bone tissue, cement fragments, or metal powder remaining at the operation site. the device we used in this study consisted of pre - filter and fat retention valve, so there were no side effects such as embolization or thrombosis after surgery. the major limitations of this study are that it is a retrospective design and transfusion was determined based on hemoglobin level alone. according to the transfusion guidelines proposed by american society of anesthesiologists, transfusion should be considered based on clinical symptoms such as hemoglobin level, blood oxygenation, and total oxygen delivery. however, in this study, hemoglobin level was the only criterion for determining transfusion ; other clinical conditions that can reflect the capacity of oxygen delivery, such as peripheral capillary oxygen saturation or dyspnea, were not taken into consideration. this may have been the cause of critical difference in the amount of transfusion with other studies where transfusion was based on physiological requirements. of the patients in whom the autologous transfusion device was used, allogeneic transfusion was not necessary for postoperative management in 24 patients. if clinical symptoms had been taken into consideration as criteria for transfusion, the amount of allogeneic transfusion could have been less, which could have enabled patient management through postoperative autotransfusion alone. furthermore, as three surgical instruments were randomly applied during surgery, it is difficult to rule out the influence of surgical instruments on the difference in postoperative blood loss. autologous transfusion using an autologous transfusion device following tka could reduce the amount of allogeneic transfusion, and there were no significant differences in the incidence of side effects between the autologous transfusion group and allogeneic transfusion group. without the risk of blood incompatibilities or unexpected events during medical treatment, autologous transfusion in tka can be a reliable alternative to reduce the amount of allogenic transfusion.	purposealthough allogeneic blood transfusion is the most common method of transfusion in total knee arthroplasty (tka), there are reports showing significant decrease in the amount of allogeneic transfusion and incidence of side effects after combined use of autologous transfusion. the purpose of this study is to investigate the efficacy of using an autologous transfusion device in tka.materials and methodspatients who underwent tka at our institution from january 2003 to january 2014 were divided into two groups : group a (n=127) who received allogeneic transfusion only in tka and group b (n=118) who received autologous transfusion via an autologous transfusion device and allogeneic transfusion. in both groups, the patients were transfused when the hemoglobin level was below 9 g / dl. in group b, blood collected by the autologous transfusion device was transfused only once after surgery. the total blood loss volume, total transfusion volume, and the presence of side effects were assessed based on medical records.resultsgroup a received 294.6 ml more allogeneic transfusion than group b (p<0.001). there were no significant differences with regard to the development of side effects between groups.conclusionsapplication of an autologous transfusion device during tka can be effective in reducing the allogeneic transfusion volume. moreover, allogeneic transfusion was not necessary after autologous transfusion in some patients.
if there were ever a competition for the most simply designed, yet most versatile biological molecule in nature, hyaluronan (ha) would be a strong contender. built of simple disaccharide sequences (d - glucuronic acid and d - n - acetylglucosamine, bound through alternating -1,4 and -1,3 glycosidic bonds), and with no known postsynthetic modification, ha was long mistaken for an inert filler of the extracellular space. in the past decades, however, it has become evident that ha possesses manifold signaling properties. a fascinating, if perhaps vexing, insight has also been that ha will often demonstrate opposing actions : it can have pro- or anti - inflammatory properties, promote or inhibit cell migration, activate, or stop cell division and differentiation. it appears that three main factors dictate the effect of ha : one is cell - specific (receptor expression, signaling pathways, and cell cycle) ; two are ha - related (size and location). this review will focus on ha sizes and their effect on ha signaling and biological effects.. why would a larger or smaller molecule of identical, monotonous molecular structure have different effects ? presumably ha receptors need a minimal molecular size to engage the ligand, but further size increase should have no effect on receptor recognition. the first concept suggests that ha size may influence affinity to receptors ; also, receptor complexes may cluster differently depending on ha size. the second concept, less well understood, is that size may affect ha uptake by the cell, and ha intracellular signaling may also modulate biological responses. an additional impediment on the elucidation of size - dependent ha signaling and biological effects is the confusing language that is used in scientific publications. while everyone seems to agree on designating ha over 1 million da high molecular weight, the nomenclature of smaller - size ha is nebulous. different papers use interchangeably terms such as short - fragment ha, low molecular weight ha, and ha oligosaccharides to describe ha molecules from a few disaccharides up to over 700 kda. for this review we are using the following nomenclature, for expediency 's sake : ha of approximately 20 monosaccharide length or less (the minimum that differentiates between monovalent and divalent interactions with cd44) are ha oligosaccharides (oha) ; ha of over 1 million da (the minimum that is thought to be native ha) is high molecular weight (hmw - ha) ; everything in between is low molecular weight (lmw - ha). for scientific papers it may be better to simply define the size or range of sizes the investigators are working with, until a clear mechanistic understanding of fragment size classification emerges. in the following we will provide brief overviews of the mechanisms of ha synthesis and degradation, which lead to the generation of different fragment sizes ; the current state of knowledge on ha size - dependent signaling ; and a conclusive discussion of implications and future directions. ha is uniquely synthesized at the plasma membrane rather than in the golgi apparatus as is typical of other glycosaminoglycans (gags). synthesis of mammalian ha is accomplished by a family of membrane - bound glycosyltransferases composed of three isozymes, hyaluronan synthases (has) 1, 2, and 3. has enzymes are evolutionarily conserved and are highly homologous (5570% protein identity) [4, 5 ], and they catalyze the addition of udp - d - glucuronic acid (glca) and udp - n - acetyl - d - glucosamine (glcnac) monomers in an alternating assembly to form ha polymers [6, 7 ]. although the three has isoforms are similar and synthesize an identical product, they exhibit differences in half - life and stability, the rate of ha synthesis, and affinity for ha substrates, all of which potentially affect the regulation of ha synthesis and biological function. of particular interest is the finding that the three has enzymes synthesized ha of varying molecular masses. in general, has3 synthesized the shortest ha polymer sizes (1 10 to 1 10 da), while has1 and has2 synthesized larger polymers (2 10 to 2 10 da), although the major population of ha produced by has2 tended to be concentrated on the longer end of the spectrum (> 2 10 da) compared to has1, which generated a wider range of ha polymers. because of the biological differences exhibited by ha of differing polymer lengths, the innate biochemical and synthetic capabilities of the has enzymes may serve an important regulatory role in development, injury, and disease. the has genes exhibit different temporal patterns of expression during morphogenesis. has2 is expressed throughout all stages of embryogenesis and is considered to be the major hyaluronan synthase during development. determined that, of the three has isoenzymes, only has2 was indispensable, with embryonic lethality due to severe cardiac and vascular defects at midgestation in has2 mice. has1 and has3 expressions, on the other hand, are restricted to early and late stages of development, respectively, although expression overlaps with has2. has1 and 3 deficient mice as well as has1 and 3 double knockouts are both viable and fertile. at the tissue level, has gene expression and subsequent ha synthesis is regulated by a wide range of cytokines and growth factors (reviewed in [2, 11 ]). all three has genes may respond similarly to a particular signal, or there may be a differential response, as demonstrated by tgf-1-mediated downregulation of has3 but upregulation of has1 expression, seen in a dose - dependent manner in human fibroblast - like synoviocytes. dysregulation of has gene expression plays important roles in disease and injury, consistent with the biological roles of ha in disease progression, wound healing, and tissue regeneration. in cancer, overexpression of hyaluronan synthases influences tumor growth, metastatic potential, and progression in several malignancies, including prostate, colon, breast, and endometrial cancers (reviewed in). ectopic expression of has genes may also functionally alter the biological responses of cells to injury in vivo [14, 15 ] (reviewed more extensively below). taken together, available studies suggest that ha synthases are critical mediators in development, injury, and disease. the mechanism of ha removal or turnover is facilitated in part by hyaluronidases (hyals), which, in mammals, consists of a family of enzymes including hyaluronidases 14 (hyal14), ph20, and hyalp1 [16, 17 ]. hyals are also found in lower organisms, like bacteria, which catabolize ha to generate primarily disaccharides and in part facilitate mobility within tissue, and in leeches and crustaceans, which produce predominately tetra- and hexasaccharide fragments. hyals as a class are highly homologous endoglycosidases, and in terms of ha catabolism, they specifically hydrolyze the -1,4 linkage of the ha molecule, which is a linear polysaccharide composed of repeating -1,4-linked d - glucuronic acid (glca) and -1,3-linked n - acetyl - d - glucosamine (glcnac) disaccharide units [1719 ]. the range of activity of mammalian hyals is not strictly limited to ha as they can also degrade chondroitin sulfates, although prokaryotic hyals specifically act on ha [17, 20 ]. hyals can be further broken down into distinct groups ; for example, hyal1hyal3 are primarily active at an acidic ph, while ph20 has optimum activity at a neutral ph [16, 21 ]. of the 6 hyal family members, hyal1 and hyal2 are the predominant isoforms functioning to catabolize ha in somatic tissues. triggs - raine. detailed a broad mrna expression pattern for hyal2 (heart, skeletal muscle, colon, spleen, kidney, liver, placenta, and lung), while hyal1 is more limited in scope but is high in liver (which is a primary location of ha degradation) and is less strongly expressed in heart, spleen, kidney, and lung ; the hyal1 protein is also found in plasma and urine. hyal3 is even more limited in its expression pattern, with low levels in brain, liver, testis, and bone marrow. finally, hyal4 is a chondroitinase with no evidence of ha catabolic activity, with expression in the placenta and skeletal muscle, and hyalp1 is an expressed pseudogene [16, 17 ]. ha degradation into smaller fragments is accomplished by hyal1 and hyal2 acting in concert to catabolize ha into tetrasaccharides. hmw - ha is anchored to the cell surface through cd44 and hyal2 and localized to lipid rafts in the cell membrane. the acidic environment necessary for hyal2 activity is provided by nhe2, facilitating the generation of ha polymers of approximately 20 kda (or 50 disaccharide units). in this model, 20 kda fragments are internalized and transported first to endosome and then to lysosomes, where lysosomal hyal1 further degrades the ha into tetrasaccharide units (reviewed in). experimental details and confirmation of this model the significance of hyal - mediated degradation of ha is demonstrated in mucopolysaccharide hyaluronidase deficiency, first described by. this disorder is characterized by elevated ha levels in the plasma (> 3890-fold increase over normal plasma levels) concomitant with lack of hyaluronidase activity. this lysosomal storage disorder is now termed mucopolysaccharidosis ix, and subsequent characterization revealed that hyaluronidase activity is specifically abrogated through mutations in hyal1. the disease has a relatively mild phenotype, limited to specific cell types (fibroblasts and histiocytes) and characterized by accumulation of ha, short stature, and multiple soft tissue masses in the joints. a further demonstration of how hyals contribute to developmental processes was shown with a mouse model of hyal2 deficiency, which, similar to mucopolysaccharidosis ix in humans, was characterized by increased plasma concentrations of ha, and a relatively mild phenotype, in this case with mild craniofacial and hematological defects. interactions with other genetic loci are suspected, as hyal2 deficiency in an outbred mouse resulted in much more severe cardiopulmonary pathology and early mortality compared to hyal2 deficiency in an inbred genetic background. increased hyal levels have also been found in several carcinomas, including prostate and bladder, as well as breast and head and neck cancer, and tend to correlate with more invasive and metastatic phenotypes (reviewed in). cumulatively, this suggests that hyals have distinctive roles in developmental and disease processes through the regulation of ha metabolism. although many diverse biological responses have been attributed to ha and its various size polymers, interpretation of experimental findings, both in vivo and in vitro, may be complicated by, for example, low levels of bacterial contamination, which may independently activate key ha receptors. developed mouse lines overexpressing hyal1, in an attempt to generate ha fragments in vivo independently of other mitigating factors. using models of constitutive overexpression of hyal1 in mouse skin (k14/hyal1) and tamoxifen - inducible expression localized to the epidermis (k14creert / hyal1), they determined that ha catabolism in the absence of injury in vivo initiated dendritic cell (dc) migration and maturation, which in turn muted the response to contact hypersensitization (chs). interestingly, hyal1-mediated catabolism of hmw - ha into size ranging between 0.5 and 27 kda (tetrasaccharides to 68 mer disaccharides) did not result in any phenotypic or inflammatory changes, so in the absence of any specific injury or challenge, catabolism of ha to smaller polymers by hyal1 did not alone induce an immune response in these models. these results were recapitulated by injection of tetrasaccharide oha into the skin of wild type mice, resulting in increased migration of dcs out of the skin and functionally, a diminished chs response. finally, they also showed that, in this context, hyal1 or oha function is dependent upon tlr4. while hyal1 overexpression or oha injection in a tlr4 background resulted in ha fragmentation, there was no change in cutaneous dc levels in either case. this effect was not seen with hyal1 on a cd44 deficient background, demonstrating specificity for tlr4 in this process. hyal1 is only active at a low ph, which is unlikely to have been present in the uninflamed skin of these mice. therefore, the exact mechanism of hyal1 activity in this case and in inflammation generally is still far from completely understood. however, it should be noted that much is unknown about hyaluronidase activity and function in vivo, and it is possible that posttranslational processing or other factors (association with proteins or salts) substantially change activity and specificity from what is found in vitro [29, 30 ]. aside from the specific enzymatic degradation pathways described above, hmw - ha can be fragmented by nonspecific pathways as well. reactive oxygen species (ros), including superoxide, hydrogen peroxide, nitric oxide and peroxynitrite, and hypohalous acids (reviewed in), are generated during the inflammatory response in sepsis, tissue inflammation, and ischemia - reperfusion injury and can degrade ha. the most direct evidence for this has been accumulated in the synovial fluid, where inflammatory oxidation leads to degradation of native hmw - ha with resulting decrease in synovial fluid viscosity and cartilage degeneration, and in the airways, where ros can degrade luminal epithelial ha. it should also be mentioned that nonenzymatically produced, ambient ros can also generate hmw - ha breakdown products. this may be most relevant in environmental lung injury ; for example, inhaled ozone and chlorine gas can generate lmw - ha from hmw - ha in vitro. no matter the origin, it seems clear that excessive generation of ros contributes to a proinflammatory status by the oxidative degradation of hyaluronan. the corollary to this is that neutralization of ros, for example, through superoxide dismutase, decreases hmw - ha degradation and inflammation [3335 ]. finally, the possibility may be entertained that ros scavenging is in fact one of the physiological functions of hmw - ha as was proposed recently ; however, no experimental support for this hypothesis exists at this point. beyond this hypothesis, it should also be noted that degradation of hmw - ha by ros may also have salutary effects, such as the promotion of ciliary beating in the airways. thus, ros may engage hyaluronan in a fine - tuned interaction rather than a monolithic response, and in fact hyaluronan may be involved in the emerging signaling pathway for these molecules [32, 38 ]. as part of the extracellular matrix (ecm), ha plays an important role in the maintenance of appropriate cell - cell communication. when ecm homeostasis is disrupted during pathological conditions (tumor invasion, inflammation, tissue remodeling, etc.) endogenous hmw - ha can be degraded by hyaluronidases and reactive oxygen species into lmw - ha, which can be further depolymerized to oha. ha and its degradation products bind several cell surface receptors such as cd44, rhamm, hare, lyve1, layilin, tlr2, and tlr4 [15, 4049 ], where the size of ha can have a significant influence on receptor activation and its downstream signaling. one theory proposes that signal transduction by ha is dependent on multivalent and cooperative interactions and/or the ability of ha to cluster the receptors on the membrane [15, 50 ]. for example the interaction of cd44 and ha is strongly influenced by cell - specific factors, cell type, state of activation, and ha size. different sizes of ha have distinct effects on cd44 clustering : hmw - ha increases clustering strength while oha has no apparent effect. however, sequential addition of oha following hmw - ha reduces the clustering strength induced by hmw - ha. long chains of ha possess multivalent sites for cd44 binding while oha have only 1 or 2 binding sites [1, 52 ] suggesting that oha binding can act as an antagonist by replacing these interactions with low affinity, low valency interactions. ha of different sizes can also signal through toll - like receptors (tlrs), either independently or in concert with other ha receptors. lmw - ha has been shown to engage in a unique receptor complex of cd44 and tlrs to induce cytokine release and airway hyperresponsiveness (ahr) [54, 55 ] in macrophages and nave mice, respectively. however, in a model of bleomycin - induced acute lung injury lmw - ha required both tlr2 and tlr4, along with myd88, to stimulate chemokine expression, which was independent of cd44. hmw - ha was protective in this model and also required tlr2, tlr4, and myd88. interestingly, receptor binding and activation by oha can even differ depending on the number of disaccharides present. for example, 4-mer oha interacts with tlr2 and tlr4, but not cd44, while 6-mer oha can bind to tlr2, tlr4, and cd44 [41, 5759 ]. additionally, 6- to 18-mer oha bind monovalently to cd44, whereas larger polymers bind multivalently, which can affect clustering and signaling of this receptor. furthermore, < 6-mer oha have low affinity for tsg-6, which is required for ha transfer to the inter - alpha - trypsin inhibitor (ii) heavy chain and optimal signaling. conversely, 8- to 21-mer oha induce an irreversible transfer of tsg-6 to the ha moiety and thus can compete with ha signaling by removing tsg-6 from the binding pool. lmw - ha induces activation and maturation of dendritic cells via tlr4, independently of rhamm, cd44, and tlr2, to induce phosphorylation of p38/p42/44 mapk and nf-b. conversely, lmw - ha stimulates macrophages independently of cd44 and tlr4 via the tlr2/myd88 pathway, and hmw - ha can act as a competitive inhibitor of this response. oligosaccharide ha can activate tlr4 and cd44 pathways in chondrocytes [57, 64, 65 ], while in synovial fibroblasts it activates tlr2 and tlr4, but not cd44, and in vascular endothelial cells it activates rhamm. in aggregate, available evidence suggests that ha size influences receptor complex formation in a size - specific manner and thus modifies downstream signaling cascades. ha is normally produced by synthases which reside at the cell membrane and is immediately extruded to the extracellular space without need for shuttling or exocytosis. however, ha has also been detected intracellularly, where it associates with the mitotic spindle, microtubules, and the receptor rhamm [68, 69 ]. two possible mechanisms by which ha may be localized intracellularly and potentially to contribute to signaling activity have been identified : intracellular (physiological or aberrant) production of ha and uptake of extracellular ha. ha synthesis can deviate from the normal pattern, especially in malignancy and cell injury. various hematologic malignancies such as monoclonal gammopathy of undetermined significance, multiple myeloma and waldenstrm 's macroglobulinemia as well as solid cancers such as bladder cancer feature cells with aberrant splice variants of has1, which are associated with cancer diagnosis, relapse and poor outcome (reviewed in). accumulating evidence has led to the hypothesis that has1 in these cancer cells may produce intracellular ha which competes with the mitotic apparatus for rhamm binding and thus protect the cells from rhamm - mediated mitotic arrest. in conditions of endoplasmic reticulum (er) stress, injured cells also produce ha cables that appear to emanate from a perinuclear region and protrude through the cytoplasm into the extracellular space. this intracellular ha directly communicates with the extracellular space and allows inflammatory cells to congregate into the inflammatory site. ha of higher molecular weight can be digested by a variety of enzymatic and nonenzymatic [18, 29, 32, 72, 73 ] pathways to 50100 long saccharide polymers and then be taken up by the cell either through receptor binding (cd44, rhamm, lyve-1, hare) [74, 75 ] or through pinocytosis. much (or most) of the endocytosed ha is localized to the endosome and lysosome, where it is digested to oligosaccharides by hyaluronidases 1 and 2, and probably further by -d - glucuronidase and -n - acetyl - d - hexosaminidase. the activities of at least hyal1 and the extracellular -hexosaminidase appear to be partly redundant. they could be recycled for the generation of glycosaminoglycans, they could be exocytosed and engage extracellular ha receptors, or they could also engage intracellular ha receptors. the presence of ha receptors (notably rhamm) in the cytoplasm strongly suggests that intracellular ha can have signaling activity. have shown that intracellular ha, rhamm, and microtubules colocalize in the cytoplasm and the nucleus and may affect the mitotic apparatus and directly or indirectly modulate rhamm - mediated signaling. although the specific mechanisms involved in the diverse signaling of ha are still poorly understood, it is known that ha can modulate many biological effects including cell adhesion, cell migration, morphogenesis, tumorigenesis, cell survival, apoptosis, and inflammation and that these biological effects can differ depending on ha size. interestingly, depending on cellular localization, endogenous hmw - ha can either protect from epithelial apoptosis in lung injury or promote an invasive fibroblast phenotype and the fibrotic process. treatment with hmw - ha can also inhibit inflammatory response in several disease models, such as in hmw - ha attenuated inflammation and lung injury in a sepsis model of ventilated rats, as well as ozone - induced ahr [54, 55, 80, 81 ]. based on recent data, hmw - ha is emerging as a viable therapeutic option in inflammatory airway disease, due to its anti - inflammatory and antiproliferative properties. lingering concerns however will have to be addressed, such as the potential of degradation of hmw - ha into lmw - ha and thus exacerbation of the inflammatory process. it is worth noting that inhaled hmw - ha has been used for several years in europe, with a remarkably good safety profile [8285 ]. certain environmental exposures and disease states have been shown to lead to breaking - down of hmw - ha into fragments (lmw - ha) that can stimulate expression of proinflammatory cytokines, chemokines, and growth factors and increase ahr. increased levels of lmw - ha have been found in many lung disorders including asthma, pulmonary fibrosis, copd, allergic alveolitis, interstitial lung disease, sarcoidosis, and pulmonary hypertension (reviewed in) and another article in this edition (lauer.), as well as other inflammatory diseases like rheumatoid arthritis. lmw - ha can also induce angiogenesis and tumor progression [50, 89 ]. in aggregate, the effect of lmw - ha in tissue injury seems to be proinflammatory and rather deleterious, and attempts to block lmw - ha signaling in disease may constitute novel treatments. interestingly, oha of different sizes (416 mers) have been shown to both stimulate and inhibit inflammatory responses depending on cell type and disease model. ha oligosaccharides have been shown to increase angiogenesis during wound healing, stimulate proinflammatory effects in synovial fibroblasts, and promote cell adhesion. conversely, there are many studies that show oha to have beneficial effects such as reducing poly(i : c)/tlr3-induced inflammation, modulating the onset and course of inflammatory demyelinating disease by interfering with lymphocyte - vascular endothelial cell slow rolling, inhibiting ha - cd44 activation of kinases, and causing disassembly of large signaling complexes [92, 93 ], as well as retarding the growth of several tumor types and sensitizing resistant cancer cells to various drugs. finally, it is worth mentioning that, unlike some experimental conditions, cells and tissues in situ are exposed to a variety of ha sizes at once. it is possible that the effects of exposure to a ha size mix may be more than the sum of its separate size effects, as has been demonstrated experimentally (de la motte c, personal communication). much is still unknown about the biological effects of ha in tissue homeostasis and the response to injury, and crucial insights into these effects will be gained by understanding the size dependence of ha signaling. mechanistically, we need to understand how ha size may affect receptor clustering and affinity to receptors (especially receptor variant forms) ; whether there is a relation between ha size and the localization of ha (intra- versus extracellular, as well as cellular compartments) ; how simultaneous engagement of different ha sizes may modulate signaling ; and the role of intracellular ha in cell behavior. undoubtedly, we can look forward to exciting developments in the field of ha biology which will help fuel translational applications and medical advances.	hyaluronan signaling properties are unique among other biologically active molecules, that they are apparently not influenced by postsynthetic molecular modification, but by hyaluronan fragment size. this review summarizes the current knowledge about the generation of hyaluronan fragments of different size and size - dependent differences in hyaluronan signaling as well as their downstream biological effects.
total knee arthroplasty (tka) involves significant blood loss that may require a blood transfusion, which carries its own risks including transmission of infection, abnormal immunological responses, febrile transfusion reactions, and allergic reactions1,2). accordingly, continuous efforts have been made to avoid complications associated with allogeneic blood transfusion, which has led to the invention of blood saving strategies including preoperative autologous donation3 - 5), treatment of preoperative anemia using oral iron therapy, subcutaneous drainage6), drain clamping7), and hypotensive anesthesia8). lower extremity alignment is one of the paramount factors determining the long - term success of tka9 - 11). a satisfactory alignment can be obtained with the use of a intramedullary or extramedullary alignment guide system. in particular, extramedullary guides are preferred for the tibial side and intramedullary guides are preferred for the femoral side. unfortunately, the use of an intramedullary guide for the femur has been associated with increased risks of fat embolism, blood loss, postoperative hypoxia, and intraoperative fractures12 - 14). despite the clinical advantages offered by the extramedullary femoral alignment system, it has been employed in limited circumstances to obtain optimal postoperative alignment and avoid technical challenges. however, tkas using extramedullary femoral alignment guides have been extensively studied in recent years. kalairajah.15) reported the results of navigation - assisted tkas and seo.16) introduced a new extramedullary guide that aligns the femoral component exactly with the coronal, sagittal, and rotational axes. the purpose of this study was to investigate whether the extramedullary femoral alignment system is advantageous over the traditional intramedullary system in terms of blood loss and transfusion after tka. our hypothesis is that the extramedullary system will result in less blood loss and fewer transfusions. this study included 80 female patients who underwent unilateral tka under the diagnosis of degenerative arthritis between october 2010 and september 2011 at our institution. exclusion criteria were a diagnosis other than degenerative arthritis, anticoagulation therapy, liver failure, and renal failure. the enrolled patients were randomized into a extramedullary (em) group (n=40) and a intramedullary (i m) group (n=40). excluding one patient in the em group in whom internal fixation was required due to an intraoperative fracture, the remaining 79 patients were included for analysis. no intergroup differences were observed in age, weight, height, body mass index, or preoperative hemoglobin concentration (table 1). the surgical protocol was the same for both groups, besides the type of the alignment guide for the distal femoral resection (extramedullary / intramedullary). in particular, all patients received epidural and spinal anesthesia and the preoperative and postoperative procedures and surgical techniques were identical. patient - controlled analgesia was implemented postoperatively using a 100 ml mixture of 90 ml of 0.125% bupivacaine and 0.5 mg/10 ml fentanyl via an epidural catheter. all operations were performed by the same surgeon (first author) using a medial parapatellar approach introduced by insall. the posterior cruciate ligament (pcl) was removed and the same pcl - sacrificing prosthesis (scorpio nrg, stryker, kalamazoo, mi, usa) was implanted in all knees. two markers indicating the center of the femoral head and the anterior margin of the greater trochanter were firmly attached to the patient 's skin in the em group prior to surgery so the surgeon could palpate the markers and identify the mechanical axis of the lower limb during surgery. a femoral cutting block was aligned along the coronal and axial axes using new surgical equipment16) and the two markers. the distal femur was cut without placing a rod into the medullary canal (fig. the angle between the anatomical axis and the mechanical axis of the femur in the i m group was measured using a pacs system on a preoperative anterioposterior long leg standing radiograph. an intramedullary alignment rod was inserted based on the preoperative measurements, and the distal femur was cut. the entrance hole for the alignment rod in the femoral medullary canal was sealed with bone fragments before inserting the implant. after wound closure, a compression dressing using gauze and a bandage was applied, and the tourniquet was deflated. blood transfusion was performed when hemoglobin was < 8 g / dl or when dyspnea, palpitation, dizziness, or abnormal vital signs were noted even if the hemoglobin was 8 - 10 g / dl. rivaroxaban, an oral anticoagulant, was administered from immediately after the drain removal until the postoperative week 2 to prevent thromboembolic disorders. the anticoagulant was discontinued if hemarthrosis due to intra - articular bleeding was present or effusion from the surgical wound continued. the continuous variables, drained blood volume and preoperative hemoglobin level, were analyzed using student 's t - test. the hemoglobin level at 5 days after surgery and the hemoglobin drop (hemoglobin level at 5 days after surgery - hemoglobin level before surgery) were compared between groups using analysis of covariance. the prevalence of allogeneic transfusion, a categorical variable, was compared between the groups using fisher 's exact test. additionally, the presence of other related symptoms were also investigated, including the duration of effusion, subcutaneous hematomas, hemarthrosis, hemorrhagic complications such as infections, painful leg swelling, acute chest pain, dyspnea, lower limb thrombosis, and pulmonary embolism. the mean drained volume via vacuum drainage was significantly less in the em group compared to that in the i m group (266.8164.1 ml vs. 482.9323.3 ml) (p=0.001). the mean hemoglobin at 5 days after surgery was significantly higher in the em group compared to that in the i m group (9.9 g / dl vs. 9.3 g / dl) (p=0.002). the hemoglobin drop at 5 days after surgery was notably lower in the em group compared to that in the i m group (2.9 g / dl vs. 3.5 g / dl) (p=0.003). the prevalence of allogeneic transfusion was remarkably lower in the em group compared to that in the i m group (8 in 39 patients, 20.5% vs. 18 in 40 patients, 45%) (p=0.026) (table 2). the transfusion was prophylactic in five in the i m group and in two in the em group. a transfusion was required in one patient with a hemoglobin level 8 g / dl in each group due to complaints of dizziness. anticoagulation therapy was discontinued due to continuous effusion from the surgical wound in two patients in the i m group, but they recovered without further complications after discontinuation. tka is commonly performed in elderly patients and substantial blood loss during tka can result in increased risks and disruption of recovery in patients with poor systemic condition17,18). accordingly, a blood transfusion is often required after tka. in an attempt to reduce the adverse effects of blood transfusion, many studies have provided various methods, including preoperative autologous donation3 - 5), use of the call saver19) and fibrinogen concentrates20), subcutaneous suction drainage6), drain clamping7), hypotensive anesthesia8), and other methods17 - 21). in this study, the em group had more favorable results than those in the i m group in terms of blood savings. the mean drained volume via the vacuum drainage was smaller in the em group, hemoglobin at 5 days after surgery was higher, the hemoglobin drop was smaller, and the prevalence of allogeneic transfusion was lower. these results are congruent with the hypothesis that the extramedullary alignment guide system reduces blood loss and transfusion rate. in this study, we measured blood volume drained via vacuum drainage and the hemoglobin but did not calculate the blood loss based on some reports22,23) in which the measured blood loss reflects calculated blood loss. the extramedullary alignment guide system and intramedullary alignment guide system have been compared in terms of blood loss in many studies. in 2005, kalairajah.15) reported that mean blood drainage volume was significantly smaller in the group in which a computer - assisted technique was used compared to that in the control group (1,351 ml vs. 1,747 ml). in 2006, kandel.14) documented that mean blood drainage volume was significantly lower in a extramedullary guide system group compared to that in a intramedullary guide system group (613 ml vs. 758 ml), and transfusion volume appeared to be less in the former group. our results were comparable with those of other studies14,15,24,25) that showed blood loss could be reduced by 145 - 396 ml when the medullary canal was not breached during tka. although the frequency of transfusion was lower in the em group in our study, it was still higher compared to some recent studies4,6). we attributed this to the broad indications for transfusion we set in this study ; a blood transfusion was performed when hemoglobin was < 8 g / dl or when dyspnea, palpitation, dizziness, or abnormal vital signs were noted, even if the hemoglobin was 8 - 10 g / dl. in addition, prophylactic transfusions that were carried out considering the patient 's history of illness were counted in the transfusion rate calculation. however, when these prophylactic cases (5 in the i m group and 2 in the em group) were not counted as transfusions, the frequency decreased to 37.1% (13 in 35 patients) in the i m group and 16.2% (6 in 37 patients) in the em group. furthermore, if a higher transfusion threshold of 7 g / dl hemoglobin was set, the frequency would have declined to 27.5% (11 in 40 patients) in the i m group and to 5.1% (2 in 39 patients) in the em group. we think that it would be difficult to extrapolate our findings to other patient groups, considering that our data were obtained only from female patients with a diagnosis of degenerative arthritis. additionally, we did not assess intergroup differences regarding fat embolisms or postoperative complications, including hypoxia, pain, or lower limb alignment accuracy. the intramedullary femoral alignment guide system can result in various complications and may not be applicable when a long - stemmed femoral component implanted during a previous surgery remains or a rod can not be inserted due to severe deformity of the femur26). it has not been clearly elucidated which system is more advantageous over the other regarding component alignment accuracy. however, some recent studies have introduced techniques to improve the accuracy of the extramedullary alignment guide system16,27 - 30). therefore, we believe that the extramedullary alignment guide system could be useful for decreasing blood loss and transfusion frequency during tka unless it is inferior to the intramedullary system in terms of component alignment accuracy. we believe the extramedullary alignment guide system can be effective for reducing blood loss and transfusion rate after tka.	purposeto compare the extramedullary femoral alignment guide system with the conventional intramedullary alignment guide system for bleeding and transfusion rate after total knee arthroplasty (tka).materials and methodsforty - nine female tka patients were randomized into two groups : intramedullary (i m) group vs. extramedullary (em) group. drained volume of blood, hemoglobin concentration, hemoglobin drop, and transfusion rate were compared between the two groups. wound problems, bleeding - related problems and thromboembolic complications were collected.resultsthe mean drained volume via vacuum drainage was less in the em group than that in the i m group (482.9 ml vs. 266.8 ml, p=0.001). hemoglobin at 5 days after surgery was higher in the em group (9.3 g / dl vs. 9.9 g / dl, p=0.002) than that in the i m group. the drop in hemoglobin after 5 days was smaller in the em group (3.5 g / dl vs. 2.9 g / dl, p=0.003) than that in the i m group. the em group had a lower prevalence of allogeneic transfusion (45.0% vs. 20.5%, p=0.026) than that in the i m group. no significant complications developed in either group.conclusionsthe results suggest that the extramedullary femoral alignment guide technique is an advantageous method that can reduce the drained volume of blood and the allogeneic transfusion rate.
the irma-2 study was a 2-year multicenter, randomized, double - blind trial in patients with type 2 diabetes and microalbuminuria comparing irbesartan (150 or 300 mg once daily) versus placebo on top of conventional antihypertensive treatment. the design of the study has been reported elsewhere (9). in brief, eligible patients had their antihypertensive agents discontinued during the run - in period and replaced by placebo. after 3 weeks, patients were randomly assigned to receive irbesartan 150, 300 mg, or matching placebo once daily. a total of 590 patients were followed for 2 years for the development of overt nephropathy. additional bp lowering medication, apart from ace inhibitors and angiotensin ii receptor blockers (arbs), was allowed to reach the target bp of 135/85 mmhg. the study protocol was in accordance with the declaration of helsinki and was approved by all local institutional review boards. irma-2 enrolled hypertensive patients with type 2 diabetes, ranging in age from 30 to 70 years. all patients had persistent microalbuminuria, which was defined as a uae rate of 20 to 200 g / min in at least two out of three consecutive, sterile, overnight urine samples. the main exclusion criteria were a serum creatinine concentration > 1.5 mg / dl (133 mol per liter) for men and > 1.1 mg / dl (97 mol per liter) for women, nondiabetic kidney disease, cancer, life - threatening disease with death expected to occur within 2 years, and an indication for ace inhibitors or arbs. the urinary albumin concentration was determined by nephelometry at a central laboratory (10). the serum creatinine concentration was determined by jaffe reaction with the use of a hoffmann laroche kit (11). estimated glomerular filtration rate (egfr) was estimated with the modification of diet in renal disease study equation (12). the lowest arterial bp during a 24-h period (korotkoff phase i / v) was measured twice in the sitting position after at least 10 min of rest. this post hoc analysis focuses on the uae and sbp change from baseline to month 6. a robust decline in uae or sbp was defined as a decline in uae or sbp more than the population median and was calculated as 100log(uae at 6 months / uae at baseline). sbp change was calculated as the difference between the month 6 and baseline value. uae change at month 6 for each patient. the month 6 values were chosen for two reasons : 1) the treatment effects were considered to be fully present at month 6, and 2) this was the earliest time point at which most variables of interest were available. patients were divided into groups according to the median of uae and sbp change from baseline to month 6. patients with both uae change and sbp change above or below the median were considered to have a concordant response, whereas patients with either a uae change or sbp change above the median were considered to have a discordant response. transition from micro- to macroalbuminuria (development of overt nephropathy) was the primary efficacy measure in the irma-2 trial, which was defined as uae > 200 g / min and at least 30% higher than baseline level of uae on at least two out of three consecutive samples. because the initial reduction in albuminuria induced by arb treatment is directly related to the primary efficacy measure (which includes the long - term change in albuminuria), we decided to use the course of decline in egfr from month 6 to end of follow - up as our primary renal end point. variables with normal distribution are presented as mean with sd, and variables with a skewed distribution are presented as median with interquartile range. graphical methods and normality tests were used to ascertain normalization of the distribution after transformation. differences between groups were tested with fisher exact test for categorical variables and anova for continuous variables, followed, where applicable, by post hoc bonferroni correction for multiple testing. a multivariate mixed model with random intercepts and random slopes was used to assess the relationship between the magnitude of sbp and uae change and the rate of egfr decline. such a model calculates renal function decline over time within and between individuals also taking into account the correlation within individuals and time. for exploration of the relationship between the month 6 change in uae and egfr decline, the change in uae was categorized according to quartiles and related to egfr decline from month 6. the multivariate mixed model included the following baseline covariates : age, sex, log - transformed uae, sbp and diastolic bp, egfr, hba1c, duration of diabetes, total cholesterol, smoking, bmi, and treatment allocation. a multivariate cox proportional hazards model was used to assess the relationship between the magnitude of uae and sbp change from baseline to 6 months and time to development of overt nephropathy from 6 months to the end of follow - up. the multivariate cox proportional hazards model included the same covariates as the above - mentioned multivariate mixed model. the initial fall in egfr after start of treatment may reflect a hemodynamic response and may be associated with long - term renoprotection (13,14). because the month 6 change in egfr was not associated with the initial change in uae and sbp, we considered the initial egfr change not a potential confounder in our analyses. relative risk reductions are described in the text as percent reductions ([1hazard ratio ] 100)., chicago, il) and sas (sas institute, cary, nc). irma-2 enrolled hypertensive patients with type 2 diabetes, ranging in age from 30 to 70 years. all patients had persistent microalbuminuria, which was defined as a uae rate of 20 to 200 g / min in at least two out of three consecutive, sterile, overnight urine samples. the main exclusion criteria were a serum creatinine concentration > 1.5 mg / dl (133 mol per liter) for men and > 1.1 mg / dl (97 mol per liter) for women, nondiabetic kidney disease, cancer, life - threatening disease with death expected to occur within 2 years, and an indication for ace inhibitors or arbs. the urinary albumin concentration was determined by nephelometry at a central laboratory (10). the serum creatinine concentration was determined by jaffe reaction with the use of a hoffmann laroche kit (11). estimated glomerular filtration rate (egfr) was estimated with the modification of diet in renal disease study equation (12). the lowest arterial bp during a 24-h period (korotkoff phase i / v) was measured twice in the sitting position after at least 10 min of rest. this post hoc analysis focuses on the uae and sbp change from baseline to month 6. a robust decline in uae or sbp was defined as a decline in uae or sbp more than the population median and was calculated as 100log(uae at 6 months / uae at baseline). the month 6 values were chosen for two reasons : 1) the treatment effects were considered to be fully present at month 6, and 2) this was the earliest time point at which most variables of interest were available. patients were divided into groups according to the median of uae and sbp change from baseline to month 6. patients with both uae change and sbp change above or below the median were considered to have a concordant response, whereas patients with either a uae change or sbp change above the median were considered to have a discordant response. transition from micro- to macroalbuminuria (development of overt nephropathy) was the primary efficacy measure in the irma-2 trial, which was defined as uae > 200 g / min and at least 30% higher than baseline level of uae on at least two out of three consecutive samples. because the initial reduction in albuminuria induced by arb treatment is directly related to the primary efficacy measure (which includes the long - term change in albuminuria), we decided to use the course of decline in egfr from month 6 to end of follow - up as our primary renal end point. variables with normal distribution are presented as mean with sd, and variables with a skewed distribution are presented as median with interquartile range. graphical methods and normality tests were used to ascertain normalization of the distribution after transformation. differences between groups were tested with fisher exact test for categorical variables and anova for continuous variables, followed, where applicable, by post hoc bonferroni correction for multiple testing. a multivariate mixed model with random intercepts and random slopes was used to assess the relationship between the magnitude of sbp and uae change and the rate of egfr decline. such a model calculates renal function decline over time within and between individuals also taking into account the correlation within individuals and time. for exploration of the relationship between the month 6 change in uae and egfr decline, the change in uae was categorized according to quartiles and related to egfr decline from month 6. the multivariate mixed model included the following baseline covariates : age, sex, log - transformed uae, sbp and diastolic bp, egfr, hba1c, duration of diabetes, total cholesterol, smoking, bmi, and treatment allocation. a multivariate cox proportional hazards model was used to assess the relationship between the magnitude of uae and sbp change from baseline to 6 months and time to development of overt nephropathy from 6 months to the end of follow - up. the multivariate cox proportional hazards model included the same covariates as the above - mentioned multivariate mixed model. the initial fall in egfr after start of treatment may reflect a hemodynamic response and may be associated with long - term renoprotection (13,14). because the month 6 change in egfr was not associated with the initial change in uae and sbp, we considered the initial egfr change not a potential confounder in our analyses. relative risk reductions are described in the text as percent reductions ([1hazard ratio ] 100). a total of 531 out of 590 randomized patients had uae and sbp measurements available at baseline and at 6 months postrandomization and were included in this post hoc analysis. the median responses in uae and sbp in the population were 18% and 11 mmhg, respectively. we subsequently divided the population according the median response in these parameters, defining a robust response as a response more than the population median. the median decline in uae and sbp in each subgroup 24.4% had a robust reduction in uae but not in sbp (discordant sbp response) and 19.3% had a robust reduction in sbp but not in uae (discordant uae response). baseline characteristics of the patients stratified by groups of change in albuminuria and sbp from baseline to month 6 negative concordant indicates no robust (i.e., more than median) response in neither uae and sbp, and positive concordant indicates no robust (i.e., more than median) response in both parameters. median uae response was 18% decline ; median sbp response was 11 mmhg decline ; # p median and patients with uae > median and sbp > median ; $ p median and patients with uae > median and sbp > median. there were no differences in baseline characteristics except that patients with a robust reduction in sbp, irrespective of the uae response, had a higher average baseline sbp and diastolic bp compared with patients without robust sbp decline. distribution of the irbesartan and conventional treatment group according to change in albuminuria and sbp from baseline to month 6 data are number of patients and (% of total). we assessed whether the degree of change in uae and sbp was associated with a different slope of renal function loss. a larger decrease in uae during the first 6 months was independently associated with a slower rate of renal function decline during the follow - up time (p = 0.0037 ; fig. 1). a robust decrease in sbp showed a similar trend of a slower rate of long - term egfr decline but was not significant (p = 0.087). long - term annual decline in egfr from 6 to 24 months, per quartile uae change from baseline to month 6 (p = 0.0037) and per group of sbp change from baseline to month 6 (divided over the median change ; p = 0.087) in 531 type 2 diabetic patients with microalbuminuria. the rate of egfr decline according to combined change in uae and sbp and adjusted for other risk variables is presented in fig. 2a. a robust uae reduction resulted in a slower rate of egfr decline, also in those patients who did not have a robust sbp reduction. the combination of a robust response in uae and sbp resulted in the lowest rate of progressive renal function loss. a : decline in egfr from 6 to 24 months for groups of uae and sbp change in 531 type 2 diabetic patients with microalbuminuria. b : hazard ratios for overt nephropathy from 6 to 24 months for groups of uae and sbp change in 531 type 2 diabetic patients with microalbuminuria. for completeness, we determined the impact of changes in uae and sbp on the risk for development of overt nephropathy in a secondary analysis. it should be reminded that the initial change in uae is directly related to the development of overt nephropathy. the risk reduction for development of overt nephropathy was 44% (95% ci 3959 ; p median and patients with uae > median and sbp > median ; $ p median and patients with uae > median and sbp > median. there were no differences in baseline characteristics except that patients with a robust reduction in sbp, irrespective of the uae response, had a higher average baseline sbp and diastolic bp compared with patients without robust sbp decline. distribution of the irbesartan and conventional treatment group according to change in albuminuria and sbp from baseline to month 6 data are number of patients and (% of total). we assessed whether the degree of change in uae and sbp was associated with a different slope of renal function loss. a larger decrease in uae during the first 6 months was independently associated with a slower rate of renal function decline during the follow - up time (p = 0.0037 ; fig. 1). a robust decrease in sbp showed a similar trend of a slower rate of long - term egfr decline but was not significant (p = 0.087). long - term annual decline in egfr from 6 to 24 months, per quartile uae change from baseline to month 6 (p = 0.0037) and per group of sbp change from baseline to month 6 (divided over the median change ; p = 0.087) in 531 type 2 diabetic patients with microalbuminuria. the rate of egfr decline according to combined change in uae and sbp and adjusted for other risk variables is presented in fig. 2a. a robust uae reduction resulted in a slower rate of egfr decline, also in those patients who did not have a robust sbp reduction. the combination of a robust response in uae and sbp resulted in the lowest rate of progressive renal function loss. a : decline in egfr from 6 to 24 months for groups of uae and sbp change in 531 type 2 diabetic patients with microalbuminuria. b : hazard ratios for overt nephropathy from 6 to 24 months for groups of uae and sbp change in 531 type 2 diabetic patients with microalbuminuria. for completeness, we determined the impact of changes in uae and sbp on the risk for development of overt nephropathy in a secondary analysis. it should be reminded that the initial change in uae is directly related to the development of overt nephropathy. the risk reduction for development of overt nephropathy was 44% (95% ci 3959 ; p 300 mg / day) and/or egfr levels below 60 ml / min/1.73 m. importantly, the results of the current study extend these findings to the patient population with levels of uae within the microalbuminuric range (uae 30300 mg / day) and egfr levels above 60 ml / min/1.73 m. an important question is whether changes in albuminuria can be used as a surrogate end point in clinical trials. the distinct advantage is that trials with surrogate end points require fewer patients, require shorter follow - up, are less expensive, and facilitate drug development. to obtain surrogacy status definitive evidence is required, demonstrating that the surrogate end point is causally related to the clinical end point. it has been pointed out that the evidence for albuminuria as a surrogate end point is reasonably robust in patients with diabetes and macroalbuminuria, but limited data are available in patients with lower uae (19,20). this study is the first to show that even in the low albuminuria range the initial antialbuminuric response to arb treatment is an important independent indicator of renoprotection. this suggests that also in patients with microalbuminuria, albuminuria may be a potential candidate as a surrogate end point. prospective randomized controlled trials will be necessary to obtain definitive evidence that an approach of targeting uae confers renoprotection within the microalbuminuria range. these trials should be designed to compare the long - term clinical effect of different predefined uae targets. such a design isolates the role of uae as an independent target for therapy and establishes the clinical relevance of targeting uae for renal or cardiovascular protection. in this respect, a recent study by ruggenenti. (21) in diabetic and nondiabetic nephropathies compared the efficacy of a treatment strategy specifically targeting uae with a historical cohort targeting only bp. the results showed that targeting uae is feasible and translates into substantial risk reductions for end - stage renal disease. interestingly, again the reduction in uae was the only variable in multivariate analyses that was associated with a lower risk of end - stage renal disease. the renoprotection of optimal antiproteinuric doses (road) study is the only randomized controlled trial testing a treatment strategy specifically targeting proteinuria. this trial showed that optimal antiproteinuric dosages of raas blockade are feasible and resulted in a substantially larger reduction in proteinuria and slower rate of renal function decline in nondiabetic patients (22). one possibility is that clinical bp and overnight albumin excretion measurements are subject to large random variability and thus do not accurately reflect true bp and uae. however, patients allocated to irbesartan more often have a robust decline in uae compared with patients treated with conventional treatment only (60 vs. 30% ; table 1). hence, a clear difference in discordant response pattern can be deduced, indicating that the arb treatment responses are not solely due to random variability. another possible explanation for the discordant treatment responses is that the intraindividual discordance in uae and sbp response is accounted for by differences in systemic and local raas activity or differences in the extent of tissue penetration of raas blockade. it is hypothesized that the uae response depends on the extent of intrarenal raas blockade, whereas the sbp response depends on systemic vasculature raas inhibition. in support of this hypothesis, preclinical studies have shown that inhibition of extrarenal raas plays an important role in mediating bp control (23). it is noteworthy that this is a post hoc analysis of clinical trial data and the results are no longer based on randomized comparisons. although we adjusted for a large range of potential confounders, unmeasured confounding may have influenced our results. our data show that arb - induced responses in bp and uae are discordant within a large proportion of patients. this underscores the recommendation of treatment guidelines of diabetes associations to regularly assess both bp and uae in individual patients with diabetes. importantly, the response in uae individually determined renal outcome, regardless of the bp response. this implies that renoprotective strategies in microalbuminuric patients with type 2 diabetes should not only target bp but also uae.	objectivewe aimed to investigate the individual impact of initial responses in urinary albumin excretion (uae) and systolic blood pressure (sbp) to angiotensin ii receptor blocker (arb) treatment on long - term renal outcome in patients with type 2 diabetes and microalbuminuria.research design and methodsin a post hoc analysis of the irbesartan in patients with type 2 diabetes and microalbuminuria (irma)-2 trial we first assessed the individual variability in uae and sbp response (06 months) in 531 subjects. subsequently, we analyzed the individual effect of both response parameters on renal outcome defined as change in estimated glomerular filtration rate (egfr) during 2 years of follow-up.resultsthe median reductions in uae and sbp in the population were 18% and 11 mmhg, respectively. in irbesartan - treated patients, 85 (24.4%) had a robust (> median) reduction in uae but not in sbp (discordant sbp response) and 67 (19.3%) had a robust (> median) reduction in sbp but not in uae (discordant uae response). the degree of reduction in uae was independently associated with the rate of egfr decline (p = 0.0037). sbp showed a similar trend (p = 0.087). the relation between a larger uae reduction and a slower rate of renal function decline was present in both cohorts with a sbp change above and below the median.conclusionswithin an individual, uae response to arb therapy may be discordant from sbp response. the initial change in uae was independently associated with egfr slope ; the more uae reduction the less egfr decline, irrespective of the sbp change. these results suggest that in microalbuminuric patients with type 2 diabetes, uae should be monitored after initiation of therapy and a separate target for renoprotective therapy.
the forkhead box family is a group of over 50 mammalian proteins that is characterized by a winged helix dna - binding domain. forkhead box m1 (foxm1) is a transcription factor that has been well studied and implicated in normal cell growth as well as carcinogenesis. a growing body of evidence suggests a role for foxm1 in various malignancies including breast, liver, lung, prostate, and colorectal cancer. more importantly, different pathways have been elucidated but the exact mechanism remains unclear as multiple transcription factors and downstream regulatory genes are being discovered. the focus of this review will be on the correlation between foxm1 and (1) hormone and growth factor receptors pathways associated with breast cancer and (2) resistance to breast cancer therapies. we will first briefly discuss the structure of foxm1 and its role in both normal cell functioning and carcinogenesis. fox proteins share a dna - binding domain, the forkhead box or winged helix domain (whd) ; it consists of three helices, three sheets, and two loops or wings forming a helix - turn helix - like motif. structural variations occur between the second and third helices, whereas the helices and sheets are highly conserved. dna binding occurs primarily through the third helix, or recognition helix, and the second wing, which bind to the major groove and minor groove of the dna, respectively. foxm1 protein binds the core consensus sequence (a / c)aaacaaac and, in some instances, requires interaction with other factors for dna - binding and transcriptional activity. additionally, foxm1 may also recruit cyclin - dependent kinase (cdk) cyclin complexes through the lxl motif. overall, there is a multitude of factors that play a role in the transcription and target gene expression of fox proteins, including histone deacetylase (hdac) and runx. it consists of seven (i vii) exons plus two alternatively expressed exons (a1 and a2) generating three alternatively spliced coding isoforms : foxm1a, foxm1b, and foxm1c. exon a2 has been shown to be a transcriptional repressor, whereas exon a1 does not have any apparent transcriptional significance. studies have shown that foxm1 exerts its effect through regulation of the cell cycle transition points. it peaks at g1/s and g2/m phases and is essential for mitosis through promotion of m phase entry [9, 13 ]. it is activated by mammalian mitotic kinase polo - like kinase-1 (plk1) through binding to carboxyl terminal domain of foxm1 and through further phosphorylation of two residues on this domain by cyclin - dependent kinase 1 (cdk1), cyclin e - cdk2, and raf - mek - erk - mediated phosphorylation. foxm1 has been shown to regulate the transcription of genes involved in cell cycle by binding to promoter of cyclin d1 and cyclin b1 genes and by direct activation of transcription of the cdc25b phosphatase promoter region. in the hepatoblasts of foxm1b / murine embryos mitosis was associated with decreased expression of the aurora b kinase and polo - like kinase 1 (plk1) and cdc25a phosphatase. wang. showed, using quantitative chromatin immunoprecipitation and expression, that foxm1 is necessary for transcription of the aurora b kinase, survivin, centromere protein a (cenpa), and cenpb. other mechanisms of action include upregulation of transcription of s phase kinase - associated protein 2 (skp2) and cyclin kinase subunit 1 (cks1), subunits of the skp1-cullin 1-f - box ubiquitin ligase complex, leading to degradation of p27 and p21. in vitro experiments in mice detected foxm1 in all proliferating cells, both adult and embryonic ; once the cell had differentiated, it was noted that the expression of foxm1 had significantly decreased. the significance of foxm1 in normal cell growth is supported by krupczak - hollis. and kim., who showed that absence of foxm1 in embryonic cells led to major developmental defects in the liver and lung and was ultimately fatal. supported this notion by demonstrating that the loss of foxm1 led to genetic mitotic spindle defects, delayed cells in mitosis, and induced mitotic catastrophe. foxm1 transcriptional activity is controlled by the small ubiquitin - like modifier-2 (sumo-2) protein. the first evidence of a correlation between foxm1 and human cancer was reported by teh. in 2002. their data revealed a high level of foxm1 expression in human basal cell carcinomas, as a downstream target of the oncogenic transcription factor, glioma - associated oncogene homolog 1 (gli1). halasi and gartel suggested several mechanisms that would explain the increased expression and activity of foxm1 in cancer : (1) foxm1 locus amplification as seen in both solid and hematologic malignancies, (2) a high level of stability or expression of foxm1 in cancer cells initiated through different pathways, (3) enhanced transcription of foxm1 through promoter binding of various factors such as e2 transcription factor (e2f), c - myc, and hypoxia - inducible factor- (hif-) 1, (4) mutations of the tumor suppressor p53 [29, 30 ], and (5) activation of foxm1 by oncogenic signaling pathways such as pi3/akt, epidermal growth factor receptor (egfr), raf / mek / mapk, and hedgehog. recently wei. demonstrated direct activation by foxm1 of snail gene, a key regulator of epithelial - mesenchymal transition (emt), and showed a direct correlation with expressions of foxm1 and snail transcription factor in human lung adenocarcinoma tissues. studies have shown that foxm1 plays a critical role in not only tumor metastasis, emt, cell motility, invasion, and premetastatic niche formation but other key cancer hallmarks as well (figure 1)reprogramming of energy metabolism ; promotion of genomic instability [34, 35 ], inflammation, cell proliferation, and angiogenesis ; evasion of growth / tumor suppression ; circumvention of apoptosis ; and enabling of replicative mortality. the effect of foxm1 on biology of breast cancer was evaluated in 236 women with breast cancer. expression of foxm1 was associated with larger tumor size, lymphovascular invasion, lymph nodes metastases, and higher stage of breast cancer. there was no significant impact of foxm1 expression on survival when all breast cancer histologies were analyzed ; however, in a subgroup of patients with estrogen receptor (er) positive tumors, low foxm1 expression correlated with better survival (hazard [low versus high ] = 7.304, 95% confidence interval [0.89759.45 ], p = 0.063). in another study of 501 er positive tumors, foxm1 was overexpressed in 20% of tumors that correlated with higher recurrence rate and shorter survival (p = 0.03), contributed to association of foxm1 with stem - cell like population, increased expression of markers of emt, and increased resistance to tamoxifen therapy. the complex and diverse ways in which foxm1 promotes breast cancer tumorigenesis are depicted in figure 2. yang. demonstrated that foxm1 promoted emt in breast cancer by binding and activation of the promoter of slug gene. xue. showed that foxm1 promotes breast cancer metastases by activation of the tgf- pathway through interaction with smad3 (this prevented e3 ubiquitin - protein ligase transcriptional intermediary factor 1 [tif1 ] binding to smad3 and protected smad4 from ubiquitination) that leads to stabilization of the smad3/smad4 complex. foxm1 also has a role in modulation of the extracellular matrix by affecting levels of upa, upar, mmp-2, mmp-9, and vegf., in which they demonstrated that foxm1 binds to forkhead response element in vegf promoter. yu. showed that binding of foxm1 to platelet derived growth factor - a promoter led to activation of the akt pathway and increased breast cancer tumorigenesis. nestal de moraes. have shown that foxm1 upregulates antiapoptotic genes xiap and survivin by interacting with their promoters, contributing to chemoresistance of breast cancer cells to docetaxel, paclitaxel, and epirubicin. moreover, coexpression of foxm1, survivin, and nuclear xiap was associated with poor outcomes of women with stage iii breast cancer with significantly reduced 5- and 10-year survival rates versus women with tumors without these features. breast cancer encompasses a heterogeneous group of entities that vary greatly in terms of histology, therapy, and prognosis. human breast malignancies can be divided into five subtypes : normal breast - like, luminal a, luminal b, her2/neu - enriched, and basal - like breast cancer (bbc). the effect of estrogen on both normal and malignant breast tissue is mediated through two types of estrogen receptors (er) : er and er. although they bind to estrogen with equal affinity, er and er respond differently to estrogen stimulation : activated er induces breast epithelium proliferation, whereas er has antiproliferative and proapoptotic effects. approximately 70% of breast cancers are estrogen receptor positive (er+) and these tumors are often better differentiated, grow slowly, and have a favorable prognosis. early research about the forkhead box family and growth factors in breast cancer emerged in 2000. provided the first evidence of a link between epidermal growth factor (egf) and a forkhead box family member, foxo3a, known at that time as fkhr. and karadedou found a positive correlation between er and foxm1 and an inverse correlation between er and foxo3a. their results showed that foxm1 is a physiologic regulator of er expression in breast cancer cells, both at the protein and at mrna levels. consistent with these findings, later studies confirmed the tumor suppressive qualities of foxo3a, including cases of er+ breast malignancies. carr. reported transcriptional repressor function of foxm1 inhibiting differentiation of luminal progenitor cells by inducing methylation of promoter of the zinc finger transcription factor gata-3 through association with dnmt3b. millour. supported the critical role of foxm1 in mitogenic functions of er and estrogen in breast malignancies. additionally, they showed that foxm1 deregulation may also contribute to antiestrogen insensitivity, opening the door to practical applications of these findings in the field of therapeutics. whereas the focus of these studies was on er, horimoto. their investigations showed that er had an antiproliferative effect through repression of foxm1 expression in breast cancer cells ; this effect was mediated by an estrogen - response element within the proximal promoter region that is also a target of er. the correlation between foxm1 and er+ breast cancer has also been shown on the genetic level. created a 70-gene signature which was found to be of prognostic value in er+ breast cancer patients in that foxm1 suggested poorer prognosis. this signature was found to be most helpful in cases of intermediate oncotype recurrence scores. other studies included genome - wide mapping by sanders. which revealed a direct relationship between foxm1 and er. the human epidermal growth factor receptor 2 (her2) is a transmembrane tyrosine kinase receptor and a member of the epidermal growth factor (egf) family. the amplification of her2 can be seen in up to 25% of breast cancer cases and has been shown to correlate with high relapse rates and poor survival. in 2008, provided the first evidence of a positive correlation between her2 status and foxm1 expression in breast cancer specimens, in comparison to normal breast tissue. additionally, their work verified the overexpression of foxm1 in breast cancer on both the rna and protein level. francis. elaborated on the concept further through various in vitro and in vivo experiments. their work suggested that foxm1 may in fact be a downstream target and marker of her2 overexpression in breast cancer. this study also had translational implications by showing a suppressive role for lapatinib (a her2 inhibitor) on foxm1 expression at the protein, mrna, and gene promoter levels. subsequent data showed a role for foxm1 in the development of trastuzumab (a her2 antibody) resistance. this is particularly important because trastuzumab resistance may develop quickly and represents a challenge in the treatment of breast cancer. basal - like breast cancer (bbc) is an aggressive phenotype of breast malignancies that is often associated with poor prognosis. bbc cells lack hormone estrogen receptors (er) and progesterone receptors (pr) and express genes that are usually seen in basal or myoepithelial cells of normal breast tissue. triple negative breast cancers (tnbc), on the other hand, are tumors that lack her2 in addition to er and pr. although differences between these two types of breast cancer have been demonstrated, bbc and tnbc overlap significantly. although tnbc accounts for only 15% of breast cancer subtypes, it causes 25% of breast cancer - related deaths due to its aggressive and refractory nature. emt has been correlated with tnbc, and nf - kappab is a transcription factor that has been shown to be essential in emt in breast cancer. they discovered that panepoxydone (pp), a nf - kappab inhibitor, halted proliferation, induced apoptosis, and reversed emt in breast cancer, in particular tnbc. interestingly, their work revealed a downregulatory effect of pp on foxm1 as well. a whole genome and transcriptome sequencing of tnbc cells by craig. the role of foxm1 has also been seen in bbc cancers, including foxm1-dependent overexpression of melk, a novel oncogenic kinase. taking into consideration the significant role of foxm1 in breast cancer biology this transcription factor could become an attractive target for cancer treatment. herein, we describe some breast cancer therapeutic strategies targeting foxm1. a foxm1-specific small interfering rna (sirna) treatment of breast cancer cells with adenoviral vector expressing short hairpin downregulating foxm1 led to inhibition of breast cancer tumor formation. overexpression of microrna mir802 led to downregulation of foxm1 and inhibited proliferation of breast cancer cells. a thiazole ring containing thiostrepton, an antibiotic with antitumor activities, was shown to induce arrest and death of breast cancer cells through downregulating foxm1 expression. as stated earlier, sumo is a posttranslational modifier that is essential for activation of foxm1. a sumoylation protease, sentrin - specific protease 2 (senp2), significantly decreased sumoylation of foxm1 and interfered with its function. casticin, an active ingredient derived from fructus viticis, the fruit of a traditional chinese medicine, has anticarcinogenic activity in breast cancer. recent experiments showed that it can induce apoptosis of breast cancer cells by reducing the expression of foxm1. foxm1 has been shown to play a critical role in development of resistance to breast cancer therapeutics. foxm1 is a downstream target of 14 - 3 - 3, a marker of endocrine therapy resistance in breast cancer malignancy. foxm1 was found to contribute to cisplatin (a platinum agent) resistance in breast cancer cells. the effect was thought to be mediated by the enhancement of dna - damage repair pathways and the promotion of cell cycle progression or inhibition of cell cycle checkpoints and apoptosis. foxm1 overexpression was implicated in the resistance to trastuzumab (a her2 monoclonal antibody) and paclitaxel (a microtubule stabilizing agent). treatment with a sirna targeting foxm1 or an alternate reading frame- (arf-) derived peptide resulted in improved therapeutic sensitivity to these agents. the nuclear factor nf - kappa - b1 (nfb1) interacted with foxm1 in the presence of doxorubicin to protect breast cancer cells from dna damage. epirubicin could activate ataxia - telangiectasia mutated (atm) that promotes e2f activity and foxm1 expression.. showed that the mechanism of epirubicin resistance was mediated by activation of mitogen - activated protein kinase - activated protein kinase 2 leading to increased phosphorylation of transcription factor e2f1 at ser-364 resulting in increased foxm1 expression. demonstrated that foxm1 reduced senescence induced by epirubicin, by increasing expression of nbs1 leading to enhanced homologous recombination dna repair. another work by khongkow. postulated that resistance to paclitaxel, a tubulin targeting agent, can be mediated by foxm1 through enhancement of promoter activity of transcriptional activity of kif20a. both foxm1 and kif20a are critical for normal formation of mitotic spindle and thus could interfere with paclitaxel activity. nestal de moraes. demonstrated that resistance to epirubicin, docetaxel, and paclitaxel was associated with activation of xiap and survivin by direct interaction of foxm1 with promoters of these antiapoptotic genes. forkhead box m1 plays a significant role in breast carcinogenesis, disease progression to metastatic stage, and development of resistance to subsequent cancer therapy, and thus it could be an attractive target for therapeutic interventions in this malignancy. however, due to incomplete understanding of the biology of the fox family of transcription factors that has complex regulatory mechanisms, it has been a challenge to develop a drug that functions specifically as a foxm1 inhibitor. in addition, druggability of foxm1 has been taxing because of lack of substrate binding pockets and hydrophobic surfaces. a recent discovery through high throughput fluorescence polarization assay of a novel small molecule that specifically inhibits foxm1 interaction with dna may be a first breakthrough in the design of selective inhibitors of this transcription factor. once foxm1 targeted agents are available, appropriate clinical testing in different breast cancer subtypes is warranted, most likely in a biomarker - driven setting. at present, however, there is no clear biomarker developed to assess sensitivity of breast cancer to foxm1 inhibition. this could be a goal of early pilot studies, especially in the most challenging breast cancer subtype, triple negative disease.	the forkhead box m1 (foxm1) is a transcription factor that has been implicated in normal cell growth and proliferation through control of cell cycle transition and mitotic spindle. it is implicated in carcinogenesis of various malignancies where it is activated by either amplification, increased stability, enhanced transcription, dysfunction of regulatory pathways, or activation of pi3k / akt, epidermal growth factor receptor, raf / mek / mapk, and hedgehog pathways. this review describes the role of foxm1 in breast cancer. this includes how foxm1 impacts on different subtypes of breast cancer, that is, luminal / estrogen receptor positive (er+), expressing human epidermal growth factor receptor 2 (her2), basal - like breast cancer (bbc), and triple negative breast cancer (tnbc). the review also describes different tested preclinical therapeutic strategies targeting foxm1. developing clinically applicable therapies that specifically inhibit foxm1 activity is a logical next step in biomarker - driven approaches against breast cancer but will not be without its challenges due to the unique properties of this transcription factor.
the kopsifolines and their unique core hexacyclic ring system were first disclosed in 2003 when kam and choo reported the initial members of this new alkaloid class (figure 1). f (16) were isolated from the leaf extracts of a previously unencountered malaysian kopsia species later identified as k. fruticosa (ker) a. dc., and their structures were established spectroscopically. subsequent exploration of k. singapurensis led to a second isolation of kopsifoline a (1) and characterization of a seventh kopsifoline (singaporentine a, 7). related to the aspidosperma alkaloids, their more complex hexacyclic core structure incorporates a previously unprecedented c3c21 carbon carbon bond linking the terminal carbon (c21) of the c5 ethyl substituent to c3 bearing a methoxycarbonyl group such that the stereochemically rich central six - membered ring incorporates five or six stereogenic centers, three or four of which are quaternary. in efforts that served to assign the natural product absolute stereochemistry, we recently disclosed a total synthesis of (+) -fendleridine (9, aspidoalbidine), enlisting an intermediate in which the c5 ethyl substituent bears an oxidized terminal c21 methyl group. closure of this alcohol onto c19 (c21o c19 bond formation) via trap of an intermediate iminium ion provided the fendleridine tetrahydrofuran ring and completed the assemblage of its hexacyclic ring system (figure 2). this same intermediate, albeit in the enantiomeric series and by virtue of conversion of the c5 ethyl group primary alcohol to a methyl dithiocarbonate, was enlisted for the total synthesis of kopsinine (10), featuring a diastereoselective smi2-mediated free radical transannular conjugate addition reaction for formation of the bicyclooctane core central to its hexacyclic ring system with c21c2 bond formation. this late - stage c21c2 bond formation not only complemented prior diels alder approaches to its bicyclooctane core, but it also represented the first synthetic approach that directly provided kopsinine from the underlying pentacyclic aspidosperma alkaloid skeleton (figure 2). herein, we report full details of the use of this same key intermediate in initial exploratory efforts that provided the parent kopsifoline skeleton (named ()-kopsifoline h, 8) and the extension of these studies to the first total synthesis of a naturally occurring kopsifoline, ()-kopsifoline d (4). by mimicking what is the likely biosynthesis of the kopsifolines, a late - stage c21c3 bond formation via a transannular enamide alkylation of an alcohol - derived c21 iodide within the aspidosperma alkaloid skeleton was developed and implemented to complete the assemblage of the kopsifoline skeleton (eq 1) in a route that directly provides the natural product in its final indolenine oxidation state. the studies further established the kopsifoline absolute configuration as enantiomeric to the aspidosperma alkaloids including fendleridine, but analogous to that found within kopsinine.1 an additional attribute of the approach is that the same key intermediate was also used herein to access natural ()-deoxoapodine (11), originally isolated from tabernae armeniaca, by virtue of c21o these efforts represent only the second total synthesis of the natural product and the first to provide 11 in optically active form, confirming the anticipated absolute configuration assignment. thus, a common aspidosperma - like pentacyclic intermediate, bearing a terminally functionalized c5 ethyl substituent (primary alcohol), was used in the divergent total synthesis of a suite of alkaloids, entailing linkage of the c21 primary alcohol oxygen to c19 (fendleridine) and c6 (deoxoapodine) or through linkage of c21 itself to c2 (kopsinine) and c3 (kopsifoline d) using the c21 functionality to conduct complementary nucleophilic or electrophilic c c bond - forming reactions (figure 3). the combined efforts represent the total syntheses of members of four different classes of natural products from a common intermediate deliberately functionalized for late - stage formation of four different key strategic bonds embedded in each unique core structure. divergent total syntheses of a series of naturally occurring alkaloids containing deep - seated structural differences from a common cascade cycloaddition product. the basis of the approach and key to the assemblage of the underlying aspidosperma skeleton is a powerful intramolecular [4 + 2]/[3 + 2 ] cycloaddition cascade of a 1,3,4-oxadiazole that provides the fully functionalized pentacyclic ring system in a single step (scheme 1). assembled in two steps from n - benzyltryptamine and subsequent n - acylation of the amino-1,3,4-oxadiazole 12 with 4-(2-tert - butyldimethylsilyloxy)pent-4-enoic acid (13), thermal cyclization of 14 (230 c, o - dichlorobenzene (o - dcb)) provided the key pentacyclic skeleton 15 (71%) as a single diastereomer. as disclosed in our initial work, the initiating intramolecular [4 + 2 ] cycloaddition is followed by a retro diels alder loss of n2 to provide an intermediate 1,3-dipole stabilized by the two substituents on the carbonyl ylide. in turn, the stabilized 1,3-dipole undergoes a subsequent diastereoselective [3 + 2 ] cycloaddition with the tethered indole exclusively through an endo transition state and with an intrinsic regioselectivity that is reinforced by the linking tether to provide the cascade cycloadduct 15. inherent in the approach, the c3 methyl ester found in the kopsifolines, deoxoapodine, and kopsinine serves as a key 1,3,4-oxadiazole substituent, activating it for participation in the initiating diels alder reaction and stabilizing the intermediate 1,3-dipole. as detailed in our preceding studies albeit with improvements in the originally disclosed conversions, diastereoselective reductive cleavage of the oxido bridge in 15 (nacnbh3, 88%) with exclusive convex face hydride reduction of an intermediate iminium ion flanked by two quaternary centers and subsequent methyl dithiocarbonate formation (nah, cs2 ; mei ; 91%) provided 17(7) in superb conversions. as our studies began, we first focused on the synthesis of 8, the parent kopsifoline skeleton. although 8 has not yet been isolated as a naturally occurring substance, lacking a -double bond and containing a saturated indoline, it possesses the parent hexacyclic skeleton and represents the tetrahydro derivative of kopsifoline d. barton mccombie deoxygenation of methyl dithiocarbonate 17 (bu3snh, cat. aibn, toluene, reflux, 1 h, 96%) cleanly provided 18 as a single diastereomer arising from exclusive convex face hydrogen atom reduction of the intermediate stabilized radical and inversion of the c3 stereochemistry (scheme 2). silyl ether cleavage of 18 (bu4nf, tetrahydrofuran (thf), 78 to 25 c, 1 h, 92%) provided the primary alcohol 19 that in turn was converted to the iodide 20 (et3n, ch3so2cl, thf, 78 c, 1 h then nai, acetone, 78 to 50 c, 12 h, 81%), setting the stage for studies on its cyclization to the kopsifoline skeleton. after exploration of several alternatives, treatment of 20 with kobu in thf (3 equiv, 78 to 0 c, 1 h) proved to be highly effective and afforded 21 (95%), whose structure and stereochemistry were confirmed with a single - crystal x - ray structure determination. initial, albeit limited, attempts to promote the cyclization of the intermediate mesylate (0%) or corresponding tosylate (3236%) were not as productive. chromatographic separation of the enantiomers of 21 (= 1.2, 10% i - proh / hexane) was carried out on a semipreparative daicel chiralcel ad column, providing (+) -21 and ent-()-21. treatment of each enantiomer of 21 with lawesson s reagent (1.1 equiv, toluene, 100 c, 0.5 h) furnished the thiolactam 22 (89%). the absolute configuration of (+) -22 was unambiguously established by an x - ray structure determination conducted on this intermediate containing a heavy atom (s). concomitant reduction of the thioamide and removal of the n - benzyl group was accomplished by the treatment of (+) -22 with raney - nickel (ra ni, h2, etoh, 80 c, 0.5 h) to furnish ()-8 (70%), which we have come to refer to as kopsifoline h, designating its hydrogenated (h) state. the additional key question addressed in initial studies was whether a late - stage c21c3 bond formation via a transannular enamide alkylation of an alcohol - derived c21 electrophile was feasible for formation of the kopsifoline core, permitting the direct introduction of the correct c2 indolenine oxidation state of the natural products (see eq 1). these efforts began with 25, originally prepared in prior studies and bearing a readily removable indoline n - carboxybenzyl (cbz) group. in efforts that impact the regioselectivity of the chugaev elimination, conversion of 16 to the corresponding cbz carbamate 23, followed by an improved methyl dithiocarbonate formation (nah, cs2 then mei, thf, 0 to 25 c, 3 h, 93%) provided 24 (scheme 3). the intermediate xanthate 24 underwent clean thermal elimination under mild reaction conditions (toluene, 100 c bath, 48 h or 150 c bath, 36 h), providing good yields (60%) of 25. here, the indoline cbz carbamate (vs n - benzyl) activates c2h for xanthate syn elimination, favoring formation of the more substituted and stable olefin. silyl ether cleavage in 25 (3 equiv bu4nf, thf, 25 c, 1 h, 98%) and conversion of the primary alcohol 26 to the primary iodide 27 (et3n, ch3so2cl, thf then nai, acetone, 50 c, 12 h, 80%) followed by reduction of the amide 27 to the tertiary amine 28 (5 equiv bh3thf, thf, 0 c, 1.5 h, 79%) set the stage for examination of the transannular cyclization. after optimization of the conditions, we were delighted to find that the treatment of 28 with bf3oet2 and dimethyl sulfide (me2s) in ch2cl2 (25 c, 15 h) proceeded smoothly not only to promote cbz deprotection but also to afford 30 directly in 75% yield as a single diastereomer, providing 6,7-dihydrokopsifoline d. shorter reaction times led to detection and isolation of the intermediate cbz deprotection product 29 that itself undergoes slow cyclization to the indolenine 30 simply upon standing at room temperature. notably, the corresponding carbinol amine derived from water addition to c2 of 30 could also be isolated if the chromatographic purification (sio2) of 30 was carried out without the presence of et3n (ethyl acetate (etoac) vs 2% et3n in etoac). reduction of 30 (nabh4, etoh, 0 to 25 c) provided 8, confirming the structure of 30 and providing an alternative synthesis of kopsifoline h. finally, limited efforts to convert 8 to 30 by oxidation (mno2, 2,3-dichloro-5,6-dicyano-1,4-benzoquinone, me2s cl, kmno4) to the c2 imine have not yet proved productive (scheme 3), suggesting that direct indolenine introduction from intermediates such as 28 may constitute a more effective approach. adoption of the approach for the total synthesis of kopsifoline d required installation of the -double bond. toward this end, -phenylselenation of 25 was achieved by treatment of the lactam enolate (lithium diisopropylamide, 78 c) with phenylselenyl chloride (phsecl, 1 equiv) to provide 31 (81%, scheme 4). the mixture of isomeric phenylselenides (8:1, :) was treated with m - chloroperoxybenzoic acid (m - cpba, 1.5 equiv) in ch2cl2 at 78 c to yield the corresponding selenoxides, and their subsequent in situ syn elimination proceeded smoothly (78 to 25 c) to afford 32 (90%). the enantiomers of 32 were easily separated (= 1.48, 20% i - proh / hexane) on a semipreparative chiralcel od column, affording natural (+) -32 and ent-()-32. clean 1,2-reductive removal of the lactam carbonyl was best accomplished by o - methylation of (+) -32 and subsequent reduction of the resulting methoxyiminium ion (3 equiv me3obf4, 2,6-t - bu2py, ch2cl2, 25 c ; nabh4, meoh, 25 c, 52%) to afford natural ()-33. nearly as effective, direct lactam reduction of (+) -32 by treatment with diisobutylaluminum hydride (dibal - h, 5 equiv, thf, 20 c, 20 min) also afforded ()-33 (48%) in a single step. alternatively, treatment of (+) -32 with lawesson s reagent (1.1 equiv, toluene, 100 c, 0.5 h, 65%), subsequent s - methylation of the intermediate thioamide (3 equiv me3obf4, 2,6-t - bu2py, ch2cl2, 25 c) and reduction of the resulting s - methyliminium ion (10 equiv nabh4, meoh, 0 c, 45 min) also provided 33 (40% overall), albeit in lower overall conversions. silyl ether cleavage (bu4nf, thf, 25 c, 1 h, 98%) followed by conversion of the primary alcohol ()-34 to the primary iodide ()-35 (et3n, ch3so2cl, thf, 78 c then nai, acetone, 90 c bath temperature, 12 h, 68%) set the stage for the key transannular cyclization. cbz deprotection and subsequent intramolecular ring closure of 35 (bf3oet2, me2s, ch2cl2, 25 c, 12 h followed by treatment with et3n, etoac, 25 c, 12 h) provided ()-kopsifoline d (4) in superb conversion (79%) and identical in all respects compared to those of the authentic material except for its distinctly different optical rotation ([]d 69 (c 0.08, chcl3) vs reported []d 27 (c 0.09, chcl3)). use of the intermediate mesylate (0%) and corresponding tosylate (22%) was less effective than the primary iodide 35, and its intramolecular ring closure to provide 4 was slower than that providing 30, requiring a longer reaction time and deliberate base treatment (et3n) for completion. initial concerns over the discrepancy in the optical rotations between our synthetic 4 versus that reported for natural 4 led us to explore several origins of the distinctions and entailed repeated preparations of synthetic material. in the course of these efforts, the water addition product to kopsifoline d (kopsifoline d hydrate, 4a) was also isolated and characterized (scheme 4). unexpectedly, the optical rotation of this synthetic hydrate, which could be deliberately prepared by simply stirring a solution of 4 in thf h2o (95%, 25 c, 3 h), perfectly matched that reported for kopsifoline d ([]d 26 (c 0.08, chcl3) vs reported []d 27 (c 0.09, chcl3)). this suggests that, while the spectroscopic characterization of natural kopsifoline d was effectively conducted with 4, the optical rotation itself was measured on a sample that had undergone hydration. the ease of kopsifoline d hydration also suggests that this corresponding hydrate is a natural product as well and that it likely will be isolated and characterized in the years ahead. the unambiguous assignment of the absolute configuration of 4 was accomplished with a single - crystal x - ray structure determination conducted on the natural enantiomer of the primary alcohol derived from 23, which was correlated with (+) -32 (see supporting information [si ]). with the natural enantiomer (+) -32 in hand, our efforts turned to the divergent total synthesis of ()-deoxoapodine (scheme 5). complementary to the approach to kopsifoline d, silyl ether deprotection prior to lactam carbonyl removal was anticipated to permit the requisite c21o silyl ether deprotection upon treatment of (+) -32 with bu4nf (3 equiv) in thf at 5 c (6 h) under basic conditions smoothly provided only the primary alcohol conjugate addition product ()-36 (70%) as a single diastereomer without the detection or isolation of the intermediate alcohol. higher reaction temperatures (025 c) led to generation of an additional minor diastereomer of the conjugate addition product, whereas lower reaction temperatures (40 c) resulted in little deprotection and cyclization. in contrast, silyl ether deprotection under acidic conditions conducted by treatment of (+) -32 with hfpyridine (3 equiv, thf, 0 c, 2 h) afforded only the deprotected primary alcohol (8495%) with little or no cyclization even under prolonged reaction times (10 h). reductive removal of the lactam carbonyl of ()-36 upon treatment with bh3thf (thf, 0 c, 1.5 h) provided ()-37 (70%) and subsequent cleavage of the cbz group (bf3oet2, me2s, 25 c, 5 h, 82%) afforded natural ()-deoxoapodine (11, []d 522 (c 0.17, chcl3) vs []d 432 (c 0.76, chcl3) and []d 593 (chcl3)), which proved identical in all respects to those of the natural product. by virtue of the crystallographic assignment of the absolute configuration of intermediates in route to 11 ((+) -23 and (+) -32), the correlation serves to unambiguously confirm the anticipated absolute configuration for the natural product. divergent total syntheses of ()-kopsifoline d and ()-deoxoapodine that unambiguously established their absolute stereochemistry are detailed from a common pentacyclic intermediate 15 with the late - stage formation of two different key strategic bonds unique to their hexacyclic ring systems, complementing its prior use in the total syntheses of kopsinine and (+) -fendleridine. the combined efforts represent the total syntheses of members of four different classes of natural products from a common intermediate functionalized for late - stage formation of four different key strategic bonds uniquely embedded in each natural product core structure. the basis of the approach and central to the assemblage of the underlying skeleton is a powerful intramolecular [4 + 2]/[3 + 2 ] cycloaddition cascade of a 1,3,4-oxadiazole. this reaction provided the c21 functionalized pentacyclic ring system 15 in a single step in which the c3 methyl ester found in the natural products served as a key 1,3,4-oxadiazole substituent, activating it for participation in the initiating diels alder reaction and stabilizing the intermediate 1,3-dipole.	divergent total syntheses of ()-kopsifoline d and ()-deoxoapodine are detailed from a common pentacyclic intermediate 15, enlisting the late - stage formation of two different key strategic bonds (c21c3 and c21o c6) unique to their hexacyclic ring systems that are complementary to its prior use in the total syntheses of kopsinine (c21c2 bond formation) and (+) -fendleridine (c21o c19 bond formation). the combined efforts represent the total syntheses of members of four classes of natural products from a common intermediate functionalized for late - stage formation of four different key strategic bonds uniquely embedded in each natural product core structure. key to the first reported total synthesis of a kopsifoline that is detailed herein was the development of a transannular enamide alkylation for late - stage formation of the c21c3 bond with direct introduction of the reactive indolenine c2 oxidation state from a penultimate c21 functionalized aspidosperma - like pentacyclic intermediate. central to the assemblage of the underlying apidosperma skeleton is a powerful intramolecular [4 + 2]/[3 + 2 ] cycloaddition cascade of a 1,3,4-oxadiazole that provided the functionalized pentacyclic ring system 15 in a single step in which the c3 methyl ester found in the natural products served as a key 1,3,4-oxadiazole substituent, activating it for participation in the initiating diels alder reaction and stabilizing the intermediate 1,3-dipole.
central serous chorioretinopathy (csc) is characterized by fluid accumulation between the neuroretina and retinal pigment epithelium (rpe).1,2 it often affects the macula, leading to central vision loss, scotoma, metamorphopsia, and/or micropsia.1,2 the accumulation of subretinal fluid (srf) results from leakage through a dysfunctional rpe with a disrupted outer blood retina barrier. dilation, congestion, and hyperpermeability of the choriocapillaris and larger choroidal vessels appear to play a pivotal role in the pathogenesis of csc.35 the etiology of csc is unknown. risk factors for csc include the use of corticosteroids, cushing s disease, pregnancy, and male sex.2,6 recent studies have also found genetic associations.7,8 there are two main subtypes of csc. acute csc is characterized by sudden vision loss, due to fluid leakage through a focal pinpoint leak in the rpe, often in association with a small rpe detachment.1,2 in most acute csc patients, the srf resolves spontaneously within 23 months.1 visual acuity (va) in these patients generally returns to (near-) normal levels.1 in the other main subtype, chronic csc (ccsc), patients have more widespread atrophic rpe abnormalities, as well as more extensive choroidal changes on fluorescein angiography (fa) and indocyanine green angiography (icga).1,2 in contrast to acute csc, most patients with ccsc have more pronounced central vision loss, and often do not show a spontaneous resolution of srf.2 although ccsc is one of the most common forms of macular degeneration,1 relatively little is known about the long - term visual consequences in these patients. the aim of this study was to evaluate the clinical outcome of patients with ccsc and assess their vision - related quality of life to provide a more accurate prognosis to patients. patients with ccsc and a follow - up of at least 1 year were included in this retrospective observational study. subjects were recruited from the department of ophthalmology of radboud university medical center (nijmegen, the netherlands) and the oxford eye hospital (oxford, uk). all patients gave written informed consent for the use of their data for this retrospective study. the diagnosis of ccsc was defined as the presence of srf longer than 3 months in at least one eye on optical coherence tomography (oct), rpe window defects on fa with at least 1 hot spot, defined as indistinct hyperfluorescent area of leakage and/or diffuse leakage in the affected eye(s), and corresponding hyperfluorescent zones on icga when available. this study adhered to the tenets of the declaration of helsinki, and approval for the study in oxford was obtained from the integrated research authority, essex 2 research ethics committee. the ethical approval for the study in nijmegen was waived by commissie mensgebonden onderzoek (cmo) of the radboud university medical center in nijmegen, as all clinical data was obtained retrospectively and the questionnaire was found to be non - invasive and not personally sensitive for the study objects. the following parameters, when available, were collected from the medical records : va, disease activity, duration of visual symptoms, (prior) use of corticosteroids, number of treatments, color fundus photography, oct, fa, and icga. only visits for which va and information about disease activity, defined as subfoveal fluid on oct or (when oct was not available) as stated by the ophthalmologist in the medical records, were included. additionally, patients were asked to complete a validated questionnaire (visual function questionnaire [vfq]-39).9 for all dutch patients, a validated dutch translation of this questionnaire was used.10 submacular rpe detachments, subfoveal srf, change in submacular srf accumulation, and retinal atrophy were scored on oct. the number of hot spots, defined as indistinct hyperfluorescent areas of leakage, seen on fa and icga were registered. central foveal thickness (cft), defined as the distance between the outer part of the internal limiting membrane and the outer part of the external limiting membrane at the central fovea, of the first and the last available oct scans was measured by two independent graders. only patients who had follow - up imaging with spectral domain oct (spectralis ; heidelberg engineering, heidelberg, germany) were included in cft measurements. the mean of the measurements of both graders was used for further analysis after no significant difference between the graders was confirmed by student s independent t - test (p>0.05). in selected patients, unaffected fellow eyes, defined as eyes the difference in cft over time was compared between the affected and unaffected eyes using student s independent t - test. the va of all first visits was compared to the va of all last known visits using an independent - sample t - test. for the va of the last visit, only patients with inactive disease, defined as absence of srf on oct, were included. additionally, a pearson s correlation test was performed to determine if the number of episodes of active disease or the number of treatments affected the overall change in va when comparing the first visit to the last visit. the scores of every individual subcategory of the vfq-39 were compared to the reference group, which was used for assessment in the original validation of the vfq-39,9,21 using the independent - sample t - test. the original validation of the vfq-39 analyzed the data of the reference group as being normally distributed ; therefore, this study also chose this approach. a pearson s correlation test was performed to analyze if the va at the end of follow - up was associated with vfq-39 score. patients with ccsc and a follow - up of at least 1 year were included in this retrospective observational study. subjects were recruited from the department of ophthalmology of radboud university medical center (nijmegen, the netherlands) and the oxford eye hospital (oxford, uk). all patients gave written informed consent for the use of their data for this retrospective study. the diagnosis of ccsc was defined as the presence of srf longer than 3 months in at least one eye on optical coherence tomography (oct), rpe window defects on fa with at least 1 hot spot, defined as indistinct hyperfluorescent area of leakage and/or diffuse leakage in the affected eye(s), and corresponding hyperfluorescent zones on icga when available. this study adhered to the tenets of the declaration of helsinki, and approval for the study in oxford was obtained from the integrated research authority, essex 2 research ethics committee. the ethical approval for the study in nijmegen was waived by commissie mensgebonden onderzoek (cmo) of the radboud university medical center in nijmegen, as all clinical data was obtained retrospectively and the questionnaire was found to be non - invasive and not personally sensitive for the study objects. the following parameters, when available, were collected from the medical records : va, disease activity, duration of visual symptoms, (prior) use of corticosteroids, number of treatments, color fundus photography, oct, fa, and icga. only visits for which va and information about disease activity, defined as subfoveal fluid on oct or (when oct was not available) as stated by the ophthalmologist in the medical records, were included. additionally, patients were asked to complete a validated questionnaire (visual function questionnaire [vfq]-39).9 for all dutch patients, a validated dutch translation of this questionnaire was used.10 submacular rpe detachments, subfoveal srf, change in submacular srf accumulation, and retinal atrophy were scored on oct. the number of hot spots, defined as indistinct hyperfluorescent areas of leakage, seen on fa and icga were registered. central foveal thickness (cft), defined as the distance between the outer part of the internal limiting membrane and the outer part of the external limiting membrane at the central fovea, of the first and the last available oct scans was measured by two independent graders. only patients who had follow - up imaging with spectral domain oct (spectralis ; heidelberg engineering, heidelberg, germany) were included in cft measurements. the mean of the measurements of both graders was used for further analysis after no significant difference between the graders was confirmed by student s independent t - test (p>0.05). in selected patients, unaffected fellow eyes, defined as eyes in which no central lesions suspect for ccsc were present, were included. the difference in cft over time was compared between the affected and unaffected eyes using student s independent t - test. the va of all first visits was compared to the va of all last known visits using an independent - sample t - test. for the va of the last visit, only patients with inactive disease, defined as absence of srf on oct, were included. additionally, a pearson s correlation test was performed to determine if the number of episodes of active disease or the number of treatments affected the overall change in va when comparing the first visit to the last visit. the scores of every individual subcategory of the vfq-39 were compared to the reference group, which was used for assessment in the original validation of the vfq-39,9,21 using the independent - sample t - test. the original validation of the vfq-39 analyzed the data of the reference group as being normally distributed ; therefore, this study also chose this approach. a pearson s correlation test was performed to analyze if the va at the end of follow - up was associated with vfq-39 score. the mean number of episodes of active disease per eye was 1.7 (range : 06). each episode was defined as a presence of documented srf followed by a period of oct - proven absence of srf. when the va of the first and last visits was compared, excluding the 21 eyes (40%) with persistent subfoveal srf at the final visit, a mean decline of 0.16 logarithm of minimum angle of resolution (logmar ; range : 0.22 to 1.3, p=0.009) was found after a mean follow - up period of 10.6 (range : 1.524) years. in the 21 eyes (40%) with persistent subfoveal srf at the final visit, a mean decline of 0.22 logmar (range : 0.35 to 0.66, p=0.003) with a mean follow - up period of 4.5 (range : 111) years was seen. in 11 of these 21 eyes (52%), neither the number of episodes of active disease nor number of treatments had a significant effect on overall change in va. an overview of oct findings is displayed in table 2 and figure 1a c. in the 13 eyes with a decrease in srf, eight (62%) the seven eyes with an increase of srf had not received treatment. in the eyes showing fluctuating srf accumulation, 18 (82%) had received treatment. the following treatments were performed : 41 micropulse therapy, 23 photodynamic therapy (pdt), one argon - laser treatment, one anecortave acetate treatment, and nine anti - vegf treatments. in 14 eyes (24%), srf was continuously present on oct until the last follow - up. in this group, this was also the case in five eyes (36%) despite receiving treatment (micropulse therapy [52% ], pdt [29% ], anti - vegf [14% ], and argon - laser treatment [5% ]). posterior cystoid retinal degeneration, described previously by piccolino, was seen in seven eyes of four patients (8% ; figure 1d g).11 in three of these seven eyes, a lesion suspected to be choroidal neovascularization developed during follow - up. in three of seven eyes, the posterior cystoid retinal degeneration disappeared completely during follow - up, in two eyes this resolution occurred spontaneously, and in one eye it occurred after pdt (figure 1h and i). the onset of posterior cystoid retinal degeneration was 2, 10, and 16 years after the reported start of the ccsc in three of the four patients in whom this was recorded (figure 1d g). in 21 eyes (41%) with srf and six eyes without srf, spectral domain oct was available for evaluation of cft on follow - up. cft showed a mean decrease of 15.1 m (standard deviation : 17.7 m) in eyes with srf, whereas no significant change was seen in eyes without srf. characteristics of hot spots of leakage on fa on follow - up are shown in table 2. of the nine eyes that showed a decrease in number of hot spots during follow - up, in seven the original hot spots disappeared after treatment, whereas the number of hot spots decreased spontaneously in the remaining two eyes. overall, the mean number of hot spots at the first visit was 1 (range : 04) compared to 1.7 (range : 07) at the last visit (figure 2a and b). an increase in atrophic rpe changes on fa was seen in 14 eyes (41%) during a mean follow - up of 9.7 years (range : 224 years). a classic gravitational tract was seen in eight eyes (15%), and was already present at the first visit in five eyes (figure 2c and d). the other three eyes developed the gravitational tract after approximately 2, 4, and 8 years, respectively. when icga was available (in 28 eyes [54% ]), hot spots and hyperfluorescent areas on icga in 27 eyes (96%), was compared with characteristics on fa that was performed on the same date. the overall hyperfluorescent area was larger on icga in eight eyes (30%), in eleven eyes (41%) the hyperfluorescent area had comparable size, in one eye (4%) the hyperfluorescent area was smaller on icga, and in eight eyes (30%) no clear hyperfluorescence was seen on icga to be compared (figure 2e and f). in eleven eyes (39%), icga showed multiple small punctate hyperfluorescent spots, without clear evidence of a leaking hot spot focus (figure 2 g and h). color fundus photography was available at baseline in 42 eyes (81%), and mild rpe atrophy in the macula was seen in 18 eyes (43%). follow - up images were available for 31 eyes (74%), with a mean follow - up of 45 months (range : 1.587 months), and evidence of development and/or progression of rpe atrophy was present in 14 eyes (45%). patients of this ccsc cohort scored significantly less on the vfq-39 questionnaire compared to the reference group on eight of the 12 subcategories (table 3). patients with a lower last recorded va scored significantly lower on the following categories of the vfq-39 : general vision, near activities, distance activities, social functioning, mental health, role difficulties, dependence, and driving (table 3). the mean number of episodes of active disease per eye was 1.7 (range : 06). each episode was defined as a presence of documented srf followed by a period of oct - proven absence of srf. when the va of the first and last visits was compared, excluding the 21 eyes (40%) with persistent subfoveal srf at the final visit, a mean decline of 0.16 logarithm of minimum angle of resolution (logmar ; range : 0.22 to 1.3, p=0.009) was found after a mean follow - up period of 10.6 (range : 1.524) years. in the 21 eyes (40%) with persistent subfoveal srf at the final visit, a mean decline of 0.22 logmar (range : 0.35 to 0.66, p=0.003) with a mean follow - up period of 4.5 (range : 111) years was seen. in 11 of these 21 eyes (52%), the srf was continuously present during follow - up. neither the number of episodes of active disease nor number of treatments had a significant effect on overall change in va. an overview of oct findings is displayed in table 2 and figure 1a c. in the 13 eyes with a decrease in srf, eight (62%) the seven eyes with an increase of srf had not received treatment. in the eyes showing fluctuating srf accumulation, 18 (82%) had received treatment. the following treatments were performed : 41 micropulse therapy, 23 photodynamic therapy (pdt), one argon - laser treatment, one anecortave acetate treatment, and nine anti - vegf treatments. in 14 eyes (24%), srf was continuously present on oct until the last follow - up. in this group, this was also the case in five eyes (36%) despite receiving treatment (micropulse therapy [52% ], pdt [29% ], anti - vegf [14% ], and argon - laser treatment [5% ]). posterior cystoid retinal degeneration, described previously by piccolino, was seen in seven eyes of four patients (8% ; figure 1d g).11 in three of these seven eyes, a lesion suspected to be choroidal neovascularization developed during follow - up. in three of seven eyes, the posterior cystoid retinal degeneration disappeared completely during follow - up, in two eyes this resolution occurred spontaneously, and in one eye it occurred after pdt (figure 1h and i). the onset of posterior cystoid retinal degeneration was 2, 10, and 16 years after the reported start of the ccsc in three of the four patients in whom this was recorded (figure 1d g). in 21 eyes (41%) with srf and six eyes without srf, spectral domain oct was available for evaluation of cft on follow - up. cft showed a mean decrease of 15.1 m (standard deviation : 17.7 m) in eyes with srf, whereas no significant change was seen in eyes without srf. characteristics of hot spots of leakage on fa on follow - up are shown in table 2. of the nine eyes that showed a decrease in number of hot spots during follow - up, in seven the original hot spots disappeared after treatment, whereas the number of hot spots decreased spontaneously in the remaining two eyes. overall, the mean number of hot spots at the first visit was 1 (range : 04) compared to 1.7 (range : 07) at the last visit (figure 2a and b). an increase in atrophic rpe changes on fa was seen in 14 eyes (41%) during a mean follow - up of 9.7 years (range : 224 years). a classic gravitational tract was seen in eight eyes (15%), and was already present at the first visit in five eyes (figure 2c and d). the other three eyes developed the gravitational tract after approximately 2, 4, and 8 years, respectively. when icga was available (in 28 eyes [54% ]), hot spots and hyperfluorescent areas on icga in 27 eyes (96%), was compared with characteristics on fa that was performed on the same date. the overall hyperfluorescent area was larger on icga in eight eyes (30%), in eleven eyes (41%) the hyperfluorescent area had comparable size, in one eye (4%) the hyperfluorescent area was smaller on icga, and in eight eyes (30%) no clear hyperfluorescence was seen on icga to be compared (figure 2e and f). in eleven eyes (39%), icga showed multiple small punctate hyperfluorescent spots, without clear evidence of a leaking hot spot focus (figure 2 g and h). color fundus photography was available at baseline in 42 eyes (81%), and mild rpe atrophy in the macula was seen in 18 eyes (43%). follow - up images were available for 31 eyes (74%), with a mean follow - up of 45 months (range : 1.587 months), and evidence of development and/or progression of rpe atrophy was present in 14 eyes (45%). patients of this ccsc cohort scored significantly less on the vfq-39 questionnaire compared to the reference group on eight of the 12 subcategories (table 3). patients with a lower last recorded va scored significantly lower on the following categories of the vfq-39 : general vision, near activities, distance activities, social functioning, mental health, role difficulties, dependence, and driving (table 3). this study shows that ccsc is a progressive chorioretinopathy with a significant impact on va and vision - related quality of life. patients in this study cohort generally showed a progressive decline in va, as well as a gradual decrease in cft on oct. wang demonstrated in the pre - oct era that persistence of srf for more than 4 months can result in foveal atrophy.12 this permanent damage to photoreceptors and the rpe can explain the often - persistent visual complaints, even after resolution of srf. the progressive nature of ccsc was demonstrated on fa, characterized by an increase of hyperfluorescent atrophic rpe changes and an increased number of hot spots during follow - up. an interesting finding in this study were the small punctate hyperfluorescent spots on icga (figure 2h). similar lesions were described by tsujikawa.13 this group described small clusters, which were located within the center of the focal hyperfluorescent area on icga and thus thought to be very small leakage spots. in this study, no clear abnormalities corresponding to these lesions on icga could be identified on oct. lehmann identified similar hyperfluorescent lesions on icga that correlated with vascular dilations on en face enhanced - depth oct.14 in contrast to the lesions seen in this study, the lesions were always located under an area of srf accumulation and/or rpe detachment. additionally, a clinical and pathogenetic overlap between csc and polypoidal choroidal vasculopathy may exist. punctate hyperfluorescent spots on icga have also been identified in polypoidal choroidal vasculopathy, in which these lesions correlated with a thicker choroid.15 we thus hypothesize that these lesions may be focal vascular dilations in the choriocapillaris that fill on icga. the blood flow in the choroid is among the highest in the human body, and the macular choriocapillaris is thicker, has a distinct lobular architecture, and higher blood flow compared to the peripheral choroid.16,17 these anatomical and functional choroidal differences, as well as anatomical differences between central and peripheral rpe and bruch s membrane,18,19 probably explain why csc principally affects the posterior pole. however, it is currently unclear why some individuals develop active csc in the case of a dysfunctional thickened choroid (pachychoroid) unlike others,20 and why in the same individual the disease can remain subclinical in the fellow eye despite the presence of similar choroidal abnormalities.5 it is likely that additional factors play a role in determining an individual s likelihood to develop csc, for instance, patient - specific differences in genetic background,7,8 local differences in rpe outer blood retina function, and possibly the interphotoreceptor matrix.21 this studies data showed a marked decrease in reported vision - related quality of life in this ccsc cohort when compared to healthy individuals. nevertheless, the impact of ccsc on vision - related quality of life seems to be less pronounced than in other common maculopathies, such as neovascular age - related macula degeneration and diabetic retinopathy.22,23 in this ccsc cohort, social functioning and dependence seemed to be most affected. differences between the vision - related quality of life impact of the different diseases may not only be due to the variable effects on macular anatomy and visual function but may also be partly explained by the difference in the mean age of the different study populations during which the vfq-39 questionnaire was taken (present study 55 years, neovascular age - related macular degeneration 77 years, diabetic retinopathy 68 years, and diabetic macular edema 62 years).23 also, mean years since diagnosis was far shorter in the neovascular age - related macular degeneration studies compared to the present study (0.6 and 7.4 years). a possible explanation could also be that younger individuals are more flexible and capable of coping with newly developed visual impairment than older individuals.24 also, the negative impact on reported vision - related quality of life appears to be higher when evaluated closer to the onset of visual impairment. surprisingly, a significant difference in the reported quality of color vision was not found, although impaired color vision has been previously reported in csc.25 it is still possible that disturbed color vision exists in this cohort, but that it remains subclinical, for example, due to a better - seeing fellow eye, and does not interfere with daily activities. ccsc is a progressive chorioretinopathy, with many ccsc patients experiencing significant vision loss, lower vision - related quality of life, and lower rating for overall health compared to healthy individuals. despite these observations, it is not uncommon for ophthalmologists to adopt a conservative approach in these patients, who are often relatively young. the findings of this study demonstrate that the possible impact of ccsc should not be underestimated, and thus an active treatment approach may be advocated. however, few high - quality randomized controlled treatment trials have been reported in ccsc, with a current lack of a gold standard for treatment of ccsc. we are currently performing a multicenter prospective randomized controlled treatment trial, comparing half - dose pdt with high - density subthreshold micropulse - laser treatment in ccsc (eudract 2012 - 004555 - 36, nct01797861) in an attempt to identify the most suitable treatment strategy in ccsc.	purposeto describe the clinical findings and long - term outcome of patients with chronic central serous chorioretinopathy (ccsc).materials and methodsthis was a retrospective case series in 52 eyes of 36 patients with a follow - up period of at least 1 year. extensive ophthalmic examination and a validated questionnaire concerning vision - related quality of life (national eye institute visual function questionnaire [nei - vfq]-39) were analyzed.resultsmean visual acuity showed a significant decline over time of 0.16 logarithm of minimum angle of resolution ([logmar ] range : 0.22 to 1.3 ; p=0.009) after a mean follow - up period of 10.6 years. also, patients reported lower vision - related quality of life based on the nei - vfq-39 for almost all categories compared to healthy controls. macular atrophy was diagnosed more often on optical coherence tomography compared to other diagnostic entities. retinal pigment epithelium detachments in the macula were documented on optical coherence tomography in 56% of the patients. a significant thinning of foveal thickness was measured over time compared to unaffected fellow eyes (p=0.002). on long - term follow - up, 13 eyes (37%) showed an increase in number of hot spots on fluorescein angiography.conclusionthis study indicates that ccsc is a progressive disease in many patients, causing a progressive decline in visual acuity, accompanied by lower reported vision - related quality of life. in deciding whether or not to treat, the progressive nature of ccsc should be taken into account in this relatively young and often still professionally active patient group.
a maxillary oral squamous cell carcinoma (scc) is generally treated with a conventional surgical excision. the resultant surgical defect often includes part of the hard and soft palates, which results in an oro - antral communication. the hard and soft palates are anatomical structures that have widely recognized roles in speech and deglutition. when these structures must be removed, partially or completely, because of malignancies, a team approach is critical. a surgical approach alone without reconstruction or obturation of the surgical defect will result in air, liquid, and food escaping into the maxillary sinus and nasal cavities, causing severe speech and swallowing dysfunction with significant reduction in quality of life. speech is often unintelligible as a result of the marked defects in articulation and nasal resonance resulting from the anatomical and structural defect. thorough pre- and postsurgical reconstructive and prosthetic treatment planning will ensure the best rehabilitation of a maxillectomy patient. numerous techniques and materials for making obturators have been suggested.[417 ] this clinical technique describes oral rehabilitation of a patient with sub - total maxillectomy with a maxillary obturator. a 55-year - old female diagnosed with scc of the right maxillary sinus had undergone hemimaxillectomy. sub - total maxillectomy was performed and after proper healing, she was referred to the department of prosthodontics, government dental college, bangalore, india. extra - oral examination revealed reduced fullness due to loss of support [figure 1 ]. on intraoral examination, a big communication was present between nasal and oral cavity except a minor part of the palatine process of maxilla and horizontal plate of palatine bone [figure 2 ]. pre - treatment photograph showing reduced fullness intra - oral photograph showing the defect speech pathology, head and neck, and maxillofacial prosthetics / dental oncology services were consulted to evaluate the extent of resection and functional deficits. a consensus among the specialists recommended an obturator to restore speech and swallowing abilities and esthetics. the impression was boxed and poured in type iv stone (ultrarock, kalabhai karson pvt. record bases were made with heat cure acrylic resin (lucitone 199, dentsply int.) [figure 5 ]. the jaw relation was recorded. after the try - in, the solid obturator was processed in heat polymerized acrylic resin (lucitone 199, dentsply int.). maxillary impression along with the defe master cast showing the resected area heat - cure acrylic record base after curing of the prosthesis, the flash was trimmed. the prosthesis was pumiced, polished, and delivered to the patient [figures 68 ]. maxillary obturator in patient 's mouth patient 's frontal view along with the prosthesis post treatment photograph with increased fullness and improved esthetics the prosthetic retention and stability were evaluated subjectively. speech intelligibility improved and the patient was then referred to the speech pathology department for further evaluation of his speech and swallowing abilities. obturator prosthesis for maxillary defects is frequently associated with problems that result from the lack of retention and stability. maxillofacial reconstruction of the partition between the nasal and oral cavities in the edentulous patient relies on anatomical undercuts and structures, such as remnants of the soft palate, palatine process of maxilla, and horizontal plate of palatine bone, for retention. an appropriate prosthetic fit and functional success ensure that the patient ultimately uses the device during daily routines. it is important that clinicians do not overlook the importance of referral to the speech pathologist, particularly in patients for whom the success of a prosthetic obturator after total or sub - totalmaxillectomy depends on the ability to adequately speak and swallow. subtotal and total bilateral maxillectomydefects represent a complex challenge for the maxillofacial prosthodontist. in this clinical report, preoperative treatment planning involving the head and neck surgeon, the maxillofacial prosthodontist, and the speech pathologist resulted in a obturator that enabled the patient to speak and swallow successfully.	this clinical report describes oral rehabilitation of a patient with sub - total maxillectomy with palatine process of maxilla and horizontal plate of palatine bone intact to retain the maxillary obturator. clinical examination has been performed to know the amount of favorable undercuts to be used for retention of the obturator for better functional efficiency. successful prosthetic reconstruction of hemimaxillectomy defect is a challenging procedure that requires multidisciplinary expertise to achieve acceptable functional speech and swallowing outcomes. this article describes the oral rehabilitation of a patient with sub - total maxillectomy with a maxillary obturator. oral rehabilitation of sub - total maxillectomy patient is a challenging task. obturation of the defect depends on volume of the defect, and positioning of remaining hard and soft tissues to be used to retain, stabilize, and support the prosthesis. a maxillary obturator for edentulous patient must provide for retention, stability, support, patient comfort, and cleanliness.
it usually occurs in middle - aged men who sustain a sudden force to a flexed arm. this injury represents only 3% of all injuries to the biceps tendon ; 96% of such injuries involve the long head, and 1% involve the short head [1, 2 ]. anatomic repair of the ruptured distal biceps tendon to the radial tuberosity is recommended to prevent the loss of strength and endurance in forearm supination and elbow flexion [2, 3, 4, 5 ]. repair of distal biceps tendon rupture using bone tunnels, suture anchors, interference screws or the endobutton has been described in many articles [2, 6, 7, 8, 9 ]. endobutton repair of distal biceps tendon ruptures was first described by bain and colleagues in 2000. subsequent clinical and biomechanical studies have demonstrated high patient satisfaction, a very low incidence of complications and higher load to failure when compared to other techniques [7, 8, 10, 11, 12, 13 ]. in this article, we describe our modified endobutton and tension slide technique for distal biceps tenodesis, along with tips and tricks aimed to avoid the possible complications associated with this procedure. we report six consecutive patients, presented with distal biceps tendon rupture (4 acute ; 2 chronic cases) between june 2013 and march 2015, who underwent single - incision, anatomical distal biceps tenodesis procedure with bicortical endobutton and tenodesis screw using tension slide technique (tst). radiographs were taken immediate post - operative to document for displacement or loosening of endobutton if any. all surgical procedures were done under general anesthesia, with the arm placed onto an arm table and the elbow extended with the tourniquet inflated. a longitudinal 4 - 5 cm anterior incision starting at the antecubital fossa and extending distally along the ulnar border of the brachioradialis, exploiting the interval between the brachioradialis and pronator teres with radial (lateral) retraction of the brachioradialis and medial retraction of the pronator teres. the lateral antebrachial cutaneous nerve is identified and protected as it exists between the biceps and the brachialis at the antecubital fossa. ligation of the recurrent branch of the radial artery minimizes hematoma formation and may protect against heterotopic ossification (ho). the lateral antebrachial cutaneous nerve and the posterior interosseous nerve (pin) flexing the elbow and using a retractor to elevate the tissue of the distal brachium anteriorly can accomplish this by allowing better exposure. once the tendon is isolated, a number 5 synthetic polyester suture (ethibond excel, ethicon inc. ; johnson and johnson, usa) is woven into the distal biceps tendon using krackow technique or whipstitch suture technique (fig. we used 4 holed endobutton without continuous loop (4.0 mm 12 mm endo - button cl ultra, smith and nephew, andover, ma, usa). one limb of the suture from the tendon end was threaded through the central two holes of the endobutton (inside out outside in). the other suture end from the tendon was passed through the terminal two holes of the endobutton in a reverse manner, i.e., from outside in inside out) (fig.. illustrations of the surgical steps in the tension - slide technique of anatomical distal biceps tendodesis using an endobutton. ((d) reaming the anterior cortex and the intramedullary canal over the guide wire using a cannulated drill. (e) drilling the posterior cortex over the guide wire using an endobutton drill. (h) tying the knot under tension after passing the suture ends through the distal end of the biceps tendon, adjacent to the bone tunnel. (i) insertion of a unicortical tenodesis screw on the radial aspect of the hole, pushing the tendon ulnarly to mimic the natural anatomy. (a) the distal end of the biceps tendon is sutured by krachow suturing technique using no.5 synthetic polyester suture (ethibond). (d) end of the procedure after tying suture knots and insertion of tenodesis screw. with the elbow in full extension and full supination, the radial tuberosity was exposed and debrided of any remaining soft tissue. under c - arm image guidance, a 2.7 mmx 15 guide wire is passed through the radial tuberosity, aiming 30 ulnar, with the forearm in maximum supination so that guide pin trajectory is away from pin (fig. 1c and 2c). fluoroscopic images were used to confirm that the guide pin is passed through center of the tuberosity and crossed only 2 - 3 mm beyond the posterior cortex. then, the size of the tendon we prepared was measured, using tendon - sizing block (fig. if the size of the tendon is 8 mm, same size endoscopic cannulated drill is passed over the guide wire to drill the anterior cortex and intramedullary canal (fig. then, a 4.5 mm endobutton drill is used over the guide wire to drill the posterior cortex (fig. the guide wire and drill were then removed and copious irrigation of the wound was done to remove bone dust and fragments. care is taken to ensure that the tendon is appropriately aligned. placing the elbow in 20 - 30 flexion and full supination, hold the looped endobutton at the tip of long thin straight artery forceps or endobutton holder and pass through both drill holes to deliver the endobutton to the dorsal aspect of the proximal radius (fig. once the button cleared the posterior cortex, the deployment instrument was removed while the button deployed itself, locking into place on the posterior cortex. the sutures were tensioned until the tendon was in contact with the posterior cortex. by placing tension onto the trailing suture fluoroscopy is useful to assess the position of the endobutton as it is advanced through the radius and locked into position (fig. knots are tied under tension after back passing one of the suture threads through the biceps tendon just adjacent to anterior cortex (fig. (a and b) intra - operative radiographic images showing ideal position of the endobutton at the end of the procedure. a 7 mm 10 mm (one size less than size of the tendon) unicortical interference screw (peek tenodesis screw, arthrex inc. naples, fl, usa) was then inserted on the radial aspect of the hole, pushing the tendon ulnar to mimic the natural anatomy and potentially increase supination strength (fig. 1i and 2d). all surgical procedures were done under general anesthesia, with the arm placed onto an arm table and the elbow extended with the tourniquet inflated. a longitudinal 4 - 5 cm anterior incision starting at the antecubital fossa and extending distally along the ulnar border of the brachioradialis, exploiting the interval between the brachioradialis and pronator teres with radial (lateral) retraction of the brachioradialis and medial retraction of the pronator teres. the lateral antebrachial cutaneous nerve is identified and protected as it exists between the biceps and the brachialis at the antecubital fossa. ligation of the recurrent branch of the radial artery minimizes hematoma formation and may protect against heterotopic ossification (ho). the lateral antebrachial cutaneous nerve and the posterior interosseous nerve (pin) flexing the elbow and using a retractor to elevate the tissue of the distal brachium anteriorly can accomplish this by allowing better exposure. once the tendon is isolated, a number 5 synthetic polyester suture (ethibond excel, ethicon inc. ; johnson and johnson, usa) is woven into the distal biceps tendon using krackow technique or whipstitch suture technique (fig. we used 4 holed endobutton without continuous loop (4.0 mm 12 mm endo - button cl ultra, smith and nephew, andover, ma, usa). one limb of the suture from the tendon end was threaded through the central two holes of the endobutton (inside out outside in). the other suture end from the tendon was passed through the terminal two holes of the endobutton in a reverse manner, i.e., from outside in inside out) (fig. illustrations of the surgical steps in the tension - slide technique of anatomical distal biceps tendodesis using an endobutton. (d) reaming the anterior cortex and the intramedullary canal over the guide wire using a cannulated drill. (e) drilling the posterior cortex over the guide wire using an endobutton drill. (f) inserting a threaded endobutton through the bone tunnel. (g) tightening the suture threads and flushing the endobutton on the posterior cortex. (h) tying the knot under tension after passing the suture ends through the distal end of the biceps tendon, adjacent to the bone tunnel. (i) insertion of a unicortical tenodesis screw on the radial aspect of the hole, pushing the tendon ulnarly to mimic the natural anatomy. (a) the distal end of the biceps tendon is sutured by krachow suturing technique using no.5 synthetic polyester suture (ethibond). (d) end of the procedure after tying suture knots and insertion of tenodesis screw. with the elbow in full extension and full supination, the radial tuberosity was exposed and debrided of any remaining soft tissue. under c - arm image guidance, a 2.7 mmx 15 guide wire is passed through the radial tuberosity, aiming 30 ulnar, with the forearm in maximum supination so that guide pin trajectory is away from pin (fig. fluoroscopic images were used to confirm that the guide pin is passed through center of the tuberosity and crossed only 2 - 3 mm beyond the posterior cortex. then, the size of the tendon we prepared was measured, using tendon - sizing block (fig. usually, the size of the tendon is around 7 - 8 mm. if the size of the tendon is 8 mm, same size endoscopic cannulated drill is passed over the guide wire to drill the anterior cortex and intramedullary canal (fig. then, a 4.5 mm endobutton drill is used over the guide wire to drill the posterior cortex (fig. the guide wire and drill were then removed and copious irrigation of the wound was done to remove bone dust and fragments. care is taken to ensure that the tendon is appropriately aligned. placing the elbow in 20 - 30 flexion and full supination, hold the looped endobutton at the tip of long thin straight artery forceps or endobutton holder and pass through both drill holes to deliver the endobutton to the dorsal aspect of the proximal radius (fig. once the button cleared the posterior cortex, the deployment instrument was removed while the button deployed itself, locking into place on the posterior cortex. the sutures were tensioned until the tendon was in contact with the posterior cortex. by placing tension onto the trailing suture, the endobutton locks into position and prevents proximal migration of the tendon (fig. fluoroscopy is useful to assess the position of the endobutton as it is advanced through the radius and locked into position (fig. knots are tied under tension after back passing one of the suture threads through the biceps tendon just adjacent to anterior cortex (fig. (a and b) intra - operative radiographic images showing ideal position of the endobutton at the end of the procedure. a 7 mm 10 mm (one size less than size of the tendon) unicortical interference screw (peek tenodesis screw, arthrex inc. naples, fl, usa) was then inserted on the radial aspect of the hole, pushing the tendon ulnar to mimic the natural anatomy and potentially increase supination strength (fig. postoperatively, patients were placed in a soft dressing, and early passive range of motion (rom) exercises started from day one and gravity - assisted flexion and extension started after 2 weeks. active strengthening exercises started at 8 weeks, with an expected return to heavy activities at 3 - 5 months, depending on the patient. all patients were satisfied with their outcome and were able to return to activities of daily living. all patients had an intact biceps tendon and had a return of grade 5 power. the average range of flexion was from 5 to 130. the average supination was 80 and average pronation 75. none of the patients suffered failure of fixation with immediate active motion. patients achieved near full rom and near complete return of strength and endurance without any complications. surgical techniques for the repair of distal biceps tendon tears using an endobutton have been previously described [7, 8, 10, 11 ]. in this article, we describe a technique in which we consider the best possible modifications of the previously described techniques, aiming to simplify the surgical procedure, achieve a very strong fixation, excellent patient outcome, and avoid the possible surgical complications. the technique, we describe, is minimally invasive as it requires only a small anterior longitudinal incision, through which the surgeon can identify and repair the biceps tendon. this single incision technique has an advantage over the double incision technique described by boyd and anderson, which has the risk of ho, radioulnar synostosis, stiffness, and pin palsy [15, 16 ]. we favor the longitudinal incision, rather than the transverse incision which has been associated with a high rate of neurologic complications. the transverse incision can be used in case of acute injuries, where the avulsed distal biceps tendon can be found without much exploration and tissue retraction. on the other hand, in chronic biceps tendon rupture, the incision has to be extended proximally to find the retracted tendon and retrieve it back to its anatomical site. moreover, having the transverse skin incision at the antecubital crease risks drilling the cortex with distally directed trajectory, which in turn increase the risk of damaging the pin. an anatomic study evaluating the safest trajectory of guide pins during biceps repair found that avoiding a radially directed trajectory reduces the risk of injury to the pin. moreover, the authors found that the best way to prevent nerve injury was aiming guide pin 30 ulnarly through center of radial tuberosity, with forearm in maximum supination, which increases the distance between the guide wire and the pin [7, 9, 18 ]. drilling over distally or radially directed guide wire, placed the pin at significant risk of injury, compared with an ulnar or direct anterior to posterior trajectory [7, 18 ]. it is important that the bony bridge between the anterior cortical window and the posterior drill hole is adequate to minimize the risk of fracture. irrigation of the wound after drilling, single incision approach, ligation of the recurrent branch of the radial artery and a minimally invasive technique, all reduce the risk of ho. we believe that the 4-holed endobutton provides a greater surface area of contact, thus allowing distribution of stress on posterior cortex through suture threads. on the other hand, two threads have to be passed through each hole in the 2-holed endobutton, which may restrict the free sliding. biomechanical studies have proven that the tst maintains the strength of a suspensory cortical button fixation and significantly reduces gap formation and motion at the repair site. conducted a biomechanical study measuring cyclical load to failure in four techniques for distal biceps brachii tendon repair. the endobutton technique had a statistically significant (p < 0.001) highest load to failure (440 n) compared with suture anchor (381 n), bone tunnel (310 n), and the interference screw (232 n). endobutton and tst have been shown to have the highest ultimate tensile load [8, 13 ]. another advantage is the ability to start early active rom exercises compared to other fixation methods. the advantages of the tst include the ability to tension the repair from an anterior incision, and there is no need to predetermine the length of suture between the button and the biceps tendon stump. we recommend using c - arm image / fluoroscopy, while passing the guide wire, drilling the cortex and confirming the position of endobutton before tightening the sutures. this avoids mal trajectory of the guide wire, blowing out the posterior cortex with first drill, intramedullary dislodgement of the endobutton just before tightening the suture threads and inter - positioning of the pin or soft tissue between the posterior cortex and the endobutton, which is depicted by a gap between the endobutton and the posterior cortex in a lateral view image. due to the fact that number 5 synthetic polyester sutures were used to secure the endobutton, and because the tendon is positioned within the intramedullary canal, allowing maximum surface area between the tendon and the bone that is why we can start early rom exercises after this procedure compared to other fixation techniques. in this technique, we use unicortical interference / tenodesis screw in addition to an endobutton, which is inserted on the radial aspect of the hole, pushing the tendon ulnar to mimic the natural anatomy and potentially increasing the supination strength. as mentioned earlier, many surgeons used a combination of an endobutton and interference screws for this procedure, in which they maintained tension on the suture after deploying the endobutton, followed by insertion of the interference screw. this technique acts as a single unit of fixation. on the contrary, in our technique, the suture knot is tied under tension after back passing the suture through the tendon itself, which itself is an independent fixation. we achieve two different independent fixations, which act synergistically and ensures the fixation is held in place, even if one of the two fixations fail. the use of an endobutton and an interference screw for repairing distal biceps tendons has been previously described. we describe a modification of originally described technique which is worth considering, as it provides two levels of fixation, whilst avoiding possible complications of such procedures. it is ideal for repairing both acute and chronic ruptures, without the need for allograft or autograft augmentation and describes detailed technical steps to avoid possible iatrogenic complications. even though our study group is small and needs further extensive research considering a larger study group, long - term evaluation along with biomechanical studies to validate our surgical technique further, we believe that these surgical tips and tricks will definitely help the surgeons dealing with such cases.	introduction : many surgical techniques have been described in the literature. in this article, we describe surgical technical details along with tips and tricks of distal biceps tendon tenodesis using the endobutton and tension slide technique, a modification of the suspensory cortical button technique, which allows the surgeon to tension and repairs the biceps tendon through the single longitudinal anterior incision. this modification in surgical technique of using dual implants, i.e., endobutton and interference screw as fixation tools and concept of tendon sliding principle made this procedure unique. in this article, we describe surgical technique along with tips and tricks of distal biceps tendon tenodesis using the endobutton and tension slide technique and also discussed about modification of endobutton technique reported in many other articles to overcome the possible complications.case report : we report six consecutive patients, presented with distal biceps tendon rupture (4 acute ; 2 chronic cases) between june 2013 and march 2015, who underwent single - incision, anatomical distal biceps tenodesis procedure with bicortical endobutton and tenodesis screw using tension slide technique. radiographs were taken immediate post - operative to document for displacement or loosening of endobutton if any.conclusion:the use of an endobutton and an interference screw for repairing distal biceps tendons have been previously described. we describe a modification of originally described technique which is worth considering, as it provides two levels of fixation, whilst avoiding possible complications of such procedures. it is ideal for repairing both acute and chronic ruptures, without the need for allograft or autograft augmentation and describes detailed technical steps to avoid possible iatrogenic complications.
diabetes mellitus affects approximately 20% of american adults over the age of 60, and diabetic retinopathy is a leading cause of blindness in older americans.1 diabetic retinopathy is also impacting younger age groups as the incidence of diabetes rises in adolescents and younger adults.2 diabetic retinopathy is therefore a significant public health problem.1 because vision loss from diabetic retinopathy is painless and usually gradual in onset, ophthalmologic evaluation is necessary to distinguish diabetic retinopathy from other causes of painless vision loss in diabetic patients, such as cataract and retinal vein occlusion. furthermore, diabetic retinopathy may be present before vision loss occurs, and progressive retinal damage may be overlooked until the disease is advanced. the incidence of diabetic retinopathy increases with duration of disease,2 and retinopathy occurs more frequently in patients with poorly controlled diabetes. it is therefore imperative that patients with diabetic retinopathy have appropriate management of both their systemic diseases and their ophthalmic condition.3 a thorough review of medical conditions and medications is important, particularly because diabetic retinopathy may be exacerbated by systemic medications such as glitazones, in which case macular edema may resolve with discontinuation.4 diabetic retinopathy occurs because the retinal vessels are abnormal, either because they proliferate (proliferative retinopathy) or because the vessels are functionally incompetent and leak fluid and lipid into the retina. visual impairment occurs when edema affects the central retina or macula (diabetic macular edema, or dme). macular edema is reversible in the early stages but chronic edema may lead to irreversible changes in the retina. in a large cohort study, diabetics with macular edema had a 50% prevalence of visual impairment and a 20% prevalence of blindness, compared to 16% and 4% respectively in diabetic patients without macular edema.5 figures 1a1b and 2a2b2c contrast the ophthalmic features of macular edema with a normal retina. most of these treatments are currently used as second - line therapy for patients with macular edema refractory to thermal laser, but an increasing number of studies have examined first - line use of these alternative therapies. this paper provides an overview of primary treatment options for diabetic macular edema, along with the rationale for each treatment modality. laser treatment is delivered at a slit lamp using a contact lens on the cornea. since the sensory retina is transparent, the laser energy is absorbed by vascular structures within the retina or by the pigmented epithelium immediately behind the sensory retina. focal laser spots may be applied to microaneurysms in order to stop leakage of fluid. a grid pattern of laser spots also may be applied lightly to the pigmented epithelium in areas of more diffuse edema. the pigmented epithelium functions as a pump to remove fluid from the retina, and stimulation with laser spots is thought to stimulate pump function and thereby decrease retinal edema.6 the early treatment of diabetic retinopathy study (etdrs), a large randomized controlled trial completed in 1985, demonstrated that prompt laser treatment of the macula reduced the frequency of vision loss by 50% in diabetic patients with macular edema compared to untreated control subjects.3 in addition, almost half of the patients with poor vision initially demonstrated some improved vision at three years of follow - up. patients typically need more than one laser treatment and long term follow - up is needed so that recurrent edema can be managed in a timely manner. diabetic macular edema is caused by a cascade of vascular permeability factors that include the production of interleukin-6 (il-6) and vascular endothelial growth factor (vegf). il-6 and vegf are present in higher concentrations in eyes with diabetic retinopathy and steroid administration reduces the concentration of these factors with concurrent clinical improvement of macular edema.7,8 there are two ways to deliver steroids into the vitreous cavity. the first is by direct injection into the vitreous cavity (intravitreal injection) ; triamcinolone is the most frequently used steroid for this purpose. in a recent prospective randomized study of more than 100 patients, intravitreal injection of triamcinolone improved visual acuity and decreased retinal thickness compared to baseline, and neither endpoint was significantly different compared to macular laser treatment alone or laser combined with triamcinolone.9 these findings suggest that triamcinolone treats dme at least as well as laser alone over six months of follow - up, although some patients in the study had received previous laser treatment.9 intravitreal triamcinolone has also been successfully combined with macular laser treatment as the initial therapy for diabetic macular edema.10,11 in addition, numerous studies have shown that intravitreal steroids reduce macular edema in cases that are refractory to laser treatment, although visual acuity does not consistently improve in these cases, probably due to permanent damage to retinal photoreceptors by chronic edema.12,13 side effects of intravitreal steroids include accelerated development of cataracts as well as elevated intraocular pressure that may lead to glaucomatous damage to the optic nerve. these complications may require surgical correction.12 a second way to deliver intravitreal steroids is by sustained - release steroid implants. the posurdex implant (dexamethasone posterior segment drug delivery system ; allergan inc, irvine, ca, usa) releases dexamethasone into the vitreous cavity for up to six months, while the retisert implant (fluocinolone acetonide intravitreal implant ; bausch and lomb inc, rochester, ny, usa) releases fluocinolone acetonide. both implants have been shown to improve vision in patients with diabetic macular edema, but both are relatively expensive and carry side effects similar to intravitreal triamcinolone injections.13 since vegf contributes to the development of macular edema, attention has recently focused on intravitreal injections of anti - vegf antibodies. antibodies delivered by intravitreal injection bind to vegf and thereby decrease downstream effects on vascular leakage. anti - vegf antibodies were first used to treat patients with exudative age - related macular degeneration (amd) with dramatic beneficial results. because of these benefits, anti - vegf antibodies are now being evaluated in the treatment of macular edema. bevacizumab (avastin ; genentech, san francisco, ca) was approved by the fda for intravenous use in cancer patients. this drug has been used off - label as an intravitreal injection, first for amd, and subsequently for a variety of other ocular diseases including macular edema.14 ranibizumab (lucentis ; genentech, san francisco, ca) is fda - approved for intravitreal injection in patients with amd, and has been used off - label for other ocular diseases including diabetic macular edema.15 pegaptanib (macugen ; osi pharmaceuticals, melville, ny) was fda - approved for intravitreal injection in patients with amd before the advent of ranibizumab and bevacizumab. pegaptanib has fallen out of favor for use in amd patients but has been studied in patients with diabetic macular edema. two prospective randomized controlled trials recently demonstrated greater improvements in visual acuity, albeit with just trhee months follow - up, in patients receiving intravitreal bevacizumab compared to controls receiving laser treatment.16,17 bevacizumab has also produced improvements in visual acuity and retinal thickness in patients who are refractory to initial laser treatment.18,19 the initial results are promising but follow up is limited. long - term follow - up may help to establish the utility of intravitreal bevacizumab for diabetic macular edema, as well as determine the regimen of treatment to maintain vision and prevent recurrence. data regarding ranibizumab for diabetic macular edema is more limited, likely due to the high price of ranibizumab compared to bevacizumab. two small prospective studies demonstrated maintenance or improvement of vision in patients treated primarily with ranibizumab.20,21 pegaptanib was shown in a phase ii randomized controlled trial to decrease vision loss and increase vision gain compared to a sham medication, with a decreased need for subsequent photocoagulation.22 however, pegaptanib was not directly compared to thermal laser in this study. side effects of intravitreal anti - vegf injections are rare and include retinal detachments, which are most likely due to the injection procedure itself rather than the pharmaceutical compound (the risk of retinal detachment is common to other intravitreal injections such as triamcinolone). similarly, endophthalmitis is a rare complication of intravitreal injection and is likely related to the injection itself rather than specific pharmaceutical characteristics.23 intravitreal bevacizumab and ranibizumab do not result in significant elevations of intraocular pressure nor have they been shown to accelerate cataract formation to any appreciable degree. pars plana vitrectomy is a surgical procedure in which the vitreous gel is removed from the eye. vitrectomy has been shown in several studies to improve vision in patients with traction - associated macular edema.24,25 vitrectomy has also been studied in patients without a tractional component, with mixed results.2628 early detection of diabetic retinopathy and prompt treatment of macular edema are important for preserving vision in the diabetic patient. thermal laser is the mainstay of treatment for diabetic macular edema, and strong evidence supports the use of laser treatment to decrease progressive vision loss. diabetic eye disease is a major cause of vision loss in working age american adults, resulting in hundreds of millions of dollars in direct and indirect medical expenses each year.29 intravitreal steroid and anti - vegf injections may be used in lieu of or in combination with thermal laser to limit vision loss and in some cases improve vision. the next few years may see a shift in the primary therapy of macular edema as more data become available about the efficacy of intravitreal steroids and anti - vegf antibodies.	diabetic macular edema (dme) is a leading cause of vision loss in older americans. thermal laser treatment remains the mainstay of treatment for dme. recently, alternative primary treatments for dme have been evaluated. these treatments include intravitreal injections of steroids as well as pharmaceuticals containing antibodies against vascular endothelial growth factor (vegf). surgical treatment has been shown to be appropriate in selected cases. we review the evidence and scientific rationale for various primary treatment options in patients with dme. regular and timely ophthalmologic evaluation remains crucial to recognition and treatment of macular edema in diabetic patients.
adjuvant radiation therapy is the standard of care for women after breast - conserving surgery for low - risk breast cancer. according to the most recent early breast cancer trialists ' collaborative group (ebctcg) meta - analysis, adjuvant radiation therapy after breast - conserving surgery better than halves the risk of local recurrence and reduces the rate of breast cancer mortality compared to surgery alone.1 however, as survival improves for breast cancer patients, the long - term effects of radiation therapy become increasingly relevant. the ebctcg reports an increased rate of mortality from heart disease in the group of women treated with radiation therapy (risk ratio = 1.27).2 even with modern radiotherapy techniques, portions of the heart may still receive doses greater than 20 gy when the left breast is irradiated depending on tumour location, the position of shielding and the use of respiratory manoeuvres.3,4 tumour laterality is an important predictor of cardiac doses. mean heart and left anterior descending coronary artery (ladca) doses are greatest when the left breast is treated, and this is reflected in an increased risk of cardiac mortality in patients receiving left breast irradiation compared to right breast irradiation.5 the increased risk of cardiac mortality and morbidity due to radiation exposure is reported to be small and dose dependent.6,7 based on a large study of breast cancer patients treated with radiation therapy in denmark and sweden,8 sardaro. estimated a 4% increase in the risk of heart disease for each 1 gy increase in mean heart dose.6 with regard to coronary heart disease, darby. calculated that the rate of major coronary events after breast radiotherapy increases by 7.4% for each 1 gy increase in mean heart dose ; this increase is with no minimum dose threshold and is independent of the presence of pre - existing cardiac risk factors.7 major coronary events were defined as myocardial infarction (heart attack), coronary revascularisation or death from ischemic heart disease.7 despite reducing the dose to cardiac structures during left breast irradiation, modern tangential techniques are not able to completely spare the heart and ladca.3 techniques that involve respiratory motion management may further decrease the exposure of cardiac structures to radiation. inspiration breath - hold strategies, including deep inspiration breath - hold (dibh), have shown the greatest promise in reducing heart doses without compromising target coverage or increasing contralateral breast dose.9 dibh causes favourable changes to the internal thoracic anatomy such that there is increased spatial separation between the heart and the target volume, which results in a decreased volume of the heart within the tangential fields.10,11 the aim of this paper was to review the available literature concerning dibh. the primary aim of was to assess the dosimetric benefits of dibh compared to standard free breathing approaches for left breast cancer patients and the estimated potential to subsequently reduce long - term cardiac morbidity and mortality. the secondary aim of this paper was to assess the reproducibility and stability of dibh. conclusions about the impact of dibh on observable clinical outcomes related to tumour control and long - term toxicities were beyond the scope of this review. even with modern radiotherapy techniques, portions of the heart may still receive doses greater than 20 gy when the left breast is irradiated depending on tumour location, the position of shielding and the use of respiratory manoeuvres.3,4 tumour laterality is an important predictor of cardiac doses. mean heart and left anterior descending coronary artery (ladca) doses are greatest when the left breast is treated, and this is reflected in an increased risk of cardiac mortality in patients receiving left breast irradiation compared to right breast irradiation.5 the increased risk of cardiac mortality and morbidity due to radiation exposure is reported to be small and dose dependent.6,7 based on a large study of breast cancer patients treated with radiation therapy in denmark and sweden,8 sardaro. estimated a 4% increase in the risk of heart disease for each 1 gy increase in mean heart dose.6 with regard to coronary heart disease, darby. calculated that the rate of major coronary events after breast radiotherapy increases by 7.4% for each 1 gy increase in mean heart dose ; this increase is with no minimum dose threshold and is independent of the presence of pre - existing cardiac risk factors.7 major coronary events were defined as myocardial infarction (heart attack), coronary revascularisation or death from ischemic heart disease.7 despite reducing the dose to cardiac structures during left breast irradiation, modern tangential techniques are not able to completely spare the heart and ladca.3 techniques that involve respiratory motion management may further decrease the exposure of cardiac structures to radiation. inspiration breath - hold strategies, including deep inspiration breath - hold (dibh), have shown the greatest promise in reducing heart doses without compromising target coverage or increasing contralateral breast dose.9 dibh causes favourable changes to the internal thoracic anatomy such that there is increased spatial separation between the heart and the target volume, which results in a decreased volume of the heart within the tangential fields.10,11 the aim of this paper was to review the available literature concerning dibh. the primary aim of was to assess the dosimetric benefits of dibh compared to standard free breathing approaches for left breast cancer patients and the estimated potential to subsequently reduce long - term cardiac morbidity and mortality. the secondary aim of this paper was to assess the reproducibility and stability of dibh. conclusions about the impact of dibh on observable clinical outcomes related to tumour control and long - term toxicities were beyond the scope of this review. a structured search was performed in pubmed from 1966 to april 2014 using the following combination of key terms ; radiation therapy. the literature search was limited to articles published in english and no attempt was made to locate unpublished material or to contact authors of unpublished studies. articles retrieved by the initial search were independently scanned by two authors to exclude irrelevant studies. all published studies involving the use of dibh for the irradiation of the left breast or left chest wall, with or without treating the axillary, supra - clavicular or internal mammary chain lymph nodes were considered for inclusion in this review. we included studies that reported the mean heart dose of a dibh treatment plan and a free - breathing treatment plan for each subject. a comparison between dibh and free breathing was required for each study due to the heterogeneity of radiation therapy techniques used between different studies at different time - points. furthermore, only studies which adopted a tangential field approach were reviewed, regardless of whether three - dimensional conformal radiation therapy (3dcrt) or intensity intensity modulated radiation therapy (imrt) was used. studies ineligible for the primary aim of this review were considered for the secondary aim. with regard to the secondary aim of this review, studies were included if they quantitatively investigated the reproducibility or stability of dibh techniques. a structured search was performed in pubmed from 1966 to april 2014 using the following combination of key terms ; radiation therapy. the literature search was limited to articles published in english and no attempt was made to locate unpublished material or to contact authors of unpublished studies. articles retrieved by the initial search were independently scanned by two authors to exclude irrelevant studies. all published studies involving the use of dibh for the irradiation of the left breast or left chest wall, with or without treating the axillary, supra - clavicular or internal mammary chain lymph nodes were considered for inclusion in this review. we included studies that reported the mean heart dose of a dibh treatment plan and a free - breathing treatment plan for each subject. a comparison between dibh and free breathing was required for each study due to the heterogeneity of radiation therapy techniques used between different studies at different time - points. furthermore, only studies which adopted a tangential field approach were reviewed, regardless of whether three - dimensional conformal radiation therapy (3dcrt) or intensity intensity modulated radiation therapy (imrt) was used. studies ineligible for the primary aim of this review were considered for the secondary aim. with regard to the secondary aim of this review, studies were included if they quantitatively investigated the reproducibility or stability of dibh techniques. of these articles, we excluded 14 studies that did not report the mean heart dose of dibh and free breathing approaches. other studies were excluded for not comparing dibh and free breathing plans for individual subjects, reporting volumetric rather than dosimetric endpoints, or having less than ten subjects. ten studies (total of 268 patients) were included to evaluate the dosimetric benefits of dibh for cardiac structures. details regarding these studies and the reported dosimetric endpoints for cardiac structures are summarised in table1. each of these studies was a case series where patients were simulated once in a free breathing state and once during dibh to produce two different treatment plans for dosimetric comparison. the largest study (n = 87) was conducted by swanson.14 and by virtue of cohort size had the greatest relative power of the ten reviewed studies. four studies14,15,17,19 used static sequence imrt to irradiate the target whilst five studies12,16,18,20,21 employed a 3dcrt approach. one study13 produced dibh and free breathing plans using both imrt and 3dcrt. summary of the studies included for dosimetric analysis 3dcrt, three - dimensional conformal radiation therapy ; imrt, intensity modulated radiation therapy ; sib, simultaneous integrated boost ; lcw, left chest wall ; ax, axilla ; scf, supra clavicular fossa ; imc, internal mammary chain. as highlighted in table2, there was a statistically significant reduction (with a significance level of p = 0.05) in mean heart and ladca dose in the dibh plans of all studies when compared with free breathing plans. borst. reported the greatest absolute reduction in mean heart dose (3.4 gy).19 stranzl. reported the smallest absolute reduction in mean heart dose (1.0 gy),21 however, free breathing plans in their study also had the lowest mean heart dose of all studies (2.3 gy) especially when compared to borst. where free breathing plans had a mean heart dose of 5.1 gy.19 in the largest study reviewed, swanson.14 reported an absolute reduction in mean heart dose of 1.7 gy between dibh and free breathing plans. studies reporting mean heart dose and mean ladca dose for free breathing versus dibh plans for left breast irradiation dibh, deep inspiration breath - hold ; ladca, left anterior descending coronary artery ; fb, free breathing. mean heart dose reductions were similar when comparing imrt and 3drct, with these techniques achieving average reductions of 2.2 gy and 1.9 gy respectively. in the only study comparing the use of dibh in the context of both imrt and 3dcrt, the dose reduction conferred by dibh was 0.3 gy greater in the 3dcrt arm compared to the imrt arm.13 in addition to reporting on mean heart dose, seven studies12,13,1519 (total of 148 patients) reported the mean ladca dose. there was considerable variability in the ladca doses reported by these studies, ranging from 11.4 to 31.7 gy in free breathing plans and 5.521.9 gy in dibh plans. as expected, the mean ladca doses were much greater than for the heart, which is reflective of the geometric relationship between the heart, ladca and target tissues during left breast irradiation. as with the mean heart dose, the mean ladca dose was smaller in dibh plans compared to free breathing plans in all of the seven studies,12,13,1519 with the greatest absolute reduction reported by wang.17 (14.1 gy). the smallest reduction in mean ladca dose was 5.9 gy, as reported by borst.19 similar to the mean heart dose, the benefit of using dibh was slightly greater for patients treated with imrt compared to 3dcrt, with average ladca dose reductions of 9.5 gy and 8.8 gy respectively. conversely, however, mast.13 demonstrated that the reduction in mean ladca dose using dibh was marginally greater in 3dcrt compared with imrt. overall, there was little difference between 3dcrt and imrt in terms of the dosimetric advantages conferred by dibh in reducing mean heart and ladca dose. a total of four studies (total of 69 subjects) investigated the reproducibility or stability of dibh techniques (see table3). all studies assessed the reproducibility of dibh, whilst betgen.22 and cervio.25 additionally assessed the stability of dibh. the largest inter - fraction translational variation in any plane was 3.1 mm in the superior inferior plane.22 however, gierga.23 and mcintosh.24 reported that inter - fraction variations in dibh set up were most prominent in the anterior posterior plane. in the studies reporting both inter - fraction and intra - fractions variations,22,25 the magnitude of intra - fraction variations (representing the stability of dibh) as assessed by external surface anatomy, was smaller than the inter - fraction variations (representing the reproducibility of dibh). the stability and reproducibility of performing dibh dibh, deep inspiration breath - hold ; cbct, cone beam computed tomography ; 3dsi, three - dimensional surface imaging ; kv portal, kilo - voltage portal imaging ; ap, anterior posterior ; si, superior inferior ; lr, left right. without visual feedback. with visual feedback. overall, inter - fraction and intra - fraction variations were modest regardless of the image matching protocol used, with intra - fraction variations notably smaller compared to inter - fraction variations. ten studies (total of 268 patients) were included to evaluate the dosimetric benefits of dibh for cardiac structures. details regarding these studies and the reported dosimetric endpoints for cardiac structures are summarised in table1. each of these studies was a case series where patients were simulated once in a free breathing state and once during dibh to produce two different treatment plans for dosimetric comparison. (n = 87) was conducted by swanson.14 and by virtue of cohort size had the greatest relative power of the ten reviewed studies. four studies14,15,17,19 used static sequence imrt to irradiate the target whilst five studies12,16,18,20,21 employed a 3dcrt approach. summary of the studies included for dosimetric analysis 3dcrt, three - dimensional conformal radiation therapy ; imrt, intensity modulated radiation therapy ; sib, simultaneous integrated boost ; lcw, left chest wall ; ax, axilla ; scf, supra clavicular fossa ; imc, internal mammary chain. as highlighted in table2, there was a statistically significant reduction (with a significance level of p = 0.05) in mean heart and ladca dose in the dibh plans of all studies when compared with free breathing plans. borst. reported the greatest absolute reduction in mean heart dose (3.4 gy).19 stranzl. reported the smallest absolute reduction in mean heart dose (1.0 gy),21 however, free breathing plans in their study also had the lowest mean heart dose of all studies (2.3 gy) especially when compared to borst. where free breathing plans had a mean heart dose of 5.1 gy.19 in the largest study reviewed, swanson.14 reported an absolute reduction in mean heart dose of 1.7 gy between dibh and free breathing plans. studies reporting mean heart dose and mean ladca dose for free breathing versus dibh plans for left breast irradiation dibh, deep inspiration breath - hold ; ladca, left anterior descending coronary artery ; fb, free breathing. mean heart dose reductions were similar when comparing imrt and 3drct, with these techniques achieving average reductions of 2.2 gy and 1.9 gy respectively. in the only study comparing the use of dibh in the context of both imrt and 3dcrt, the dose reduction conferred by dibh was 0.3 gy greater in the 3dcrt arm compared to the imrt arm.13 in addition to reporting on mean heart dose, seven studies12,13,1519 (total of 148 patients) reported the mean ladca dose. there was considerable variability in the ladca doses reported by these studies, ranging from 11.4 to 31.7 gy in free breathing plans and 5.521.9 gy in dibh plans. as expected, the mean ladca doses were much greater than for the heart, which is reflective of the geometric relationship between the heart, ladca and target tissues during left breast irradiation. as with the mean heart dose, the mean ladca dose was smaller in dibh plans compared to free breathing plans in all of the seven studies,12,13,1519 with the greatest absolute reduction reported by wang.17 (14.1 gy). the smallest reduction in mean ladca dose was 5.9 gy, as reported by borst.19 similar to the mean heart dose, the benefit of using dibh was slightly greater for patients treated with imrt compared to 3dcrt, with average ladca dose reductions of 9.5 gy and 8.8 gy respectively. conversely, however, mast.13 demonstrated that the reduction in mean ladca dose using dibh was marginally greater in 3dcrt compared with imrt. overall, there was little difference between 3dcrt and imrt in terms of the dosimetric advantages conferred by dibh in reducing mean heart and ladca dose. a total of four studies (total of 69 subjects) investigated the reproducibility or stability of dibh techniques (see table3). all studies assessed the reproducibility of dibh, whilst betgen.22 and cervio.25 additionally assessed the stability of dibh. the largest inter - fraction translational variation in any plane was 3.1 mm in the superior inferior plane.22 however, gierga.23 and mcintosh.24 reported that inter - fraction variations in dibh set up were most prominent in the anterior posterior plane. in the studies reporting both inter - fraction and intra - fractions variations,22,25 the magnitude of intra - fraction variations (representing the stability of dibh) as assessed by external surface anatomy, was smaller than the inter - fraction variations (representing the reproducibility of dibh). the stability and reproducibility of performing dibh dibh, deep inspiration breath - hold ; cbct, cone beam computed tomography ; 3dsi, three - dimensional surface imaging ; kv portal, kilo - voltage portal imaging ; ap, anterior posterior ; si, superior overall, inter - fraction and intra - fraction variations were modest regardless of the image matching protocol used, with intra - fraction variations notably smaller compared to inter - fraction variations. there are no studies to date investigating the clinical outcomes of using dibh for left breast irradiation. therefore, there are no data available to assess the impact of dibh on the rate of late cardiac toxicities. with the long period of latency associated with late cardiac morbidity and mortality, the relative infancy of dibh and the limited number of published clinical series regarding its use, it may be many years before data are available to assess the impact of dibh on cardiac toxicity. as such, a theoretical and dosimetric approach to estimating the benefits of dibh is necessary in the interim to determine whether the clinical implementation of dibh is worthwhile. sardaro.6 estimate that a 1 gy increase in mean heart dose equates to a 4% increase in the risk of late heart disease and darby.7 estimate that a 1 gy increase in mean heart dose equates to a 7.4% increase in the rate of major coronary events, such as myocardial infarction or death from ischemic heart disease. in the ten studies reviewed, free breathing treatment plans were associated with mean heart doses ranging from 2.3 gy21 to 6.9 gy,15 depending on the specific radiotherapy technique used and whether additional lymph node groups were included in the target volume. based on the estimate made by sardaro.6 and darby.,7 this represents a 9.2% to 27.6% increase in long - term heart disease risk from baseline risk levels and a 1751% increase in the rate of major coronary events. in all ten studies, dibh produced a statistically significant reduction in the mean heart dose from free breathing plans, which would lead to a notably smaller increase in the risk of late cardiac morbidity for these women. the mean heart dose in the dibh plans ranged from 1.3 gy21 to 3.9 gy,15 which may equate to an increased heart disease risk of only 5.215.6% and an increased rate of major coronary events of only 9.628.9%. the exact mean heart dose reduction is dependent on the specific radiation therapy technique used, whether the internal mammary chain lymph nodes are irradiated and the prescribed dose to the target volume. data from the available studies are not sufficient to conclude whether dibh is more beneficial for imrt or 3dcrt. there are no studies to date that assess the relationship between ladca dosimetric endpoints and late coronary events. as such, an estimate of the reduction in late coronary morbidity as a result of using dibh to spare the ladca can not be made. nevertheless, the literature recognises the importance of considering the ladca in left breast radiation due to its anatomic location and spatial relationship to the target tissue.4,26 given the physiological significance of this structure and the mean ladca doses in both free breathing and dibh plans reported in this review, clinicians would be well advised to consider the ladca as an organ at risk. future studies are necessary to evaluate normal tissue complication probabilities for the ladca.27 the benefits of dibh for the heart compared to free breathing seem clear, however, these estimates regarding the increase in cardiac morbidity risk are based on dosimetric studies. the dosimetry of these plans must be accurately translated to the delivered dosimetry during treatment in order for these benefits to be realised. a limited number of studies reporting on small cohorts have investigated the reproducibility and stability of dibh. these studies agree that the inter - fraction and intra - fraction variability in set up position when using dibh is small. betgen.22 investigated the role of online image guidance to correct for inter - fraction variations. using a pre - treatment online correction protocol, they found that inter - fraction variability prior to set up could be markedly reduced from 1.2, 3.1 and 1 mm to 0.3, 0.4 and 0.1 mm in anterior posterior, superior inferior and left therefore, online image guidance may play an important role in ensuring that dibh is reproducible on a daily basis. combined with sub - millimetre intra - fraction variability, betgen.22 showed that the set up for dibh is both reproducible and stable. additionally, cervio.25 explore the role of a visual feedback system to supplement audio - based coaching of patients. their findings suggest that the stability and reproducibility of dibh can be further increased with real - time visual feedback, which together confer sub - millimetre inter - fraction and intra - fraction variations in chest - wall excursion. in terms of the dosimetric impact of dibh reproducibility on cardiac sparing, mcintosh.24 found that in 10 patients the difference between the planned and treated mean heart dose was insignificant when compared to the mean heart dose in free breathing plans. this study found that the average difference in mean heart dose and mean ladca dose between planning and treatment was 8% and 9% of the same dosimetric endpoints in the free breathing plan. thus, the available data from the four studies assessed in this review demonstrate that dibh is reproducible and stable and that the dosimetric impact of inter - fraction variations is insignificant. however, from these studies it is not possible to directly draw a comparison with the stability and reproducibility of left breast irradiation during free breathing. the available data suggest that imaging technology may play an important role in the clinical implementation of dibh, however, further studies will be required to determine the optimal imaging protocol to reproduce and monitor dibh treatments. multi - leaf collimation is an obvious alternative to dibh when it comes to protecting the heart during left breast irradiation. in a study of 67 left breast patients, bartlett.28 investigated the impact of shielding the heart with multi - leaf collimation on target tissue coverage. they found that the average mean heart dose across 67 subjects was 0.8 gy when using multi - leaf collimation to shield the heart, which is less than the smallest reported average mean heart dose achieved using dibh (1.3 gy).21 however, completely shielding the heart from irradiation in tangential fields will simultaneously shield a portion of the medial and inferior part of the breast tissue, depending on the exact positioning of the collimator leaves. in the study conducted by bartlett.,28 35% of patients had less than 90% of the whole breast target volume covered by 95% of the prescription dose. of the ten studies primarily assessed in this review, two reported on coverage of the planning target volume (ptv) coverage.16,18 in both of these studies, 99% of the ptv received 95% isodose coverage when dibh was used to spare the heart and there was no significant difference between dibh and free breathing plans.16,18 therefore, although multi - leaf collimation provides slightly better sparing of the heart, the available data suggest that the cardiac sparing conferred by dibh does not come at the expense of ptv coverage. because local recurrence is most likely to occur in the region close to the original tumour, the location of the original tumour should be considered when deciding which cardiac sparing strategy is most appropriate. the main limitation of this review is that it is based on dosimetric rather than clinical studies. as such, the reported reduction in the risk of late cardiac morbidity and mortality is an estimate rather than an observation. as discussed previously, the aim of this review was to provide an estimate to inform decisions regarding the implementation of dibh in future clinical practice. however, this will require randomised studies with long - term follow - up to observe the late cardiac effects related to left breast irradiation. one limitation of basing estimates on dosimetric studies is that the planned dosimetry does not always accurately represent the delivered dosimetry. for this reason, the reproducibility and stability of dibh was secondarily assessed in this review. however, the impact of respiratory motion on the free breathing plans could not be assessed. dose plans created from free breathing scans do not account for respiratory motion, and therefore, there is uncertainty about how accurately these free breathing plans were delivered. as such, the mean heart dose delivered to the patient may be greater or lesser than planned. this will depend on the respiratory phase of the patient at the moment that the free breathing planning scan was taken. however, a study conducted by frazier.29 suggests that this uncertainty due to normal respiratory motion is minimal. they super - imposed free breathing - based dose plans for breast irradiation onto scans taken at the end of normal expiration and inspiration to assess the difference in dosimetry for the breast target volume and lung due to normal respiratory motion. these differences were insignificant for the ipsilateral lung and target breast tissue.29 finally, it must be stressed that the majority of studies included in this review had small sample sizes. thus, their results are subject to either the low probability of finding a true effect, a low positive predicative value when an effect was claimed or even the potential to exaggerate the estimate of the magnitude of the effect.30 furthermore, this review was limited by the non - randomised nature of the included studies and the variable methods of patient selection. this made a number of the reviewed studies susceptible to selection bias,31 leading to a possible overestimation of the reduction in mean heart dose conferred by dibh. wang.17 only produced dibh dose plans for patients with unfavourable cardiac anatomy on the original free breathing plan, where greater than 10 cm of the heart would have received greater than 50% of the prescription dose. only these patients (20 of 53) were included for analysis in their study, and as such, the reduction in heart dose reported by this study may be exaggerated as it only applies to this subset of patients with unfavourable cardiac anatomy. in the study conducted by swanson.,14 only dibh plans that showed improvements in cardiac dose relative to free breathing plans were included for analysis. as a result, 12 dibh dose plans that failed to improve the cardiac dose were excluded from further analysis. sardaro.6 estimate that a 1 gy increase in mean heart dose equates to a 4% increase in the risk of late heart disease and darby.7 estimate that a 1 gy increase in mean heart dose equates to a 7.4% increase in the rate of major coronary events, such as myocardial infarction or death from ischemic heart disease. in the ten studies reviewed, free breathing treatment plans were associated with mean heart doses ranging from 2.3 gy21 to 6.9 gy,15 depending on the specific radiotherapy technique used and whether additional lymph node groups were included in the target volume. based on the estimate made by sardaro.6 and darby.,7 this represents a 9.2% to 27.6% increase in long - term heart disease risk from baseline risk levels and a 1751% increase in the rate of major coronary events. in all ten studies, dibh produced a statistically significant reduction in the mean heart dose from free breathing plans, which would lead to a notably smaller increase in the risk of late cardiac morbidity for these women. the mean heart dose in the dibh plans ranged from 1.3 gy21 to 3.9 gy,15 which may equate to an increased heart disease risk of only 5.215.6% and an increased rate of major coronary events of only 9.628.9%. the exact mean heart dose reduction is dependent on the specific radiation therapy technique used, whether the internal mammary chain lymph nodes are irradiated and the prescribed dose to the target volume. data from the available studies are not sufficient to conclude whether dibh is more beneficial for imrt or 3dcrt. there are no studies to date that assess the relationship between ladca dosimetric endpoints and late coronary events. as such, an estimate of the reduction in late coronary morbidity as a result of using dibh to spare the ladca can not be made. nevertheless, the literature recognises the importance of considering the ladca in left breast radiation due to its anatomic location and spatial relationship to the target tissue.4,26 given the physiological significance of this structure and the mean ladca doses in both free breathing and dibh plans reported in this review, clinicians would be well advised to consider the ladca as an organ at risk. the benefits of dibh for the heart compared to free breathing seem clear, however, these estimates regarding the increase in cardiac morbidity risk are based on dosimetric studies. the dosimetry of these plans must be accurately translated to the delivered dosimetry during treatment in order for these benefits to be realised. a limited number of studies reporting on small cohorts have investigated the reproducibility and stability of dibh. these studies agree that the inter - fraction and intra - fraction variability in set up position when using dibh is small. betgen.22 investigated the role of online image guidance to correct for inter - fraction variations. using a pre - treatment online correction protocol, they found that inter - fraction variability prior to set up could be markedly reduced from 1.2, 3.1 and 1 mm to 0.3, 0.4 and 0.1 mm in anterior posterior, superior inferior and left therefore, online image guidance may play an important role in ensuring that dibh is reproducible on a daily basis. combined with sub - millimetre intra - fraction variability, betgen.22 showed that the set up for dibh is both reproducible and stable. additionally, cervio.25 explore the role of a visual feedback system to supplement audio - based coaching of patients. their findings suggest that the stability and reproducibility of dibh can be further increased with real - time visual feedback, which together confer sub - millimetre inter - fraction and intra - fraction variations in chest - wall excursion. in terms of the dosimetric impact of dibh reproducibility on cardiac sparing, mcintosh.24 found that in 10 patients the difference between the planned and treated mean heart dose was insignificant when compared to the mean heart dose in free breathing plans. this study found that the average difference in mean heart dose and mean ladca dose between planning and treatment was 8% and 9% of the same dosimetric endpoints in the free breathing plan. thus, the available data from the four studies assessed in this review demonstrate that dibh is reproducible and stable and that the dosimetric impact of inter - fraction variations is insignificant. however, from these studies it is not possible to directly draw a comparison with the stability and reproducibility of left breast irradiation during free breathing. the available data suggest that imaging technology may play an important role in the clinical implementation of dibh, however, further studies will be required to determine the optimal imaging protocol to reproduce and monitor dibh treatments. multi - leaf collimation is an obvious alternative to dibh when it comes to protecting the heart during left breast irradiation. in a study of 67 left breast patients, bartlett.28 investigated the impact of shielding the heart with multi - leaf collimation on target tissue coverage. they found that the average mean heart dose across 67 subjects was 0.8 gy when using multi - leaf collimation to shield the heart, which is less than the smallest reported average mean heart dose achieved using dibh (1.3 gy).21 however, completely shielding the heart from irradiation in tangential fields will simultaneously shield a portion of the medial and inferior part of the breast tissue, depending on the exact positioning of the collimator leaves. in the study conducted by bartlett.,28 35% of patients had less than 90% of the whole breast target volume covered by 95% of the prescription dose. of the ten studies primarily assessed in this review, two reported on coverage of the planning target volume (ptv) coverage.16,18 in both of these studies, 99% of the ptv received 95% isodose coverage when dibh was used to spare the heart and there was no significant difference between dibh and free breathing plans.16,18 therefore, although multi - leaf collimation provides slightly better sparing of the heart, the available data suggest that the cardiac sparing conferred by dibh does not come at the expense of ptv coverage. because local recurrence is most likely to occur in the region close to the original tumour, the location of the original tumour should be considered when deciding which cardiac sparing strategy is most appropriate. the main limitation of this review is that it is based on dosimetric rather than clinical studies. as such, the reported reduction in the risk of late cardiac morbidity and mortality as discussed previously, the aim of this review was to provide an estimate to inform decisions regarding the implementation of dibh in future clinical practice. however, this will require randomised studies with long - term follow - up to observe the late cardiac effects related to left breast irradiation. one limitation of basing estimates on dosimetric studies is that the planned dosimetry does not always accurately represent the delivered dosimetry. for this reason, the reproducibility and stability of dibh was secondarily assessed in this review. however, the impact of respiratory motion on the free breathing plans could not be assessed. dose plans created from free breathing scans do not account for respiratory motion, and therefore, there is uncertainty about how accurately these free breathing plans were delivered. as such, the mean heart dose delivered to the patient may be greater or lesser than planned. this will depend on the respiratory phase of the patient at the moment that the free breathing planning scan was taken. however, a study conducted by frazier.29 suggests that this uncertainty due to normal respiratory motion is minimal. they super - imposed free breathing - based dose plans for breast irradiation onto scans taken at the end of normal expiration and inspiration to assess the difference in dosimetry for the breast target volume and lung due to normal respiratory motion. these differences were insignificant for the ipsilateral lung and target breast tissue.29 finally, it must be stressed that the majority of studies included in this review had small sample sizes. thus, their results are subject to either the low probability of finding a true effect, a low positive predicative value when an effect was claimed or even the potential to exaggerate the estimate of the magnitude of the effect.30 furthermore, this review was limited by the non - randomised nature of the included studies and the variable methods of patient selection. this made a number of the reviewed studies susceptible to selection bias,31 leading to a possible overestimation of the reduction in mean heart dose conferred by dibh. wang.17 only produced dibh dose plans for patients with unfavourable cardiac anatomy on the original free breathing plan, where greater than 10 cm of the heart would have received greater than 50% of the prescription dose. only these patients (20 of 53) were included for analysis in their study, and as such, the reduction in heart dose reported by this study may be exaggerated as it only applies to this subset of patients with unfavourable cardiac anatomy. in the study conducted by swanson.,14 only dibh plans that showed improvements in cardiac dose relative to free breathing plans were included for analysis. as a result, 12 dibh dose plans that failed to improve the cardiac dose were excluded from further analysis. the current evidence base regarding the benefits of dibh for left breast cancer patients is exclusively limited to dosimetric studies. based on a review of these studies, using dibh rather than free breathing plans for left breast radiation therapy may reduce the mean heart dose by up to 3.4 gy and mean ladca dose by up to 14.1 gy. in light of the reported reproducibility and stability of dibh, these dosimetric benefits should be preserved when the treatment is delivered. according to current estimates of the excess cardiac toxicity associated with radiation therapy, dibh can reduce the projected increased risk of heart disease by 13.6% and reduce the projected percentage increase in the rate of major coronary events by 25.2%. the reduction in mean heart and ladca dose for a given patient is dependent on the specific radiation therapy technique used, the prescription dose and whether the internal mammary chain lymph nodes require irradiation. the limitations inherent to this systematic review indicate the need for future studies with long - term follow up so that the estimated benefits of dibh for cardiac toxicity can be confirmed.	introductiondespite technical advancements in breast radiation therapy, cardiac structures are still subject to significant levels of irradiation. as the use of adjuvant radiation therapy after breast - conserving surgery continues to improve survival for early breast cancer patients, the associated radiation - induced cardiac toxicities become increasingly relevant. our primary aim was to evaluate the cardiac - sparing benefits of the deep inspiration breath - hold (dibh) technique.methodsan electronic literature search of the pubmed database from 1966 to july 2014 was used to identify articles published in english relating to the dosimetric benefits of dibh. studies comparing the mean heart dose of dibh and free breathing treatment plans for left breast cancer patients were eligible to be included in the review. studies evaluating the reproducibility and stability of the dibh technique were also reviewed.resultsten studies provided data on the benefits of dibh during left breast irradiation. from these studies, dibh reduced the mean heart dose by up to 3.4 gy when compared to a free breathing approach. four studies reported that the dibh technique was stable and reproducible on a daily basis. according to current estimates of the excess cardiac toxicity associated with radiation therapy, a 3.4 gy reduction in mean heart dose is equivalent to a 13.6% reduction in the projected increase in risk of heart disease.conclusiondibh is a reproducible and stable technique for left breast irradiation showing significant promise in reducing the late cardiac toxicities associated with radiation therapy.
rpe is a rare but potentially fatal complication, often after thoracentesis of pleural fluid as described in this case. the range reported in the literature varies significantly, however, ranging from 0.3 to 32.5% (1, 2). this is likely due to differences in definition (clinical vs. radiographic), small sample sizes, and different patient populations. taira. only studied 40 patients retrospectively, and they all had spontaneous pneumothorax prior to rpe. that particular study found rpe in 13 of the 40, or 32.5%, and concluded that the incidence may be higher than previously reported. also, strict ct - based criteria for diagnosis were used that were more sensitive than radiographic or clinical criteria (2). conducted a prospective study of 185 individuals with varying amounts of pleural fluid removed by thoracentesis with an incidence of clinical rpe at 0.5% and radiographic rpe at 2.2% (3). yoon. again cited the incidence higher in a population with spontaneous pneumothorax at 16% (4). as the higher numbers tend to be based on radiographic data, a more accurate and useful assessment would be that clinically significant rpe is an occurrence of less than 1%. the mortality rate is often cited at 20% ; however, that number appears to have come from one study of 53 patients and is likely an overestimation (5). more recent studies have found it to be much lower (2, 4). with such low prevalence, prognosis is difficult to estimate. although the pathophysiology at this time is not fully elucidated, there are risk factors to be aware of. taira. found the presence of pleural effusion in conjunction with a pneumothorax increased risk but duration of symptoms, size of pneumothorax, and location had minimal effect (2). in the 185 patients studied prospectively by feller - kopman. in 2007, they found rpe was independent of the volume removed, pleural pressures, and pleural elastance ; instead recommending that large pleural effusions be drained completely (3). yoon. showed in a retrospective study of 306 patients that rpe was more common in patients with diabetes and tension pneumothorax, and found those with rpe tended to have a larger pneumothorax than those without (4). another study of 146 cases of spontaneous pneumothorax found the rate of rpe to be higher in persons aged 2039 than in those over 40 and again correlated with size of pneumothorax (6). after a review of 233 papers, echevarria and colleagues determined that the patients at highest risk are those with large pneumothoraces, young patients, those with lung collapse for more than 1 week, and those needing 3 l of fluid drained (7). excessive negative pleural pressures have long been thought of as a major contributor to rpe. several authors have suggested avoiding pleural pressures less than -20 cm h2o for this reason. other authors since have found it less important or that rpe may be completely independent of the pleural pressure (3, 10). a paper describing two cases of rpe showed marked increases in polymorphonuclear neutrophils (pmns) and pmn - elastase with elevated fluid to plasma protein concentration. interleukin (il)-8 and leukotriene b-4 have been observed in edema fluid (as well as pmn - elastase again and p - selectin) (12). experimental rabbit models for rpe showed up - regulation of pro - inflammatory cytokines as well but no significant difference in capillary permeability (13). as opposed to prior studies that had suggested a mechanism based primarily on increased capillary permeability (11, 12, 1416), sue. found using edema to plasma protein ratios in a retrospective analysis that hydrostatic mechanisms may play a more significant role (17). sohara asserts histological abnormalities of the pulmonary microvasculature result from a collapsed lung in the order of days, as well as mechanical stress during reexpansion, are the mechanisms driving rpe (18). these histologic changes may be mediated by inflammatory cytokines as previously suggested as well as oxidative stress. experimental studies in rats were able to demonstrate, with statistical significance, higher levels of a marker for oxidative stress, malondialdehyde (mda), in the experimental groups (19). additional studies on rats also suggest tissue reperfusion, hypoxia, and free radical damage by lipid peroxidation in a collapsed lung as a mechanism for injury and edema (16). it is likely these all contribute in varying degrees depending on the particular clinical scenario. when clinically significant, signs and symptoms may include a new cough generally lasting more than 20 min, dyspnea, tachypnea, hypoxia, tachycardia, chest pain, or hemodynamic instability (3, 10, 20, 21). most patients are symptomatic within an hour after pleural drainage, although it can occur at any point within 24 h (10, 21). ct findings most commonly include ipsilateral ground - glass opacities, septal thickening, consolidation, and persistent areas of atelectasis (22). edema most commonly occurs in the ipsilateral lung, but can present in the contralateral lung or bilaterally (22, 23). currently, the mainstay treatment for rpe is supportive care with supplemental oxygen and diuretics. steroids and hemodynamic support are sometimes used in severe cases, although rare (2, 18, 24). other clinicians recommend positioning the patient in lateral decubitus with the affected side down with noninvasive positive pressure or orotracheal intubation with mechanical ventilation if the rpe is more severe (10). there have been a few case studies demonstrating successful outcomes of severe rpe with alternative treatment methods. one case study by cho demonstrated a very severe case of rpe requiring a double - lumen endobronchial tube with asynchronous differential lung ventilation for 48 h before ventilation profusion mismatch was restored (25). pretreatment with il-8 neutralizing antibody to combat overproduction of il-8 during lung collapse and reexpansion has shown promise in rabbits (15). similarly alpha - lipoic acid (ala) has been suggested as a treatment option by reduction of oxidative stress, demonstrated in rat models (19). although these small animal studies are promising, they have limited clinical utility thus far in clinical medicine. most cases require only minimal supportive care. as severe rpe is exceedingly rare, more emphasis has been placed on prevention. avoidance of pleural pressures less than 20 cm h2o and removing less than 1.8 l are often cited methods (3, 10, 26), though the data is limited. the range reported in the literature varies significantly, however, ranging from 0.3 to 32.5% (1, 2). this is likely due to differences in definition (clinical vs. radiographic), small sample sizes, and different patient populations. taira. only studied 40 patients retrospectively, and they all had spontaneous pneumothorax prior to rpe. that particular study found rpe in 13 of the 40, or 32.5%, and concluded that the incidence may be higher than previously reported. also, strict ct - based criteria for diagnosis were used that were more sensitive than radiographic or clinical criteria (2). conducted a prospective study of 185 individuals with varying amounts of pleural fluid removed by thoracentesis with an incidence of clinical rpe at 0.5% and radiographic rpe at 2.2% (3). yoon. again cited the incidence higher in a population with spontaneous pneumothorax at 16% (4). as the higher numbers tend to be based on radiographic data, a more accurate and useful assessment would be that clinically significant rpe is an occurrence of less than 1%. the mortality rate is often cited at 20% ; however, that number appears to have come from one study of 53 patients and is likely an overestimation (5). more recent studies have found it to be much lower (2, 4). with such low prevalence, prognosis is difficult to estimate. although the pathophysiology at this time is not fully elucidated, there are risk factors to be aware of. taira. found the presence of pleural effusion in conjunction with a pneumothorax increased risk but duration of symptoms, size of pneumothorax, and location had minimal effect (2). in the 185 patients studied prospectively by feller - kopman. in 2007, they found rpe was independent of the volume removed, pleural pressures, and pleural elastance ; instead recommending that large pleural effusions be drained completely (3). yoon. showed in a retrospective study of 306 patients that rpe was more common in patients with diabetes and tension pneumothorax, and found those with rpe tended to have a larger pneumothorax than those without (4). another study of 146 cases of spontaneous pneumothorax found the rate of rpe to be higher in persons aged 2039 than in those over 40 and again correlated with size of pneumothorax (6). after a review of 233 papers, echevarria and colleagues determined that the patients at highest risk are those with large pneumothoraces, young patients, those with lung collapse for more than 1 week, and those needing 3 l of fluid drained (7). excessive negative pleural pressures have long been thought of as a major contributor to rpe. several authors have suggested avoiding pleural pressures less than -20 cm h2o for this reason. other authors since have found it less important or that rpe may be completely independent of the pleural pressure (3, 10). a paper describing two cases of rpe showed marked increases in polymorphonuclear neutrophils (pmns) and pmn - elastase with elevated fluid to plasma protein concentration. interleukin (il)-8 and leukotriene b-4 have been observed in edema fluid (as well as pmn - elastase again and p - selectin) (12). experimental rabbit models for rpe showed up - regulation of pro - inflammatory cytokines as well but no significant difference in capillary permeability (13). as opposed to prior studies that had suggested a mechanism based primarily on increased capillary permeability (11, 12, 1416), sue. found using edema to plasma protein ratios in a retrospective analysis that hydrostatic mechanisms may play a more significant role (17). sohara asserts histological abnormalities of the pulmonary microvasculature result from a collapsed lung in the order of days, as well as mechanical stress during reexpansion, are the mechanisms driving rpe (18). these histologic changes may be mediated by inflammatory cytokines as previously suggested as well as oxidative stress. experimental studies in rats were able to demonstrate, with statistical significance, higher levels of a marker for oxidative stress, malondialdehyde (mda), in the experimental groups (19). additional studies on rats also suggest tissue reperfusion, hypoxia, and free radical damage by lipid peroxidation in a collapsed lung as a mechanism for injury and edema (16). it is likely these all contribute in varying degrees depending on the particular clinical scenario. when clinically significant, signs and symptoms may include a new cough generally lasting more than 20 min, dyspnea, tachypnea, hypoxia, tachycardia, chest pain, or hemodynamic instability (3, 10, 20, 21). most patients are symptomatic within an hour after pleural drainage, although it can occur at any point within 24 h (10, 21). ct findings most commonly include ipsilateral ground - glass opacities, septal thickening, consolidation, and persistent areas of atelectasis (22). edema most commonly occurs in the ipsilateral lung, but can present in the contralateral lung or bilaterally (22, 23). currently, the mainstay treatment for rpe is supportive care with supplemental oxygen and diuretics. steroids and hemodynamic support are sometimes used in severe cases, although rare (2, 18, 24). other clinicians recommend positioning the patient in lateral decubitus with the affected side down with noninvasive positive pressure or orotracheal intubation with mechanical ventilation if the rpe is more severe (10). there have been a few case studies demonstrating successful outcomes of severe rpe with alternative treatment methods. one case study by cho demonstrated a very severe case of rpe requiring a double - lumen endobronchial tube with asynchronous differential lung ventilation for 48 h before ventilation profusion mismatch was restored (25). pretreatment with il-8 neutralizing antibody to combat overproduction of il-8 during lung collapse and reexpansion has shown promise in rabbits (15). similarly alpha - lipoic acid (ala) has been suggested as a treatment option by reduction of oxidative stress, demonstrated in rat models (19). although these small animal studies are promising, they have limited clinical utility thus far in clinical medicine. avoidance of pleural pressures less than 20 cm h2o and removing less than 1.8 l are often cited methods (3, 10, 26), though the data is limited. this is a classic case of rpe and demonstrates the potential for its sudden onset and severity. the condition does appear to be more benign than previously thought, and clinically significant episodes are rare. in severe cases such as this one, proper support with mechanical ventilation, diuretics, and hemodynamic support are pillars of treatment. our particular patient benefited significantly from lateral decubitus position ventilation with the affected side up. many studies investigating the pathophysiology and preventative techniques are too small to have adequate power. there are some general guiding principles that appear to help minimize risk, including the avoidance of extreme negative pleural pressures, limiting the amount taken off at one time, stopping with symptoms like chest pain and cough, and recognizing patients at higher risk. irrespective of the cutoffs used, this rare complication can be managed effectively with supportive care most of the time. further research, especially larger studies, may be beneficial in understanding and further minimizing the risks associated with rpe.	reexpansion pulmonary edema (rpe) is a rare complication that can occur after rapid reinflation of the lung following thoracentesis of a pleural effusion or chest tube drainage of pneumothorax. the severity in clinical presentation can be widely varied from radiographic changes only to rapidly progressive respiratory failure requiring mechanical ventilation. the quick nature of onset and potential for serious decline in a previously stable patient makes it important to prepare, recognize, diagnose, and appropriately manage patients who develop rpe. the standard treatment for rpe consists of supportive care, and there are certain measures that may be taken to reduce the risk, including limiting the amount drained and avoiding excessive negative pleural pressure. exactly how to prevent rpe remains unclear, however, and varying recommendations exist. this is a case report of rpe after thoracentesis for a pleural effusion and a brief review of literature to date, including potential preventative strategies.
running is a popular sports discipline and can be performed over several different distances.1,2 an abundant variety of physiological, anthropometrical and training variables showed an association with running performances depending upon gender, the length and the duration of performance.3 - 5 apart from physiological parameters, a number of different anthropometric variables were related to endurance running performance such as body mass,6,7 body height,8,9 body mass index,10 - 12 body fat,10 the sum of skin - fold thickness6, single skin - fold thicknesses at the upper and lower body,6,13 - 16 length of legs17,18 and circumferences of limbs.7,12,18,19 these anthropometric properties had different associations regarding running distances and gender. body height was associated with race time in both male and female marathon runners,8 body mass index was related to marathon running time in females,10 and body fat was positively associated with marathon race times in females.10 the relationship between selected skin - fold thicknesses and running performance has been investigated in several studies. hagan. demonstrated that apart from other variables, the sum of skin - fold thicknesses was correlated to marathon race time in males.20 bale. reported the sum of skin - fold thicknesses, the type and frequency of training and the number of years running were the best predictor variables for 10-km race time in males.6 in recent studies, a relationship between the thicknesses of selected skin - folds and running performance has been demonstrated for high - level runners.13,14 in these studies, elite male and female runners of distances from 100 m to 10 km and the marathon had been investigated.13,14 high correlations were found between the front thigh and medial calf skin - fold and 10-km race times for male runners, and between iliac crest and abdominal skin - fold and marathon times for female runners.13 it was supposed that the reduced thickness of skin - folds of the lower limb were a result of high intensity in running training.14 legaz & eston concluded from their study of high - level runners that running training led to a decrease in the sum skin - folds and the skin - fold thickness at the abdominal, front thigh and medial calf sites.14 further, the lower limb skin - fold thicknesses might be a useful predictor variable of running performance.13,14 apart from anthropometry, volume and intensity in training also seemed to influence running performance in long - distance runners up to the marathon distance. regarding volume, in marathon finishers, the longest distance ran per training unit was the best predictor variable for marathon race time.21 male runners completing more than 100 km per week had significantly faster race times over 10 to 90 km than athletes covering less than 100 km22 and elite runners with a higher training frequency, higher weekly training volume and longer running experience competed faster in a 10 km run.6 in female marathoners, the number of weekly training sessions and the number of years training were the best predictors for race time.23 in long - distance runners, training units of moderate intensity were related to race performance.24 in female marathoners, a faster marathon time was associated with higher aerobic capacity and years of training rather than with body dimensions.25 male and female top class marathon runners trained for more total kilometres per week and at a higher velocity than runners at a lower level,26 and peak running velocity during training was highly related to 5 km run times for both male and female athletes.27 when training in runners was analysed in details, several parameters such as training days, total training sessions, total kilometres, mean kilometres per training session, longest mileage covered per training session, total training minutes, maximal kilometres ran per week, mean kilometres per week and mean kilometres per day, were related to marathon race time.10,20,21 what this background shows, is that the relationship between skin - fold thicknesses and running performance has been investigated in running distances from 100 m to the marathon distance, except the half marathon distance.13,14 the intention was therefore to investigate whether a relationship exists between selected anthropometric variables including skin - fold thicknesses and training variables with half marathon race performance in recreational female runners. it was hypothesised that significant relationships would be found between upper body skin - fold thicknesses (suprailiacal and abdominal site) and half marathon race times for recreational female runners. we also intended to create an equation to predict a half marathon race time for future novice female half marathoners, based upon basic measurements any athlete could determine for himself without the need for highly sophisticated equipment. we performed a cross - sectional observational research at a half marathon race during the half marathon basel in switzerland. the organiser contacted all participants of the half marathon basel in 2010 via a separate newsletter, three months before the race, in which they were asked to participate in the study. half marathon basel took place on 12 september 2010, in the city of basel, switzerland. the athletes started at 11:00 a.m. and had to run one flat lap on asphalt. the temperature was 13 celsius at the start and the relative humidity was at 63%. a total of 396 female athletes started in the half marathon ; 42 female runners were interested in participating in our investigation. half marathon basel within the time limit of 2:30 h : min. before the start of the race body mass, body height, and thicknesses of skin - folds were measured. with this data we calculated body mass index and percent body fat using an anthropometric method. body mass was measured to the nearest 0.1 kg using a beurer bf15 scale (beurer gmbh, ulm, germany). body mass index (kg / m) was calculated from body mass and body height. skin - fold thicknesses were measured by the same investigator at the following eight sites : pectoral, mid - axilla, triceps, subscapular, abdominal, suprailiac, front thigh and medial calf. skin - fold data was obtained using a skin - fold calliper (gpm - hautfaltenmessgert, siber & hegner, zurich, switzerland) and recorded to the nearest 0.2 mm. the measurements were made three times on the right side and the mean of the three measurements was used for the analyses. the timing of the taking of the skin - fold measurements was standardised to ensure reliability. one trained investigator took all the skin fold measurements as intertester variability is a major source of error in skin - fold measurements. an intratester reliability check was conducted on 27 male and 11 female runners prior to testing. intra - class correlation (icc) within the two judges was excellent for both men and women for all anatomical measurement sites (icc>0.9).28 readings were performed 4 s after applying the calliper, according to becque.29 percent body fat was calculated using the formula : percent body fat = -6.40665 + 0.41946 (3sf) 0.00126 (3sf) + 0.12515 (hip) + 0.06473 (age) using the formula of ball.30 3sf was taken as the sum of the three skin - fold thickness of the triceps, suprailiac and front thigh skin - fold thicknesses. volunteers were asked to maintain a comprehensive training diary during the 3-month period before the race. the training records consisted of the number of training units with duration, kilometres and pace, weekly kilometres ran, weekly hours ran, and minimal and maximal kilometres ran per week. the athletes recorded their running speed during training in min / km and reported on the number of years that they had actively participated in running. the coefficient of variation of performance (cv% = 100 sd / mean) for total race time was calculated. in a first step, the association of the variables of anthropometry, training and pre race experience with total race time was investigated using bivariate correlation analysis. given the multiple tests, bonferroni correction was applied for n = 22 variables (p = 0.0022). in a second step, multiple linear regression analysis was used to further investigate the relationship of variables with significance in the bivariate analysis to race time. a probability value of less than 0.05 was accepted as significant for the multiple linear regression analysis. the half marathon basel took place on 12 september 2010, in the city of basel, switzerland. the athletes started at 11:00 a.m. and had to run one flat lap on asphalt. the temperature was 13 celsius at the start and the relative humidity was at 63%. a total of 396 female athletes started in the half marathon ; 42 female runners were interested in participating in our investigation. before the start of the race body mass, body height, and thicknesses of skin - folds were measured. with this data we calculated body mass index and percent body fat using an anthropometric method. body mass was measured to the nearest 0.1 kg using a beurer bf15 scale (beurer gmbh, ulm, germany). body mass index (kg / m) was calculated from body mass and body height. skin - fold thicknesses were measured by the same investigator at the following eight sites : pectoral, mid - axilla, triceps, subscapular, abdominal, suprailiac, front thigh and medial calf. skin - fold data was obtained using a skin - fold calliper (gpm - hautfaltenmessgert, siber & hegner, zurich, switzerland) and recorded to the nearest 0.2 mm. the measurements were made three times on the right side and the mean of the three measurements was used for the analyses. the timing of the taking of the skin - fold measurements was standardised to ensure reliability. one trained investigator took all the skin fold measurements as intertester variability is a major source of error in skin - fold measurements. an intratester reliability check was conducted on 27 male and 11 female runners prior to testing. intra - class correlation (icc) within the two judges was excellent for both men and women for all anatomical measurement sites (icc>0.9).28 readings were performed 4 s after applying the calliper, according to becque.29 percent body fat was calculated using the formula : percent body fat = -6.40665 + 0.41946 (3sf) 0.00126 (3sf) + 0.12515 (hip) + 0.06473 (age) using the formula of ball.30 3sf was taken as the sum of the three skin - fold thickness of the triceps, suprailiac and front thigh skin - fold thicknesses. volunteers were asked to maintain a comprehensive training diary during the 3-month period before the race. the training records consisted of the number of training units with duration, kilometres and pace, weekly kilometres ran, weekly hours ran, and minimal and maximal kilometres ran per week. the athletes recorded their running speed during training in min / km and reported on the number of years that they had actively participated in running. normally distributed data are presented as mean and standard deviation (sd). the coefficient of variation of performance (cv% = 100 sd / mean) for total race time was calculated. in a first step, the association of the variables of anthropometry, training and pre race experience with total race time was investigated using bivariate correlation analysis. given the multiple tests, bonferroni correction was applied for n = 22 variables (p = 0.0022). in a second step, multiple linear regression analysis was used to further investigate the relationship of variables with significance in the bivariate analysis to race time. a probability value of less than 0.05 was accepted as significant for the multiple linear regression analysis. the 42 athletes finished the half marathon basel within 119 (15) min (cv = 12.6%), running at a mean speed of 10.8 (1.4) km / h. for the anthropometric characteristics, body mass, body mass index and body fat percentage (see table 1) as well as pectoral, mid - axilla, triceps, subscapular, abdominal, suprailiac, and medial calf skin - folds (see table 2) correlated to race time in the bivariate analysis. for the training characteristics, the mean speed of the training sessions was highly significantly and positively related to race time (see table 3). when the variables with significant association in the bivariate analysis were inserted into a linear regression model (see table 4), mid - axilla skin - fold and mean speed of the training sessions were related to race time. race time in a half marathon might be predicted by the following equation (r = 0.71) for recreational female runners : race time (min) = 166.7 + 1.7x (mid - axilla skin - fold thickness, mm) - 6.4x (speed in training, km / h). mean running speed during training was related to mid - axilla, subscapular, abdominal, and suprailiacal skin - fold thicknesses, the sum of eight skin - folds and percent body fat (see table 5). the aim of this study was to find predictor variables for half marathon race time in recreational female runners in order to create an equation to predict race time for novice future runners. we hypothesised that, according to the existing literature of arrese & ostriz, significant relationships will be found between the upper body skin - fold thicknesses at suprailiacal and abdominal site and the half marathon race times of recreational female runners.13 as we hypothesised, an association between upper body skin - fold thicknesses and race performance was found in the bivariate analysis ; pectoral, mid - axilla, triceps, subscapular, abdominal and suprailiac skin - folds were related to half - marathon running times. arrese & ostriz described in their 11 female high - level marathon runners a significant relationship between iliac crest (r = 0.62, p = 0.042) and abdominal skin - fold thickness (r = 0.61, p = 0.046) with marathon race times.13 we can confirm their findings and in addition, we also found for pectoral, mid - axilla, triceps, and subscapular sites significant associations. the female top class marathoners of arrese & ostriz with a body mass of 45.6 kg and a body height of 1.58 m had a body mass index of 18.3 kg / m compared to the 21.2 kg / m of our subjects.13 we must assume that our recreational females with a higher body mass index also had a higher body fat percentage, and consequently thicker skin - folds compared to the high - level marathoners of arrese & ostriz.13 in the bivariate analysis, mean speed of the training sessions was highly significantly and negatively correlated to half marathon race times. these findings confirm recent findings where peak running velocity during training was highly related to 5 km run times for both male and female athletes.27 according to hagan., however, both variables of anthropometry and training seem to account for marathon race time in females.10 in their sample of 35 female distance runners, marathon performance time was related to body mass index, maximal oxygen uptake, previous marathons completed, number of weekly training sessions, training session per two days, total number of training sessions, total training duration, training pace and distance in training. we can confirm their findings that both variables of anthropometry and training were associated with half marathon running times in our subjects after multivariate analysis and not only anthropometric or training variables. based upon previous studies of runners up to the marathon distance, we expected to find an association between lower body skin - fold thicknesses and variables of training. legaz & eston described in a sample of 24 male and female endurance runners a significant association between the decrease in front thigh skin - fold thickness and improvement in performance due to training (r = -0.74, p<0.001).14 we found, however, an association of mid - axilla, subscapular, abdominal and suprailiac skin - fold thickness with running speed during training. legaz & eston investigated 16 male and eight female spanish high - level runners covering distances between 100 m and the marathon.14 their athletes trained six or seven days a week, for 20 to 25 hours. in contrast, our recreational runners trained for only three hours a week. also in the study of arrese & ostriz, where iliac crest and abdominal skin - fold thicknesses were related to marathon performance in females, high - level runners had been investigated.13 furthermore we must assume that our runners were older and had a higher body fat percentage compared to the elite runners in legaz & eston14 and arrese & ostriz.13 the subjects in the study of arrese & ostriz were between 21 years (100 m) and 30 years (marathon).13 the mean age of our subjects was 38 years, and we might expect that older runners would have more body fat. a cross - sectional study is limited regarding the influence and effects of anthropometric and training characteristics on race time in runners, since only an intervention trial can answer this question. unfortunately we have no data about energy deficit31 or disorder in fluid or electrolyte metabolism32 which also might affect performance. however, in existing literature, smaller samples were investigated. in the study of arrese & ostriz,13 11 female marathoners were included and legaz & eston14 investigated a total of eight female endurance runners. a cross - sectional study is limited regarding the influence and effects of anthropometric and training characteristics on race time in runners, since only an intervention trial can answer this question. unfortunately we have no data about energy deficit31 or disorder in fluid or electrolyte metabolism32 which also might affect performance. however, in existing literature, smaller samples were investigated. in the study of arrese & ostriz,13 11 female marathoners were included and legaz & eston14 investigated a total of eight female endurance runners. to summarize, both variables of anthropometry and training were related to half marathon race time in these recreational female runners and an association between upper body skin - fold thicknesses and speed in running during training was found. intensity in training seemed to be of importance for a fast half marathon race time and high training intensity may lead to reduced thickness of upper body skin - folds in recreational female half marathoners.	introduction : the relationship between skin - fold thickness and running performance has been investigated from 100 m to the marathon distance, except the half marathon distance.objective:to investigate whether anthropometry characteristics or training practices were related to race time in 42 recreational female half marathoners to determine the predictor variables of half - marathon race time and to inform future novice female half marathoners.methods:observational field study at the half marathon basel in switzerland.results:in the bivariate analysis, body mass (r = 0.60), body mass index (r = 0.48), body fat (r = 0.56), skin - fold at pectoral (r = 0.61), mid - axilla (r = 0.69), triceps (r = 0.49), subscapular (r = 0.61), abdominal (r = 0.59), suprailiac (r = 0.55) medial calf (r = 0.53) site, and speed of the training sessions (r = -0.68) correlated to race time. mid - axilla skin - fold (p = 0.04) and speed of the training sessions (p = 0.0001) remained significant after multi - variate analysis. race time in a half marathon might be predicted by the following equation (r2 = 0.71) : race time (min) = 166.7 + 1.7x (mid - axilla skin - fold, mm) - 6.4x (speed in training, km / h). running speed during training was related to skin - fold thickness at mid - axilla (r = -0.31), subscapular (r = -0.38), abdominal (r = -0.44), suprailiacal (r = -0.41), the sum of eight skin - folds (r = -0.36) and percent body fat (r = -0.31).conclusion : anthropometric and training variables were related to half - marathon race time in recreational female runners. skin - fold thicknesses at various upper body locations were related to training intensity. high running speed in training appears to be important for fast half - marathon race times and may reduce upper body skin - fold thicknesses in recreational female half marathoners.
fragile x syndrome (fxs) is the most frequent form of inherited intellectual disability, and is the most common known cause of autism or autism - like behaviors. this syndrome shares clinical behavioral features with mental retardation, learning disorders, attention deficit disorder, hyperactivity disorder, anxiety, and epilepsy [24 ]. recent studies report an association between the therapeutic potential of physical exercise and autistic individuals with fragile x syndrome. physical exercise as an aspect of autism therapy could lead to increased neuronal survival and counter brain injury in asd patients. in addition, a variety of activities ranging from light leisure activities to heavy aerobic exercise promotes brain vascularization and stimulates neuronal and glial genesis. some studies report that specific molecular factors contribute in multiple ways to the beneficial effects of exercise on brain function in patients with autism - like diseases [1012 ]. neurotrophic factors, such as nerve growth factor (ngf), fibroblast growth factor 2 (fgf-2), and brain - derived neurotrophic factor (bdnf) are generated in the hippocampus in response to exercise. thus this review discusses the effects of physical exercise intervention on stereotypic behavior and physical capability in people with ads. a systematic search of articles and review papers in the pubmed digital library database from 1995 to 2011 was conducted. the criteria of the article search mainly focused on physical exercise correlated with autism spectrum disorder. only papers published in english in international journals the main key search words were physical exercise, rehabilitation, and autism spectrum disorder. additionally, we inserted some synonyms of autism - like diseases to search a greater number of articles containing more and deeper results for the present review. individuals with asd tend to have balance problems in gait, posture instability, and difficulties in joint flexibility. in addition, they have deficits and delays in the development of motor behaviors [1520 ]. these difficulties in motor abilities may be further impaired by reduced opportunities to engage in physical exercise and behavioral intervention. moreover, game exercise that combines physical and mental activities has decreased stereotypies in individuals with autism and has improved their cognitive function. in the case of aerobic exercise, improved academic function has been found in children with asd. in addition, enhanced motor ability and sensory integrative function due to physical activity may be consistently maintained in patients with asd. the difficulties that arise in community interactions with asd mainly pertain to the lack of understanding that results from an incapability to effectively interpret asd behaviors. physical exercise is confirmed to be an effective means to prevent these problems between general and asd populations. mild exercise programs involving walking and jogging decrease stereotypic behaviors in the short term, but this state of behavior is shown to return to previous levels after 90 minutes following the exercise. some studies confirm that walking exercise not only improves the physical condition, but also reduces the bmi (body mass index) of patients with severe autism. following physical exercise, stereotypic and maladaptive behaviors in asd patients have been shown to improve, and sleep disorders and physical capabilities involving muscular strength have also been enhanced. stimulation of the fragile x mental retardation 1 (fmr1) gene at postsynaptic sites is associated with the neuronal plasticity of dendritic spines. therefore, an absence of fmr1 genes is expressed in neuronal dysmorphology and the defection of dendritic spines. a great deal of evidence suggests that synaptic dysfunction is a cause of autism. some research points out that autism neurotrophic factors such as brain - derived neurotrophic factor (bdnf), nerve growth factor (ngf), fibroblast growth factor 2 (fgf-2), and other molecules contribute to synaptic transmission. moreover, neurotrophic factors play a role in the regulation of synaptogenesis, synaptic plasticity, and neural survival. studies show that synaptic plasticity, neurotransmission, and neural growth factor are increased in rat and mouse brains due to physical exercise. brain - derived neurotrophic factor (bdnf) binds to receptors trkb and p75ntr, resulting in the regulation of neuronal differentiation and survival in embryos. in adults, bdnf may be necessary for both the proliferation and long - term survival of newborn neurons in the forebrain. these neurotrophic factors tend to increase with physical exercise, and expression of bdnf is reported in parts of the brain particularly the hippocampus as a result of physical activity. it is suggested that exercise increases levels of neurotrophic factors in the brains of humans. levels of bdnf may be a factor in clinical diagnosis and intervention for the pathophysiology of autism is patients. previous studies show that bdnf levels in autism patients under six years of age are significantly higher than bdnf levels in autism patients over six years of age. asd reduces volume in the forebrain, including the size of the amygdala and hippocampus, relative to total brain volume. this finding reflects the neural connections of limbic systems with other parts of the cerebral cortex. on the other hand, exercise has been associated with increases in hippocampal tissue, and with boosting the growth of new brain cells by increasing bdnf in the brain cortex. it is probable that the growth of new brain cells in the hippocampus, together with the modulation of remaining connections in the brain cortex are responsible for improvements in brain function due to physical exercise. the field of autism is linked with extensive research in areas of molecular biology and has infinite potential for targeted treatment. our review researches the characterization of autism and investigates preventive and therapeutic approaches to fxs in asd. we explore the clinical application of physical exercise - based therapeutics, leading to effective treatment and clear benefits in the prevention of disease. however, additional studies are needed to examine the mechanism of melatonin associated with autism and fxs. as a result of the positive influence of exercise, neurotrophic factors and the growth of new brain cells further research is required to investigate the mechanisms of interaction between improvement due to exercise therapy for various asd behaviors and newly generated brain cells through increased neurotrophic factors.	fragile x syndrome (fxs) is the most common known genetic cause of autism spectrum disorder, and is also linked to other neurologic and psychiatric disorders. the purpose of this review article is to examine a variety of recent studies on the correlation between physical exercise and autistic behavior in individuals with fragile x syndrome. additionally, we discuss promising approaches for further investigation of the benefits of physical exercise for autism spectrum disorder (asd) patients. a systematic search of the pubmed digital library database for pertinent articles published from 1995 to 2011 was conducted. individuals with asd who experience exercise tend to exhibit improvement in physical function. in addition, exercise promotes neurotrophic factors and boosts the growth of new brain cells. the collected review articles describe how physical exercise has particular effects on stereotypic behavior and cognition among asd patients. finally, physical exercise may benefit patients with autism spectrum disorder through the improvement of muscular strength for increased physical capability.
traditionally, femoral and tibial cuts in total knee arthroplasty (tka) have been made perpendicular to the mechanical axis of the knee using either an intramedullary (i m) or an extramedullary alignment guide system. however, there is no consensus on the differences and relative superiority of the two systems1,2). hungerford3) and hungerford and krackow4) suggested that the proximal tibia be cut in 3 of varus since tibial plateau is in 3 varus relative to the tibial axis and the distal femur in 3 of valgus with respect to the mechanical axis. however, it has been of limited use because of the likelihood of damage to the lateral collateral ligament or the popliteus tendon caused by excessive varus cutting of the tibia or extensive resection of the lateral femoral condyle. on the other hand, in tka using an i m alignment guide system, the use of a fixed length i m rod that can not be adjusted to the length of the femur during surgery may cause a gap between the medullary canal and the rod, and thus affect the femoral component position. in particular, deep insertion of the rod for additional femoral bone resection in knees with flexion contracture often results in abnormal placement of the femoral component with angle under 94 or over 98. in this study, we investigated the relationship between inappropriate femoral resection in the coronal plane and clinical outcome in im - guided tka. of the patients who underwent tka using the nexgen legacy posterior stabilised (lps ; zimmer, warsaw, in, usa) and an i m alignment guide system for 6 of valgus correction, 101 patients (154 knees) who were available for 3 years of follow - up between april 2005 and may 2008 (mean, 4.4 years ; range, 3 to 6.8 years) were included in this study. the exclusion criteria there were 5 males and 96 females with a mean age of 69.64 years (range, 53 to 85 years). after soft tissue balancing, distal femoral resection was performed using an intramedullary cutting guide, which was followed by the proximal tibial cut using an extramedullary guide. an intramedullary rod for distal femoral cut was inserted in 6 of valgus so that the cutting guide was positioned perpendicular to the mechanical axis of the femur. after resection of the anterior aspect of the distal femur, distal femoral cut and proximal tibial cut were performed using a device that allows for creation of a rectangular extension gap until medial - lateral and flexion - extension gaps were balanced. patellar resurfacing was performed in only 10 patients (13 knees). on the clinical assessment, american knee society knee score (ksks) and american knee society functional score (ksfs) were evaluated preoperatively and at the last follow - up. on the radiographic assessment, mechanical axis deviation (mad) that was defined as the angular difference between the femoral and tibial mechanical axes patellar tilt angle (pta) on the merchant view was measured preoperatively and at the last follow - up (fig., the angle created by the mechanical axis and anatomical axis of the femur was measured on the preoperative long leg ap radiograph to assess the adequacy of 6 of valgus insertion of the intramedullary rod. the location of the femoral component (angle) was assessed on the postoperative plain radiograph (fig. 3). based on previous studies5,6), we arbitrarily took 2 as an acceptable margin of error for angle. the patients were divided according to the angle into two groups : group 1, 94 angle, 98. intergroup differences in the mad, pta, ksks, ksfs, and pre- and postoperative changes for each parameter were analyzed. the relationship between the angle and the mad, pta, ksks, and ksfs, and the cut - off value that yields the greatest clinical and radiological difference were assessed. 9.3 (sas institute inc., cary, nc, usa) with statistical significance set at p, 98. intergroup differences in the mad, pta, ksks, ksfs, and pre- and postoperative changes for each parameter were analyzed. the relationship between the angle and the mad, pta, ksks, and ksfs, and the cut - off value that yields the greatest clinical and radiological difference were assessed. 9.3 (sas institute inc., cary, nc, usa) with statistical significance set at p 2 of mad was not significantly different between the groups, 65 in group 1 and 24 in group 2 (p=0.12) (table 3). the mean pta was 9.03 (5.09) preoperatively and 10.47 (5.69) at the last follow - up (p=0.004) (table 1). there was no significant difference in the pta between the groups, 10.17 (5.39) in group 1 and 11.58 (6.65) in group 2 (p=0.45) (table 2). there was a negative correlation between the angle and the mad (p=0.01 ; correlation value, -0.20), whereas no correlation was found with the other parameters (table 4). on the correlation between the mad and the angle, the cut - off value that yields the greatest difference in the mad was 3. therefore, intergroup difference in the mad was most distinctive when the patients were divided into a group with 93 99, although the difference was not at a statistically significant level (p=0.12) (table 5). this is because malalignment caused by improper bone resection results in component wear and loosening, which is the primary cause of tka failure2,7 - 9). postoperative component alignment is essential for predicting the failure of tka and the need for revision surgery. distal femoral resection in primary tka is vital to achieving normal alignment in the coronal and sagittal planes. in particular, bone resection in the coronal plane has been considered as the most important procedure in tka10). most of the contemporary tka systems use intramedullary alignment guides along the anatomical axis of the femur. femoral and tibial resections are performed separately in general and postoperative mechanical alignment depends on the accuracy of each bone resection. it has been thought that 2 of deviation from the normal femorotibial angle on the coronal plane is acceptable8,9,11,12). studies have reported various important factors that are associated with femoral bone resection for accurate alignment on the coronal plane and the intramedullary alignment guide entry point is one of those. in this study, an intramedullary rod was inserted into the center of the femoral notch as opposed to medial insertion of a cutting guide. medial insertion of the cutting guide has been associated with a varus alignment and lateral insertion with a valgus alignment. reed and gollish13) suggested that the proper entry point of an intramedullary rod was 6.6 mm medial to the center of the femoral notch. jiang and insall14) reported that rotation of the femur on the ap radiograph was associated with the knee alignment. however, such a relationship could be observable only when an excessive degree of femoral bowing was present and rotation tends to have no significant influence on the anatomical - mechanical axis. accordingly, we believe that intramedullary guide insertion along the anatomical axis can be more effective in reducing malalignment error than the conventional insertion along the center of the femoral notch. in this study, the intramedullary rod was inserted in 6 valgus position to make the cutting guide be perpendicular to the mechanical axis of the femur, based on the assumption that the angular difference between the mechanical axis of the femur and the anatomical axis is 6. however, the value can be different depending on the patient, in which a 6 valgus insertion can result in malalignment. accordingly, we measured the angle on the preoperative long leg ap radiograph in each patient and included the patients in whom the distal femoral cutting guide was perpendicular to the mechanical axis when inserted in 6 valgus position in this study. the length of an intramedullary rod which can not be adjusted to the length of the femur results in errors due to the gap between the medullary canal and the rod. in our study, the number of knees with >2 of varus or valgus resection error in the coronal plane was 34 out of 154 (22%), which was high compared to 24 out of 146 (16.4%) in the study by longstaff.6). in particular, an intramedullary cutting guide needs to be inserted deeply for extensive femoral resection in knees with severe flexion contracture. during this procedure, the device we used in this study which using a hammer for cutting guide insertion instead of hands often results in a valgus knee because of applying valgus force on the femur even after the medial condyle of the distal femur in contact with the cutting guide. when the patients were divided with 96 set as the standard angle, more patients had valgus alignment (85 patients, 55.2%) than varus alignment. thereafter, we have designed an intramedullary rod that is 2.5 cm longer than the traditional device with a length of 22 cm (fig., we investigated the relationship between the angle and the clinical and radiographic results and found a negative correlation between the angle and the mad. the greater the angle was, the more valgus the femoral component position was, which eventually led to valgus alignment. therefore, the cut off value of an acceptable angle was determined based on the mad. initially, the patients were divided into two groups for comparisons by taking 2 as the acceptable margin of error of the angle based on previous studies. however, there were no significant differences between the groups in the mad, pta, ksks, and ksfs (mad, p=0.63 ; pta, p=0.45 ; ksks, p=0.67 ; ksfs, p=0.56) (table 2). therefore, we investigated the cut - off value that would show intergroup differences in the mad. when 3 was taken as the cut - off value, or the patients were divided into those with 93> angle 99, the difference in the mad was most notable between the groups, which, however, did not show statistical significance (p=0.12) (table 5). although the number of outliers with > 2 mad was higher in group 1 than group 2 (65 vs. 24) when grouping was done with 2 chosen as the acceptable margin of error for angle, there was no statistical significance in the difference (p=0.12) (table 3). in addition, no significant intergroup difference was found in the number of outliers when other cut - off values were used for grouping (table 3). we attributed this to the use of a device that enabled creation of a rectangular extension gap following soft tissue balancing, which allowed distal femoral cutting error to be partially compensated during proximal tibial cutting. there was no statistically significant difference between the groups in the clinical outcome assessed by ksks and ksfs. the pta and mad were not significantly different between the groups, which resulted in no remarkable difference in the coronal plane alignment. however, the greatest intergroup difference in the mad could be observed when 3 was taken as the cut - off value, which should be confirmed in a study with a longer follow - up period and a larger study population before clinical application. we have made various attempts to improve the coronal plane alignment in im - guided tka. currently, our method of choice is to use an intramedullary rod that is longer than the conventional rod in knees with the distance between the distal femur and the contact point between the femoral condyles is longer on the preoperative radiograph. in addition, we think that excessive varus or valgus resection can be prevented by predicting the proper thickness of the distal femoral resection based on the measurement of the length of a line that is tangent to the medial and lateral femoral condyles and perpendicular to the anatomical axis of the femur on the preoperative long leg plain ap radiograph. there have been many studies on methods to reduce coronal plane alignment error and mad. in particular, computer - aided navigation tka has been reported effective in improving mad in some studies15 - 17). the limitations of this study include that the study population was small (n=154), radiographic assessment was done by one surgeon, and the follow - up period was short for establishing clinical end radiological outcome. furthermore, we think that the influence of the relationship between the length of the femur and the intramedullary rod on the clinical and radiological outcome should be addressed in future studies. in tka using a fixed length intramedullary guide for femoral resection, the number of outliers with > 2 of varus or valgus resection error in the coronal plane was relatively high (34 in 154 knees, 22%). however, there was no statistically significant difference in clinical results between the knees with a resection error of > 2 and those with 2 (ksks, p=0.67 ; ksfs, p=0.56).	purposethe purpose of this study was to evaluate clinical results and accuracy of femoral cutting in the coronal plane in total knee arthroplasty (tka) using a fixed length intramedullary guide.materials and methodsfrom 2005 to 2008, 101 patients (154 knees) underwent tka (nexgen lps implant). the minimal follow - up period was 3 years (mean, 4.4 years). the patients were divided into two groups (group 1, 94 angle, 98). mechanical axis deviation (mad), patellar tilting angle (pta), knee society knee score (ksks), and knee society function score (ksfs) were measured in both groups.resultsthere were 120 knees in group 1 and 34 knees in group 2. there was no significant intergroup difference in the postoperative mad (group 1, 1.59 ; group 2, 1.91). the number of outliers with 2 mad was 65 in group 1 and 24 in group 2. the mean pta, ksks, and ksfs were 10.17, 96.0, and 96.6, respectively, in group 1 and 11.58, 84.5, and 85.5, respectively, in group 2.conclusionsthe percentage of coronal alignment outliers was relatively high (34 in 154 cases, 22%) after tka using a fixed length intramedullary guide. however, there was no statistically significant intergroup difference in clinical results (ksks, p=0.67 ; ksfs, p=0.56).
numerous single - nucleotide polymorphisms (snps) have been discovered and deposited in public databases (e.g. the national center for biotechnology information [http://www.ncbi.nlm.nih.gov ], ensembl [http://asia.ensembl.org/index.html ], and the mext integrated database project [http://dbcls.rois.ac.jp ]) through international snp discovery projects such as the human genome project,1 the international hapmap project (http://hapmap.ncbi.nlm.nih.gov/index.html.en), and the 1000 genomes project (www.1000genomes.org). together with the development of technologies for large - scale snp genotyping, genome - wide association studies (gwass) using hundreds of thousands of snps allow the identification of candidate genetic loci for multifactorial diseases. disease - associated snps have also been deposited in public databases, such as the database of genotypes and phenotypes (www.ncbi.nlm.nih.gov/gap). moreover, a number of snps have been reported to be associated with complex genetic traits, such as body mass index,2 height,3 and hair thickness.4 in the national human genome research institute (nhgri) gwas catalog (www.genome.gov), more than 8,800 trait- or disease - associated snps with genome - wide significance (p<510) have been archived from a total of 1,551 published gwas (through march, 2013).5 here, we describe a gwas strategy to identify disease - associated snps, including snp genotyping technologies for both the gwas stage and the following replication stage. based on this gwas strategy, we have identified associations of genetic variations with diseases related to hepatitis b and c viruses (hbv and hcv), including drug response in patients with chronic hcv infection,6 susceptibility to primary biliary cirrhosis (pbc),7 and hbv - related hepatocellular carcinoma (hcc).8 a number of snp typing methods have been used to analyze a single snp, or snps at multiple sites of a template or templates simultaneously. most of the methods employ single or multiple site - specific amplifications and a genotyping step based on various types of chemical reactions, including sanger direct sequencing, 5 exonuclease fluorescence - based assay (taqman),9 pyrosequencing,10 digitag2 assay,11 single - base extension,12 and matrix - assisted laser desorption / ionization time - of - flight mass spectrometry (maldi - tof).13 together with technology developments in large - scale snp genotyping, the most recent versions of commercially available genotyping platforms allow the simultaneous analysis of more than one million snps across the whole genome in a single experiment. two platforms are commercially available and widely used for genome - wide snp typing : affymetrix snp genechip arrays14 and illumina beadarray genotyping technology.15 the number of snps embedded in both platforms has been gradually increasing since 2003, when the first commercial genome - wide snp genotyping platform was released by affymetrix.16 the first platform of the affymetrix genechip mapping 10k array included 14,548 snps, which enabled the performance of whole - genome linkage analyses and was indeed used to identify a disease - associated missense mutation in the hoxd10 gene with charcot marie - tooth disease through a family - based linkage study.17 the current versions of the commercial platforms from affymetrix and illumina include more than 900,000 snps (genome - wide human snp array 6.0) and 4.3 million snps (humanomni5-quad beadchip), respectively. a newly released genome - wide snp typing platform, named the affymetrix axiom genome - wide asi 1 array, has a probe set for snps (including rare and common variants) that are optimized for asian populations. these platforms open a new approach for researchers to perform gwass with hundreds of thousands of snps, allowing the identification of candidate genetic loci for multifactorial diseases. in 2002, the first gwas using 92,788 gene - based snps was reported by a japanese group (riken), which identified the lymphotoxin- gene as being associated with susceptibility to myocardial infarction.18 the riken group developed its own platform to perform a gwas based on the invader assay19 with multiplex polymerase chain reaction (pcr).20 since 2002, the number of published genome - wide associations with genome - wide significance (p<510) has increased annually, reaching 1,551 publications in the nhgri gwa catalog (through march, 2013).5 for a replication study following a gwas stage, several candidate genetic regions that have been detected in the initial gwas need to be analyzed. suitable platforms for replication analyses have the ability to perform multiplex detections in a single reaction, such as the mini - sequencing (snapshot) technique,21 chip - based genotyping by mass spectrometry (sequenom),22 and the digitag2 assay.11 the digitag2 assay is our own technology for multiplex snp typing, and represents a simple and cost - effective approach by combining multiplex pcr to enrich genetic regions including the target snps with an oligonucleotide ligation assay to determine the genotype of the target locus. for a single locus analysis the taqman assay would be more commonly used to determine the genotype of the target locus, as opposed to conventional sanger sequencing, which is more commonly used when a large number of samples need to be analyzed. association(s) of certain variants with a particular trait by examining differences in allele and/or genotype frequency of all snps, which exist across the entire genome. gwas enables the effective detection of associated variations in strong linkage disequilibrium with the causal variants and genes, and the following replication analysis and high - density mapping identify the causal variants and genes using an independent set of participants with a larger number of samples. however, the association of snps with low minor allele frequency (below 15% ; known as rare variants) would be difficult to detect in a snp - based gwas because of insufficient statistical power due to the limitation of sample number.23 fig. the emerging strategy of gwas has revealed disease - causing alleles, or variants that lead to susceptibility to complex polygenic diseases with small additive or multiplicative effects on the disease phenotype. for example, a recent gwas and subsequent meta - analyses in populations of european descent identified human leukocyte antigen (hla) and 21 non - hla susceptibility loci, most of which are involved in interleukin (il)-12/il-12 receptor (il-12r) signaling, tumor necrosis factor (tnf)/toll - like receptor (tlr)nuclear factor (nf)-b signaling, and b - cell differentiation in the development of pbc.2427 pbc is a chronic cholestatic liver disease characterized by chronic non - suppurative destructive cholangitis of the intrahepatic small bile ducts. a high concordance rate in monozygotic twins and familial clustering of patients with pbc indicates the involvement of strong genetic factors in the development of pbc.28 to identify susceptibility loci for pbc in the japanese population, we conducted a gwas and subsequent replication study using a total of 1,327 pbc patients and 1,120 healthy controls.7 in addition to the most significant susceptibility region at hla, two significant susceptibility loci (tnfsf15 and pou2af1) with p - values < 510 were identified (table 1). moreover, of the 21 non - hla susceptibility loci that were identified in populations of european descent, three loci (ikzf3, cd80, and il7r) showed significant associations and two loci (nfkb1 and stat4) showed suggestive associations with pbc in the japanese population. five other loci (cxcr5, tnfaip2, map3k7ip1, rs6974491, and dennd1b) also showed marginal associations (p<0.05) with pbc in the japanese population (table 1). these results indicate that additional important disease pathways (via tnfsf15 and pou2af1) differentiation to t - helper 1 (th1) cells (via tnfsf15, cd80, il12, il12r, and stat4 ; fig. 2), b - cell differentiation (via pou2af1, cxcr5, spib, il7r, and ikzf3), and nf-b signaling in addition to the previously reported disease pathways have a role in the development of pbc in japanese populations. a proportion of susceptibility genes associated with pbc (cd80, il12a, il12rb2, stat4, and tnfsf15) are related to t - cell proliferation via both th1 and th17 cells. in another study that aimed to identify host genetic factors related to drug response to pegylated interferon- plus ribavirin treatment for hcv infected patients, comparatively small number of samples were analyzed in a gwas, including samples from 154 japanese hcv patients undergoing pegylated interferon-/ribavirin treatment, 78 null virologic responders, and 64 virologic responders.6 despite the small number of samples in the gwas in comparison with other studies in european descendants (european american,29 australian,30 and swiss31), the same disease - causing locus of il28b was identified with the strongest association in the japanese population. in general, the number of samples affects the statistical power of detection in a gwas. moreover, false - positive associations can increase when low - quality genotype data are incorporated in the analysis, presumably caused by accidental errors in genotyping steps or low - quality genomic dna. the japanese gwas was able to successfully identify the risk factors in a small number of samples because : (1) il28b is a strong host risk factor for drug response in asian and white populations ; and (2) quality controls were used in sample collection in terms of clinical characteristics, and the genotype data were checked for quality.14 as for hbv - related hcc, a gwas using chronic hbv carriers with and without hcc in five independent chinese samples found that one snp (rs17401966) in kif1b was associated with susceptibility to hbv - related hcc.32 moreover, in the most recent report on this topic, genetic variants in the stat4 and hla - dq genes were identified as genetic susceptibility loci for hbv - related hcc in the chinese population.33 we performed snp genotyping of rs17401966 on the kif1b gene in japanese, korean, and hong kong populations for the purpose of replication analysis of a previous gwas report.8 we first examined two independent japanese hbv - related hcc populations and healthy controls, including 179 patients and 769 controls from biobank japan, and142 patients and 251 controls from various hospitals. we also detected no association of the snp with hbv - related hcc in korean and hong kong populations using 164 patients and 144 controls, and 94 patients and 187 controls, respectively. in a recent report from another group, no significant association of the kif1b gene was observed in hbv - related hcc patients of saudi arabian ethnicity.34 these results may be explained by genetic diversity among the chinese, japanese, korean, hong kong, and saudi arabian populations. the complexity of multivariate interactions in the pathogenesis of hcc may lead to difficulties in identifying the gene(s) associated with hbv - related hcc. in a previous report that studied 179 japanese individuals with chronic hbv infection (chb) and 934 control participants, a gwas identified significant associations of chb with a region including hla - dpa1 and hla - dpb1.35 the same group was also conducted a second gwas with a total of 2,667 japanese patients with persistent hbv and 6,496 controls, which confirmed significant associations between the hla - dp locus and chb, in addition to associations with another two snps located in a genetic region including the hla - dq gene.36 we performed a gwas using samples from japanese hbv carriers, healthy controls, and individuals who spontaneously resolved hbv infections (hepatitis b surface antigen [hbsag ] negative and hepatitis b core antibody [anti - hbc ] positive), in order to confirm or identify the host genetic factors related to chb and viral clearance.37 in the subsequent replication analysis, we validated the associated snps in the gwas using two independent sets of japanese and korean individuals. in our study, healthy controls with no clinical evidence of hbv exposure were randomly selected ; therefore, hbv - resolved individuals were prepared to clearly identify the host genetic factors related to chb or hbv clearance. an association analysis conducted with the japanese and korean data identified the hla - dpa1 and hla - dpb1 genes with pmeta=1.8910 for rs3077 and pmeta=9.6910 for rs9277542. we also found that the hla - dpa1 and hla - dpb1 genes were significantly associated with protective effects against chb in asian populations including japanese, korean, chinese, and thai individuals (pmeta=1.2610 for rs3077 and pmeta=1.1010 for rs9277535) (fig. 3).3541 the snp rs9277535 was located about 4 kb upstream from rs9277542 and showed strong linkage disequilibrium of r 0.955 in the hapmap jpt (japanese in tokyo, japan) population. these results suggest that the associations between the hla - dp locus and the protective effects against persistent hbv infection and with clearance of hbv are widely replicated in east asian populations ; however, there are few reports of gwass in caucasian or african populations. further studies are necessary to clarify the pathogenesis of chb and the mechanisms of hbv clearance, including functional analyses of the hla - dp molecule. meta - analysis using the random - effects model across seven independent studies, including six additional published data sets, showed pmeta=1.2610, odds ratio (or) 0.55 for rs3077, and pmeta=1.1010, or 0.63 for rs9277535. the gwass described above have successfully identified disease - associated genes or snps using different types of genome - wide snp tying platforms. the embedded snps are varied among platforms by selecting the tagging snps and the suitable snps for their own genotyping strategy ; however, the genome coverage among platforms revealed no differences over 60% between the hapmap ceu samples and the hapmap jpt+chb samples.42 moreover, the genome coverage of the current version of the affymetrix genome - wide human snp array 6.0 platform has been estimated to reach 75% in the japanese population.14 together with technology developments, gwass are a promising strategy with which to identify host genetic factors for multactorial diseases, including common liver diseases, and various host genetic traits recently, new strategies and emerging technologies for massive parallel sequencing (also termed next - generation sequencing) have allowed whole - genome analysis to identify single - nucleotide variations and structural variations (including insertion, deletion, duplication, translocation, and transposition events). the costs of using these emerging technologies are currently high ; therefore, common snp - based gwass using the genome - wide snp analysis technologies introduced in this paper still have an important potential role in the fields of clinical and basic research.	a number of disease - associated genetic markers for common liver diseases have been identified using genome - wide association studies (gwass). the gwas strategy is based on genome - wide single - nucleotide polymorphism typing technologies, which are now commercially available, accompanied by statistical methods to identify host genetic factors that are associated with target diseases or complex genetic traits. one of the most striking features of the gwas strategy is the ability to identify unexpected disease - associated genetic markers across the entire human genome. here, we describe the technological aspects of the gwas strategy with examples from actual gwas reports related to hepatitis research, including drug response for patients with chronic hepatitis c, susceptibility to primary biliary cirrhosis, and hepatitis - b - related hepatocellular carcinoma.
intra - abdominal fibromatosis are uncommon benign neoplasms which originate from musculoaponeurotic structures of the body and primarily affects the mesentery or retroperitoneum. however, rarely, they can arise from the intestinal wall and mimic a gastrointestinal malign neoplasm.1 acute appendicitis is a common clinical condition, however, < 1% of patients with acute appendicitis have an accompanying malignant process.2 acute appendicitis cases associated with benign lesions in the cecum are very rare. this is a report of a 25-year - old female patient with a cecal fibromatosis causing acute appendicitis. the purpose of this report is to remind health care practitioners that some very rare etiologies may be involved in a common clinical condition and to underscore the place of laparoscopic approach and preoperative computed tomography in this disease. a 25-year - old female patient presented to our emergency department because of abdominal pain, nausea, and vomiting within the last 24 hours. the pain started as a diffuse abdominal pain and eventually localized in the right lower quadrant. she had fever (38.5c), 90 beats per minute heart rate, and 110/70 blood pressure. ultrasonography demonstrated a noncompressed tubular lesion in the right lower quadrant which was reported to be consistent with acute appendicitis. while the cecum was observed to be normal, a solid mass of 2.5 cm diameter was palpated in the appendiceal base. the mass was obstructing the appendix lumen and cecum resection was decided (figure 1). immunohistochemically, the lesion was diffuse strong positive with vimentin and actin, and negative for desmin, cd34, cd117, and s-100, which was reported to be a fibromatosis of 2.5 cm diameter. fibromatosis, also known as desmoid tumors, are local aggressive benign neoplasms which originate from musculoaponeurotic structures of the body fibromatosis may be associated with gardner s syndrome and familial polyposis coli.3 colonoscopy can show the colonic polyps whereas fundoscopy can display multiple pigmented lesions in the retina. while intra - abdominal fibromatosis generally originates from the mesentery of the small intestine, there are also involvements reported in the retroperitoneum, transverse mesocolon, and omentum.4 rarely, it can also stem from the intestinal wall like submucosal benign lesions such as inflammatory fibroid polyp, leimyoma, etc, and gastrointestinal stromal tumor (gist), and mimic a gastrointestinal malign neoplasia. gists are the most common mesenchymal tumor of the gastrointestinal tract and the treatment strategy is important because of its malignant potential. as a result, surgeons give special attention to gists and a specimen with this suspected diagnosis is important to pathologists. absence of reaction with cd117 and s-100 is the key point of differential diagnosis between fibromatosis and gist as in the present case. while most cases have an asymptomatic course, urgent surgical interventions have been reported due to reasons such as intestinal obstruction, perforation, and abscess formation.5 in women and the elderly, establishing an acute appendicitis diagnosis is problematic. the appendix has been found to be normal among 32%45% of appendectomies performed on women between 15 and 45 years of age.6 pelvic inflammatory disease, graft follicle rupture, ectopic pregnancy rupture, and ovarian cyst pathologies may be hard to differentiate from acute appendicitis. less than 1% of patients with acute appendicitis have an accompanying malignant process, most commonly appendiceal carcinoids. less frequently, acute appendicitis may be the initial symptom of a cecal malignancy and acute appendicitis cases associated with benign lesions in the cecum are very rare. a search of pubmed7 with the keywords intra - abdominal fibromatosis, desmoid tumour, and cecal fibromatosis, found only one case about a cecal fibromatosis causing appendicitis8 and this report seems to be the first case in the english language. since the advent of video - assisted technology in general surgery, many operations have been performed by laparoscopy. laparoscopic approach has several obvious advantages over open surgery such as less postoperative pain, reduced duration of hospitalization and incision - site infection, and achievement of a better cosmetic appearance.9 many health care centers perform laparoscopic appendectomy routinely. although the laparoscopic approach has significant advantages over open surgery, since the advent of the technique the most important criticism about it has been lack of tactile sensation by palpation. excessive fat, adhesions occurring due to earlier surgeries, and lack of tactile sensation may cause some lesions to go unnoticed. shayani10 performed colonoscopy on a patient due to detection of mucin islets on the appendiceal base by histopathological examination after the laparoscopic appendectomy. a submucosal mass was seen in the cecum during the colonoscopy and the patient was subjected to right hemicholectomy via laparotomy. in the histopathological examination, however, the lesion in the cecum was not detected in the first intervention. in the current case the cecum appeared to be totally normal and ileocecal resection was decided due to detection of a mass only by palpation. if this intervention had been performed laparoscopically, this lesion would probably not have been detected. however, the application of a preoperative computed tomography (ct) could have revealed the lesion of 2.5 cm diameter prior to the procedure. multislice ct benefits detection of ileocecal region diseases such as mesentery lymphadenopathy, ischemic colitis, cecal tumors, crohn s disease, and, particularly, acute appendicitis.11 clinical evaluation of right lower quadrant pain is carried out with scorings (alvarado12) and most of the surgeons resort to ct imaging in cases with low scores or suspected symptoms. a recently published meta - analysis study reported that the negative appendectomy rate is 8.7% when using ct compared with 16.7% when using clinical evaluation alone (p < 0.001).13 although routine use of ct in all patients presenting with suspected appendicitis may lead to delay in surgery, this delay is not associated with increased appendiceal perforation of morbidity rates.10 however, it is important to know whether routine use of ct is cost - effective in patients with suspected right lower quadrant pain. morse and colleagues demonstrated that multislice ct reduces the negative appendectomy rates and savings averaged us$1412 per patient.14 in conclusion, surgeons should be aware that some very rare etiologies may be involved in acute appendicitis management. cecal fibromatosis is one of the least common reasons for acute appendicitis and preoperative diagnosis is difficult. ct can be helpful in both detection of unpredicted coexisting lesions and reduction of negative appendectomy.	although acute appendicitis is a common clinical condition in general surgical practice, < 1% of them are associated with malignancies. appendiceal carcinoids make up most of those malignancies and acute appendicitis cases associated with benign cecal neoplasias are very uncommon. in this study, a 25-year - old female patient who presented with distinct acute appendicitis symptoms is reported. the patient was operated on via open technique. exploration revealed an appendix with advanced edema and hyperemia. while the cecum was observed to be normal, a solid mass of 2.5 cm diameter was palpated in the appendiceal base. following the ileocecal resection, histopathological examination revealed the mass as a fibromatosis. the goals of this report are to remind health care professionals that some very rare etiologies may be involved in acute appendicitis diagnosis and treatment, and to underscore the place of laparoscopic approach and preoperative computed tomography in this disease.
technology is beginning to permeate health care and is being used in multiple ways including helping patients seek out health information via the web, scheduling appointments, refilling medications, sending and receiving secure messages, managing chronic conditions, keeping personal health records, performing self - management, using remote monitoring devices, and building social networks based on similar health concerns. if technology interventions are integrated into existing delivery systems, the potential exists to increase access to care and subsequently improve the health of individuals and populations. though the use of individual technologies has been effective in improving patient outcomes, this knowledge has not translated into the development of an integrated system to enhance the delivery of primary care. technology is developing in piecemeal and most applications use one methodology, treat or monitor one illness, and are incompatible with one another or existing electronic health records (ehr). in addition, evidence is lacking about provider input into the early stages of software development. provider input from the beginning stages of development of emerging technologies would give opportunities to build, shape, and apply the technology. thus, this input would potentially enhance future sustainability and scalability in real world delivery systems. a new platform that integrates multiple technologies for primary health care called mi smart (mobile improvement of self - management ability through rural technology) is developed and is being evaluated for feasibility in persons with chronic conditions. the integrated technologies of mi smart combine a hipaa compliant, web - based, system of mhealth sensors, and mobile devices to treat and monitor multiple chronic conditions. the mi smart system allows patients to track diagnoses, medications, and lab results, receive reminders for self - management, perform self - monitoring, obtain feedback in real time, engage in education, and attend e - visits (video conferencing). the system displays a record database to patients and providers that will be integrated into existing electronic health records. the study protocol, development, and feasibility of the mi smart are published and available for review [46 ]. the creation of mi smart was guided by the model for developing complex nursing interventions. the model suggests a process for building and informing interventions with the intention of making interventions effective, sustainable, and scalable. the steps are as follows : problem identification, practice analysis, identification of the overall objective of the intervention, identification of theory or key principles to guide the intervention, building the intervention and planning the delivery of the intervention, modelling the intervention and seeking expert review, and developing the study protocol. this paper presents the findings related to seeking expert review which occurred in the form of provider focus groups. we sought information to inform the development and implementation of mi smart to improve understanding of providers ' needs when using technology to improve health. we assessed preferences and perceived obstacles of multidisciplinary healthcare team members through 3 activities : (1) demonstration of the mi smart platform, (2) group discussion, and (3) confidential questionnaire. the purpose of this paper is to report the findings of the focus groups which were used to revise mi smart prior to feasibility testing. a focus group is an interview technique that uses purposive sampling to select participants, who are of a specific population, share similar characteristics, and have something to say about the topic. one of the distinct features of focus group interviews is group dynamics. the type and range of data generated through the interaction of the group members are often deeper and richer than those obtained from a one - on - one interview. focus groups were completed in three different populations including academic healthcare providers (group 1), interprofessional outpatient healthcare providers (group 2), and community stakeholders (group 3). the setting was in north central west virginia, the location where mi smart will be implemented. these populations were selected so that the population of future users of the mi smart platform would be able to participate in the focus groups. each focus group was held after a regularly scheduled meeting of the group, was arranged with the assistance of the organizer, and was announced a month prior to the planned focus group. a total of 37 participants attended one of the three demonstrations of the mi smart platform. of those who attended the demonstration, 31 participated in the group discussion and focus group participants included medical assistants, registered nurses, front desk staff, physicians, nurse practitioners, physician 's assistants, pharmacists, social workers, administrators, and community board members. the demonstration of mi smart was done through a combination of powerpoint slides with screen shots, displaying the platform, and demonstrating the actual equipment to be used. participants were also able to try the equipment and platform by using a tablet and bluetooth enabled self - monitoring devices including a glucometer, blood pressure cuff, and weight scale. the demonstration of mi smart system included a mock e - visit, viewing examples of planed distance education materials, and viewing a mock patient record which included current prescriptions with a links to patient education, a list of current diagnosis with links to patient education, a database of self - monitoring results color coded to normal, above normal, and critical, automated responses for self - monitoring results, patient reminders / notifications, lab results, survey collection links, and short education videos related to common chronic illness control. open ended questions were posed to each group to elicit responses and conversation from the healthcare team members. the following open ended questions were asked to each group : what do you think your biggest obstacle is when thinking about prescribing mhealth tools?as a patient yourself, what do you think your biggest obstacle is when thinking about using mhealth tools?what did you like best about the mi smart platform?what would you like to see removed from the mi smart platform?what would you like to see added to the mi smart platform?which outcome measures do you think would be most helpful to measure when treating patients with chronic illness using technology?what outcome measures do you think are most important to patients?what else would you like to tell me about mi smart ? what do you think your biggest obstacle is when thinking about prescribing mhealth tools ? as a patient yourself, what do you think your biggest obstacle is when thinking about using mhealth tools ? what would you like to see removed from the mi smart platform ? what would you like to see added to the mi smart platform ? which outcome measures do you think would be most helpful to measure when treating patients with chronic illness using technology ? what outcome measures do you think are most important to patients ? after all discussion points were covered, the facilitator asked for any additional comments or questions. lastly, the participants were asked to complete a questionnaire with demographic information and their past, current, and future use of technology (figures 1, 2, and 3 and tables 1 and 2). due to purposive sampling to select participants, data saturation was not the aim of data collection. the researchers identified recurring themes found in the deidentified data and counted all instances of a given theme. several procedures were employed to maximize the transcription quality and to ensure that quality standards were maintained. multiple team members participated in the analysis of the transcriptions by identifying, discussing, and agreeing on the main points in participant responses. the demonstration of mi smart was attended by 37 participants (group 1 n = 15, group 2 n = 12, and group 3 n = 10). thirty - one participants stayed for group discussion of the mi smart platform (group 1 n = 14, group 2 n = 11, and group 3 n = 6) and 29 participants, both prescribing (n = 8) and nonprescribing healthcare team members (n = 21), completed the questionnaire (group 1 n = 12, group 2 n = 12, and group 3 n = 5). there are 22 full time employees, an executive board of 8, and 14 volunteers from the local university. all of the participants had used technology such as ehr. however, only 2 participants had previously used technology tools, other than the ehr. nineteen providers (65%) reported that they would definitely use mi smart in the future. providers identified the following attributes as what they liked best about the specific technology, mi smart : capability to provide virtual visits, readability, connectivity of several different monitors in one system, ease of use, real - time feedback, and enhanced access. providers in all three focus groups reported that the patient home screen was simple and easy to use. an example provider comment was i can look at the home screen and tell what will be in each tab. other providers liked that the system gave real - time feedback stating, it 's great that they get immediate feedback on their readings. it 's good that they get a messages telling them if their glucose was normal or not and what to do if it 's not normal. multiple providers agreed that mi smart would help their patients who traveled long distances and that the technology was more patient - centered than traditional visits. this would save so much time for my patients that drive from far away. lastly, the ability to use any device that connects to the internet was given as a positive attribute of mi smart as well as the incorporation of common parts of a primary care visit integrated into one platform. several themes related to barriers of using this type of technology in their practice emerged across all focus groups including patient ability / willingness, lack of reimbursement for services, and lack of integration into the electronic medical record (emr). the biggest obstacle that healthcare providers perceived when prescribing technology tools is patient ability or willingness to use the technology. one provider statedi'd really have to think about which patients would use and benefit from this technology before i would consider offering it to them. you know ? i think the equipment would be a lot of money and then there is the time we would spend teaching them how to use it. which patients would use it and which patients would be able to use it and then which patients would want to use it. i 'd really have to think about which patients would use and benefit from this technology before i would consider offering it to them. you know ? i think the equipment would be a lot of money and then there is the time we would spend teaching them how to use it. which patients would use it and which patients would be able to use it and then which patients would want to use it. the majority of the providers said they would need more information on how the use of systems like this would be reimbursed prior to using it in their clinic. one provider said i do not think this will catch on in practice unless it 's in the ehr. it would just be another thing i 'd have to check and i already have enough to do. in another group, a provider asked would you be able to see all of the patient 's self - monitoring readings in the chart ? if not, i 'm not going to remember to look at them. providers identified several potential obstacles for patients including time, alarm fatigue, ability, technical support, and having multiple chronic illnesses. across all three focus groups, providers described time to do the required self - monitoring as the a significant barrier for using technology. an example comment was you would really have to prioritize using this system daily. i 'm not sure, if i was the patient, i would have enough time to do it all. other providers talked about the concept of alarm fatigue or being overwhelmed by constant reminders for monitoring. however, some liked the notifications and wondered if flexibility to set reminders based on individual preference or need was possible. one provider commented i 'm not sure patients much older than me would have the ability to use all of this technology. this led others to ask about what would happen if there were technical glitches and the need technology support. maybe this would make caring for complex chronic illness more manageable for me as a patient, or maybe it would make it more complex. another joined in sayingperhaps it would help patients realize that some of their illness are tied together improving one, often improves the other. perhaps it would help patients realize that some of their illness are tied together improving one, often improves the other. adding symptom monitoring and more devices to provided home patient monitoring were the themes that emerged related to what should be added to mi smart. the biophysical outcomes such as glucose, a1c, blood pressure, weight, lipids, inr (international normalized ratio), and pulmonary function tests were the most frequently requested outcome measures. providers commented that adding a box where patients could type how they feel may offer more detailed symptom monitoring and insight into how to better help them. six providers reported wanting to see patient self - reported outcomes such as mood, symptoms, patient perceived health changes, quality of life, depression symptoms, suicidal ideation, patient satisfaction, and self - efficacy. additionally, adding information on adherence to lifestyle behaviors was mentioned in two groups one provider said i 'd like to know how they are sticking to their diet, exercise and medications. if they could log this, i would be able to identify what education and support was needed. two providers commented that the platform focuses on metabolic disorders and that they would like to see other chronic illnesses monitoring for illness like chf and copd. another provider commented it would be nice if other self - monitoring devices like their fit bit would be incorporated. yet another asked is it possible for patients to add additional information about themselves in the system like labs from another healthcare system or previous medical records ? the participation of 37 individuals, both prescribing and nonprescribing, demonstrates interest in including technology into routine care of patients. no research literature can be found related to the numbers of primary care providers who are using technology other than ehrs in primary care practices. in 2014, the american telemedicine association reported that there were about 200 telemedicine networks in the us, with 3,500 service sites. while the vast majority of participants in these focus groups have not used technology in the past, they were optimistic about mi smart and contributed ideas about how to improve the system for both providers and patients. the clinic of interest where the first trail of mi smart will be conducted is a free clinic that is transitioning to federally qualified health centers (fqhc) accepting centers for medicare & medicaid services (cms) payment. hence, some of the providers and the executive board are concerned regarding the lack of reimbursement for services provided via electronic means. payment and coverage for services delivered via technology is one of the biggest challenges for adoption by practices. in the united states, the mi smart team has been working with the clinic to determine appropriate billing procedures for offering this type of technology. additionally, we will be completing a cost and time investment analysis during the first trial of the system. the lack of integration into existing ehrs and capability to include personal fitness monitors and expanding self - monitoring to include symptoms, quality of life, and chronic illness other than metabolic disorders remains a challenge. we have developed mi smart so that it is meets the standards for transferring healthcare information, hl7, into multiple health records. the last step of the first mi smart trial will be integrating the data into the existing ehr. however, the first trial will only address the most common chronic illnesses found in the clinic, due to cost of equipment. additionally, the first trial of mi smart will not include incorporation of personal fitness monitors, symptom monitoring, or a food diary. however, plans for incorporating this monitoring are being developed with the input of the clinicians and patients in this clinic. additionally, we have added measures of quality of life, depression, loneliness, and self - management ability to the first trial in order to gain baseline data. currently, technology innovations have emerged in silos of incompatible systems with entrenched company legacies to overcome. future work related to technology development should consider the preferences of providers and patients, need for policy changes related to reimbursement for care, cost effectiveness of technology for patients, and future potential for integrating with existing technologies. technology interventions have the potential to improve access and outcomes but will not be successful without the input of users. findings from these focus groups provided essential information that informed the continued development of the mi smart system. the demonstration of the mi smart platform to the healthcare providers who are intended to use it was a critical step prior to feasibility testing. once feasibility testing is complete, the next logical step will be a larger clinical trial.	used as integrated tools, technology may improve the ability of healthcare providers to improve access and outcomes of care. little is known about healthcare teams ' preferences in using such technology. this paper reports the findings from focus groups aimed at evaluating a newly developed primary care technology platform. focus groups were completed in academic, outpatient, and community settings. focus groups were attended by 37 individuals. the participants included professionals from multiple disciplines. both prescribing (n = 8) and nonprescribing healthcare team members (n = 21) completed the focus groups and survey. the majority were practicing for more than 20 years (44.8%) in an outpatient clinic (62%) for 2040 hours per week (37.9%). providers identified perceived obstacles of patient use as ability, willingness, and time. system obstacles were identified as lack of integration, lack of reimbursement, and cost. the positive attributes of the developed system were capability for virtual visits, readability, connectivity, user - friendliness, ability to capture biophysical measures, enhanced patient access, and incorporation of multiple technologies. providers suggested increasing capability for biophysical and symptom monitoring for more common chronic conditions. technology interventions have the potential to improve access and outcomes but will not be successful without the input of users.
while epidermal growth factor receptor (egfr) inhibitors have improved progression - free survival in patients with non - small cell lung cancer (nsclc), one of the most common adverse effects is papulopustular skin eruption, which is frequently severe enough to be treated with oral minocycline or doxycycline. we present a case of an 87-year - old man who developed a severe papulopustular skin eruption secondary to erlotinib therapy for nsclc. control of the eruption with 100 mg of minocycline twice daily for 8 months eventually led to blue - gray skin hyperpigmentation. after 30 months, this side effect was recognized as minocycline drug deposition, which was confirmed with skin biopsy. compliance with egfr inhibitor therapy in nsclc is often challenging due to common side effects, most notably cutaneous skin eruptions. use of minocycline to treat the most common cutaneous side effect (papulopustular eruption) can in turn cause blue - black skin, eye, or tooth discoloration that can nullify its benefits, resulting in suboptimal patient adherence to cancer therapy. although this adverse effect is well known in dermatology literature as a risk when using minocycline to treat acne, rosacea, or blistering disorders, it is less well documented in oncology literature. we present this case to highlight the need for greater consideration of unique patient characteristics in selecting an oral antibiotic as a treatment modality for egfr inhibitor skin toxicities. the use of epidermal growth factor receptor (egfr) tyrosine kinase inhibitor, erlotinib, as a first- or second - line therapy in egfr - positive non - small cell lung cancer has increased progression - free survival. the cutaneous side effects of this treatment include papulopustular eruption, xerosis, photosensitivity, alopecia, paronychia, onycholysis, and brittle nails. about 80% of patients treated with egfr inhibitors develop a papulopustular eruption and up to 32% of these are severe enough to be treated with minocycline or doxycycline. inadequate control of cutaneous side effects leads to impaired quality of life, decreased compliance, and dose reduction. in addition, the tetracyclines used to treat these side effects have their own toxicities. in dermatology literature, minocycline for the treatment of acne, rosacea, and blistering diseases is well known to cause blue - black pigmentation of the skin, eyes, bones, existing scars, and teeth. however, in oncology literature, there are rare reports of minocycline hyperpigmentation when used to treat egfr inhibitor - induced papulopustular eruptions. an 87-year - old man with egfr - positive non - small cell lung adenocarcinoma stage iv (t1an3m1b) was started on erlotinib 150 mg daily in october 2013. he presented with inflammatory follicular - based papules and pustules over the face after 3 weeks of treatment. two months later, the eruption involved greater than 50% of his body and he self - discontinued erlotinib. subsequently, he was treated with minocycline 100 mg twice daily and was restarted on a decreased dose of erlotinib (50 mg daily). after 8 months of minocycline, he developed new blue - gray patches over his shins that eventually spread to thighs, arms, hands, existing scars, sclera, and teeth (fig. 1, fig., the discoloration was attributed to erlotinib, although hyperpigmentation is not a common side effect of this therapy. upon consultation to dermatology, his medication was changed to doxycycline and the pigmentation was treated with laser therapy, eventually fading. optimization and treatment of skin - related side effects remains paramount for patient adherence to egfr inhibitor therapy to prevent dose reduction or discontinuation. however, in current oncology literature, there is little distinction between which tetracycline is favored in the treatment of papulopustular eruptions. we present this case to illustrate a common side effect of prolonged minocycline use that is not well reported in oncology literature. pigmentation most commonly occurs on shins, ankles, hands, or forearms, but may involve eyes, bones, existing scars, mucosa, and teeth. minocycline pigmentation occurs in a dose - dependent fashion, with longer use increasing the risk. 3). once recognized, discontinuation of therapy results in eventual fading of pigmentation over months to years. to hasten resolution, q - switch laser is an effective treatment option. with targeted therapies, progression - free survival in non - small cell lung cancer is extending, leading to greater use of minocycline and greater opportunity for such hyperpigmentation. to protect patient compliance to egfr inhibitors, we recommend greater consideration of the risks and benefits of each tetracycline class, in combination with patient characteristics, when treating erlotinib - based papulopustular eruption. this material has not been published previously, in total or in part, in print or electronic format and is not under consideration by another publication or electronic medium. the views expressed in this article are those of the authors and do not reflect the official policy of the department of defense or us government. this research did not receive any specific grant from funding agencies in the public, commercial, or not - for - profit sectors.	introductionwhile epidermal growth factor receptor (egfr) inhibitors have improved progression - free survival in patients with non - small cell lung cancer (nsclc), one of the most common adverse effects is papulopustular skin eruption, which is frequently severe enough to be treated with oral minocycline or doxycycline.casewe present a case of an 87-year - old man who developed a severe papulopustular skin eruption secondary to erlotinib therapy for nsclc. control of the eruption with 100 mg of minocycline twice daily for 8 months eventually led to blue - gray skin hyperpigmentation. after 30 months, this side effect was recognized as minocycline drug deposition, which was confirmed with skin biopsy.discussioncompliance with egfr inhibitor therapy in nsclc is often challenging due to common side effects, most notably cutaneous skin eruptions. treatment of cutaneous toxicities is important to preserve patient compliance with targeted cancer therapy. use of minocycline to treat the most common cutaneous side effect (papulopustular eruption) can in turn cause blue - black skin, eye, or tooth discoloration that can nullify its benefits, resulting in suboptimal patient adherence to cancer therapy. although this adverse effect is well known in dermatology literature as a risk when using minocycline to treat acne, rosacea, or blistering disorders, it is less well documented in oncology literature. we present this case to highlight the need for greater consideration of unique patient characteristics in selecting an oral antibiotic as a treatment modality for egfr inhibitor skin toxicities.
the discovery cohort and first replication cohort were both ascertained from the diabetes audit and research tayside study (darts)23. validated prescribing data, biochemistry data as well as clinical phenotypes back to 1992 can be retrieved from central databases for all the darts patients. all patients with diabetes have been invited to give written informed consent to dna as part of the wellcome trust united kingdom type 2 diabetes case control collection. as of june 2009, 8000 cases and 7000 controls of european ancestry have participated in this genetics of darts (godarts) study. as part of the wtccc2, 4134 godarts cases were selected primarily for a genome wide association study of statin response, but also for a study of response to oral hypoglycaemic agents (oha). following the wtccc2 genotyping quality control (see below), 1024 patients were identified who were initiated on metformin and had a definable metformin response (discovery cohort). of the godarts cases not in the wtccc2 discovery cohort metformin response could be defined in 1783 patients and this was used for the first round replication. the second replication cohort was patients with type 2 diabetes either randomised to metformin, or treated with metformin as per protocol, in the uk prospective diabetes study (ukpds)24. a total number of 1113 white european patients, who were exposed to metformin and passed the genotyping quality control, were identified in the ukpds cohort. these were either primarily randomized to metformin (n=284), were randomized second line as add - in to sulphonylureas (n=231), or had metformin added to sulphonylurea per protocol for symptomatic hyperglycaemia or when fasting glucose was greater than 15mmol / l (n=598). because over 92% of the oha prescriptions issued in godarts cohort are either metformin (51.4%) or sulphonylurea (41.2%), we focused on two treatment schemes of metformin monotherapy (metformin added in following failure of dietary control) or dual therapy (following initiation of oral hypoglycaemic agents in type 2 diabetes, there is an initial reduction in hba1c, followed by a gradual deterioration. this can be seen in both the ukpds study and other diabetes trials such as adopt25. the gradual deterioration in hba1c will reflect both drug efficacy (or inefficacy) to control hba1c, and the underlying diabetes progression. to target the drug response alone we focused on the first 18 months of metformin therapy to minimize the response window but ensure minimal exclusion due to lack of hba1c data. in this observational study, the patient s physician will be treating to achieve an hba1c target, which over the majority of the study period would have been 7%. we therefore defined our primary drug response phenotype as a dichotomous trait of treatment success which was the ability to achieve an hba1c below 7% in the first 18 months of treatment, with censoring if diabetes therapy was changed prior to this. our secondary analysis took the quantitive phenotype as treatment hba1c which was the lowest hba1c observed between 1 and 18 months after metformin treatment or prior to a change in therapy (cessation of metformin or addition of further oral hypoglycaemic therapy). the two phenotypes were modelled with multiple linear or logistic regression using the same set of covariates which includes baseline hba1c, adherence, daily dose, creatinine clearance, baseline gap and treatment group. more details of the models and covariates were provided in supplementary methods. in the ukpds the hba1c was measured yearly, and the hba1c measure closest to 1 year after commencing metformin was taken as the treatment hba1c. in the cases randomized to metformin this would have been approximately one year after starting metformin ; in those starting metformin as per protocol due to hyperglycaemia the time between starting metformin and the treatment hba1c was variable so an additional covariate (treatment gap) was included in the model. other covariates included were as for the godarts models, except adherence and dose were not included due to missing data. samples were genotyped at affymetrix s service laboratory on the genome - wide human snp array 6.0. genotype data quality control was via the standard protocol that was established for the wtccc2 studies26 (supplementary methods). specifically, concordance check was performed on 116 snps by 1779 individuals overlapped between this gwa data and the wtccc1 t2d case control study27. based on the concordance rate of 99.73%, individuals with more than 10% discordance were removed from the current study. after such stringent qc, the clean data set included 705125 autosomal snps on 3736 samples, of whom 1024 have definable metformin response. in the first round replication, following assay optimization of the top two snps from the gwa (rs11212617 and rs624366, r=0.997 in 3736 godarts samples), rs11212617 had better genotyping performance and was genotyped by the standard taqman - based allelic discrimination method (applied biosystems) in the whole godarts cohort, which included 7000 non - diabetic controls. the overall call rate was 98.3% with a concordance rate of 99.9% to the gwa genotypes. taqman was also used for genotyping the ukpds replication sample. following optimization of 4 highly correlated snps (rs11212617 and rs624366 as well as two proxies rs609261 and rs2345801) in a subset of ukpds samples, snp rs609261 was selected and genotyped based on better genotyping performance than the other snps. rs609261 is almost a perfect proxy for rs11212617 (r=0.997 in 5197 wtccc2 controls) and as such, results are presented as for rs11212617 in the ukpds cohort. genotyping of all available ukpds dnas (n=3400) was carried out in duplicate using standard conditions. discrepancy between the duplicate genotyping runs was 0.4%, and only samples for which the duplicate genotypes were concordant the final ukpds replication cohort included 1113 caucasian patients and the minor allele frequency was 42.1% with no deviation from hwe (p=0.98). logistic and linear regression modelling was performed with plink and snptest28,29 assuming an additive genetic model. all the results presented were unadjusted for population stratification as a genomic inflation factor of 1.003 and 0.998 was observed for logistic and linear regression analyzes respectively. data from the discovery cohort were also imputed to the hapmap ii ceu panel of 2.2 million snps with program impute and tested for association with snptest taking into account the imputed genotype probabilities30. as the two snps rs11212617 and rs609261 that were genotyped in the go - darts sample and the ukpds sample respectively were in near complete ld (r=0.997), we directly combined the single marker association test results from the discovery cohort and the two replication cohorts with the inverse variance fixed effect method as implemented in the r package of genabel31. ku-55933 is a cell - permeable atp - competitive inhibitor of atm (ic50 = 13 nm ; ki = 2.2 nm) with selectivity over other pikk family kinases (ic50 = 2.5, 9.3, 16.6 m for dna - pk, mtor, pi 3-k, respectively ; ic50 > 100 m for pi 4-k and atr)32. it is reported to have little activity towards a panel of 70 conventional serine / threonine kinases. it has been shown to inhibit atm - dependent cellular protein phosphorylation following ionizing radiation (ir) and sensitizes cells that express wild - type atm (but not mutant atm) to the cytotoxic effects of ir and dna - damaging agents33,34. h4iie cells were cultured in dulbecco s modified eagle s medium containing 5% (v / v) fetal bovine serum to 80% confluence. for ampk assays, cells were pretreated with 10 m ku55933 (30 min) prior to treatment with various concentrations of metformin for 1 hr. lysates were prepared (rapid lysis method35), centrifuged (4c, 10 min 21,000 x g), and the supernatants frozen for later analysis. ampk activity was assayed in immunoprecipitates as described35 using the amara peptide36. to establish that the change in ampk activity was due to regulation of phosphorylation of ampk and that this translated into changes in substrate phosphorylation, we used western blotting to compare the phosphorylation status of thr-172 of ampk and ser-79 of acc (a well characterized marker of ampk activation). in both cases for these experiments, h4iie cells were cultured to 80% confluence as above, then serum starved for 16 hr, pretreated with ku55933 or vehicle (dmso) for 1 hr then treated for 3 hr with or without metformin. the membrane was blocked in tbst containing 5% milk for 1 hour, then incubated overnight at 4c with anti - phospho - thr172-ampk antibody (cell signaling, beverly, ma), anti - phospho - ser79 acc antibody (cell signaling) or anti--actin antibody (sigma, dorset, uk). membranes were washed in tbst, incubated with goat anti - rabbit igg conjugated to horseradish peroxidase (pierce, chester, uk) then washed again in tbst. the membrane was then developed using an ecl western blotting detection kit (amersham biosciences) before exposure to x - ray film (thermo scientific, waltham, ma). the discovery cohort and first replication cohort were both ascertained from the diabetes audit and research tayside study (darts)23. validated prescribing data, biochemistry data as well as clinical phenotypes back to 1992 can be retrieved from central databases for all the darts patients. all patients with diabetes have been invited to give written informed consent to dna as part of the wellcome trust united kingdom type 2 diabetes case control collection. as of june 2009, 8000 cases and 7000 controls of european ancestry have participated in this genetics of darts (godarts) study. as part of the wtccc2, 4134 godarts cases were selected primarily for a genome wide association study of statin response, but also for a study of response to oral hypoglycaemic agents (oha). following the wtccc2 genotyping quality control (see below), 1024 patients were identified who were initiated on metformin and had a definable metformin response (discovery cohort). of the godarts cases not in the wtccc2 discovery cohort metformin response could be defined in 1783 patients and this was used for the first round replication. the second replication cohort was patients with type 2 diabetes either randomised to metformin, or treated with metformin as per protocol, in the uk prospective diabetes study (ukpds)24. a total number of 1113 white european patients, who were exposed to metformin and passed the genotyping quality control, were identified in the ukpds cohort. these were either primarily randomized to metformin (n=284), were randomized second line as add - in to sulphonylureas (n=231), or had metformin added to sulphonylurea per protocol for symptomatic hyperglycaemia or when fasting glucose was greater than 15mmol / l (n=598). because over 92% of the oha prescriptions issued in godarts cohort are either metformin (51.4%) or sulphonylurea (41.2%), we focused on two treatment schemes of metformin monotherapy (metformin added in following failure of dietary control) or dual therapy (metformin added to stable sulphonylurea treatment). following initiation of oral hypoglycaemic agents in type 2 diabetes, there is an initial reduction in hba1c, followed by a gradual deterioration. this can be seen in both the ukpds study and other diabetes trials such as adopt25. the gradual deterioration in hba1c will reflect both drug efficacy (or inefficacy) to control hba1c, and the underlying diabetes progression. to target the drug response alone we focused on the first 18 months of metformin therapy to minimize the response window but ensure minimal exclusion due to lack of hba1c data. in this observational study, the patient s physician will be treating to achieve an hba1c target, which over the majority of the study period would have been 7%. we therefore defined our primary drug response phenotype as a dichotomous trait of treatment success which was the ability to achieve an hba1c below 7% in the first 18 months of treatment, with censoring if diabetes therapy was changed prior to this. our secondary analysis took the quantitive phenotype as treatment hba1c which was the lowest hba1c observed between 1 and 18 months after metformin treatment or prior to a change in therapy (cessation of metformin or addition of further oral hypoglycaemic therapy). the two phenotypes were modelled with multiple linear or logistic regression using the same set of covariates which includes baseline hba1c, adherence, daily dose, creatinine clearance, baseline gap and treatment group. more details of the models and covariates were provided in supplementary methods. in the ukpds the hba1c was measured yearly, and the hba1c measure closest to 1 year after commencing metformin was taken as the treatment hba1c. in the cases randomized to metformin this would have been approximately one year after starting metformin ; in those starting metformin as per protocol due to hyperglycaemia the time between starting metformin and the treatment hba1c was variable so an additional covariate (treatment gap) was included in the model. other covariates included were as for the godarts models, except adherence and dose were not included due to missing data. samples were genotyped at affymetrix s service laboratory on the genome - wide human snp array 6.0. genotype data quality control was via the standard protocol that was established for the wtccc2 studies26 (supplementary methods). specifically, concordance check was performed on 116 snps by 1779 individuals overlapped between this gwa data and the wtccc1 t2d case control study27. based on the concordance rate of 99.73%, individuals with more than 10% discordance were removed from the current study. after such stringent qc, the clean data set included 705125 autosomal snps on 3736 samples, of whom 1024 have definable metformin response. in the first round replication, following assay optimization of the top two snps from the gwa (rs11212617 and rs624366, r=0.997 in 3736 godarts samples), rs11212617 had better genotyping performance and was genotyped by the standard taqman - based allelic discrimination method (applied biosystems) in the whole godarts cohort, which included 7000 non - diabetic controls. the overall call rate was 98.3% with a concordance rate of 99.9% to the gwa genotypes. taqman was also used for genotyping the ukpds replication sample. following optimization of 4 highly correlated snps (rs11212617 and rs624366 as well as two proxies rs609261 and rs2345801) in a subset of ukpds samples, snp rs609261 was selected and genotyped based on better genotyping performance than the other snps. rs609261 is almost a perfect proxy for rs11212617 (r=0.997 in 5197 wtccc2 controls) and as such, results are presented as for rs11212617 in the ukpds cohort. genotyping of all available ukpds dnas (n=3400) was carried out in duplicate using standard conditions. discrepancy between the duplicate genotyping runs was 0.4%, and only samples for which the duplicate genotypes were concordant were analysed. the final ukpds replication cohort included 1113 caucasian patients and the minor allele frequency was 42.1% with no deviation from hwe (p=0.98). logistic and linear regression modelling was performed with plink and snptest28,29 assuming an additive genetic model. all the results presented were unadjusted for population stratification as a genomic inflation factor of 1.003 and 0.998 was observed for logistic and linear regression analyzes respectively. data from the discovery cohort were also imputed to the hapmap ii ceu panel of 2.2 million snps with program impute and tested for association with snptest taking into account the imputed genotype probabilities30. as the two snps rs11212617 and rs609261 that were genotyped in the go - darts sample and the ukpds sample respectively were in near complete ld (r=0.997), we directly combined the single marker association test results from the discovery cohort and the two replication cohorts with the inverse variance fixed effect method as implemented in the r package of genabel31. samples were genotyped at affymetrix s service laboratory on the genome - wide human snp array 6.0. genotype data quality control was via the standard protocol that was established for the wtccc2 studies26 (supplementary methods). specifically, concordance check was performed on 116 snps by 1779 individuals overlapped between this gwa data and the wtccc1 t2d case control study27. based on the concordance rate of 99.73%, individuals with more than 10% discordance were removed from the current study. after such stringent qc, the clean data set included 705125 autosomal snps on 3736 samples, of whom 1024 have definable metformin response. in the first round replication, following assay optimization of the top two snps from the gwa (rs11212617 and rs624366, r=0.997 in 3736 godarts samples), rs11212617 had better genotyping performance and was genotyped by the standard taqman - based allelic discrimination method (applied biosystems) in the whole godarts cohort, which included 7000 non - diabetic controls. the overall call rate was 98.3% with a concordance rate of 99.9% to the gwa genotypes. following optimization of 4 highly correlated snps (rs11212617 and rs624366 as well as two proxies rs609261 and rs2345801) in a subset of ukpds samples, snp rs609261 was selected and genotyped based on better genotyping performance than the other snps. rs609261 is almost a perfect proxy for rs11212617 (r=0.997 in 5197 wtccc2 controls) and as such, results are presented as for rs11212617 in the ukpds cohort. genotyping of all available ukpds dnas (n=3400) was carried out in duplicate using standard conditions. discrepancy between the duplicate genotyping runs was 0.4%, and only samples for which the duplicate genotypes were concordant were analysed. the final ukpds replication cohort included 1113 caucasian patients and the minor allele frequency was 42.1% with no deviation from hwe (p=0.98). logistic and linear regression modelling was performed with plink and snptest28,29 assuming an additive genetic model. all the results presented were unadjusted for population stratification as a genomic inflation factor of 1.003 and 0.998 was observed for logistic and linear regression analyzes respectively. data from the discovery cohort were also imputed to the hapmap ii ceu panel of 2.2 million snps with program impute and tested for association with snptest taking into account the imputed genotype probabilities30. as the two snps rs11212617 and rs609261 that were genotyped in the go - darts sample and the ukpds sample respectively were in near complete ld (r=0.997), we directly combined the single marker association test results from the discovery cohort and the two replication cohorts with the inverse variance fixed effect method as implemented in the r package of genabel31. ku-55933 is a cell - permeable atp - competitive inhibitor of atm (ic50 = 13 nm ; ki = 2.2 nm) with selectivity over other pikk family kinases (ic50 = 2.5, 9.3, 16.6 m for dna - pk, mtor, pi 3-k, respectively ; ic50 > 100 m for pi 4-k and atr)32. it is reported to have little activity towards a panel of 70 conventional serine / threonine kinases. it has been shown to inhibit atm - dependent cellular protein phosphorylation following ionizing radiation (ir) and sensitizes cells that express wild - type atm (but not mutant atm) to the cytotoxic effects of ir and dna - damaging agents33,34. h4iie cells were cultured in dulbecco s modified eagle s medium containing 5% (v / v) fetal bovine serum to 80% confluence. for ampk assays, cells were pretreated with 10 m ku55933 (30 min) prior to treatment with various concentrations of metformin for 1 hr. lysates were prepared (rapid lysis method35), centrifuged (4c, 10 min 21,000 x g), and the supernatants frozen for later analysis. ampk activity was assayed in immunoprecipitates as described35 using the amara peptide36. to establish that the change in ampk activity was due to regulation of phosphorylation of ampk and that this translated into changes in substrate phosphorylation, we used western blotting to compare the phosphorylation status of thr-172 of ampk and ser-79 of acc (a well characterized marker of ampk activation). in both cases metformin induced phosphorylation was partially reduced (figure 3). for these experiments, h4iie cells were cultured to 80% confluence as above, then serum starved for 16 hr, pretreated with ku55933 or vehicle (dmso) for 1 hr then treated for 3 hr with or without metformin. the membrane was blocked in tbst containing 5% milk for 1 hour, then incubated overnight at 4c with anti - phospho - thr172-ampk antibody (cell signaling, beverly, ma), anti - phospho - ser79 acc antibody (cell signaling) or anti--actin antibody (sigma, dorset, uk). membranes were washed in tbst, incubated with goat anti - rabbit igg conjugated to horseradish peroxidase (pierce, chester, uk) then washed again in tbst. the membrane was then developed using an ecl western blotting detection kit (amersham biosciences) before exposure to x - ray film (thermo scientific, waltham, ma).	metformin is the most commonly used pharmacological therapy for type 2 diabetes. we carried out a gwa study on glycaemic response to metformin in 1024 scottish patients with type 2 diabetes. replication was in two cohorts consisting of 1783 scottish patients and 1113 patients from the uk prospective diabetes study. in a meta - analysis (n=3920) we observed an association (p=2.9 109) for a snp rs11212617 at a locus containing the ataxia telangiectasia mutated (atm) gene with an odds ratio of 1.35 (95% ci 1.22 to 1.49) for treatment success. in a rat hepatoma cell line, inhibition of atm with ku-55933 attenuated the phosphorylation and activation of ampk in response to metformin. we conclude that atm, a gene known to be involved in dna repair and cell cycle control, plays a role in the effect of metformin upstream of ampk, and variation in this gene alters glycaemic response to metformin.
cardiac calcification is not uncommon, but massive left ventricle endocardial calcification is a rare entity. endomyocardial fibrosis is a restrictive cardiomyopathy of unknown etiology that occurs almost exclusively in tropical and subtropical regions, particularly in some countries of africa, india, and brazil. the disease is characterized by irregular fibrous thickening of the endocardium in the apex and inflow tract of one or both ventricles. herein, we report of a rare case of endomyocardial fibrosis associated with massive calcification of the left ventricle in a female patient presenting with progressive dyspnea and congestive heart failure. an 18-year - old female presented with progressive shortness of breath since the last 6 months ; she had an audible left ventricular (lv) s3, grade iii / vi pansystolic murmur, severe pulmonary hypertension, and elevated jugular venous pressure with giant c - v wave. mild cardiomegaly, dilated main pulmonary artery segment, and dense ringed calcification within the cardiac silhoutte were noted on the x - ray chest [figure 1a ]. (a) heterogeneous opacification in the cardiac silhouette. fluoroscopy in right anterior oblique view, 64-slice computed tomography thorax sagittal, (c) and transverse view, (d) massive calcification of left ventricle endocardium anterolateral and inferior segment and calcification of left atrium superior margin electrocardiogram revealed right axis deviation with biatrial enlargement. transthoracic and transesophageal echocardiography revealed biatrial dilatation, normal left ventricle systolic function, and a hyperechoic, glittering appearance of the left ventricle endocardium with fibrocalcific infiltration and obliteration of the left ventricle apex [video 1 ]. the calcification extended up to the mitral valve apparatus [figure 2a and b ]. transmitral and tissue doppler flows confirmed a restrictive physiology [figure 2c and d ]. fluoroscopy revealed scattered massive calcification on the cardiac silhouette along the left ventricle region [figure 1b ]. a 64-slice cardiac computed tomography (ct) confirmed extensive myocardial calcification along the left ventricle anterolateral and inferior segments, mitral annulus, and the left atrium [figure 1c and d ]. transthoracic (a) and transesophageal echocardiography, (b) revealed biatrial dilatation, hyperechoic left ventricular endocardium with fibrocalcific infiltration of the left ventricular apex ; the calcification extended up to the mitral valve apparatus, transmitral (c) and tissue doppler flflows, (d) confirmed a restrictive physiology her blood investigation showed hemoglobin of 14 g / dl, total leukocyte count of 8200/mm with 65% neutrophils, 28% lymphocytes, 5% monocytes, and 2% eosinophils. the blood chemistry including serum creatinine, bilirubin, total protein, albumin, calcium, magnesium, phosphate, and thyroid and parathyroid hormone levels was normal. features of progressive heart failure, restrictive cardiac physiology, and fibrocalcific infiltration / obliteration of left ventricle apex and inflow region strongly suggested a possibility of endomyocardial fibrosis. cardiac calcification is not uncommon, but massive left ventricle endocardial calcification is a rare entity. this rare condition is associated with metastatic deposition, infarction, or other endocrine disorders. metastatic calcification is due to deposition of calcium salts in previously normal tissue due to disturbance in calcium / phosphorus metabolism. most commonly, it occurs due to persistently elevated calcium levels as in primary hyperparathyroidism, chronic renal disease, hypervitaminosis d, widespread bone destruction from metastases, or myeloma. dystrophic calcification is deposition of calcium salts in previously damaged tissue with normal calcium metabolism. patients with myeloproliferative disorder may have associated loeffler endocarditis secondary to chronic prolonged eosinophilia which may be complicated by calcification of the endocardial sclerotic lesions. in hyperparathyroid condition, there is an increased prevalence of cardiac structural abnormalities such as left ventricle hypertrophy, calcification, and deranged functional properties of the heart. endomyocardial fibrosis is a disease of unknown etiology. in 1984, silver. described the first case of massive endocardial calcification of the left ventricle, suggesting it was a different entity causing restrictive cardiomyopathy. this suggestion was refuted by lengyel., who suggested that the endocardial calcification was a clue for the diagnosis of endomyocardial fibrosis. most of the patients of endomyocardial fibrosis present with symptoms of heart failure, generalized weakness, and fever. there is no definitive treatment of endomyocardial fibrosis, and symptomatic therapy is usually given for heart failure and prevention of thrombus. however, the prognosis of endomyocardial fibrosis is poor after manifestation of heart failure symptoms. in our patient, the blood biochemistry profile revealed normal blood counts, renal profile, and parathyroid hormone. hence, calcification secondary to chronic renal failure, myeloproliferative disease, and ischemic heart disease were unlikely. features of progressive heart failure, restrictive cardiac physiology, and fibrocalcific infiltration / obliteration of lv apex and inflow region strongly suggested a possibility of endomyocardial fibrosis. endomyocardial fibrosis is characterized by progressive heart failure, and a restrictive physiology secondary to subendocardial fibrosis involving ventricular apices and inflow tracts and multimodality imaging is often useful in such situations. this shows that a rare disease such as endomyocardial fibrosis associated with massive calcification of the left ventricle may be suspected on a simple chest x - ray and confirmed by ct.	massive endocardial calcification is a rare entity. we describe a rare disease endomyocardial fibrosis associated with massive calcification of the left ventricle suspected on a chest x - ray and confirmed by echocardiography and computed tomography in an 18-year - old female presenting with breathlessness and congestive heart failure.
much of our understanding of bacteria is from studies of pure cultures. in some cases, single strains of single species have come to represent what scientists know about diverse bacterial lineages. yet in nature, bacteria almost always exist in complex communities of other species that can be predators or prey, as well as competitors, commensals, mutualists, or pathogens. despite our appreciation of the ubiquity and importance of bacterial communities, little molecular and cellular biologists have unraveled some of the key mechanisms that modulate bacterial interactions, but there is little understanding of the relative importance of cooperation and conflict in these associations. it is critical to understand the potential for cooperation among bacteria if we are to successfully manage multispecies infections (whether harmful or beneficial to the host) or if we are to engineer bacterial consortia to perform valuable biological tasks. it is also important to discern how and when natural selection can shape cooperation among bacteria. toward these ends, the opinion piece by morris, lenski, and zinser (1) offers a new perspective on the ecology and evolution of bacterial cooperation. they present a model that reshapes a decades - old idea : that cooperation among individuals can be automatic and based on little more than the production of by - products (e.g., see references 2 and 3). beyond this core, the model has clear and testable predictions germane to bacterial genomic evolution, the design of novel in vitro methods, and the assembly and stability of bacterial communities. to appreciate the utility of this new model, it is important to introduce a recent controversy over bacterial cooperation. one school of thought is that cooperation among bacteria has produced emergent properties in bacterial communities. biofilms are clonal or multispecies groups of cells defined by their secreted polymers, which help to glue cells together (4). biofilm communities exhibit several properties that are hypothesized to require molecular communication and cooperation among participating bacteria, including differentiation among cells to produce highly resilient biofilm structures, species stratification to optimize productivity, and the formation of channels to maximize nutrient and oxygen flow (5). in a similar vein, emergent group properties have been highlighted in studies of bacterial quorum sensing, a process in which bacteria secrete and detect diffusible molecular signals to express traits in a context - dependent fashion, usually among members of the same species (4). detection of released signals in cell groups is thought to allow bacteria to perform functions that would be beneficial only in large populations of cells. classic examples include the group release of plant - cell - degrading compounds by bacterial phytopathogens and group secretion of virulence compounds in animal pathogens. the idea is that quorum sensing allows the pathogenic traits to be expressed only when the bacterial population numbers are high enough to overwhelm host defenses (4). thus, both in biofilms and in quorum sensing, the paradigm has been that evolution has shaped cooperation to maximize the greater good of the group. yet the central evolutionary argument is that the relentless action of natural selection favors selfish behavior (6) and that group - level adaptations should be analyzed with care and invoked only when necessary (7). for instance, individual - based models of bacterial movement and attachment can lead to biofilm - like structures in computer simulations, inconsistent with the hypothesis that cell - cell communication is needed to create them (8). moreover, quorum - sensing traits have also been modeled on the simple terms of individual cell action. under the diffusion - sensing model, single cells selfishly monitor levels of costly diffused products as opposed to coordinating their activities with other cells (9). a difficulty for clarifying predictions for cooperation in bacterial communities has been the lack of predictive models specific to microbe - microbe interactions. the black queen hypothesis by morris and colleagues (1) makes two basic assumptions about bacterial biology that build a foundation for cooperative community evolution. specifically, bacteria unavoidably produce publicly usable resources, which become available to their local community. this assumption closely mirrors classic models of by - product mutualism (2, 3), in which one individual produces a by - product as an automatic effect of its biology and thus enhances the fitness of other individuals that can use that product. by - product mutualism might not seem like a typical form of cooperation, since the cooperative phenotype carries no cost and because the trait need not evolve in the context of the interaction. yet by - products fit well into a broad definition of cooperation : a trait in one individual that increases another s fitness (10). the second assumption of the model is that a bacterium that uses another s by - products will benefit from deleting its own costly pathways for those products. adaptive genome streamlining is known to be common in parasites and symbionts, both of which can benefit from deleting costly functions that are provided by their hosts. when gene loss of vital functions occurs, this builds dependency into the interactions (11). importantly, these assumptions and the model as a whole can be applied to cooperation within or among bacterial species. nonetheless, by - product mutualisms based upon resources most often occur among species, since they tend to exhibit divergent resource usage. morris and colleagues support their assumptions with a diversity of sources. for instance, many empirical studies have shown that bacteria produce by - products that are useful to other cells. examples of leaky systems include catalase - peroxidase activity (enzymes that break down toxic hydrogen peroxide), production of iron chelators (which make iron soluble for metabolism), and the reduction of sulfur (which is often growth limiting). such leakiness might be unavoidable because of the nature of diffusion gradients and the need for bacteria to maintain membrane permeability. morris and colleagues also highlight potential links between by - product utilization and adaptive gene loss. this species is fascinating because it can not grow in its own natural habitat (sunlit seawater) as a pure culture (12). prochlorococcus becomes poisoned under these conditions by hydrogen peroxide (which is generated by photooxidation). however, poisoning fails to occur in natural habitats because prochlorococcus grows commensally with catalase - peroxidase - producing species, such as synechococcus, which brings to the table enzyme activity that permeates its membranes and eliminates hydrogen peroxide in the local environment (13). phylogenetic data suggest that the prochlorococcus lineage ancestrally exhibited catalase - peroxidase function but that it has been subsequently lost, consistent with the hypothesis of adaptive streamlining (1). is this combination of by - product utilization and genome streamlining common in bacterial communities ? if so, are most bacteria tied into obligate dependence upon partners in coevolved consortia ? morris and colleagues suggest that such interdependence is common and hypothesize that the failure of many bacteria to grow in pure culture is caused by obligate interspecies dependencies. they further suggest that whole bacterial communities could depend on rare keystone taxa that provide vital resources for many other species. the extinction of such a species would have dramatic effects on bacterial communities (8). this degree of bacterial cooperation might seem farfetched to some, but extensions of the classic by - product mutualism models suggest a clear route to the expanded evolution of such interdependence. one such extension, termed by - product reciprocity, predicts that natural selection will shape receivers of by - products to maximize these benefits by being cooperative to by - product producers (10, 14). coupled with the two key assumptions of the black queen hypothesis, this suggests a third step in which simple by - product interactions evolve into more - complex associations based upon costly resource exchange. thus, the predicted evolutionary steps are as follows : (i) selfish usage of another species by - products, (ii) genome streamlining to minimize production of resources that can be gotten from others, and finally (iii) the evolution of costly cooperative traits to maximize vital functions produced by others (14). the black queen hypothesis has implications for bacterial genome evolution, for the development of new in vitro techniques, and finally for the origins and stability of bacterial consortia. in terms of gene content, the model suggests that bacterial genomes should exhibit signatures of deletion mutations in gene pathways that produce transferable resources. loss - of - function mutations are predicted to be most widespread among intimately interacting bacteria, such as in biofilm communities and rhizosphere consortia. the model suggests that within these communities deletions might often occur in a complementary fashion among interacting bacteria. moreover, specific taxa might exhibit different loss - of - function deletions among populations that vary in community content. in terms of studying bacteria in vitro, taxa that can not be grown in pure culture might be successfully cocultured with key interacting helper species (e.g., see reference 15). finally, in terms of bacterial community assembly and stability, the model suggests that obligate interspecific interactions should be much more common in bacteria than previously suspected. although these interactions are predicted to originate via simple selfish steps, such as by - product production and genome streamlining, these prerequisites can begin an evolutionary cascade towards more - intimate interactions. once dependency has evolved through the deletion of a vital pathway provided by another taxon the evolution of costly cooperative phenotypes to maintain and optimize these interactions becomes much more likely (14). the black queen hypothesis reminds us of darwin s dictum that natural selection favors selfishness above all else.	abstractbacteria live in complex multispecies communities. intimately interacting bacterial cells are ubiquitous on biological and mineral surfaces in all habitats. molecular and cellular biologists have unraveled some key mechanisms that modulate bacterial interactions, but the ecology and evolution of these associations remain poorly understood. one debate has focused on the relative importance of cooperation among cells in bacterial communities. some researchers suggest that communication and cooperation, both within and among bacterial species, have produced emergent properties that give such groups a selective advantage. evolutionary biologists have countered that the appearance of group - level traits should be viewed with caution, as natural selection almost invariably favors selfishness. a recent theory by morris, lenski, and zinser, called the black queen hypothesis, gives a new perspective on this debate (j. j. morris, r. e. lenski, and e. r. zinser, mbio 3(2):e00036 - 12, 2012). these authors present a model that reshapes a decades - old idea : cooperation among species can be automatic and based upon purely selfish traits. moreover, this hypothesis stands in contrast to the red queen hypothesis, which states that species are in constant evolutionary conflict. two assumptions serve as the core of the black queen model. first, bacterial functions are often leaky, such that cells unavoidably produce resources that benefit others. second, the receivers of such by - products will tend to delete their own costly pathways for those products, thus building dependency into the interactions. although not explicitly required in their model, an emergent prediction is that the initiation of such dependency can favor the spread of more obligate coevolved partnerships. this new paradigm suggests that bacteria might often form interdependent cooperative interactions in communities and moreover that bacterial cooperation should leave a clear genomic signature via complementary loss of shared diffusible functions.
synapses transfer information from sensory cells or neurons to other neurons or distinct target cell types, such as muscle cells. a plethora of presynaptic proteins orchestrate neurotransmitter release at the presynaptic active zone (az). these proteins are organized into three main compartments, which are ultrastructurally defined and classically referred to as (1) the cytomatrix at the active zone (caz) with (2) presynaptic electron dense projections that are clustering (3) synaptic vesicles (zhai and bellen 2004). the presynaptic dense projections appear highly variable in size and shape, which have been hypothesized to follow the function of a given synapse type. they seem to be present at all neuronal azs but differ greatly in terms of order, density and morphology as well as molecular composition (zhai and bellen 2004). for example, rather small structures of less than 100 nm height are found at mammalian conventional central nervous system (cns) synapses where they form a presynaptic grid, also termed a remarkably regularly arranged structures can be observed at neuromuscular junctions of the frog (harlow. 2012). moreover, presynaptic dense projections are not an evolutionary invention of vertebrates, as insects such as the fruitfly drosophila melanogaster also harbor elaborated dense projections termed t - bars, which are found at almost every synapse type (for review, see wichmann and sigrist 2010). the anatomical hallmark of tonically releasing sensory mammalian photoreceptor synapses, a huge plate - like dense projection that tethers hundreds of synaptic vesicles (schmitz. 2000), was discovered in the 1950s (de robertis and franchi 1956), when transmission electron microscopy started to become a commonly used technique. electron microscopy allowed researchers to visualize the ultrastructure of cells in detail for the first time (de robertis and bennett 1955), bringing exciting new knowledge about morphology, organization and communication of cells in general and synapses in particular (see, for example : de robertis and bennett 1955 ; de robertis and franchi 1956). at this time, synaptic vesicles were discovered at guinea pig retinal synapses, where they were called synaptic vesicle was coined by de robertis and bennett (1955), who were inspecting bullfrog and earthworm synapses. in parallel, the work of de robertis and franchi (1956) on photoreceptors of light- or dark - exposed rabbits provided the first experimental evidence correlating synaptic vesicle numbers and presynaptic activity. a few years later, the large presynaptic dense structures of these synapses were named ribbons, when their characteristic shape with extended longitudinal axis was recognized in serial 3d reconstructions of guinea pig retinas (sjostrand 1958). subsequently, synaptic ribbons were also found to decorate cochlear afferent hair cell synapses (smith and sjostrand 1961). golgi or horseradish peroxidase labeling in combination with transmission electron microscopy were also and still are, widely used to visualize neurons (meller. 1968 ; levay 1973 ; white and rock 1980 ; defelipe. 1986) and to understand the anatomy of the inner ear. for example, the afferent spiral ganglion neurons (sgns) of the cochlear nerve, which carry the information about an acoustical signal from the inner ear to the brainstem, were studied intensely in various mammals such as guinea pig, mouse or cat (spoendlin 1972, 1975, 1979 ; paradiesgarten and spoendlin 1976 ; bodian 1978 ; kiang. 1982 ; liberman 1982a ; ginzberg and morest 1984 ; ryugo and rouiller 1988 ; liberman. these studies revealed that inner and outer hair cells are innervated by different sgn types (kiang. 1982), outer hair cells (ohcs) by unmyelinated (5 %) and inner hair cells (ihcs) by myelinated (95 %) afferent fibers (spoendlin 1969, 1975). each of the myelinated, bipolar type i sgns sends a peripheral unmyelinated and unbranched neurite to form a synapse with a single ihc ribbon synapse (liberman 1980 ; liberman. ; reviewed in meyer and moser 2010). therefore, recordings from sgns enable the investigation of the function of individual azs within an ihc. type i sgns show different intensity thresholds and dynamic ranges in cat (liberman and kiang 1978). paired recordings from hair cells and postsynaptic neurons have provided insight into synaptic sound encoding and its presynaptic determinants (palmer and russell 1986). finally, observations of postsynaptic excitatory potentials by recordings from near the synapse revealed the first information on the presynaptic release mechanism (furukawa. each ihc contains 530 azs, dependent on species and tonotopic position along the cochlea, generally peaking at the region with the greatest sound sensitivity for the particular species (francis. liberman and co - workers were among the pioneers coupling structural investigations of the mammalian auditory system to its function. in his seminal study, liberman s (1982b) functional characterization of cat single auditory nerve fibers was followed by horseradish peroxidase labeling to individually back - trace the innervation location at the respective ihc azs. this approach allowed the author to relate functional parameters such as spontaneous firing rates and firing thresholds to morphology of type i sgns, described, for example, by the dimension and location of their unmyelinated terminals on the ihcs. these studies together led to the hypothesis that ribbon synapses within a given ihc are structurally and functionally heterogeneous (which will be discussed later in this review) and pointed to the further need for detailed structure function analyses. horseradish peroxidase labeling combined with electron microscopy also provided insights into presynaptic vesicle cycling in hair cells (siegel and brownell 1986). more recently, hair cell synapses have increasingly attracted research activity and novel as well as classical methods have been employed for assessing their structure and function in combination with genetic or pharmacological manipulation of the synapses or noise exposure. quantitative electron microscopy analysis employing electron tomography of different functional states as well as freeze - fracture and subsequent electron microscopy have been introduced by roberts and others for studies of hair cell synapses (roberts. molecular manipulations involving germline mutagenesis as well as virus - mediated gene transfer were established. further, patch - clamp recordings have characterized ca currents (e.g., lewis and hudspeth 1983 ; fuchs. 2000 ; brandt. 2003) and membrane turnover (e.g., parsons. 1994 ; moser and beutner 2000 ; schnee. technically very challenging postsynaptic patch - clamp recordings have provided insight into the excitatory postsynaptic currents (glowatzki and fuchs 2002) and, combined with presynaptic recordings, have elucidated hair cell synaptic mechanisms with superb resolution (e.g., keen and hudspeth 2006 ; goutman and glowatzki 2007 ; li. 2009). immunohistochemistry combined with high - resolution microscopy as well as transcriptomic and proteomic analyses have informed on the molecular composition of hair cell synapses (khimich. finally, fluorescence imaging has been implemented for studies of hair cell synapse function (tucker and fettiplace 1995 ; issa and hudspeth 1996 ; zenisek. 2003 ; griesinger. 2005 ; frank. 2009 ; revelo. 2014). ribbon - type azs cope with a demanding task : synaptic vesicles need to be released indefatigably and rapidly recycled at individual synapses in order to maintain high firing rates of sgns that fire at hundreds of hz even during continued stimulation (reviewed in matthews and fuchs 2010 ; pangri. 2012). sustained exocytosis amounts to up to 70 hz from each release site, of which about a dozen comprise the readily - releasable vesicle pool (rrp). this was demonstrated in mouse ihcs (pangri. 2010) and is to our knowledge one of the highest release rates per site described to date (pangri. this process requires very efficient means of clearing previously exocytosed membrane and proteins from the site followed by immobilization and priming of new vesicles for the next round of release. moreover, the release of the neurotransmitter must exhibit both rapid on and off kinetics to accurately follow acoustic stimuli with a periodicity of 1 ms or less (kiang. 1967 ; palmer and russell 1986 ; kppl 1997 ; goutman 2012 ; li. 2014). how the molecular machinery of ihc azs meets these requirements is just starting to emerge. it is becoming clear that ultrastructural assessment of functional synapse states is required in addition to the powerful combination of molecular manipulation and physiological characterization. in this review, we will emphasize recent approaches coupling functional and structural investigations of release at the level of ihcs and their ribbon synapses, as well as recent findings regarding vesicular recycling after transmitter release. how does the subcellular organization of sensory ihcs enable mechanotransduction and transmitter release at high rates ? ihcs are epithelial cells by origin and exhibit several characteristics that distinguish them from neurons. most notably, they show a strong polarization with respect to both long and short cell axes. the polarization along the apicobasal axis follows a clear compartmentalization, e.g., apparent by the hair bundle harboring the mechanotransduction apparatus of the apical membrane. graded receptor potentials are formed by mechanoelectrical (apical) and voltage - gated (basal) conductances (corey and hudspeth 1979 ; roberts. actin - filled stereocilia protrude into the endolymph in a highly organized manner and their sophisticated supramolecular mechanotransduction apparatus enables ultrasensitive detection of sound - born vibrations of the cochlear partition (reviewed in kazmierczak and mller 2012). while the molecular identity of the mechanotransducer channel still awaits definitive demonstration, recent work indicates the transmembrane channel - like proteins (tmc)-1 and -2 as promising candidates (pan. 2013). opening of the apical mechanotransducer channels depolarizes the ihc, subsequently activating cav1.3 ca channels (platzer. 2004) at the presynaptic az in the basolateral membrane, where the incoming ca triggers neurotransmitter release. the density of ribbon synapses shows a strong basoapical gradient, with the supranuclear portion of the hair cell being devoid of azs (francis., the cuticular plate likely serves as an anchor for the stereociliar actin bundles, containing a rich protein network with cytoskeletal proteins such as actin, -actinin and tropomyosin (slepecky and chamberlain 1985 ; zine and romand 1993). moreover, the striated organelle, located underneath the cuticular plate, likely modulates the stereociliar actin bundles (vranceanu. microtubules are primarily found beneath the cuticular plate (slepecky and chamberlain 1985 ; steyger. 1990) but appear connected to cytoskeletal proteins in the cuticular plate, for example via acf7a (actin crosslinking family protein 7a), as suggested for zebrafish neuromast hair cells (antonellis. 2014). 1990), providing the mechanical strength of hair cells (szarama. 2012) and tracks for efficient cargo protein trafficking along the apicobasal axis (furness. 1990). in addition to the cellular apicobasal polarity, hair cells also show planar cell polarity, which is reflected in the orderly orientation of their hair bundles (reviewed in ezan and montcouquiol 2013 ; sienknecht. 2014). whether the basolateral organization of the hair cells is similarly instructed by planar cell polarity remains to be tested. in the next sections, we will focus on the organization of the basal portion of ihcs and discuss structure and function of hair cell ribbon synapses. emphasis will be on the molecular machinery of the synapse, synapse development, synaptic heterogeneity and synaptic vesicle recycling. phylogenetically, ribbons in sensory cells are old structures that occur not only in mammals but also in fishes, amphibians and birds. in the mammalian organ of corti, they were first described by smith and sjstrand (1961) and are found in both sensory cell types, i.e., ihcs and ohcs (sobkowicz. 1982). the discovery of the protein ribeye, initially purified from bovine retina, (schmitz. 2000), as the main and structure - yielding component of ribbons in rat photoreceptors (schmitz. (zenisek. 2003), zebrafish photoreceptors and bipolar cells (wan. 2005) and mouse cochlear hair cells (khimich. 2005 ; see also immunogold labeling in fig. 1a) highlights the conservation of the ribbon in vertebrate evolution (schmitz 2009). nonetheless, ribbons still vary greatly in size and shape (lenzi and von gersdorff 2001 ; moser. 2006 ; matthews and fuchs 2010), likely reflecting structural adaptation to the specific needs of the respective synaptic connection for sensory coding.fig. a ribeye is the main component of the ribbon as shown by pre - embedding immunogold labeling of a p14 ihc synaptic ribbon using an anti - ctbp2 antibody (courtesy of susann michanski, innerearlab, university medical center, gttingen, germany) ; a representative image of an electron micrograph of a round - shaped p9 immature ribbon exhibiting a dotted pattern possibly caused by ribeye arrangement (contrast enhanced image in a), see also schematic representation (a). b a p14 mature ribbon with the typical multi - lamellar pattern (contrast enhanced image in b), see also scheme in b c a serial 3d reconstruction of a mature ribbon with two distinct morphological vesicle pools (yellow : ribbon - associated vesicles ; orange : membrane - proximal vesicles ; red : ribbon ; blue : az membrane ; magenta : presynaptic density). c the membrane - proximal vesicles (orange) are arranged around the presynaptic density (magenta) that is containing the scaffolding protein bassoon as shown by the pre - embedding immunogold labeling in (d), scale bar (d) 100 nm (courtesy of susann michanski, innerearlab, university medical center, gttingen, germany) ; d 2-color sted image of immunolabeled bassoon (magenta) and cav1.3 channel clusters (green) in mature ihcs : stripelike morphology and closely aligned immunofluorescence of bassoon and cav1.3 can be observed. scale image:700 700 nm ; e, e mathematic model showing the total mean steady state [ca ] profile at the az membrane (e) ; e effective number of cav1.3 channels contributing to total mean steady state [ca ] as shown in (e). (c, c, d, e, e modified from wong. 2014, embo j ; reprinted with permission 2014 wong.). f schematic summary of the protein arrangement at mature ihc ribbon synapses spatial distribution of ihc az proteins. a ribeye is the main component of the ribbon as shown by pre - embedding immunogold labeling of a p14 ihc synaptic ribbon using an anti - ctbp2 antibody (courtesy of susann michanski, innerearlab, university medical center, gttingen, germany) ; a representative image of an electron micrograph of a round - shaped p9 immature ribbon exhibiting a dotted pattern possibly caused by ribeye arrangement (contrast enhanced image in a), see also schematic representation (a). b a p14 mature ribbon with the typical multi - lamellar pattern (contrast enhanced image in b), see also scheme in b. scale bars (a, a, b) 100 nm. c a serial 3d reconstruction of a mature ribbon with two distinct morphological vesicle pools (yellow : ribbon - associated vesicles ; orange : membrane - proximal vesicles ; red : ribbon ; blue : az membrane ; magenta : presynaptic density). c the membrane - proximal vesicles (orange) are arranged around the presynaptic density (magenta) that is containing the scaffolding protein bassoon as shown by the pre - embedding immunogold labeling in (d), scale bar (d) 100 nm (courtesy of susann michanski, innerearlab, university medical center, gttingen, germany) ; d 2-color sted image of immunolabeled bassoon (magenta) and cav1.3 channel clusters (green) in mature ihcs : stripelike morphology and closely aligned immunofluorescence of bassoon and cav1.3 can be observed. scale image:700 700 nm ; e, e mathematic model showing the total mean steady state [ca ] profile at the az membrane (e) ; e effective number of cav1.3 channels contributing to total mean steady state [ca ] as shown in (e). (c, c, d, e, e modified from wong. 2014, embo j ; reprinted with permission 2014 wong.). f schematic summary of the protein arrangement at mature ihc ribbon synapses ribeye is composed of two major domains : while the a domain organizes the assembly of the synaptic ribbon and is unique in structure, the b domain is structurally nearly identical to the transcription repressor ctbp2, which is encoded by the same gene but uses a different transcription initiation site (schmitz. the b domain is also assumed to be involved in tethering of synaptic vesicles to the ribbon (schmitz. 2000 ; schmitz 2009), though the proteins that form tethers remain to be identified. ribeye appears to organize ribbon shape directly based on its domain structure (schmitz 2009) and its aggregation properties (magupalli. a trifold lamellar pattern has been described for photoreceptor ribbons and assigned to the polarized arrangement of ribeye (schmitz 2009). also at mature ihc ribbons, a lamellar substructure is observed that harbors multiple lamellar foldings (sobkowicz. this effect can be attributed to the differences in the ribbon shape in ihcs and photoreceptors. in immature ihc ribbons, the lamellar pattern is not prominent but instead a dotted pattern can be observed (fig. recently, at zebrafish hair cell ribbon synapses, it was found that the ribeye a and b domain segregate along the vertical axis of the ribbons, with the b domain more located towards the basal end (sheets. ribeye, in contrast to other az proteins, is not found in invertebrates such as the fruitfly drosophila melanogaster ; however, bruchpilot (brp), the homolog of the vertebrate az protein cast (caz - associated structural protein)/erc2, functions as the main building block of t - bars (kittel. in fact, only the n - terminus is conserved and shows sequence homologies to cast, whereas the c - terminus is only found in dipteran insects and rather resembles cytoskeletal elements such as plectin due to its numerous coiled - coil domains (wagh. 2006). moreover, the c - terminus mediates vesicle tethering to the t - bar (hallermann. the structurally related proteins bassoon and piccolo as well as cast, elks, rab3-interacting molecule (rim) and munc13 are present (betz. 1998 ; fenster. 2000 ; dresbach. 2001 ; deguchi - tawarada. additionally, ctbp2 and ctbp1 have also been found at conventional azs (tom dieck. 2005). except for piccolo, which at ribbon synapses is solely expressed as a shorter splice variant nicknamed as piccolino (regus - leidig. 2013), these proteins also largely form the caz at photoreceptor ribbon synapses (wang. the components of hair cell azs, on the other hand, are still largely unexplored, except for bassoon (khimich. 2005) and piccolo / piccolino (khimich. 2005 ; regus - leidig. in fact, a recent study indicates that ihc synapses operate without munc13-like priming factors (vogl. bassoon, together with ribeye, is responsible for the ribbon shape and, hence, might contribute to its function. studies of ihcs from bassoon mutant mice indicated an anchoring function of bassoon (khimich. 2005 ; frank. 2013), in line with findings at photoreceptor ribbon synapses (dick. 2003 ; tom dieck. the fraction of ribbon - occupied synapses remaining in bassoon - deficient ihcs seems to depend on age and residual levels of full - length bassoon (khimich. the fraction of ribbonless synapses increased from 50 % at postnatal day 11 (p11) up to 88 % at p70. a lower and relatively constant fraction of 56 % ribbonless synapses was found in a gene - trap bassoon mutant (bsn) likely due to a weak residual synaptic expression of bassoon (jing.. however, the anchorage of the remaining ribbons seems impaired (frank. maintained az ultrastructure, bsn animals exhibited a larger number of ca channels at ihc synapses compared to bsn mice and also displayed an intermediate phenotype regarding sustained ihc exocytosis (jing. in contrast, the size of the readily releasable vesicle pool (rrp) was strikingly reduced in both mutants. moreover, single unit recordings of the sgns show comparably severe defects in bsn and bsn mice, as both genotypes had impaired sound onset coding and lower evoked and spontaneous spike rates. taken together, these results indicate that the remaining, loosely anchored ribbons might function inadequately (jing. this further suggests that the mere presence of the ribbon, even with tethered vesicles, is not sufficient to maintain normal transmitter release and sustain the rrp at ihc ribbon synapses. moreover, it seems that bassoon contributes to organizing the ihc az beyond anchoring the ribbon. this has been concluded from impaired clustering of ca channels at bsn ribbon - occupied synapses (frank. 2010) and indicates a potential direct contribution of bassoon in organizing the az (frank. 2010 ; hallermann and silver 2013). in contrast to conventional synapses, where often only the combined knockdown of bassoon and its homolog piccolo causes synaptic defects (altrock. 2008 ; mukherjee. 2010 ; waites. 2011, 2013), these proteins seem not to act redundantly at ribbon synapses. as mentioned above, at hair cell and photoreceptor ribbons, only the short isoform of piccolo, piccolino, is expressed, which lacks a large c - terminal part (regus - leidig. binding sites for the proteins abp1, pra1, git1 and profilin (wang. 1999 ; fenster. 2003) are still present, whereas binding sites for, e.g., bassoon or rim are lacking (regus - leidig. accordingly, piccolino exhibits a different spatial distribution at ribbon synapses of photoreceptors, where it is found directly on the ribbon, as indicated by pre - embedding immunogold - labelings, using an antibody recognizing the n - terminus of the protein (limbach. (2014) revealed a striking impairment in the ribbon structure upon piccolino rnai - based knockdown. instead, a high proportion of attached spherical ribbons was found that resemble ribbon precursors of photoreceptor ribbons suggesting a role of piccolino in structural ribbon maturation (regus - leidig. ribeye and presumably piccolino as well as bassoon present the main structural components of the ribbon and/or the anchorage of the ribbon to the az. in order to resolve the function of ribbons, the ribbon was suggested to (1) promote a large readily releasable pool of vesicles via establishing / stabilizing many ca channels and vesicular release sites (khimich. 2010), (2) facilitate vesicle replenishment at the az (conveyor belt model, e.g., bunt 1971 ; gray and pease 1971 ; vollrath and spiwoks - becker 1996 ; lenzi and von gersdorff 2001 ; snellman. 2011), (3) facilitate multivesicular release (edmonds 2004 ; fuchs 2005), or (4) serve as a diffusion barrier to enable high local ca concentrations (graydon. functional interpretations have also been provided for the morphologically distinct populations of synaptic vesicles at ribbon synapses but in each case remain to be validated. ribbon - associated vesicles structurally attached through filamentous protein tethers the ones at the base of the ribbon face the presynaptic plasma membrane (membrane - proximal vesicles ; fig. 1c, c) and are often tethered to the membrane and/or to the presynaptic density (tethered vesicles). lateral to this subpopulation of ribbon - associated vesicles, there are few additional membrane - proximal and even membrane - tethered vesicles that are not in close vicinity to the ribbon. while further testing is required, current evidence suggests that the membrane - proximal vesicles comprise the rrp (e.g., in retinal bipolar cells : von gersdorff. 1996 ; zenisek. 2000 ; frog saccular hair cells : lenzi. 1999, 2002 ; rutherford and roberts 2006 ; and mouse inner hair cells : khimich. support for this hypothesis comes from the approximate matching between the morphologically estimated number of membrane - proximal vesicles and the functionally defined size of the rrp, i.e., the fast component of exocytosis upon depolarization - evoked ca influx (von gersdorff. 1996 ; pangri. 2010 ; frank. 2010), as well as from the observation that these vesicles are most heavily depleted upon stimulation (lenzi. 2002 ; pangri. furthermore, the tethering of vesicles to the az membrane might reflect a structural correlate of fusion competence (siksou. new high - resolution imaging approaches using rapid freezing methods and/or electron tomography have revealed that synaptic vesicles in proximity to the membrane exhibit several morphologically distinct stages. tethers of different numbers and lengths connecting synaptic vesicles to the az membrane could be observed at conventional synapses and synaptosome preparations (siksou. 2007, 2009 ; fernndez - busnadiego. 2010, 2013) but also at ihc ribbon synapses (frank. 2010). moreover, cryo - electron tomography, a method allowing visualization of hydrated and unstained tissue, revealed that tethering of synaptic vesicles in synaptosomes prepared from hippocampal tissue precedes the full contact of a synaptic vesicle with the membrane (fernndez - busnadiego. single long tethers initially seem to be formed and synaptic vesicles likely enter the rrp via the formation of several short tethers (< 5 nm). in line with this hypothesis, this fraction of vesicles could be depleted by application of hypertonic sucrose, that is thought to trigger rrp release (rosenmund and stevens 1996). moreover, the formation of short tethers could be inhibited using tetanus toxin, pointing towards the fact that neuronal soluble nsf attachment protein receptors (snare) proteins are involved in this process (fernndez - busnadiego. 2013) play a crucial role in tethering vesicles to the membrane at conventional cns synapses. interestingly, munc13 and caps priming factors seem not to operate at the ihc ribbon synapse (vogl. some authors even argue that the entire ribbon - associated vesicle population is fusion - competent and, therefore, can be released within a few milliseconds or less (heidelberger. 1994 ; edmonds 2004). evidence for a priming function of the ribbon has recently been presented (snellman. in contrast to caz proteins, which are, at least in part, conserved at ihc azs, the molecular machinery involved in the regulation of synaptic vesicle fusion seems to deviate strongly from that of as mentioned above, neurotransmitter release at conventional synapses is mediated by neuronal snares, namely snap-25, syntaxin 1 and synaptobrevin 1 or 2 (reviewed in jahn and fasshauer 2012). snare activity can be blocked by neurotoxin - mediated cleavage or genetic manipulations (schiavo. ribbon synapses, the snare protein machinery appears to be present and functional (brandsttter. 1996 ; morgans. 1996 ; morgans 2000 ; von kriegstein and schmitz 2003 ; uthaiah and hudspeth 2010 ; cooper. in contrast, ihc exocytosis seems to be insensitive to neurotoxins and genetic ablation of neuronal snares (nouvian. 2011) and, hence, a functional role of syntaxin 1, synaptobrevin 1 and 2 as well as snap-25 in ihc exocytosis is questionable. while some studies detected snare mrnas and proteins in the sensory epithelium and hair cells (safieddine and wenthold 1999 ; uthaiah and hudspeth 2010 ; nouvian. 2011), neither snap-25, synaptobrevin 13 nor syntaxin 1 could be detected by immunofluorescence imaging in mouse ihcs (nouvian. 2010 ; reisinger. 2011) and complexins (strenzke. 2009 in contrast, the multi - c2 domain protein otoferlin plays a central role for hair cell exocytosis (roux. the absence or mutation of otoferlin causes deafness or temperature - sensitive hearing impairment in humans (yasunaga. 1999 ; varga. 2008) and rodents (roux. 2006 ; longo - guess. 2007 ; ultrastructurally, otoferlin is also found at ribbon synapses, mostly but not exclusively at synaptic vesicles and the az membrane (roux. sufficient amounts of otoferlin appear to be required for correct vesicular fusion and replenishment (roux. otoferlin is suggested to act as the ca sensor in ihcs (roux. 2006 ; johnson and chapman 2010), due to its ca - binding capabilities (roux. 2006 ; ramakrishnan. 2010) and facilitates snare - mediated liposome fusion (johnson and chapman 2010). in its absence, no depolarization - evoked rrp exocytosis is observed in ihcs (roux. 2010). transgenic expression of synaptotagmin 1, the major ca sensor of neuronal synaptic vesicle exocytosis, failed to restore ihc exocytosis and hearing in otoferlin ko mice, which may not be too surprising given the overall low conservation of the molecular composition between conventional and ihc synapses (reisinger. next to proteinaceous exocytosis machineries, the actual mechanisms of vesicle fusion as well as the transport of vesicles to the ihc release site are still largely unknown. the large and, in terms of amplitude and shape, heterogeneous excitatory postsynaptic currents (epscs) measured at postsynaptic afferent boutons of sgns have been interpreted to result from multivesicular (multiquantal) release at ihc azs (glowatzki and fuchs 2002). large epscs ensure rapid and temporal precise spike generation of sgns (rutherford. 2012) and the relevance of such large epscs for achieving a high synchronization index of postsynaptic firing (i.e., better phase locking precision) has recently been shown in the frog papilla (li. several multiquantal release scenarios at ihc ribbons have been discussed : (1) synchronized vesicle fusion of several single vesicles as well as (2) compound fusion, following homotypic vesicle - to - vesicle fusion and (3) sequential fusion involving homotypic vesicle - to - vesicle fusion while release occurs (glowatzki and fuchs 2002 ; edmonds 2004 ; neef. (2014) indicated that univesicular (uniquantal) release can explain the large size of sgn epscs and that the control of release by a dynamic vesicular fusion pore can account for the observed epsc heterogeneity. at this point, none of the above discussed mechanisms can definitively be ruled out or confirmed and future work, including detailed morphological analysis using electron microscopy of defined functional states, will be required to advance our understanding of exocytosis mechanism at ihc ribbon synapses. in case vesicles do not homotypically fuse with other vesicles at the ribbon while releasing, a transport mechanism of the vesicles to the membrane has to exist. the conveyor belt model, transporting the vesicle actively along the ribbon to the membrane, was one of the first models to be introduced (bunt 1971 ; gray and pease 1971 ; vollrath and spiwoks - becker 1996 ; lenzi and von gersdorff 2001). accordingly, a kinesin polypeptide, kif3a, was identified on photoreceptor ribbons that could serve as a motor for vesicle transport involving the filamentous tethers observed at the ribbon (muresan. 1999), which were also proposed to function as stepping stones for synaptic vesicles (usukura and yamada 1987 ; parsons and sterling 2003). recently, the tethers at the ribbon were suggested to be directly involved in coordinating vesicle transport towards the membrane via crowd surfing, based on passive diffusion following the gradient established by exocytic vesicle consumption at the base of the ribbon (graydon. the tethers simply need to bind the vesicles and prevent them from detaching until they reach the az membrane, where release maintains the diffusion gradient (graydon. 2014). however, future experiments involving mutant analyses will be necessary to identify the proteins mediating vesicular tethering to the ribbon and estimate their affinity to the vesicles and the functional relevance of the tethers for synaptic transmission. moreover, it will be interesting to investigate whether and how the tethering can be influenced by factors such as activity or even developmental stage. for example, maturation from pre - hearing to hearing significantly determines structure and function of the ribbon synapses and the spatial arrangement of az proteins such as the ca channels or bassoon, as will be emphasized in the next section. during maturation of the organ of corti,. how do morphological alterations during the transition from a pre - hearing to a hearing state correlate to functional maturation of ribbon synapses ? generally, synaptic contacts are ultrastructurally defined as pre- and postsynaptic electron - dense membranes that are closely aligned. the postsynaptic density (psd) is clearly visible as an electron - dense structure beneath the postsynaptic membranes directly juxtaposed to the presynaptic az. the innervation pattern of sgn fibers at hair cells within the immature rodent cochlea is significantly different from the mature configuration and massive rearrangements of the fibers that occur before the onset of hearing. hereby, type i sgn fibers retract from the ohcs, whereas type ii sgn fibers disappear from the ihcs (perkins and morest 1975 ; echteler 1992 ; simmons 2002). these developmental processes of fiber innervation take place in the first postnatal week in three distinct phases : (1) in e18-p0 animals, fibers of both afferent types extend towards all hair cells ; (2) between p0 and p3 a refinement occurs, where the outer spiral bundle forms that innervate the ohcs ; and (3) the type i fibers retract from the ohcs around p3p6, accompanied by synaptic pruning, while they keep their projections on the ihcs (huang. ampa - typed glutamate receptors and scaffold proteins like bassoon and shank1 disappear during the maturation process from ohcs. glua2/3 subunits remain stable throughout development and into adulthood, while glua4 subunit expression significantly increase in adult type i fibers (huang. recently, the molecular arrangement of afferent synapses in relation to functional changes at the ihcs has been addressed in more detail using a combination of confocal, stimulated emission depletion (sted) and electron microscopy, as well as ihc presynaptic physiology and computational modeling (wong. it is known that, in the early pre - hearing stages between p6 and p9, several small apposing pre- and postsynaptic densities mark nascent synapses. some of the presynaptic densities are occupied by synaptic ribbons, which are small and round in shape and attached via two triangular - shaped proteinaceous anchors (sobkowicz. however, floating ribbons were also frequently observed in close proximity to az areas at these developmental stages (wong. serial 3d electron microscopic reconstructions corroborated the notion of several discontinuous pre- and postsynaptic specializations. such synaptic sites are organized as loose suprastructures on the bouton surface and are likely functional, as immunohistochemistry indicates the presence of presynaptic ca channels and postsynaptic ampa receptors (wong. 2000 ; hell 2007), revealed that cav1.3 channels are arranged in small round spots (wong. 2014) rather than the stripes previously described for mature azs (frank. 2010 in addition, a huge number of extrasynaptic cav1.3 channels can be observed in immature ihcs (zampini. 2014), which enable the cells to fire ca action potentials (kros. 1998 ; brandt. these action potentials evoke exocytosis in the pre - hearing stage (beutner and moser 2001 ; glowatzki and fuchs 2002 ; johnson. 2005) but show lower ca efficiency (beutner and moser 2001 ; brandt. 2003 ; johnson. 2005) and a supra - linear ca dependence (johnson. 2005). the pre - sensory ihc activity appears to drive bursting activity in the developing auditory system (glowatzki and fuchs 2002 ; tritsch. 2007, 2010 ; wong. 2013 ; clause. 2014). in this context, the regulation of presynaptic firing by paracrine and/or efferent synaptic control is being subject to intense research (glowatzki and fuchs 2000 ; tritsch. efferent innervation, moreover, seems to play an important role in the maturation process of ihcs (glowatzki and fuchs 2000 ; marcotti 2004 ; goutman. efferent fibers originate from the superior olivary complex and, before onset of hearing, form transient axosomatic contacts with ihcs (simmons. later, they largely retract from ihcs and rather form axodendritic contacts to the afferent terminals (pujol. the transient efferent inhibition is thought to counteract the ihc depolarization resulting from the resting mechanotransducer current (gloc and holt 2003 ; waguespack. 2007 ; lelli. 2009). upon genetically induced impairment of the efferent input, the linearization of ca dependent exocytosis is affected (johnson. 2007) and the maturation of ihc afferent synapses is also disturbed (johnson. 2013b). around the onset of hearing (at around p11 ; mikaelian and ruben 1965), when graded receptor potentials start governing transmitter release, extrasynaptic cav1.3 channels get pruned and spatial coupling of ca channels and vesicular release sites is tightened. ca efficiency of exocytosis and a near - linear ca dependence of rrp exocytosis when probed with changes in the number of open ca channels (wong. 2014). therefore, while the intrinsic ca dependence of exocytosis apparently does not change upon the onset of hearing, experimental data and biophysical modeling of exocytosis at mature and immature az topographies support the notion of a developmental switch from the more ca microdomain - like control of exocytosis by several ca channels per vesicle to a more ca nanodomain - like control of exocytosis (wong. 2014 ; fig. 1e, e). interestingly, in adult gerbils, the open probability of ca channels in ihcs increased due to a preference of the ca channel for the bursting mode (zampini. structurally, alongside ca channels, other presynaptic az components become reorganized such as the bassoon containing presynaptic density (fig. these alterations are accompanied by changes of the postsynaptic glutamate receptor fields that also develop to one continuous ring - like cluster (wong. moreover, ribbons increase in size and undergo striking changes of shape. at the ultrastructural level, their cross - sectional shape changes from predominantly round (fig. 1a) to a rather oval-, droplet- or wedge - like shape between p14 and p20 (wong. 2014 the two rootlets seem to merge to a continuous presynaptic density that contains the scaffolding protein bassoon, as revealed by immunogold labeling (wong. shortly after onset of hearing at p14, a large proportion of ribbons with two rootlets can still be found, whereas about a week later the morphological maturation appears to be completed (wong. factors that participate in the maturation of ihc synapses, next to the efferent olivocochlear transmission (see above ; johnson. 2013a), are thyroid hormone (sendin. 2007) and myosin 6 (heidrych. 2009 ; roux. 2009). for both, a higher proportion of morphological immature ribbons have been observed in genetic deletion models. in conclusion, during development from pre - hearing to hearing, ihc ribbon synapses undergo major morphological and functional refinements, resulting in tighter spatial coupling between ca influx and exocytosis (wong. the number of ca channels, vesicular release sites and ribbon - associated vesicles seems to scale with the size and number of ribbons at the az (martinez - dunst. ; graydon. 2011 ; kantardzhieva. 2013 ; wong. 2013, 2014). strengthening of presynaptic transmitter release might therefore be accomplished by increasing ribbon or az size and/or ribbon numbers per az. moreover, finally, lateral olivocochlear efferent fibers might modulate postsynaptic excitability and thereby affect afferent synaptic strength. to establish which of these mechanisms contribute to determining and regulating synaptic strength of hair cell synapses interestingly, the size and shape of ribbons appear to be highly variable and dynamic. in fact, in photoreceptors, these parameters strongly correlate with activity in light (silent) or dark (active) conditions (spiwoks - becker. similarly, in ihcs, a diverse spectrum of ribbons has also been observed (bodian 1978 ; sobkowicz. the specific ultrastructural properties seem to depend on several factors : (1) the maturation / age (see section above), (2) position within the inner hair cell and maybe also (3) dynamic adaptation to activity. a pioneering study in cats was one of the first to identify the correlation between structural heterogeneity of ribbon synapses and functional characteristics of auditory nerve fibers (merchan - perez and liberman 1996). surprisingly, large azs with big and/or several ribbons, supposedly reflecting large presynaptic strength, seem to drive sgns with low spontaneous rate and high thresholds (see also scheme in fig. whereas this conundrum remains unsolved, the mechanisms of functional presynaptic heterogeneity are now beginning to be understood. evidence for such heterogeneity within individual ihcs was obtained using confocal imaging of presynaptic ca influx (frank. this study showed that presynaptic ca signals varied substantially in amplitude and voltage - dependence among the azs within individual ihcs. the amplitude of the ca signal scaled with ribbon size as approximated by simultaneous imaging of a fluorescently tagged ribeye - binding peptide (frank. 2009) and seemed to be greater at the neural side of the ihcs (meyer. linking such estimates to the functional and morphological properties of the postsynaptic neurons will be an important task for future studies. so far, correlative arguments based on coincidental changes in maximal strength of presynaptic ca influx and postsynaptic spiking during development and upon genetic disruption as well as modeling have been brought forward to argue that strong synapses drive sgns that have high spontaneous rates and low thresholds (wong. interestingly, an inverse correlation of pre- and postsynaptic parameters of synaptic strength has recently been reported for mouse ihcs : liberman. the authors favored the interpretation that the sgns inserting at the neural (modiolar) face of ihcs exhibit low spontaneous rates and high thresholds despite their corresponding large ihc azs, because they have a smaller complement of ampa receptors than those at the neural (pillar) side. this would agree with the conclusion of the classical study, which showed a neural abneural gradient of az size using electron microscopy for cat ihcs whereby large azs faced sgns with low spontaneous rates and high thresholds (merchan - perez and liberman 1996). in a laborious approach, the authors traced 11 functionally - characterized fibers to the ihcs using serial 3d reconstructions of ultrathin sections. in this way, it was possible to directly correlate morphological parameters such as ribbon length, fiber contact area, synaptic plaque area and synaptic vesicle numbers to the functional parameters determined prior to fiber labeling using single unit recordings. recently, such a gradient was also suggested for mouse ihcs and reported to be influenced by the lateral olivocochlear innervation (yin. the segregation of nerve fibers on neural and abneural sides was further observed in a study investigating the abundance of mitochondria in postsynaptic terminals. here, postsynaptic boutons facing the abneural side seem to harbor more mitochondria (francis. 2004). monitoring epscs from single afferent boutons, which is a suitable method to address synaptic function on the level of individual release sites (glowatzki and fuchs 2002), further showed differences among synapses. in these experiments, varying fractions of multiphasic epscs were observed and proposed to underlie the diverse firing properties of sgns (grant. a schematic of an organ of corti showing afferent and efferent innervations at ihcs. modified from meyer and moser 2010, curr opin otolaryngol head neck surg, reprinted with permission from 2010 wolters kluwer health. b mean and sd of f (gray) as a function of depolarizing potential (v m), obtained from spot - detection experiments at the center of the ca microdomain ; f was averaged over the last 15 ms of a 20-ms stimulus. f (mean gray) and i ca (mean black) show a similar voltage dependence (thin lines corresponding sds). b heterogeneous voltage dependence and ca channel number of synaptic ca channel clusters in ihcs. pronounced variability in the voltage dependence of activation, even within the same cell (dashed traces individual data curves from 3 ca microdomains in an ihc). modified from frank. c, c colorized spatial distribution of vesicles and cisterns around the ribbon in low- and high - spontaneous rate (sr) fibers. sections through a high - sr (c) and a low - sr (c) synapse containing the synaptic ribbon are shown with cisternal (maroon) and vesicular (green) profiles. d mean density (se) of docked vesicles, i.e., within 20 nm of the presynaptic density along the presynaptic membrane. d mean number (se) of cisterns within 20 nm of the presynaptic density versus distance along the presynaptic membrane is shown for all synapses. 2013, j comp neurol, reprinted with permission from 2013 wiley periodicals) principle of functional heterogeneity in ihcs. a schematic of an organ of corti showing afferent and efferent innervations at ihcs. modified from meyer and moser 2010, curr opin otolaryngol head neck surg, reprinted with permission from 2010 wolters kluwer health. b mean and sd of f (gray) as a function of depolarizing potential (v m), obtained from spot - detection experiments at the center of the ca microdomain ; f was averaged over the last 15 ms of a 20-ms stimulus. f (mean gray) and i ca (mean black) show a similar voltage dependence (thin lines corresponding sds). b heterogeneous voltage dependence and ca channel number of synaptic ca channel clusters in ihcs. pronounced variability in the voltage dependence of activation, even within the same cell (dashed traces individual data curves from 3 ca microdomains in an ihc). modified from frank. c, c colorized spatial distribution of vesicles and cisterns around the ribbon in low- and high - spontaneous rate (sr) fibers. sections through a high - sr (c) and a low - sr (c) synapse containing the synaptic ribbon are shown with cisternal (maroon) and vesicular (green) profiles. d mean density (se) of docked vesicles, i.e., within 20 nm of the presynaptic density along the presynaptic membrane. d mean number (se) of cisterns within 20 nm of the presynaptic density versus distance along the presynaptic membrane is shown for all synapses. 2013, j comp neurol, reprinted with permission from 2013 wiley periodicals) further insights into the morphological heterogeneity require modern 3d reconstructions of larger volumes such as serial block face scanning electron microscopy (denk and horstmann 2004) or focused ion beam scanning electron microscopy (see, e.g., knott. 2008 ; kreshuk. using these methods, a detailed 3d view of single ihcs including afferent and efferent innervations would be possible, finally allowing correlation of parameters, such as presynaptic ribbon size and postsynaptic bouton diameter, as a function of position on the ihc for a large number of synapses, together with the functional assessment of pre- and postsynaptic properties in cochlear explants by electrophysiology (goutman and glowatzki 2007) and/or imaging of ihc ca and exocytosis (e.g., frank. tight coupling between exo- and endocytosis is a prerequisite for maintaining the enormous vesicle turnover rates at ribbon synapses clathrin - coated structures but also large cisterns without clathrin - coats, are observed close to synaptic ribbons (siegel and brownell 1986 ; sendin. 2007 ; frank. 2010 ; kantardzhieva. 2013 ; neef. 2014 ; revelo. 2014). 2013) set out to determine whether such cisterns participate in vesicle reformation and what differences can be observed in correlation to the functional properties of high and low spontaneous rate fibers (fig. an extensive quantitative analysis of ribbons, vesicles and cisterns from serial sections of cat ihc ribbon synapses suggested a fewer cisterns and more synaptic vesicles are found, which indeed points towards a contribution of cisterns to locally restricted vesicle formation. other studies used membrane capacitance measurements to provide an initial functional assessment of endocytic membrane retrieval at ihc azs (moser and beutner 2000 ; beutner and moser 2001 ; neef. 2014). moreover, ph - sensitive gfp (phluorin ; miesenbck. 1998) targeted to the intraluminal face of vesicle membranes by attachment to vesicular glutamate transporters (zhu. 2009) has become an important tool in studying exo- and endocytosis, not only from neurons but also ihcs (neef. additionally, a novel membrane tracer specifically tailored to use in the organ of corti has been devised and applied to investigate endocytosis (revelo. 2014), as the commonly used styryl dye fm1 - 43 penetrates stereociliar mechanotransduction channels and hence is of limited use to study endocytosis, in ihcs (gale., expression analysis and immunohistochemistry have revealed the presence of several important molecular players of endocytosis such as dynamins, amphiphysin, clathrin (neef. 2014) and adaptor protein 2 (ap-2) (duncker. a very recent dna microarray study investigating ihc and ohc transcriptomes might even give more insight into proteins involved in vesicle recycling (liu. currently, in ihcs, three distinct mechanisms are considered to mediate endocytosis : slow cme, fast bulk endocytosis and potentially kiss - and - run or cme is the main pathway of membrane retrieval for mild stimulation and proceeds at a constant rate ; it represents the linear component of endocytosis following exocytosis of the rrp (fig. this mechanism is not only inhibited by the clathrin - inhibitor pitstop-2 but also by disruption of dynamin 1 via pharmacological and genetic means (neef. in contrast, a different study reported inhibition of sustained exocytosis by the presumptive dynamin inhibitor dynasore but did not investigate endocytic membrane retrieval (duncker. finally, when exocytosis exceeds three to four rrp equivalents, ihcs additionally recruit a faster mode of membrane retrieval, which proceeds with an exponential time course within a few seconds. 3a) and, indeed, there is plenty of evidence for the invagination and fission of large stretches of plasma membrane in the vicinity of hair cell azs (lenzi. 2002 ; frank. 2010 ; pangri. 2010 ; kamin. 2014 ; neef. 2014 ; revelo. both mechanisms seem to engage in different phases of release : cme supports vesicle cycling during mild stimulation but bulk endocytosis finally occurs after prolonged stimulation, providing a mechanism that assures the balance between exo- and endocytosis in ihcs and thus, assures high release rates (neef. 3endocytosis in inner hair cells. a, a representative recordings in response to 20 ms (a) or 200 ms (a) depolarizations. after the c m increase upon 20 ms depolarization, the slope - corrected c m traces (middle) typically showed a linear decay (a). the 200-ms - long depolarization resulted in a combination of exponential and linear decay (a). b 3d reconstructions of resting (b), stimulated (b) and recovered ihcs (b, b). most organelles, including the tubular ones, are replaced by small vesicles during the recovery periods. insets magnified regions from the four different cell regions (cuticular plate, top, nuclear and basal regions). note the increased number of endosome - like organelles at the base of the cell after stimulation and during recovery. c mcling - labeled organs of corti were immunostained for vglut3 and otoferlin (first row), for vglut3 and syntaxin 6 (sx 6, second row), for otoferlin and syntaxin 16 (sx 16, third row) and finally for syntaxin 6 and syntaxin 16 (fourth row). the samples were cut into 20-nm sections and were imaged using an epifluorescence microscope. dashed white lines the plasma membrane of the ihcs. d graphic representation of pearson s correlation coefficients : otoferlin and syntaxin 6 (or syntaxin 16) correlate in the mcling - labeled organelles at the top and nuclear levels. organelles with a different molecular composition recycle membrane in different regions, taking up mcling. apical endocytosis takes up the membrane into round organelles, a sizeable proportion of which is similar to late endosomes (light blue). endocytosis in the top and nuclear regions reaches tubular organelles containing otoferlin and two endosome markers, syntaxin 16 and syntaxin 6. this suggests that these organelles participate in constitutive pathways, probably by maintaining membrane traffic between the plasma membrane and the trans - golgi. at the base of the cell, stimulation induces the formation of membrane infoldings and cisterns that are characterized by the presence of vglut3, rab3 and also otoferlin. endocytosis in inner hair cells. a, a representative recordings in response to 20 ms (a) or 200 ms (a) depolarizations. after the c m increase upon 20 ms depolarization, the slope - corrected c m traces (middle) typically showed a linear decay (a). the 200-ms - long depolarization resulted in a combination of exponential and linear decay (a). b 3d reconstructions of resting (b), stimulated (b) and recovered ihcs (b, b). most organelles, including the tubular ones, are replaced by small vesicles during the recovery periods. insets magnified regions from the four different cell regions (cuticular plate, top, nuclear and basal regions). note the increased number of endosome - like organelles at the base of the cell after stimulation and during recovery. c mcling - labeled organs of corti were immunostained for vglut3 and otoferlin (first row), for vglut3 and syntaxin 6 (sx 6, second row), for otoferlin and syntaxin 16 (sx 16, third row) and finally for syntaxin 6 and syntaxin 16 (fourth row). the samples were cut into 20-nm sections and were imaged using an epifluorescence microscope. dashed white lines the plasma membrane of the ihcs. d graphic representation of pearson s correlation coefficients : otoferlin and syntaxin 6 (or syntaxin 16) correlate in the mcling - labeled organelles at the top and nuclear levels. organelles with a different molecular composition recycle membrane in different regions, taking up mcling. apical endocytosis takes up the membrane into round organelles, a sizeable proportion of which is similar to late endosomes (light blue). endocytosis in the top and nuclear regions reaches tubular organelles containing otoferlin and two endosome markers, syntaxin 16 and syntaxin 6. this suggests that these organelles participate in constitutive pathways, probably by maintaining membrane traffic between the plasma membrane and the trans - golgi. at the base of the cell, stimulation induces the formation of membrane infoldings and cisterns that are characterized by the presence of vglut3, rab3 and also otoferlin. 2014, reprinted with permission from 2014 revelo.) but where does the retrieval of synaptic vesicle membrane take place and where and how are synaptic vesicles regenerated following membrane retrieval in hair cells ? does synaptic endocytosis comply with the apicobasal compartmentalization of the hair cell ? at photoreceptor ribbons, a periactive zone, marked by the presence of endocytic proteins, was identified directly adjacent to the az area (in a range of 120250 nm from the ribbon) using high - resolution and electron microscopy (wahl. does this also apply to ihc ribbons ? at the resolution of confocal microscopy, a similar perisynaptic accumulation of endocytic proteins has, so far, not been found (neef. clearly, electron micrographs indicate that both bulk and cme endocytosis take place near the az (siegel and brownell 1986 ; lenzi. 2002 ; frank. 2010 ; kamin. 2014 ; neef. a radically different model of ihc endocytosis has been sketched based on life imaging of fm1 - 43 uptake into ihcs, whereby exocytosed membrane was postulated to move towards the ihc apex for endocytosis and recycling via the golgi apparatus (griesinger. recent elaborative studies using various styryl dyes and more suitable fluorescent membrane markers lead us to reconsider this hypothesis. first, it was corroborated that many styryl dyes including fm1 - 43 permeate into the cytosol of ihcs via the mechanotransducer channels (kamin. therefore, on the ultrastructural level, the photo - oxidation technique revealed a fuzzy dark diaminobenzidin (dab) smear inside the cytosol in addition to the precipitate in membrane - bound organelles likely resulting from internalized fm1 - 43. the stimulation - induced endocytic uptake of fm1 - 43 could still be followed by observing an electron - dense precipitate within vesicular structures, which allows the determination of the presence or absence as well as the localization of stained structures under resting (fig. 3b) and recovery (fig. 3b, b) conditions in serial 3d reconstructed ihcs (kamin. 2014). at rest, endosome - like organelles were detected in the apex of the ihcs, whereas larger tubulo - cisternal organelles dominated at the nuclear region. at the basal region, only a few labeled structures were present. stimulation massively increased the amount of basolateral membrane trafficking, reflected by the appearance of labeled small vesicles and endosome - like vacuoles ; however, no changes in the apical and nuclear regions could be observed (kamin. strikingly, the basolateral cisterns were replaced in the basal region by small, synaptic - like vesicles during a few minutes of recovery from stimulation, suggesting a highly efficient mechanism of vesicle regeneration from cisternal membranes internalized by bulk endocytosis. the combination of fm1 - 43 uptake and photo - oxidation therefore suggests that synaptic vesicle recycling takes place at the basal part, close to ribbons at least during synaptic activity (kamin. recently, a novel membrane tracer, named membrane - binding fluorophore - cysteine - lysine - palmitoyl group (mcling), which does not permeate the mechanotransducer channel, tightly binds biological membranes and can be fixed, has been developed (revelo. 2014). in combination with super - resolution light microscopy (i.e., sted), the spatial organization and pathways of endocytosis in ihcs could be further investigated. in order to improve the spatial resolution of sted microscopy in the axial direction, thin sections of ihcs were imaged after embedding the organs of corti in melamine, which maintains the fluorescence (revelo. the apical, nuclear and basal regions under conditions of rest, stimulation and recovery from stimulation, were investigated and the uptake of mcling monitored. in order to reveal the molecular identity of mcling - labeled structures and thereby identify the respective endocytic pathways, samples were co - stained with different protein markers for the endoplasmic reticulum (er) and golgi as well as synaptic vesicle endo- and exocytosis. in these experiments, a strong correlation for basolateral mcling localization with vglut3, rab3 and otoferlin immunofluorescence was found. otoferlin as well as syntaxin 16, a late endosomal marker, colocalized with apical and nuclear mcling (fig. moreover, the lysosomal - associated membrane protein 1 (lamp1) colocalized with mcling in the apical region of the ihc. stimulation led to a selective uptake of mcling at the base of the ihc, corroborating the notion of local recycling of synaptic vesicles that was postulated based on electron microscopy and photo - oxidation (kamin. the local recycling hypothesis was further supported by the finding that exogenous vglut1-phluorin fluorescence not only transiently appeared at ribbon - type azs but remained there for tens of seconds after stimulation (neef. finally, the mcling experiments revealed large membranous organelles near synapses, which were replaced by small organelles a few minutes after stimulation, thereby providing direct evidence of bulk endocytosis and vesicle regeneration from the internalized plasma membrane. the association of otoferlin with all three putative membrane recycling pathways suggests a more general role of this protein in endocytosis. otoferlin has recently been assigned a role in vesicle endocytosis due to its interaction with ap-2. using a high - resolution liquid chromatography coupled with a mass spectrometry approach, multiple subunits of ap-2 were identified as interaction partners of otoferlin in the mammalian cochlea and the proposed interactions were biochemically confirmed by co - immunoprecipitation (duncker. 2013). ap-2 plays a role in clathrin - mediated endocytosis via binding to clathrin - coated vesicles budding from the plasma membrane (keyel. 2010) and has been shown to be expressed in ihcs (duncker. future work is required to clarify the role of ap-2 in hair cell endocytosis and the relevance of its interaction with otoferlin. recently, major progress has been made towards dissecting the molecular anatomy and physiology of hair cell ribbon synapses. this includes powerful single synapse techniques such as (1) patch - clamp of postsynaptic afferent terminals of sgns, (2) high resolution -functional imaging of presynaptic ihc ca dynamics and membrane turnover, as well as (3) super - resolution light microscopy and electron tomography following high - pressure freezing. however, in order to investigate the release mechanisms of ihcs and firmly correlate structure and function, the development of new functional and morphological approaches is required. functional and morphological analysis of single synapses will be necessary and some questions require reading out both pre- and postsynaptic properties at the same time. the commonly used k stimulation of cochlear tissue likely mimics strong physiological steady - state stimulation. but this stimulation does not provide the temporal resolution to allow the observation of the release kinetics at ihc ribbon synapses. especially, knowledge about short - term plastic changes is lacking, since it is not possible to apply very short stimuli (i.e., millisecond range) and investigate the cells during and at defined times after stimulation. therefore, approaches are needed that meet two requirements : (1) a precise stimulation protocol combined with (2) rapid immobilization of the sample, e.g., by using high - pressure freezing. one emerging tool that promises to fulfill these requirements is the combination of optogenetic stimulation with high - pressure freezing. this could involve the expression of a light - sensitive ion channel such as channelrhodopsin-2 (chr-2) from the green algae chlamydomonas reinhardii (nagel. 2003) in hair cells and stimulation would ideally be performed within a chamber that should be mounted in a freezing machine in order to minimize the time delay before freezing. recently, synaptic recovery of motoneurons from c. elegans was analyzed using optogenetic stimulation in combination with high - pressure freezing (kittelmann. moreover, after a single light stimulus, docked vesicles fused along a broad az on c. elegans motoneurons expressing chr-2. these vesicles were replenished with a time constant of about 2 s. further, endocytosis occurred within 50 ms adjacent to the dense projection and after 1 s adjacent to adherens junctions (watanabe. moreover, a study on optically stimulated cultured hippocampal neurons revealed an ultrafast endocytosis mechanism at central synapses (watanabe. these initial experiments indicate that optogenetics, in combination with high - pressure freezing (flash and freeze ; watanabe. 2013b) and subsequent electron tomography, might provide sufficient resolution to study the ultrastructure of spatiotemporally defined functional states and thus provide a completely new view on the release mechanism of ihc ribbon synapses. how does the subcellular organization of sensory ihcs enable mechanotransduction and transmitter release at high rates ? ihcs are epithelial cells by origin and exhibit several characteristics that distinguish them from neurons. most notably, they show a strong polarization with respect to both long and short cell axes. the polarization along the apicobasal axis follows a clear compartmentalization, e.g., apparent by the hair bundle harboring the mechanotransduction apparatus of the apical membrane. graded receptor potentials are formed by mechanoelectrical (apical) and voltage - gated (basal) conductances (corey and hudspeth 1979 ; roberts. 1990). actin - filled stereocilia protrude into the endolymph in a highly organized manner and their sophisticated supramolecular mechanotransduction apparatus enables ultrasensitive detection of sound - born vibrations of the cochlear partition (reviewed in kazmierczak and mller 2012). while the molecular identity of the mechanotransducer channel still awaits definitive demonstration, recent work indicates the transmembrane channel - like proteins (tmc)-1 and -2 as promising candidates (pan. opening of the apical mechanotransducer channels depolarizes the ihc, subsequently activating cav1.3 ca channels (platzer. ; dou. 2004) at the presynaptic az in the basolateral membrane, where the incoming ca triggers neurotransmitter release. the density of ribbon synapses shows a strong basoapical gradient, with the supranuclear portion of the hair cell being devoid of azs (francis., the cuticular plate likely serves as an anchor for the stereociliar actin bundles, containing a rich protein network with cytoskeletal proteins such as actin, -actinin and tropomyosin (slepecky and chamberlain 1985 ; zine and romand 1993). moreover, the striated organelle, located underneath the cuticular plate, likely modulates the stereociliar actin bundles (vranceanu. microtubules are primarily found beneath the cuticular plate (slepecky and chamberlain 1985 ; steyger. 1990) but appear connected to cytoskeletal proteins in the cuticular plate, for example via acf7a (actin crosslinking family protein 7a), as suggested for zebrafish neuromast hair cells (antonellis. 2014). 1990), providing the mechanical strength of hair cells (szarama. 2012) and tracks for efficient cargo protein trafficking along the apicobasal axis (furness. 1990). in addition to the cellular apicobasal polarity, hair cells also show planar cell polarity, which is reflected in the orderly orientation of their hair bundles (reviewed in ezan and montcouquiol 2013 ; sienknecht. 2014). whether the basolateral organization of the hair cells is similarly instructed by planar cell polarity, we will focus on the organization of the basal portion of ihcs and discuss structure and function of hair cell ribbon synapses. emphasis will be on the molecular machinery of the synapse, synapse development, synaptic heterogeneity and synaptic vesicle recycling. phylogenetically, ribbons in sensory cells are old structures that occur not only in mammals but also in fishes, amphibians and birds. in the mammalian organ of corti, they were first described by smith and sjstrand (1961) and are found in both sensory cell types, i.e., ihcs and ohcs (sobkowicz. 1982). the discovery of the protein ribeye, initially purified from bovine retina, (schmitz. 2000), as the main and structure - yielding component of ribbons in rat photoreceptors (schmitz. 2003), zebrafish photoreceptors and bipolar cells (wan. 2005) and mouse cochlear hair cells (khimich. 2005 ; see also immunogold labeling in fig. 1a) highlights the conservation of the ribbon in vertebrate evolution (schmitz 2009). nonetheless, ribbons still vary greatly in size and shape (lenzi and von gersdorff 2001 ; moser. 2006 ; matthews and fuchs 2010), likely reflecting structural adaptation to the specific needs of the respective synaptic connection for sensory coding.fig. a ribeye is the main component of the ribbon as shown by pre - embedding immunogold labeling of a p14 ihc synaptic ribbon using an anti - ctbp2 antibody (courtesy of susann michanski, innerearlab, university medical center, gttingen, germany) ; a representative image of an electron micrograph of a round - shaped p9 immature ribbon exhibiting a dotted pattern possibly caused by ribeye arrangement (contrast enhanced image in a), see also schematic representation (a). b a p14 mature ribbon with the typical multi - lamellar pattern (contrast enhanced image in b), see also scheme in b. scale bars (a, a, b) 100 nm. c a serial 3d reconstruction of a mature ribbon with two distinct morphological vesicle pools (yellow : ribbon - associated vesicles ; orange : membrane - proximal vesicles ; red : ribbon ; blue : az membrane ; magenta : presynaptic density). c the membrane - proximal vesicles (orange) are arranged around the presynaptic density (magenta) that is containing the scaffolding protein bassoon as shown by the pre - embedding immunogold labeling in (d), scale bar (d) 100 nm (courtesy of susann michanski, innerearlab, university medical center, gttingen, germany) ; d 2-color sted image of immunolabeled bassoon (magenta) and cav1.3 channel clusters (green) in mature ihcs : stripelike morphology and closely aligned immunofluorescence of bassoon and cav1.3 can be observed. scale image:700 700 nm ; e, e mathematic model showing the total mean steady state [ca ] profile at the az membrane (e) ; e effective number of cav1.3 channels contributing to total mean steady state [ca ] as shown in (e). (c, c, d, e, e modified from wong. 2014, embo j ; reprinted with permission 2014 wong.). f schematic summary of the protein arrangement at mature ihc ribbon synapses spatial distribution of ihc az proteins. a ribeye is the main component of the ribbon as shown by pre - embedding immunogold labeling of a p14 ihc synaptic ribbon using an anti - ctbp2 antibody (courtesy of susann michanski, innerearlab, university medical center, gttingen, germany) ; a representative image of an electron micrograph of a round - shaped p9 immature ribbon exhibiting a dotted pattern possibly caused by ribeye arrangement (contrast enhanced image in a), see also schematic representation (a). b a p14 mature ribbon with the typical multi - lamellar pattern (contrast enhanced image in b), see also scheme in b. scale bars (a, a, b) 100 nm. c a serial 3d reconstruction of a mature ribbon with two distinct morphological vesicle pools (yellow : ribbon - associated vesicles ; orange : membrane - proximal vesicles ; red : ribbon ; blue : az membrane ; magenta : presynaptic density). c the membrane - proximal vesicles (orange) are arranged around the presynaptic density (magenta) that is containing the scaffolding protein bassoon as shown by the pre - embedding immunogold labeling in (d), scale bar (d) 100 nm (courtesy of susann michanski, innerearlab, university medical center, gttingen, germany) ; d 2-color sted image of immunolabeled bassoon (magenta) and cav1.3 channel clusters (green) in mature ihcs : stripelike morphology and closely aligned immunofluorescence of bassoon and cav1.3 can be observed. scale image:700 700 nm ; e, e mathematic model showing the total mean steady state [ca ] profile at the az membrane (e) ; e effective number of cav1.3 channels contributing to total mean steady state [ca ] as shown in (e). (c, c, d, e, e modified from wong. 2014, embo j ; reprinted with permission 2014 wong.). f schematic summary of the protein arrangement at mature ihc ribbon synapses ribeye is composed of two major domains : while the a domain organizes the assembly of the synaptic ribbon and is unique in structure, the b domain is structurally nearly identical to the transcription repressor ctbp2, which is encoded by the same gene but uses a different transcription initiation site (schmitz. 2000) and exhibits enzymatic activity (schwarz. the b domain is also assumed to be involved in tethering of synaptic vesicles to the ribbon (schmitz. 2000 ; schmitz 2009), though the proteins that form tethers remain to be identified. ribeye appears to organize ribbon shape directly based on its domain structure (schmitz 2009) and its aggregation properties (magupalli. a trifold lamellar pattern has been described for photoreceptor ribbons and assigned to the polarized arrangement of ribeye (schmitz 2009). also at mature ihc ribbons, a lamellar substructure is observed that harbors multiple lamellar foldings (sobkowicz. this effect can be attributed to the differences in the ribbon shape in ihcs and photoreceptors. in immature ihc ribbons, the lamellar pattern is not prominent but instead a dotted pattern can be observed (fig. recently, at zebrafish hair cell ribbon synapses, it was found that the ribeye a and b domain segregate along the vertical axis of the ribbons, with the b domain more located towards the basal end (sheets. ribeye, in contrast to other az proteins, is not found in invertebrates such as the fruitfly drosophila melanogaster ; however, bruchpilot (brp), the homolog of the vertebrate az protein cast (caz - associated structural protein)/erc2, functions as the main building block of t - bars (kittel. 2006 ; wagh. in fact, only the n - terminus is conserved and shows sequence homologies to cast, whereas the c - terminus is only found in dipteran insects and rather resembles cytoskeletal elements such as plectin due to its numerous coiled - coil domains (wagh. moreover, the c - terminus mediates vesicle tethering to the t - bar (hallermann. the structurally related proteins bassoon and piccolo as well as cast, elks, rab3-interacting molecule (rim) and munc13 are present (betz. 1998 ; fenster. 2000 ; dresbach. 2001 ; deguchi - tawarada. 2006 ; wang. 2009 ; sdhof 2012). additionally, ctbp2 and ctbp1 have also been found at conventional azs (tom dieck. 2005). except for piccolo, which at ribbon synapses is solely expressed as a shorter splice variant nicknamed as piccolino (regus - leidig. 2013), these proteins also largely form the caz at photoreceptor ribbon synapses (wang. the components of hair cell azs, on the other hand, are still largely unexplored, except for bassoon (khimich. 2005) and piccolo / piccolino (khimich. 2005 ; regus - leidig. in fact, a recent study indicates that ihc synapses operate without munc13-like priming factors (vogl. bassoon, together with ribeye, is responsible for the ribbon shape and, hence, might contribute to its function. studies of ihcs from bassoon mutant mice indicated an anchoring function of bassoon (khimich. 2005 ; frank. 2010 ; jing. 2013), in line with findings at photoreceptor ribbon synapses (dick. 2003 ; tom dieck. the fraction of ribbon - occupied synapses remaining in bassoon - deficient ihcs seems to depend on age and residual levels of full - length bassoon (khimich. the fraction of ribbonless synapses increased from 50 % at postnatal day 11 (p11) up to 88 % at p70. a lower and relatively constant fraction of 56 % ribbonless synapses was found in a gene - trap bassoon mutant (bsn) likely due to a weak residual synaptic expression of bassoon (jing. maintained az ultrastructure, bsn animals exhibited a larger number of ca channels at ihc synapses compared to bsn mice and also displayed an intermediate phenotype regarding sustained ihc exocytosis (jing. in contrast, the size of the readily releasable vesicle pool (rrp) was strikingly reduced in both mutants. moreover, single unit recordings of the sgns show comparably severe defects in bsn and bsn mice, as both genotypes had impaired sound onset coding and lower evoked and spontaneous spike rates. taken together, these results indicate that the remaining, loosely anchored ribbons might function inadequately (jing. this further suggests that the mere presence of the ribbon, even with tethered vesicles, is not sufficient to maintain normal transmitter release and sustain the rrp at ihc ribbon synapses. moreover, it seems that bassoon contributes to organizing the ihc az beyond anchoring the ribbon. this has been concluded from impaired clustering of ca channels at bsn ribbon - occupied synapses (frank. 2010) and indicates a potential direct contribution of bassoon in organizing the az (frank. 2010 ; hallermann and silver 2013). in contrast to conventional synapses, where often only the combined knockdown of bassoon and its homolog piccolo causes synaptic defects (altrock. 2003 ; leal - ortiz. 2008 ; mukherjee. 2010 ; waites. 2011, 2013), these proteins seem not to act redundantly at ribbon synapses. as mentioned above, at hair cell and photoreceptor ribbons, only the short isoform of piccolo, piccolino, is expressed, which lacks a large c - terminal part (regus - leidig. binding sites for the proteins abp1, pra1, git1 and profilin (wang. 2003) are still present, whereas binding sites for, e.g., bassoon or rim are lacking (regus - leidig. accordingly, piccolino exhibits a different spatial distribution at ribbon synapses of photoreceptors, where it is found directly on the ribbon, as indicated by pre - embedding immunogold - labelings, using an antibody recognizing the n - terminus of the protein (limbach. (2014) revealed a striking impairment in the ribbon structure upon piccolino rnai - based knockdown. instead, a high proportion of attached spherical ribbons was found that resemble ribbon precursors of photoreceptor ribbons suggesting a role of piccolino in structural ribbon maturation (regus - leidig. ribeye and presumably piccolino as well as bassoon present the main structural components of the ribbon and/or the anchorage of the ribbon to the az. in order to resolve the function of ribbons, the ribbon was suggested to (1) promote a large readily releasable pool of vesicles via establishing / stabilizing many ca channels and vesicular release sites (khimich. 2010), (2) facilitate vesicle replenishment at the az (conveyor belt model, e.g., bunt 1971 ; gray and pease 1971 ; vollrath and spiwoks - becker 1996 ; lenzi and von gersdorff 2001 ; snellman. 2011), (3) facilitate multivesicular release (edmonds 2004 ; fuchs 2005), or (4) serve as a diffusion barrier to enable high local ca concentrations (graydon. functional interpretations have also been provided for the morphologically distinct populations of synaptic vesicles at ribbon synapses but in each case remain to be validated. ribbon - associated vesicles structurally attached through filamentous protein tethers the ones at the base of the ribbon face the presynaptic plasma membrane (membrane - proximal vesicles ; fig. 1c, c) and are often tethered to the membrane and/or to the presynaptic density (tethered vesicles). lateral to this subpopulation of ribbon - associated vesicles, there are few additional membrane - proximal and even membrane - tethered vesicles that are not in close vicinity to the ribbon. while further testing is required, current evidence suggests that the membrane - proximal vesicles comprise the rrp (e.g., in retinal bipolar cells : von gersdorff. 1996 ; zenisek. 2000 ; frog saccular hair cells : lenzi. 1999, 2002 ; rutherford and roberts 2006 ; and mouse inner hair cells : khimich. 2005 ; frank. support for this hypothesis comes from the approximate matching between the morphologically estimated number of membrane - proximal vesicles and the functionally defined size of the rrp, i.e., the fast component of exocytosis upon depolarization - evoked ca influx (von gersdorff. 1996 ; pangri. 2010 ; frank. 2010), as well as from the observation that these vesicles are most heavily depleted upon stimulation (lenzi. 2002 ; pangri. furthermore, the tethering of vesicles to the az membrane might reflect a structural correlate of fusion competence (siksou. new high - resolution imaging approaches using rapid freezing methods and/or electron tomography have revealed that synaptic vesicles in proximity to the membrane exhibit several morphologically distinct stages. tethers of different numbers and lengths connecting synaptic vesicles to the az membrane could be observed at conventional synapses and synaptosome preparations (siksou. 2007, 2009 ; fernndez - busnadiego. 2010, 2013) but also at ihc ribbon synapses (frank. 2010). moreover, cryo - electron tomography, a method allowing visualization of hydrated and unstained tissue, revealed that tethering of synaptic vesicles in synaptosomes prepared from hippocampal tissue precedes the full contact of a synaptic vesicle with the membrane (fernndez - busnadiego. single long tethers initially seem to be formed and synaptic vesicles likely enter the rrp via the formation of several short tethers (< 5 nm). in line with this hypothesis, this fraction of vesicles could be depleted by application of hypertonic sucrose, that is thought to trigger rrp release (rosenmund and stevens 1996). moreover, the formation of short tethers could be inhibited using tetanus toxin, pointing towards the fact that neuronal soluble nsf attachment protein receptors (snare) proteins are involved in this process (fernndez - busnadiego. 2013) play a crucial role in tethering vesicles to the membrane at conventional cns synapses. interestingly, munc13 and caps priming factors seem not to operate at the ihc ribbon synapse (vogl. 2015). some authors even argue that the entire ribbon - associated vesicle population is fusion - competent and, therefore, can be released within a few milliseconds or less (heidelberger. 1994 ; edmonds 2004). evidence for a priming function of the ribbon has recently been presented (snellman. in contrast to caz proteins, which are, at least in part, conserved at ihc azs, the molecular machinery involved in the regulation of synaptic vesicle fusion seems to deviate strongly from that of as mentioned above, neurotransmitter release at conventional synapses is mediated by neuronal snares, namely snap-25, syntaxin 1 and synaptobrevin 1 or 2 (reviewed in jahn and fasshauer 2012). snare activity can be blocked by neurotoxin - mediated cleavage or genetic manipulations (schiavo. ribbon synapses, the snare protein machinery appears to be present and functional (brandsttter. 1996 ; morgans. 1996 ; morgans 2000 ; von kriegstein and schmitz 2003 ; uthaiah and hudspeth 2010 ; cooper. in contrast, ihc exocytosis seems to be insensitive to neurotoxins and genetic ablation of neuronal snares (nouvian. 2011) and, hence, a functional role of syntaxin 1, synaptobrevin 1 and 2 as well as snap-25 in ihc exocytosis is questionable. while some studies detected snare mrnas and proteins in the sensory epithelium and hair cells (safieddine and wenthold 1999 ; uthaiah and hudspeth 2010 ; nouvian. 2011), neither snap-25, synaptobrevin 13 nor syntaxin 1 could be detected by immunofluorescence imaging in mouse ihcs (nouvian. moreover, snare regulators such as synaptotagmins 13 (beurg. 2010 ; reisinger. 2011) and complexins (strenzke. in contrast, the multi - c2 domain protein otoferlin plays a central role for hair cell exocytosis (roux. the absence or mutation of otoferlin causes deafness or temperature - sensitive hearing impairment in humans (yasunaga. 1999 ; varga. 2008) and rodents (roux. 2006 ; longo - guess. 2007 ; ultrastructurally, otoferlin is also found at ribbon synapses, mostly but not exclusively at synaptic vesicles and the az membrane (roux. sufficient amounts of otoferlin appear to be required for correct vesicular fusion and replenishment (roux. otoferlin is suggested to act as the ca sensor in ihcs (roux. 2006 ; johnson and chapman 2010), due to its ca - binding capabilities (roux. 2006 ; ramakrishnan. 2009, 2014 ; johnson and chapman 2010 ; pangri. 2010) and facilitates snare - mediated liposome fusion (johnson and chapman 2010). in its absence, no depolarization - evoked rrp exocytosis is observed in ihcs (roux. 2010). transgenic expression of synaptotagmin 1, the major ca sensor of neuronal synaptic vesicle exocytosis, failed to restore ihc exocytosis and hearing in otoferlin ko mice, which may not be too surprising given the overall low conservation of the molecular composition between conventional and ihc synapses (reisinger. next to proteinaceous exocytosis machineries, the actual mechanisms of vesicle fusion as well as the transport of vesicles to the ihc release site are still largely unknown. the large and, in terms of amplitude and shape, heterogeneous excitatory postsynaptic currents (epscs) measured at postsynaptic afferent boutons of sgns have been interpreted to result from multivesicular (multiquantal) release at ihc azs (glowatzki and fuchs 2002). large epscs ensure rapid and temporal precise spike generation of sgns (rutherford. 2012) and the relevance of such large epscs for achieving a high synchronization index of postsynaptic firing (i.e., better phase locking precision) has recently been shown in the frog papilla (li. several multiquantal release scenarios at ihc ribbons have been discussed : (1) synchronized vesicle fusion of several single vesicles as well as (2) compound fusion, following homotypic vesicle - to - vesicle fusion and (3) sequential fusion involving homotypic vesicle - to - vesicle fusion while release occurs (glowatzki and fuchs 2002 ; edmonds 2004 ; neef. recent findings suggest an alternative candidate mechanism for ihc exocytosis. combining experimental approaches and mathematical modeling chapochnikov. (2014) indicated that univesicular (uniquantal) release can explain the large size of sgn epscs and that the control of release by a dynamic vesicular fusion pore can account for the observed epsc heterogeneity. at this point, none of the above discussed mechanisms can definitively be ruled out or confirmed and future work, including detailed morphological analysis using electron microscopy of defined functional states, will be required to advance our understanding of exocytosis mechanism at ihc ribbon synapses. in case vesicles do not homotypically fuse with other vesicles at the ribbon while releasing, a transport mechanism of the vesicles to the membrane has to exist. the conveyor belt model, transporting the vesicle actively along the ribbon to the membrane, was one of the first models to be introduced (bunt 1971 ; gray and pease 1971 ; vollrath and spiwoks - becker 1996 ; lenzi and von gersdorff 2001). accordingly, a kinesin polypeptide, kif3a, was identified on photoreceptor ribbons that could serve as a motor for vesicle transport involving the filamentous tethers observed at the ribbon (muresan. 1999), which were also proposed to function as stepping stones for synaptic vesicles (usukura and yamada 1987 ; parsons and sterling 2003). recently, the tethers at the ribbon were suggested to be directly involved in coordinating vesicle transport towards the membrane via crowd surfing, based on passive diffusion following the gradient established by exocytic vesicle consumption at the base of the ribbon (graydon. the tethers simply need to bind the vesicles and prevent them from detaching until they reach the az membrane, where release maintains the diffusion gradient (graydon. 2014). however, future experiments involving mutant analyses will be necessary to identify the proteins mediating vesicular tethering to the ribbon and estimate their affinity to the vesicles and the functional relevance of the tethers for synaptic transmission. moreover, it will be interesting to investigate whether and how the tethering can be influenced by factors such as activity or even developmental stage. for example, maturation from pre - hearing to hearing significantly determines structure and function of the ribbon synapses and the spatial arrangement of az proteins such as the ca channels or bassoon, as will be emphasized in the next section. during maturation of the organ of corti, ribbon synapses and sgn fibers undergo drastic morphological changes. how do morphological alterations during the transition from a pre - hearing to a hearing state correlate to functional maturation of ribbon synapses ? generally, synaptic contacts are ultrastructurally defined as pre- and postsynaptic electron - dense membranes that are closely aligned. the postsynaptic density (psd) is clearly visible as an electron - dense structure beneath the postsynaptic membranes directly juxtaposed to the presynaptic az. the innervation pattern of sgn fibers at hair cells within the immature rodent cochlea is significantly different from the mature configuration and massive rearrangements of the fibers that occur before the onset of hearing. hereby, type i sgn fibers retract from the ohcs, whereas type ii sgn fibers disappear from the ihcs (perkins and morest 1975 ; echteler 1992 ; simmons 2002). these developmental processes of fiber innervation take place in the first postnatal week in three distinct phases : (1) in e18-p0 animals, fibers of both afferent types extend towards all hair cells ; (2) between p0 and p3 a refinement occurs, where the outer spiral bundle forms that innervate the ohcs ; and (3) the type i fibers retract from the ohcs around p3p6, accompanied by synaptic pruning, while they keep their projections on the ihcs (huang. ampa - typed glutamate receptors and scaffold proteins like bassoon and shank1 disappear during the maturation process from ohcs. glua2/3 subunits remain stable throughout development and into adulthood, while glua4 subunit expression significantly increase in adult type i fibers (huang. recently, the molecular arrangement of afferent synapses in relation to functional changes at the ihcs has been addressed in more detail using a combination of confocal, stimulated emission depletion (sted) and electron microscopy, as well as ihc presynaptic physiology and computational modeling (wong. 2014). it is known that, in the early pre - hearing stages between p6 and p9, several small apposing pre- and postsynaptic densities mark nascent synapses. some of the presynaptic densities are occupied by synaptic ribbons, which are small and round in shape and attached via two triangular - shaped proteinaceous anchors (sobkowicz. however, floating ribbons were also frequently observed in close proximity to az areas at these developmental stages (wong. serial 3d electron microscopic reconstructions corroborated the notion of several discontinuous pre- and postsynaptic specializations. such synaptic sites are organized as loose suprastructures on the bouton surface and are likely functional, as immunohistochemistry indicates the presence of presynaptic ca channels and postsynaptic ampa receptors (wong. 2000 ; hell 2007), revealed that cav1.3 channels are arranged in small round spots (wong. 2014) rather than the stripes previously described for mature azs (frank. in addition, a huge number of extrasynaptic cav1.3 channels can be observed in immature ihcs (zampini. 2014), which enable the cells to fire ca action potentials (kros. 1998 ; brandt. these action potentials evoke exocytosis in the pre - hearing stage (beutner and moser 2001 ; glowatzki and fuchs 2002 ; johnson. 2005) but show lower ca efficiency (beutner and moser 2001 ; brandt. 2003 ; johnson. 2005) and a supra - linear ca dependence (johnson. 2005). the pre - sensory ihc activity appears to drive bursting activity in the developing auditory system (glowatzki and fuchs 2002 ; tritsch. 2007, 2010 ; wong. 2013 ; clause. 2014). in this context, the regulation of presynaptic firing by paracrine and/or efferent synaptic control is being subject to intense research (glowatzki and fuchs 2000 ; tritsch. efferent innervation, moreover, seems to play an important role in the maturation process of ihcs (glowatzki and fuchs 2000 ; marcotti 2004 ; goutman. efferent fibers originate from the superior olivary complex and, before onset of hearing, form transient axosomatic contacts with ihcs (simmons. later, they largely retract from ihcs and rather form axodendritic contacts to the afferent terminals (pujol. the transient efferent inhibition is thought to counteract the ihc depolarization resulting from the resting mechanotransducer current (gloc and holt 2003 ; waguespack. 2009). upon genetically induced impairment of the efferent input, the linearization of ca dependent exocytosis is affected (johnson. 2007) and the maturation of ihc afferent synapses is also disturbed (johnson. 2013b). around the onset of hearing (at around p11 ; mikaelian and ruben 1965), when graded receptor potentials start governing transmitter release, extrasynaptic cav1.3 channels get pruned and spatial coupling of ca channels and vesicular release sites is tightened. ca efficiency of exocytosis and a near - linear ca dependence of rrp exocytosis when probed with changes in the number of open ca channels (wong. 2014). therefore, while the intrinsic ca dependence of exocytosis apparently does not change upon the onset of hearing, experimental data and biophysical modeling of exocytosis at mature and immature az topographies support the notion of a developmental switch from the more ca microdomain - like control of exocytosis by several ca channels per vesicle to a more ca nanodomain - like control of exocytosis (wong. 2014 ; fig. 1e, e). interestingly, in adult gerbils, the open probability of ca channels in ihcs increased due to a preference of the ca channel for the bursting mode (zampini. structurally, alongside ca channels, other presynaptic az components become reorganized such as the bassoon containing presynaptic density (fig. these alterations are accompanied by changes of the postsynaptic glutamate receptor fields that also develop to one continuous ring - like cluster (wong. moreover, ribbons increase in size and undergo striking changes of shape. at the ultrastructural level, their cross - sectional shape changes from predominantly round (fig. 1a) to a rather oval-, droplet- or wedge - like shape between p14 and p20 (wong. 2014 the two rootlets seem to merge to a continuous presynaptic density that contains the scaffolding protein bassoon, as revealed by immunogold labeling (wong. shortly after onset of hearing at p14, a large proportion of ribbons with two rootlets can still be found, whereas about a week later the morphological maturation appears to be completed (wong.. factors that participate in the maturation of ihc synapses, next to the efferent olivocochlear transmission (see above ; johnson. 2013a), are thyroid hormone (sendin. 2007) and myosin 6 (heidrych. 2009 ; roux. 2009). for both, a higher proportion of morphological immature ribbons have been observed in genetic deletion models. in conclusion, during development from pre - hearing to hearing, ihc ribbon synapses undergo major morphological and functional refinements, resulting in tighter spatial coupling between ca influx and exocytosis (wong. the number of ca channels, vesicular release sites and ribbon - associated vesicles seems to scale with the size and number of ribbons at the az (martinez - dunst. ; graydon. 2011 ; kantardzhieva. 2013 ; wong. 2013, 2014). strengthening of presynaptic transmitter release might therefore be accomplished by increasing ribbon or az size and/or ribbon numbers per az. moreover, finally, lateral olivocochlear efferent fibers might modulate postsynaptic excitability and thereby affect afferent synaptic strength. to establish which of these mechanisms contribute to determining and regulating synaptic strength of hair cell synapses interestingly, the size and shape of ribbons appear to be highly variable and dynamic. in fact, in photoreceptors, these parameters strongly correlate with activity in light (silent) or dark (active) conditions (spiwoks - becker. a diverse spectrum of ribbons has also been observed (bodian 1978 ; sobkowicz. the specific ultrastructural properties seem to depend on several factors : (1) the maturation / age (see section above), (2) position within the inner hair cell and maybe also (3) dynamic adaptation to activity. a pioneering study in cats was one of the first to identify the correlation between structural heterogeneity of ribbon synapses and functional characteristics of auditory nerve fibers (merchan - perez and liberman 1996). surprisingly, large azs with big and/or several ribbons, supposedly reflecting large presynaptic strength, seem to drive sgns with low spontaneous rate and high thresholds (see also scheme in fig. whereas this conundrum remains unsolved, the mechanisms of functional presynaptic heterogeneity are now beginning to be understood. evidence for such heterogeneity within individual ihcs was obtained using confocal imaging of presynaptic ca influx (frank. this study showed that presynaptic ca signals varied substantially in amplitude and voltage - dependence among the azs within individual ihcs. the amplitude of the ca signal scaled with ribbon size as approximated by simultaneous imaging of a fluorescently tagged ribeye - binding peptide (frank. 2009) and seemed to be greater at the neural side of the ihcs (meyer. linking such estimates to the functional and morphological properties of the postsynaptic neurons will be an important task for future studies. so far, correlative arguments based on coincidental changes in maximal strength of presynaptic ca influx and postsynaptic spiking during development and upon genetic disruption as well as modeling have been brought forward to argue that strong synapses drive sgns that have high spontaneous rates and low thresholds (wong. 2013). interestingly, an inverse correlation of pre- and postsynaptic parameters of synaptic strength has recently been reported for mouse ihcs : liberman. the authors favored the interpretation that the sgns inserting at the neural (modiolar) face of ihcs exhibit low spontaneous rates and high thresholds despite their corresponding large ihc azs, because they have a smaller complement of ampa receptors than those at the neural (pillar) side. this would agree with the conclusion of the classical study, which showed a neural abneural gradient of az size using electron microscopy for cat ihcs whereby large azs faced sgns with low spontaneous rates and high thresholds (merchan - perez and liberman 1996). in a laborious approach, the authors traced 11 functionally - characterized fibers to the ihcs using serial 3d reconstructions of ultrathin sections. in this way, it was possible to directly correlate morphological parameters such as ribbon length, fiber contact area, synaptic plaque area and synaptic vesicle numbers to the functional parameters determined prior to fiber labeling using single unit recordings. recently, such a gradient was also suggested for mouse ihcs and reported to be influenced by the lateral olivocochlear innervation (yin. the segregation of nerve fibers on neural and abneural sides was further observed in a study investigating the abundance of mitochondria in postsynaptic terminals. here, postsynaptic boutons facing the abneural side seem to harbor more mitochondria (francis. monitoring epscs from single afferent boutons, which is a suitable method to address synaptic function on the level of individual release sites (glowatzki and fuchs 2002), further showed differences among synapses. in these experiments, varying fractions of multiphasic epscs were observed and proposed to underlie the diverse firing properties of sgns (grant. a schematic of an organ of corti showing afferent and efferent innervations at ihcs. modified from meyer and moser 2010, curr opin otolaryngol head neck surg, reprinted with permission from 2010 wolters kluwer health. b mean and sd of f (gray) as a function of depolarizing potential (v m), obtained from spot - detection experiments at the center of the ca microdomain ; f was averaged over the last 15 ms of a 20-ms stimulus. f (mean gray) and i ca (mean black) show a similar voltage dependence (thin lines corresponding sds). b heterogeneous voltage dependence and ca channel number of synaptic ca channel clusters in ihcs. pronounced variability in the voltage dependence of activation, even within the same cell (dashed traces individual data curves from 3 ca microdomains in an ihc). modified from frank. c, c colorized spatial distribution of vesicles and cisterns around the ribbon in low- and high - spontaneous rate (sr) fibers. sections through a high - sr (c) and a low - sr (c) synapse containing the synaptic ribbon are shown with cisternal (maroon) and vesicular (green) profiles. d mean density (se) of docked vesicles, i.e., within 20 nm of the presynaptic density along the presynaptic membrane. d mean number (se) of cisterns within 20 nm of the presynaptic density versus distance along the presynaptic membrane is shown for all synapses. rectangle the area of significant differences between low- and high - sr synapses. se standard error ; sr spontaneous rate. 2013, j comp neurol, reprinted with permission from 2013 wiley periodicals) principle of functional heterogeneity in ihcs. a schematic of an organ of corti showing afferent and efferent innervations at ihcs. modified from meyer and moser 2010, curr opin otolaryngol head neck surg, reprinted with permission from 2010 wolters kluwer health. b mean and sd of f (gray) as a function of depolarizing potential (v m), obtained from spot - detection experiments at the center of the ca microdomain ; f was averaged over the last 15 ms of a 20-ms stimulus. f (mean gray) and i ca (mean black) show a similar voltage dependence (thin lines corresponding sds). b heterogeneous voltage dependence and ca channel number of synaptic ca channel clusters in ihcs. pronounced variability in the voltage dependence of activation, even within the same cell (dashed traces individual data curves from 3 ca microdomains in an ihc). modified from frank. c, c colorized spatial distribution of vesicles and cisterns around the ribbon in low- and high - spontaneous rate (sr) fibers. sections through a high - sr (c) and a low - sr (c) synapse containing the synaptic ribbon are shown with cisternal (maroon) and vesicular (green) profiles. d mean density (se) of docked vesicles, i.e., within 20 nm of the presynaptic density along the presynaptic membrane. d mean number (se) of cisterns within 20 nm of the presynaptic density versus distance along the presynaptic membrane is shown for all synapses. 2013, j comp neurol, reprinted with permission from 2013 wiley periodicals) further insights into the morphological heterogeneity require modern 3d reconstructions of larger volumes such as serial block face scanning electron microscopy (denk and horstmann 2004) or focused ion beam scanning electron microscopy (see, e.g., knott. 2008 using these methods, a detailed 3d view of single ihcs including afferent and efferent innervations would be possible, finally allowing correlation of parameters, such as presynaptic ribbon size and postsynaptic bouton diameter, as a function of position on the ihc for a large number of synapses, together with the functional assessment of pre- and postsynaptic properties in cochlear explants by electrophysiology (goutman and glowatzki 2007) and/or imaging of ihc ca and exocytosis (e.g., frank. tight coupling between exo- and endocytosis is a prerequisite for maintaining the enormous vesicle turnover rates at ribbon synapses clathrin - coated structures but also large cisterns without clathrin - coats, are observed close to synaptic ribbons (siegel and brownell 1986 ; sendin. 2007 ; frank. 2010 ; kantardzhieva. 2013 ; neef. 2014 ; revelo. 2014). 2013) set out to determine whether such cisterns participate in vesicle reformation and what differences can be observed in correlation to the functional properties of high and low spontaneous rate fibers (fig. an extensive quantitative analysis of ribbons, vesicles and cisterns from serial sections of cat ihc ribbon synapses suggested a fewer cisterns and more synaptic vesicles are found, which indeed points towards a contribution of cisterns to locally restricted vesicle formation. other studies used membrane capacitance measurements to provide an initial functional assessment of endocytic membrane retrieval at ihc azs (moser and beutner 2000 ; beutner and moser 2001 ; neef. moreover, ph - sensitive gfp (phluorin ; miesenbck. 1998) targeted to the intraluminal face of vesicle membranes by attachment to vesicular glutamate transporters (zhu. 2009) has become an important tool in studying exo- and endocytosis, not only from neurons but also ihcs (neef. additionally, a novel membrane tracer specifically tailored to use in the organ of corti has been devised and applied to investigate endocytosis (revelo. 2014), as the commonly used styryl dye fm1 - 43 penetrates stereociliar mechanotransduction channels and hence is of limited use to study endocytosis, in ihcs (gale., expression analysis and immunohistochemistry have revealed the presence of several important molecular players of endocytosis such as dynamins, amphiphysin, clathrin (neef. 2014) and adaptor protein 2 (ap-2) (duncker. 2013) in ihcs. a very recent dna microarray study investigating ihc and ohc transcriptomes might even give more insight into proteins involved in vesicle recycling (liu. currently, in ihcs, three distinct mechanisms are considered to mediate endocytosis : slow cme, fast bulk endocytosis and potentially kiss - and - run or cme is the main pathway of membrane retrieval for mild stimulation and proceeds at a constant rate ; it represents the linear component of endocytosis following exocytosis of the rrp (fig. this mechanism is not only inhibited by the clathrin - inhibitor pitstop-2 but also by disruption of dynamin 1 via pharmacological and genetic means (neef. 2014). in contrast, a different study reported inhibition of sustained exocytosis by the presumptive dynamin inhibitor dynasore but did not investigate endocytic membrane retrieval (duncker. finally, when exocytosis exceeds three to four rrp equivalents, ihcs additionally recruit a faster mode of membrane retrieval, which proceeds with an exponential time course within a few seconds. 3a) and, indeed, there is plenty of evidence for the invagination and fission of large stretches of plasma membrane in the vicinity of hair cell azs (lenzi. both mechanisms seem to engage in different phases of release : cme supports vesicle cycling during mild stimulation but bulk endocytosis finally occurs after prolonged stimulation, providing a mechanism that assures the balance between exo- and endocytosis in ihcs and thus, assures high release rates (neef. 3endocytosis in inner hair cells. a, a representative recordings in response to 20 ms (a) or 200 ms (a) depolarizations. after the c m increase upon 20 ms depolarization, the slope - corrected c m traces (middle) typically showed a linear decay (a). the 200-ms - long depolarization resulted in a combination of exponential and linear decay (a). b 3d reconstructions of resting (b), stimulated (b) and recovered ihcs (b, b). most organelles, including the tubular ones, are replaced by small vesicles during the recovery periods. insets magnified regions from the four different cell regions (cuticular plate, top, nuclear and basal regions). note the increased number of endosome - like organelles at the base of the cell after stimulation and during recovery. c mcling - labeled organs of corti were immunostained for vglut3 and otoferlin (first row), for vglut3 and syntaxin 6 (sx 6, second row), for otoferlin and syntaxin 16 (sx 16, third row) and finally for syntaxin 6 and syntaxin 16 (fourth row). the samples were cut into 20-nm sections and were imaged using an epifluorescence microscope. dashed white lines the plasma membrane of the ihcs. d graphic representation of pearson s correlation coefficients : otoferlin and syntaxin 6 (or syntaxin 16) correlate in the mcling - labeled organelles at the top and nuclear levels. organelles with a different molecular composition recycle membrane in different regions, taking up mcling. apical endocytosis takes up the membrane into round organelles, a sizeable proportion of which is similar to late endosomes (light blue). endocytosis in the top and nuclear regions reaches tubular organelles containing otoferlin and two endosome markers, syntaxin 16 and syntaxin 6. this suggests that these organelles participate in constitutive pathways, probably by maintaining membrane traffic between the plasma membrane and the trans - golgi. at the base of the cell, stimulation induces the formation of membrane infoldings and cisterns that are characterized by the presence of vglut3, rab3 and also otoferlin. a, a representative recordings in response to 20 ms (a) or 200 ms (a) depolarizations. after the c m increase upon 20 ms depolarization, the slope - corrected c m traces (middle) typically showed a linear decay (a). the 200-ms - long depolarization resulted in a combination of exponential and linear decay (a). b 3d reconstructions of resting (b), stimulated (b) and recovered ihcs (b, b). most organelles, including the tubular ones, are replaced by small vesicles during the recovery periods. insets magnified regions from the four different cell regions (cuticular plate, top, nuclear and basal regions). note the increased number of endosome - like organelles at the base of the cell after stimulation and during recovery. c mcling - labeled organs of corti were immunostained for vglut3 and otoferlin (first row), for vglut3 and syntaxin 6 (sx 6, second row), for otoferlin and syntaxin 16 (sx 16, third row) and finally for syntaxin 6 and syntaxin 16 (fourth row). the samples were cut into 20-nm sections and were imaged using an epifluorescence microscope. dashed white lines the plasma membrane of the ihcs. d graphic representation of pearson s correlation coefficients : otoferlin and syntaxin 6 (or syntaxin 16) correlate in the mcling - labeled organelles at the top and nuclear levels. organelles with a different molecular composition recycle membrane in different regions, taking up mcling. apical endocytosis takes up the membrane into round organelles, a sizeable proportion of which is similar to late endosomes (light blue). endocytosis in the top and nuclear regions reaches tubular organelles containing otoferlin and two endosome markers, syntaxin 16 and syntaxin 6. this suggests that these organelles participate in constitutive pathways, probably by maintaining membrane traffic between the plasma membrane and the trans - golgi. at the base of the cell, stimulation induces the formation of membrane infoldings and cisterns that are characterized by the presence of vglut3, rab3 and also otoferlin. (c e modified from revelo. 2014, reprinted with permission from 2014 revelo.) but where does the retrieval of synaptic vesicle membrane take place and where and how are synaptic vesicles regenerated following membrane retrieval in hair cells ? does synaptic endocytosis comply with the apicobasal compartmentalization of the hair cell ? at photoreceptor ribbons, a periactive zone, marked by the presence of endocytic proteins, was identified directly adjacent to the az area (in a range of 120250 nm from the ribbon) using high - resolution and electron microscopy (wahl. does this also apply to ihc ribbons ? at the resolution of confocal microscopy, a similar perisynaptic accumulation of endocytic proteins has, so far, not been found (neef. clearly, electron micrographs indicate that both bulk and cme endocytosis take place near the az (siegel and brownell 1986 ; lenzi. a radically different model of ihc endocytosis has been sketched based on life imaging of fm1 - 43 uptake into ihcs, whereby exocytosed membrane was postulated to move towards the ihc apex for endocytosis and recycling via the golgi apparatus (griesinger. recent elaborative studies using various styryl dyes and more suitable fluorescent membrane markers lead us to reconsider this hypothesis. first, it was corroborated that many styryl dyes including fm1 - 43 permeate into the cytosol of ihcs via the mechanotransducer channels (kamin. therefore, on the ultrastructural level, the photo - oxidation technique revealed a fuzzy dark diaminobenzidin (dab) smear inside the cytosol in addition to the precipitate in membrane - bound organelles likely resulting from internalized fm1 - 43. the stimulation - induced endocytic uptake of fm1 - 43 could still be followed by observing an electron - dense precipitate within vesicular structures, which allows the determination of the presence or absence as well as the localization of stained structures under resting (fig. b) conditions in serial 3d reconstructed ihcs (kamin. 2014). at rest, endosome - like organelles were detected in the apex of the ihcs, whereas larger tubulo - cisternal organelles dominated at the nuclear region. at the basal region, only a few labeled structures were present. stimulation massively increased the amount of basolateral membrane trafficking, reflected by the appearance of labeled small vesicles and endosome - like vacuoles ; however, no changes in the apical and nuclear regions could be observed (kamin. strikingly, the basolateral cisterns were replaced in the basal region by small, synaptic - like vesicles during a few minutes of recovery from stimulation, suggesting a highly efficient mechanism of vesicle regeneration from cisternal membranes internalized by bulk endocytosis. the combination of fm1 - 43 uptake and photo - oxidation therefore suggests that synaptic vesicle recycling takes place at the basal part, close to ribbons at least during synaptic activity (kamin. recently, a novel membrane tracer, named membrane - binding fluorophore - cysteine - lysine - palmitoyl group (mcling), which does not permeate the mechanotransducer channel, tightly binds biological membranes and can be fixed, has been developed (revelo. 2014). in combination with super - resolution light microscopy (i.e., sted), the spatial organization and pathways of endocytosis in ihcs could be further investigated. in order to improve the spatial resolution of sted microscopy in the axial direction, thin sections of ihcs were imaged after embedding the organs of corti in melamine, which maintains the fluorescence (revelo. the apical, nuclear and basal regions under conditions of rest, stimulation and recovery from stimulation, were investigated and the uptake of mcling monitored. in order to reveal the molecular identity of mcling - labeled structures and thereby identify the respective endocytic pathways, samples were co - stained with different protein markers for the endoplasmic reticulum (er) and golgi as well as synaptic vesicle endo- and exocytosis. in these experiments, a strong correlation for basolateral mcling localization with vglut3, rab3 and otoferlin immunofluorescence was found. otoferlin as well as syntaxin 16, a late endosomal marker, colocalized with apical and nuclear mcling (fig. moreover, the lysosomal - associated membrane protein 1 (lamp1) colocalized with mcling in the apical region of the ihc. stimulation led to a selective uptake of mcling at the base of the ihc, corroborating the notion of local recycling of synaptic vesicles that was postulated based on electron microscopy and photo - oxidation (kamin. the local recycling hypothesis was further supported by the finding that exogenous vglut1-phluorin fluorescence not only transiently appeared at ribbon - type azs but remained there for tens of seconds after stimulation (neef. finally, the mcling experiments revealed large membranous organelles near synapses, which were replaced by small organelles a few minutes after stimulation, thereby providing direct evidence of bulk endocytosis and vesicle regeneration from the internalized plasma membrane. the association of otoferlin with all three putative membrane recycling pathways suggests a more general role of this protein in endocytosis. otoferlin has recently been assigned a role in vesicle endocytosis due to its interaction with ap-2. using a high - resolution liquid chromatography coupled with a mass spectrometry approach, multiple subunits of ap-2 were identified as interaction partners of otoferlin in the mammalian cochlea and the proposed interactions were biochemically confirmed by co - immunoprecipitation (duncker. 2013). ap-2 plays a role in clathrin - mediated endocytosis via binding to clathrin - coated vesicles budding from the plasma membrane (keyel. 2010) and has been shown to be expressed in ihcs (duncker. future work is required to clarify the role of ap-2 in hair cell endocytosis and the relevance of its interaction with otoferlin. recently, major progress has been made towards dissecting the molecular anatomy and physiology of hair cell ribbon synapses. this includes powerful single synapse techniques such as (1) patch - clamp of postsynaptic afferent terminals of sgns, (2) high resolution -functional imaging of presynaptic ihc ca dynamics and membrane turnover, as well as (3) super - resolution light microscopy and electron tomography following high - pressure freezing. however, in order to investigate the release mechanisms of ihcs and firmly correlate structure and function, the development of new functional and morphological approaches is required. functional and morphological analysis of single synapses will be necessary and some questions require reading out both pre- and postsynaptic properties at the same time. the commonly used k stimulation of cochlear tissue likely mimics strong physiological steady - state stimulation. but this stimulation does not provide the temporal resolution to allow the observation of the release kinetics at ihc ribbon synapses. especially, knowledge about short - term plastic changes is lacking, since it is not possible to apply very short stimuli (i.e., millisecond range) and investigate the cells during and at defined times after stimulation. therefore, approaches are needed that meet two requirements : (1) a precise stimulation protocol combined with (2) rapid immobilization of the sample, e.g., by using high - pressure freezing. one emerging tool that promises to fulfill these requirements is the combination of optogenetic stimulation with high - pressure freezing. this could involve the expression of a light - sensitive ion channel such as channelrhodopsin-2 (chr-2) from the green algae chlamydomonas reinhardii (nagel. 2003) in hair cells and stimulation would ideally be performed within a chamber that should be mounted in a freezing machine in order to minimize the time delay before freezing. recently, synaptic recovery of motoneurons from c. elegans was analyzed using optogenetic stimulation in combination with high - pressure freezing (kittelmann. moreover, after a single light stimulus, docked vesicles fused along a broad az on c. elegans motoneurons expressing chr-2. these vesicles were replenished with a time constant of about 2 s. further, endocytosis occurred within 50 ms adjacent to the dense projection and after 1 s adjacent to adherens junctions (watanabe. moreover, a study on optically stimulated cultured hippocampal neurons revealed an ultrafast endocytosis mechanism at central synapses (watanabe. these initial experiments indicate that optogenetics, in combination with high - pressure freezing (flash and freeze ; watanabe. 2013b) and subsequent electron tomography, might provide sufficient resolution to study the ultrastructure of spatiotemporally defined functional states and thus provide a completely new view on the release mechanism of ihc ribbon synapses.	in the mammalian cochlea, sound is encoded at synapses between inner hair cells (ihcs) and type i spiral ganglion neurons (sgns). each sgn receives input from a single ihc ribbon - type active zone (az) and yet sgns indefatigably spike up to hundreds of hz to encode acoustic stimuli with submillisecond precision. accumulating evidence indicates a highly specialized molecular composition and structure of the presynapse, adapted to suit these high functional demands. however, we are only beginning to understand key features such as stimulus secretion coupling, exocytosis mechanisms, exo endocytosis coupling, modes of endocytosis and vesicle reformation, as well as replenishment of the readily releasable pool. relating structure and function has become an important avenue in addressing these points and has been applied to normal and genetically manipulated hair cell synapses. here, we review some of the exciting new insights gained from recent studies of the molecular anatomy and physiology of ihc ribbon synapses.
you need : a vu - ams5fs ambulatory recording device (including an infrared interface cable that either connects to the rs232 serial port of a pc or to a usb port).7 electrodes (we used conmed 1690 - 003).2 charged aa-batteries.an empty compactflash memory card (the vu - ams5fs has been extensively tested with the 1 gb 80x cf card from transcend (ts1gcf80), but other cf cards should work too).a laptop or pc with flash card reader and the data analysis management software (dams) suite installed.a stopwatch.music player with children 's stories and headphones and a small self - inflatable air mattress are optional. a vu - ams5fs ambulatory recording device (including an infrared interface cable that either connects to the rs232 serial port of a pc or to a usb port). 7 electrodes (we used conmed 1690 - 003). 2 charged aa - batteries. an empty compactflash memory card (the vu - ams5fs has been extensively tested with the 1 gb 80x cf card from transcend (ts1gcf80), but other cf cards should work too). a laptop or pc with flash card reader and the data analysis management software (dams) suite installed. music player with children 's stories and headphones and a small self - inflatable air mattress are optional. check the time and date settings on the laptop / pc, since these will be recorded as metadata on your files. put the empty memory card and full batteries in the vu - ams device (successful placement is signalled by a triple beep). when the device is on standby, the green light will flash twice every ten seconds. now connect the device to the laptop using the provided cable and start up the dams program. initiate communication with the device (select the tab ' device ' and choose the appropriate connection mode, infrared cable or bluetooth). where the electrodes will be placed, clean the skin with alcohol - wipes, and place the seven electrodes on the chest and back (figure 1). then attach the lead wires following the color scheme, and connect them to the device. check the battery type and battery voltage indication (this should be about 3.4 v for alkaline and about 2.4 v for rechargeable nimh batteries). fill out the identification field. measure the distance between the two chest electrodes in millimeters, and fill this out in the field ' icg - v distance '. then click ' send settings ' to send the current settings / id to the device. now, the ' online ' option of the program should be used to display the ecg, z (this is the respiration) and dz / dt (this is the icg). the z signal reflects the base impedance across the thorax, which after appropriate filtering can be used to extract the respiration signal with high fidelity. the dz / dt signal is the z differentiated over time and reflects rapid changes in z linked to the ejection of blood from the ventricle into the aorta.. the r - wave should be upward and it should be the peak with the largest (absolute) amplitude in either direction.the z should be within -0.5 and + 0.5 most of the time and dz / dt between -1 and + 1 /sec.z0 should always stay within an 8 to 20 range. this variation reflects the fact that the thorax impedance signal depends on the distance between the measuring electrodes which is a function of the child 's height, and the ' wetness ' of the thorax column enclosed by the measuring electrodes, differences in body composition (e.g. bmi) can affect the amplitude of the dz / dt signal (fat mass containing less water than muscle). individual differences in absolute z0 amplitude are also reflected in the z signal but this does not affect the determination of systolic time intervals, which are amplitude-independent.the z signal should reflect deep breathing of the subject clearly (instruct the child to take a slow deep breath and exhale slowly).in the icg the typical upward waveform reflecting the cardiac ejection phase should be clearly detectable. if these criteria are not met, re - clean the skin and re - attach the electrodes until satisfactory signals are obtained. a clear qrst - complex should be detectable in the ecg. the r - wave should be upward and it should be the peak with the largest (absolute) amplitude in either direction. the z should be within -0.5 and + 0.5 most of the time and dz / dt between -1 and + 1 /sec. this variation reflects the fact that the thorax impedance signal depends on the distance between the measuring electrodes which is a function of the child 's height, and the ' wetness ' of the thorax column enclosed by the measuring electrodes, differences in body composition (e.g. bmi) can affect the amplitude of the dz / dt signal (fat mass containing less water than muscle). individual differences in absolute z0 amplitude are also reflected in the z signal but this does not affect the determination of systolic time intervals, which are amplitude - independent. the z signal should reflect deep breathing of the subject clearly (instruct the child to take a slow deep breath and exhale slowly). in the icg the typical upward waveform reflecting the cardiac ejection phase should be clearly detectable. if these criteria are not met, re - clean the skin and re - attach the electrodes until satisfactory signals are obtained. when good signals are attained, start data recording by pressing the ' start ' button. you will hear a beep acknowledging the start of the recording and the green light will start flashing once every three seconds. the registration has now started. once the registration has started, ask the child to lie down for the first experimental condition. when the child has been in the supine position (without head - up tilt) for two minutes, you shortly (< 2 s) press the small black button on top of the device. pressing this button marks a special event, and will later on help you identify the start of this condition in your data. now have the child sit up and repeat the procedure for this second condition. press the children are instructed to rest quietly during these conditions. to stop the measurement, press and the light will flash every 10 sec to indicate it has stopped and is in ' stand by ' mode. once the device has stopped, you may disconnect the lead wire plug from the connector and the lead wires from the electrodes. remove the batteries and flash card form the vu - ams device and place the flash card in the reader unit. move the acquired files to a designated directory (typically the name of the directory will be identical to the subject identifier used in the identification field). upon opening the data with vu - dams program, the data will be automatically converted from raw data format (extension.5fs) to a new format (extension.amsdata). this is the data file that vu - dams will be using in the ensuing steps. an automated algorithm will detect all r - peaks in the ecg signal and select (if present) periods with very low ecg quality for removal. in the upper left hand corner the number of blue (correct), yellow (medium suspicious) or red (highly suspicious) is indicated. by pressing '. ' (dot) the cursor is moved to the next suspicious r - peak and the user can delete or add markers for r - waves by hand. the main aim is to obtain a mean value for the heart rate, the preejection period (pep) and measures of respiratory sinus arrhythmia (rsa, hf, rmssd) across the experimental conditions used. therefore proceed by indicating which periods in the raw data correspond to these conditions. this process two panels show the heart rate signal and movement signal respectively, as well as the actual time of the recording. place the mouse cursor in the top bar where it says " click and drag to add labels " at around the start time of your first condition and drag the mouse to the end time of that condition. these times are either obtained from a written record of start and stop times (that you noted down during data collection) or you can use the start and stop markers obtained from pressing the button at the start and end of each condition, which are the vertical lines running across the hr and movement graphs. each label vu - dams needs to be made aware of the experimental design by a so - called label configuration file (label.cfg). this is an ascii file that can be opened with most text editors and, for example, looks like this : # exp_condition 10 lying down 11 sitting up by placing the label.cfg file in the directory of the.amsdata files, it will be automatically loaded by the vu - dams program. once a label has been made, a pop up screen will appear with the categories / values listed in the label.cfg file. select ' lying down ' for the first label and ' sitting up ' for the second label. after labeling, select the ' impedance scoring ' tab to score the pep in the impedance cardiogram. for each of the conditions an ensemble averaged dz / dt waveform place the four vertical cursors in the correct positions : ecg q - wave onset (start of electrical activity), icg b - point (start of the ejection phase), icg dz / dt - min (maximal ejection speed), and icg x - point (aortic valve closure - end of ejection phase). next, select the ' respiration scoring ' tab to score the peak - valley rsa using the respiratory and ecg signals. automated breath - to - breath scoring of the respiratory interval and the shortest interbeat interval during inspiration and the longest interbeat interval during expiration can now be inspected. typically the automated detection algorithm should not classify more than 15% of the breaths as deviant - otherwise inspect the respiration signal and tune the parameters of the detection algorithm as needed. guidelines for visual inspection of the ecg, icg and respiration signals and interactive pep and rsa scoring can be found on the vu - ams website, www.vu-ams.nl. each row represents the average value of a series of physiological parameters (heart rate, pep, rsa, rr) for each labeled time period. the last column indicates the values of all categories used during labeling (here only a single category ' experimental condition ' with two values, ' sitting up ' and ' lying down '). spectral powers of the interbeat interval times series are given only for labels with a minimum length of 4 min (otherwise the missing code is displayed). the spreadsheet in this display can be exported to ascii or excel for further statistical analyses. you need : a vu - ams5fs ambulatory recording device (including an infrared interface cable that either connects to the rs232 serial port of a pc or to a usb port).7 electrodes (we used conmed 1690 - 003).2 charged aa-batteries.an empty compactflash memory card (the vu - ams5fs has been extensively tested with the 1 gb 80x cf card from transcend (ts1gcf80), but other cf cards should work too).a laptop or pc with flash card reader and the data analysis management software (dams) suite installed.a stopwatch.music player with children 's stories and headphones and a small self - inflatable air mattress are optional. a vu - ams5fs ambulatory recording device (including an infrared interface cable that either connects to the rs232 serial port of a pc or to a usb port). 7 electrodes (we used conmed 1690 - 003). 2 charged aa - batteries. an empty compactflash memory card (the vu - ams5fs has been extensively tested with the 1 gb 80x cf card from transcend (ts1gcf80), but other cf cards should work too). a laptop or pc with flash card reader and the data analysis management software (dams) suite installed. music player with children 's stories and headphones and a small self - inflatable air mattress are optional. check the time and date settings on the laptop / pc, since these will be recorded as metadata on your files. put the empty memory card and full batteries in the vu - ams device (successful placement is signalled by a triple beep). when the device is on standby, the green light will flash twice every ten seconds. now connect the device to the laptop using the provided cable and start up the dams program. initiate communication with the device (select the tab ' device ' and choose the appropriate connection mode, infrared cable or bluetooth). where the electrodes will be placed, clean the skin with alcohol - wipes, and place the seven electrodes on the chest and back (figure 1). then attach the lead wires following the color scheme, and connect them to the device. check the battery type and battery voltage indication (this should be about 3.4 v for alkaline and about 2.4 v for rechargeable nimh batteries). fill out the identification field. measure the distance between the two chest electrodes in millimeters, and fill this out in the field ' icg - v distance '. then click ' send settings ' to send the current settings / id to the device. now, the ' online ' option of the program should be used to display the ecg, z (this is the respiration) and dz / dt (this is the icg). the z signal reflects the base impedance across the thorax, which after appropriate filtering can be used to extract the respiration signal with high fidelity. the dz / dt signal is the z differentiated over time and reflects rapid changes in z linked to the ejection of blood from the ventricle into the aorta.. the r - wave should be upward and it should be the peak with the largest (absolute) amplitude in either direction.the z should be within -0.5 and + 0.5 most of the time and dz / dt between -1 and + 1 /sec.z0 should always stay within an 8 to 20 range. this variation reflects the fact that the thorax impedance signal depends on the distance between the measuring electrodes which is a function of the child 's height, and the ' wetness ' of the thorax column enclosed by the measuring electrodes, differences in body composition (e.g. bmi) can affect the amplitude of the dz / dt signal (fat mass containing less water than muscle). individual differences in absolute z0 amplitude are also reflected in the z signal but this does not affect the determination of systolic time intervals, which are amplitude-independent.the z signal should reflect deep breathing of the subject clearly (instruct the child to take a slow deep breath and exhale slowly).in the icg the typical upward waveform reflecting the cardiac ejection phase should be clearly detectable. if these criteria are not met, re - clean the skin and re - attach the electrodes until satisfactory signals are obtained. a clear qrst - complex should be detectable in the ecg. the r - wave should be upward and it should be the peak with the largest (absolute) amplitude in either direction. the z should be within -0.5 and + 0.5 most of the time and dz / dt between -1 and + 1 /sec. this variation reflects the fact that the thorax impedance signal depends on the distance between the measuring electrodes which is a function of the child 's height, and the ' wetness ' of the thorax column enclosed by the measuring electrodes, differences in body composition (e.g. bmi) can affect the amplitude of the dz / dt signal (fat mass containing less water than muscle). individual differences in absolute z0 amplitude are also reflected in the z signal but this does not affect the determination of systolic time intervals, which are amplitude - independent. the z signal should reflect deep breathing of the subject clearly (instruct the child to take a slow deep breath and exhale slowly). in the icg the typical upward waveform reflecting the cardiac ejection phase should be clearly detectable. if these criteria are not met, re - clean the skin and re - attach the electrodes until satisfactory signals are obtained. when good signals are attained, start data recording by pressing the ' start ' button. you will hear a beep acknowledging the start of the recording and the green light will start flashing once every three seconds. the registration has now started. once the registration has started, ask the child to lie down for the first experimental condition. when the child has been in the supine position (without head - up tilt) for two minutes, you shortly (< 2 s) press the small black button on top of the device. pressing this button marks a special event, and will later on help you identify the start of this condition in your data. after four minutes, press the event button again. now have the child sit up and repeat the procedure for this second condition. press the children are instructed to rest quietly during these conditions. to stop the measurement, press and the light will flash every 10 sec to indicate it has stopped and is in ' stand by ' mode. once the device has stopped, you may disconnect the lead wire plug from the connector and the lead wires from the electrodes. remove the batteries and flash card form the vu - ams device and place the flash card in the reader unit. move the acquired files to a designated directory (typically the name of the directory will be identical to the subject identifier used in the identification field). upon opening the data with vu - dams program, the data will be automatically converted from raw data format (extension.5fs) to a new format (extension.amsdata). this is the data file that vu - dams will be using in the ensuing steps. an automated algorithm will detect all r - peaks in the ecg signal and select (if present) periods with very low ecg quality for removal. in the upper left hand corner the number of blue (correct), yellow (medium suspicious) or red (highly suspicious) is indicated. by pressing '. ' (dot) the cursor is moved to the next suspicious r - peak and the user can delete or add markers for r - waves by hand. the main aim is to obtain a mean value for the heart rate, the preejection period (pep) and measures of respiratory sinus arrhythmia (rsa, hf, rmssd) across the experimental conditions used. therefore proceed by indicating which periods in the raw data correspond to these conditions. this process two panels show the heart rate signal and movement signal respectively, as well as the actual time of the recording. place the mouse cursor in the top bar where it says " click and drag to add labels " at around the start time of your first condition and drag the mouse to the end time of that condition. these times are either obtained from a written record of start and stop times (that you noted down during data collection) or you can use the start and stop markers obtained from pressing the button at the start and end of each condition, which are the vertical lines running across the hr and movement graphs. each label can be given a (unique) identifier to signal a particular condition. in our case vu - dams needs to be made aware of the experimental design by a so - called label configuration file (label.cfg). this is an ascii file that can be opened with most text editors and, for example, looks like this : # exp_condition 10 lying down 11 sitting up by placing the label.cfg file in the directory of the.amsdata files, it will be automatically loaded by the vu - dams program. once a label has been made, a pop up screen will appear with the categories / values listed in the label.cfg file. select ' lying down ' for the first label and ' sitting up ' for the second label. after labeling, select the ' impedance scoring ' tab to score the pep in the impedance cardiogram. for each of the conditions an ensemble averaged dz / dt waveform place the four vertical cursors in the correct positions : ecg q - wave onset (start of electrical activity), icg b - point (start of the ejection phase), icg dz / dt - min (maximal ejection speed), and icg x - point (aortic valve closure - end of ejection phase). next, select the ' respiration scoring ' tab to score the peak - valley rsa using the respiratory and ecg signals. automated breath - to - breath scoring of the respiratory interval and the shortest interbeat interval during inspiration and the longest interbeat interval during expiration can now be inspected. typically the automated detection algorithm should not classify more than 15% of the breaths as deviant - otherwise inspect the respiration signal and tune the parameters of the detection algorithm as needed. guidelines for visual inspection of the ecg, icg and respiration signals and interactive pep and rsa scoring can be found on the vu - ams website, www.vu-ams.nl. each row represents the average value of a series of physiological parameters (heart rate, pep, rsa, rr) for each labeled time period. the last column indicates the values of all categories used during labeling (here only a single category ' experimental condition ' with two values, ' sitting up ' and ' lying down '). spectral powers of the interbeat interval times series are given only for labels with a minimum length of 4 min (otherwise the missing code is displayed). the spreadsheet in this display can be exported to ascii or excel for further statistical analyses. in the amsterdam born children and their development study, a dutch prospective, longitudinal birth cohort, the measurement protocol was started in 3,097 children. approval was obtained from the academic medical center medical ethical committee, the vu university medical center medical ethical committee and the registration committee of amsterdam. all participating mothers gave written informed consent for themselves and their children. as the monitors are lightweight and unobtrusive, the children tolerated these measurements very well. we do not have data on the refusal rate, but experience taught us that only a few children resisted the placement of the electrodes and thereby obstructed further assessment. of the 3,097 registrations, 0.7% were lost due to either equipment failure or misplacement of files. out of the 3,074 registrations left, 98.7% were of children who completed the entire protocol (n = 3,056). within each of the labelled time periods (we originally labelled four time periods, but later summarized these to two), we encountered unclear icg signals, meaning pep could not be determined. this led to a loss of 1.5% in the first out of four labelled periods, 2.4% in the second, 2.8% in the third and 4.1% in the fourth period. complete data on pep in all time periods was available in 2,797 cases (91.5%, thus 8.5% loss due to unclear icg signals). complete data on heart rate (hr), pre - ejection period (pep) and respiratory sinus arrhythmia (rsa), as well as sex and age, was available from 2,761 children ; in this final step, 1.3% data loss occurred, due to unknown reasons. the mean age of the children was 5.7 years (sd 0.5 ; interquartile range 5.0:6.5), and their bmi was 15.5 kg / m (sd 1.5 ; interquartile range 13.9:17.2). the mean values of the major outcome variables hr, pep, and rsa are given in table 1 and graphically depicted in figure 3, separately for boys and girls. hr (both lying down and sitting up) and pep (only sitting up) were higher in girls than in boys (both postures). the higher values for hr in girls are likely to be caused by the lower vagal (parasympathetic) cardiac control. their sympathetic cardiac control was not different or even lower than that in boys (sitting up). in both sexes, hr was higher when sitting up compared to lying down, whereas rsa was lower when sitting up. this effect was also as expected, and it reflects the outcome of opposite processes : lower sympathetic activity (lengthens pep) while lying down with increased preload (shortens pep). table 1. cardiac autonomic nervous system measures in boys and girls, by posture on posture difference. p < 0.05 for one sample t - test on sex difference. p < 0.05 for paired samples t - test. the seven electrodes should be placed on the participant 's chest and back. the first ecg electrode (v-) is placed slightly below the right collar bone 4 cm to the right of the sternum. the second ecg electrode (v+) is placed at the apex of the heart over the ninth rib on the left lateral margin of the chest approximately at the level of the processus xiphodius. the third ecg electrode (gnd) is a ground electrode and is placed on the right side, between the lower two ribs at the right abdomen. the first icg measuring electrode (v1) is placed at the top end of the sternum, between the tips of the collar bones. the second icg measuring electrode is placed at the xiphoid complex of the sternum, where the ribs meet. the two current electrodes are placed on the back : i- on the spine over the cervical vertebra c4, at least 3 cm (1 in) above the icg measuring elec - trode v-, and i+ between thoracic vertebrae t8 and t9 on the spine, at least 3 cm (1 ") below the icg measuring elec - trode v2. the icg electrode placement takes into account that the largest part of the left ventricle driven change in thorax impedance is captured by the column between the suprasternal notch and the processus xiphoideus. the typical settings used for a recording as displayed by the dams software after connecting to the vu - ams5fs device. figure 3. cardiac autonomic nervous system measures in boys and girls, by posture. we used an ambulatory recording device to measure cardiac autonomic control in 3097 children, aged between 5 and 7 years. seven electrodes sufficed to measure the ecg and icg from which the heart rate, heart rate variability and the systolic time intervals were extracted. heart rate variability in the respiratory frequency band (rsa) is a valid indicator of cardiac parasympathetic activity. the systolic time interval, pep, by reflecting cardiac contractility, is a valid indicator of cardiac sympathetic activity. the mean values obtained for hr, pep and rsa, the effects of posture changes and the differences between boys and girls were in line with what would be expected from the literature. as ambulatory monitoring removed the necessity of assessment in a laboratory our recordings could be done in various locations (e.g. school, sports center, science museum) without differences in signal recording quality. however, it is of crucial importance to standardize within or between subject comparisons for posture and physical load, as afterload and preload effects can co - determine the pep without any changes in cardiac sympathetic drive. we conclude that ambulatory recording of the ecg and icg in large samples of children is highly feasible and propose the current standardized study design as a useful template for future assessments of cardiac autonomic control in children.	the autonomic nervous system (ans) controls mainly automatic bodily functions that are engaged in homeostasis, like heart rate, digestion, respiratory rate, salivation, perspiration and renal function. the ans has two main branches : the sympathetic nervous system, preparing the human body for action in times of danger and stress, and the parasympathetic nervous system, which regulates the resting state of the body.ans activity can be measured invasively, for instance by radiotracer techniques or microelectrode recording from superficial nerves, or it can be measured non - invasively by using changes in an organ 's response as a proxy for changes in ans activity, for instance of the sweat glands or the heart. invasive measurements have the highest validity but are very poorly feasible in large scale samples where non - invasive measures are the preferred approach. autonomic effects on the heart can be reliably quantified by the recording of the electrocardiogram (ecg) in combination with the impedance cardiogram (icg), which reflects the changes in thorax impedance in response to respiration and the ejection of blood from the ventricle into the aorta. from the respiration and ecg signals, respiratory sinus arrhythmia can be extracted as a measure of cardiac parasympathetic control. from the ecg and the left ventricular ejection signals, the preejection period can be extracted as a measure of cardiac sympathetic control. ecg and icg recording is mostly done in laboratory settings. however, having the subjects report to a laboratory greatly reduces ecological validity, is not always doable in large scale epidemiological studies, and can be intimidating for young children. an ambulatory device for ecg and icg simultaneously resolves these three problems.here, we present a study design for a minimally invasive and rapid assessment of cardiac autonomic control in children, using a validated ambulatory device 1 - 5, the vu university ambulatory monitoring system (vu - ams, amsterdam, the netherlands, www.vu-ams.nl).
one - third of men aged 3060 and one - fifth of women over 40 years of age are overweight or obese (body mass index (bmi) 25 kg / m and over). although the proportions of overweight and obese people are less than those in europe and the united states, cardiovascular disease risk in the japanese population has greatly increased, even in those with bmi less than 30 kg / m [3, 4 ]. losing weight has favorable effects for overweight / obese people ; in particular, sustaining a loss of as little as 510% of initial body weight is associated with significant improvement of obesity - related comorbidities [5, 6 ]. therefore, to date, a large number of weight loss programs that incorporate a combination of dietary, physical activity, and behavior modification approaches have been conducted. however, the difficulty of long - term weight maintenance has remained a serious problem. according to the nih technology assessment conference panel, one - third to two - thirds of the lost weight was regained within one year. moreover, meta - analysis regarding the effect of weight loss programs reported that, as a whole, only 23% of the lost weight was maintained after 4 - 5 years of followup. therefore previously, comparisons of weight gainers and weight maintainers were primarily conducted ; however, inconsistent criteria of weight regain made it difficult to make comparisons across these studies. on the other hand, recently, with evidence that at least 5% loss of initial body weight is enough to improve obesity - related comorbidities, focus has been placed on maintaining this criterion for one year or longer, rather than preventing any regain. although comparison between successful weight maintainers according to this definition and unsuccessful weight maintainers has been conducted, there have still been only a few studies focused on this topic. teixeira. compared pretreatment variables between successful participants (who maintained 5% loss or more of their initial weight after a weight loss program) and unsuccessful participants and concluded that pretreatment psychological and behavioral variables such as dieting history, outcome evaluations, exercise self - efficacy, and quality of life were the predictors of successful weight maintenance. however, in this study, there is a possibility that other psychological and behavioral factors during the intervention or follow - up period affected weight maintenance. wing and hill found specific behaviors of successful weight loss maintainers among the subjects of the national weight control registry (nwcr). they shared common behaviors including eating a diet low in fat, frequent self - monitoring of body weight and food intake, and high levels of regular physical activity. compared successful weight maintainers and unsuccessful weight maintainers and reported that more successful weight maintainers ate more fruits / vegetables, did more exercise, kept records, made plans for exercise, and felt fewer barriers than unsuccessful weight maintainers. however, participants ' weight was self - reported, response rate of the survey was only 47% and weight maintenance for less than one year was included in the analysis. in addition, as far as we know, there have been no studies focused on this topic in japan. saku control obesity program (scop) is a behavioral approach - based weight loss program developed in japan. in this study, we followed the participants for one year and conducted a questionnaire survey about weight control behaviors, stress, obstacles, and support from people during the follow - up period and their confidence in continuing weight control behaviors in the future (self - efficacy). we also assessed stress, obstacles, support, self - efficacy, changes in energy intake, the number of steps walked per day, and eating behavior during the weight loss program. the participants were divided into either successful weight maintainers or unsuccessful weight maintainers and the factors that are important for successful weight maintenance were examined. subjects were the participants of a one - year weight loss program (saku control obesity program (scop)) held in nagano prefecture in japan in 2007. recruitment was conducted for people aged 40 to 64 and with a body mass index of 28.4 kg / m or more. exclusion criteria were psychiatric conditions or physical conditions that would preclude full participation in the study. a total of 116 (57 men and 59 women) met the study criteria and participated in the program. written information including the purpose of the study, assurance of refusal, and security of personal information was provided to each participant and written informed consent was obtained from all participants. the study protocol was approved by the ethics committee of the national institute of health and nutrition. the program and physical measurement were conducted at a dock center in nagano prefecture. in the program, the participants received individual counseling sessions about energy restriction and group sessions for exercise at the baseline and at 1, 3, 6, and 9 months. in the individual counseling, the participants found lifestyle habits (diet, dietary habit and physical activities) that needed improvement and set objectives to modify them with the support of trained registered dietitians and exercise instructors. in addition, they were instructed to self - monitor weight daily, diet and implementation of the plans using a self - monitoring sheet. the months between these five face - to - face counseling sessions (i.e., at 2, 4, 5, 7, 8, 10, and 11 months), participants reported their progress for the previous month and their new plans for the following month by mailing the records to the dietitians. after the program, the participants significantly lost weight from 81.0 13.5 kg to 75.6 12.2 kg (p < 0.001 by paired t - test, data not shown). height was measured with shoes off, and body weight was measured with light clothes in the fasting state in the morning. body weight was measured by bioelectrical impedance analysis (tb-220, tanita co., japan). bmi was calculated from the body weight (kg) divided by the height (m) of each subject. the participants answered a diet history questionnaire (dhq) [1416 ] and a questionnaire about eating behavior at the baseline and at the end of the program. energy intake was calculated using an ad hoc computer program for dhq, which was based on the food composition table in japan. the questionnaire about eating behavior was made by the japan society for the study of obesity. this included 51 items based on the statements given by the obese participants in a clinical survey. a four - point likert scale (strongly disagree / disagree / agree / strongly agree) was used in the questionnaire. based on these items, scores were calculated for the following eight eating behavior categories : (1) irregularity of eating, (2) perception gap about the feelings of fullness and hunger, (3) preference for unhealthy foods, (4) eating on unexpected occasions, (5) perception gap about constitution and weight, (6) eating quickly, (7) motivation for eating, and (8) total score. daily step counts were assessed using a uniaxial accelerometer (suzuken co., japan). each participant wore a uniaxial accelerometer on his or her belt from the time of waking to going to bed for two weeks before the baseline assessment and the assessment at the end of the program. participants were unable to view the data so that they would not consequently alter their normal routines of physical activity. we used the mean step counts for two weeks in the analysis. only at the end of the program, the participants answered a questionnaire about stress, obstacles, and support during weight loss program and confidence to continue weight control behaviors (practicing dietary objectives, increasing physical activity, and self - monitoring for their weight) (see table 3). they visited the dock center again at the end of the followup and physical measurement (height and weight), assessment of daily step counts for two weeks, and a questionnaire survey were conducted. in the questionnaire, the participants were asked whether or not they had any weight control strategies that they had learned in the program (setting objectives for diet and physical activity and practice, and self - monitoring for such practice, weight, and diet), whether or not they experienced any stress or obstacles, and also whether they had any support from people, and finally if they had confidence to continue the weight control behaviors in the future (see table 4). they answered all the questions with yes or no. a question on the frequency of weight measuring (times / week) was also included in the questionnaire. because among the 111 subjects who completed the program, 21 subjects did not attend follow - up physical measurement, analyses were based on 90 participants (44 men and 46 women). using the institute of medicine (iom) definition, successful weight maintainers (swm) were defined as those who maintained a weight loss of 5% or more of their initial weight at one year 's follow - up assessment and the others were defined as unsuccessful weight maintainers (uswms). the percentage of men was compared between swm and uswm by chi - square test. age, height, weight, and bmi at the baseline and weight and bmi at the end of the program and followup were compared by t - test. mean energy intake, the number of steps walked per day, and the scores for eating behavior at the end of the program between swm and uswm were compared using analysis of covariance (ancova) adjusting for age, sex, and baseline values. stress, obstacles, and support during weight loss program and confidence to continue weight control behaviors in the future between swm and uswm were compared by logistic regression analysis both without any adjustment and after adjusting for age, sex, and baseline weight. weight control activities, stress, obstacles, and support during the follow - up period and confidence in the future between swm and uswm were also compared by logistic regression analysis both without any adjustment and after adjusting for age, sex, and baseline weight. frequency of measuring weight was compared by ancova adjusting for age, sex, and baseline weight. the number of steps walked per day at the end of the followup between swm and uswm was compared using ancova adjusting for age, sex, and the value at the end of the program (or value at the baseline). all statistical analyses were carried out using spss for windows (version 16.0 ; spss inc., among the 90 participants, 36 participants were classified into successful weight maintainers (swms) and 54 were classified into unsuccessful weight maintainers (uswms). baseline characteristics and changes in weight and bmi in each swm and uswm group are shown in table 1. there were no significant differences in the proportion of men, baseline age, height, weight, and bmi between the groups. at the end of the program and followup, bmi values in the swm group were significantly lower than those in the uswm group. on average, the swm group had lost 12.0% from the baseline weight at the end of the program and still maintained 9.9% loss from the baseline at the end of the followup. on the other hand, the uswm group had lost 3.1% weight at the end of the program and maintained only 0.7% loss at the end of the followup. comparisons of the changes in energy intake, the number of steps walked per day, and the scores for eating behavior in swm and uswm are shown in table 2. the baseline score for perception gap about the feelings of fullness and hunger in uswm was significantly higher than that in swm (p = 0.019 by t - test, data not shown), but no significant differences were seen in the other scores, energy intake, or the number of steps walked in a day. at the end of the program, although no significant difference was seen in the energy intake, the swm participants were found to walk significantly more than the uswm participants after adjusting for age, sex, and baseline values. the score for irregularity of eating in swm was also lower than that of uswm (namely., swm had less irregularity in their eating habits) at the end of the program. table 3 shows the results of comparisons between swm and uswm in terms of stress, obstacles, and support during weight loss program and the confidence of continuing weight control behavior in the future. on the whole, fewer swm participants felt stress and obstacles in regard to carrying out the dietary objectives, increasing physical activity, and regularly weighing themselves than uswm. in addition, more swm were confident about carrying out such weight control behavior than uswm. significant differences were seen in the items regarding the stress associated with increasing their physical activity (odds ratio (95% confident interval) after adjusting for age, sex, and baseline weight was 0.36 (0.140.94)). comparisons of weight control practice, stress, obstacles, and support during the follow - up period and confidence to continue weight control behaviors in the future (self - efficacy) between swm and uswm are shown in table 4. significantly more swm participants answered yes to the questions about self - efficacy for practicing dietary objective, measuring weight, and assessing the practice and keeping records than the uswm participants both without adjustment and after adjusting for age, sex, and baseline weight. odds ratios (95% confident interval) after adjusting for age, sex, and baseline weight were 5.45 (1.9215.45), 2.79 (1.067.34), and 2.71 (1.086.81), respectively. in addition, significantly more swm participants answered yes to the questions about measuring weight and keeping records for assessing their practice (odds ratios after adjusting for age, sex, and baseline weight were 5.84 (1.1130.88) and 3.26 (1.298.22), resp.). the frequency of weighing tended to be higher in swm than in uswm (0.83 0.35 times / week versus 0.65 0.41 times / week, p = 0.052 by ancova after adjusting for age, sex, and baseline weight (data not shown)). the swm participants felt significantly less stress about measuring weight than the uswm participants (odds ratio after adjusting for age, sex, and baseline weight was 0.05 (0.010.55)). they also felt less stress regarding assessing their practice and keeping records every day (odds ratio without any adjustment was 0.35 (0.130.96)), but significance was not seen after adjusting for age, sex, and baseline weight. there were no significant differences in the other items such as obstacles, support from people, increasing physical activity, and self - monitoring for diet. the number of steps walked per day at the end of the followup was also not significantly different between the groups after adjusting for age, sex, and the values at the end of the program (8894 2867 in uswm and 10281 3202 in swm, respectively, p = 0.664 (data not shown)), but the walking steps in swm were significantly greater than that in uswm after adjusting for age, sex, and baseline values (p = 0.048 (data not shown)). in this study, we examined what behavioral factors were different between successful weight maintainers and unsuccessful weight maintainers. in addition to their having been only a few studies examining this topic using the iom definition, there were some limitations in each previous study. in our study, there are various strengths, such as that the weight was actually measured, follow - up rate was relatively high (81%), and successful weight maintainers were compared with unsuccessful weight maintainers. in our study, the swm participants showed a substantial weight loss during the weight loss program ; they lost mean of 12.0% from their initial weight. they also showed a greater improvement in their regularity of eating, walked more, and felt less stress associated with increasing their physical activity than the uswm participants. a previous study also reported that successful weight loss participants perceived fewer barriers for exercise. and because the change in energy intake was not significantly different between the groups, whether or not a person can increase his / her physical activity might be an important factor which distinguishes those who demonstrate greater weight loss from those with less weight loss, and this factor may affect subsequent weight maintenance. during the follow - up period, the swm participants had higher self - efficacy about weight control behaviors (such as activities to achieve the dietary objective, measuring weight, and assessing activities and keeping records), and actually kept records and measured weight. for example, a review study that examined psychological factors of weight maintenance and relapse in obesity suggested that higher self - efficacy was one of the important factors that affect weight maintenance. in addition, study of the national weight control registry members showed that frequent self - weighing was related to weight maintenance [12, 13 ]. in this study, more swm participants measured their body weight, and weighing frequency tended to be greater than that in the uswm participants. the participants might have been able to identify weight gain at an early stage by self - monitoring of weight and could control it. keeping records was also identified in a previous study. assessing practice and keeping records may be related to increased motivation. if the participants consistently undertook activities to meet the objective and kept records, a sense of achievement could develop, which is a great motivator of weight control practice and may contribute to weight maintenance. higher physical activity was often reported to be a factor related to sustaining lost weight [1113 ]. however, in this study, there were no significant differences in the items of physical activity including setting objectives, stress, obstacle and support. one possible reason for this inconsistent result is that because we asked the participants regarding increasing physical activity ; the participants who had already increased physical activity during the weight loss program and kept it during the follow - up period might not answer yes to the questions. considering that swm maintained significantly higher walking step counts than the uswm at the end of the followup after adjusting for age, sex, and the baseline values, keeping higher physical activity may, related to weight maintenance. however, more studies are needed to examine the role of physical activity in weight maintenance. in this study, we defined successful weight maintainers (swms) as those who maintained a weight loss of 5% or more of their initial weight at the one - year follow - up assessment, and the others were defined as unsuccessful weight maintainers (uswms). uswms included the participants who had not succeeded in achieving a 5% weight loss (we named this group uswm1) and those who initially reached a 5% weight loss but could not maintain it (we named this group uswm2). we therefore analyzed these two groups (data not shown) ; at the end of the program, uswm2 walked significantly more than uswm1. the results of the increased number of steps walked per day in uswm2 were consistent with the results of swm. in addition, more uswm2 participants felt less stress and difficulty in carrying out their dietary objectives than the participants in uswm1. during the follow - up period, significantly fewer participants in uswm2 set dietary objectives than the participants in uswm1. this might be one of the causes of decreased motivation and weight gain. in this study, we examined which behaviors were important for successful weight maintenance. however, there are limitations in this study ; as already discussed, in the followup survey, because we assessed only whether or not the participants increased their physical activity, there is a possibility that this affected our results. another limitation is that the questionnaire surveys were conducted at the end of the program / follow - up. thus, the participants already knew the amount of weight loss and this might affect the participants ' answer to the questionnaires. however, in this study, we identified the difference between successful weight maintainers and unsuccessful weight maintainers and some of these results were consistent with the results of previous studies. because there have still been only a few studies focused on this topic, more studies are needed to accumulate evidence. the results of this study suggested that in addition to a substantial initial weight loss due to an increased amount of physical activity, having a higher self - efficacy for weight control behaviors, as well as keeping records of the activities and regularly weighing themselves may be important factors for successful weight maintenance. these results may be useful to provide advice for individuals who have completed weight loss programs.	purpose. to examine behavioral factors related to successful weight maintenance. methods. subjects were 90 middle - aged participants who attended a weight loss program and were followed for one year. the subjects were classified into either successful weight maintainers (maintained a weight loss of 5% or more from their initial weight for one year) (swm) or unsuccessful weight maintainers (uswm), and weight control practice, stress, obstacles, support, and self - efficacy during the program and follow - up period were compared. results. swm had mean loss of 12% from their initial weight during the program. they showed a greater improvement in their regularity of eating, walked more, and felt less stress regarding their increased physical activity than the uswm. during the follow - up period, significantly more swm participants had self - efficacy (for measuring weight, practicing dietary objective, and assessing the practice and keeping records), actually kept records and measured weight more than the uswm participants. in contrast, more uswm participants felt stress about measuring weight. conclusion. in addition to a substantial initial weight loss due to an increased amount of physical activity, having a higher self - efficacy and consistently keeping records of one 's activities, as well as regularly weighing themselves, may be important for successful weight maintenance.
a decade ago, we (a.h.n. and m.a.n.) wrote a perspective entitled dopamine d3 receptor partial agonists and antagonists as potential drug abuse therapeutics.(1) we posited that, as all drugs of abuse either directly or indirectly increase dopamine (da) levels in the mesolimbic region of the brain, da receptors were one obvious target for medication development. we highlighted the supporting literature up to that time, focusing on the da d3 receptor (d3r), and further emphasized that despite tremendous progress in identifying mechanistic underpinnings of the psychoactive actions of addictive drugs, particularly for the psychostimulants such as cocaine and methamphetamine (meth), not a single pharmacological treatment had been approved by the fda. as millions of people in the united states and worldwide suffer from psychostimulant abuse and addiction, the public health need to develop medications to treat these substance use disorders was and remains significant. since 2005, more than 6000 papers have been published on basic, preclinical, and clinical cocaine abuse research in an effort to develop effective treatments. nonetheless, despite our best efforts with hypothesis - driven investigations producing promising results in animal models of psychostimulant addiction, we have not yet succeeded in identifying a single medication that can meet fda safety and efficacy requirements in this population of patients. a narrower question, and the focus of this perspective, relates directly to the development of medications that target the d3r. in this review, we once again set the stage for the d3r as a lead target for medication discovery for psychostimulant addiction and highlight the application of small molecule structure activity relationships (sar), with the aid of structure - based design using the d3r crystal structure, toward novel drug - like molecules. we then illustrate some of the challenges of moving our basic hypotheses through the rigors of drug development using our lead molecule 1 (pg648, chart 1) as an example. all known addictive drugs enhance da signaling within key corticolimbic circuits of the brain that control reward, emotion, cognition, and motivation. although the molecular targets of addictive drugs vary widely, they all appear to directly or indirectly enhance da signaling in the ventral striatum, particularly the nucleus accumbens, and activate neural circuitry that normally mediates reward responses to natural stimuli such as food and sex. other dopaminergic pathways that project into the dorsal striatum and frontal cortex have also been identified as contributing to drug reward and may be especially affected in the progression to addiction. specifically, psychostimulant drugs directly increase synaptic da by altering the function of the da transporter (dat). dat blockers (e.g., cocaine, methylphenidate) inhibit da removal from the synaptic cleft ; dat substrates (e.g., amphetamines) have more complex actions and can induce nonvesicular da release into the synapse. the commonality between these mechanistically distinct drugs of abuse is that rapid and profound increases in synaptic da lead to stimulation of da receptors, producing the stimulant and rewarding euphoric effects that can lead to abuse and addiction. what makes cocaine unique, though, is the rapid reversal of elevated da, leading to compulsive drug taking. for example, cocaine and methylphenidate have similar regional distributions in the striatum, but the rapid pharmacokinetics of cocaine likely lead to its higher abuse potential. da signaling is mediated by a family of g protein - coupled receptors (gpcrs). the five known da receptors (d1r d5r) are classified into two families on the basis of sequence similarity and second messenger activity. d1-like receptors (d1r and d5r) signal through gs and enhance the production of intracellular cyclic amp (camp) ; d2-like receptors (d2r, d3r, and d4r) signal through gi / o and inhibit the production of intracellular camp. in comparison d3rs have relatively small shifts in agonist binding affinity in response to guanyl nucleotides, which may indicate relatively poor receptor coupling to g proteins or, alternatively, that the receptor structure is relatively rigid and only modestly affected by g protein association ; relatedly, d3r receptor high- and low - affinity states are reported to differ only 510-fold in heterologous systems. d3rs do couple to g proteins in heterologous systems, but not exclusively to gi / o (some signaling through gq has been reported), and the adenylate cyclase v isozyme is required for agonist - mediated inhibition of camp production. furthermore, recent evidence indicates that d3rs likely form functional heteromers with d1rs in the striatum. the functional consequences of this interaction in vivo have yet to be elucidated, but it may play an important role in a variety of neuropsychiatric disorders. the d3r has long been a target of interest in addiction pharmacotherapy due to its relatively focal localization within the ventral striatum and its enhanced expression in drug - exposed brains. several research groups have discovered highly selective d3r antagonists, partial agonists, and full agonists using small molecule sar (for recent reviews, see refs (2022)) and more recently using the d3r crystal structure, computational methods, and molecular pharmacology. many of these d3r - selective ligands have served as essential research tools for pharmacological investigations at the molecular, cellular, and behavioral levels. herein, we briefly discuss the history of d3r as a target for addiction treatment, including a preview of limited clinical studies. we discuss the viability of identifying a novel translational candidate for psychostimulant addiction, practical concerns for future development of d3r - targeted pharmacotherapies, and general obstacles to medication development for addiction. translation of hypotheses based on preclinical findings has proven to be challenging due to the lack of clinically available, d3r - preferential compounds. one concern is that failure in the clinic of a single lead molecule could prematurely eliminate the d3r as a medication target for addiction pharmacotherapy. over the past decade, we have discovered many d3r - selective ligands with varying efficacies as research tools that have high affinity (ki 100-fold over d2r). one of our lead compounds, n-(4-(4-(2,3-dichlorophenyl)piperazin-1-yl)-3-hydroxybutyl)-1h - indole-2-carboxamide (1), is a high - affinity d3r - selective antagonist first reported in 2009. computational studies following the publication of the d3r crystal structure identified key interactions within the orthosteric (da) binding site and the 2,3-dicl - phenylpiperazine moiety of 1, as well as a secondary binding pocket that together likely contribute to the 4-phenylpiperazine class of compounds affinity, subtype selectivity, and efficacy. d3r subtype selectivity of 1 and related compounds depended critically on a divergent glycine residue in extracellular loop 1. given the success of earlier and structurally related compounds 2 (ngb2904),3 (pg01037), and 4 (sb277011a) in animal models of addiction, compound 1 s favorable receptor selectivity profile made it an enticing preclinical candidate. hence, we highlight the development and preclinical evaluation of 1 as an instructive example of a d3r - selective antagonist that ultimately will not make it to the clinic ; identifying pitfalls in the medications development process and areas of need in behavioral models of addiction. as a substantial cause of morbidity and mortality, drug addiction exerts major sociological and financial costs on individuals and society at large. a recent estimate of $ 500 billion in economic costs to the u.s. alone has been used to inspire further support and development of addiction medicine programs. however, while there are presently pharmacotherapeutic options to treat dependence on nicotine, alcohol, and opiates, there are currently no fda - approved medications for psychostimulant addiction. this deficit in our medical armamentarium significantly reduces what clinicians can do for psychostimulant abusers, which underscores the public health need to develop medications for treatment. it is well - established that prolonged exposure to psychostimulant drugs leads to adaptive neurobiological changes. key observations that encouraged the development of d3r - specific compounds for psychostimulant addiction treatment came from human postmortem studies showing elevated expression of d3rs following acute or chronic exposure to cocaine. the phenomenon of increased d3r expression has been confirmed and extended to other drugs, including meth and alcohol, via animal and human studies using various molecular biology techniques and pet imaging. as the focus of this perspective is targeting d3r for psychostimulant addiction treatment, we encourage interested readers to consult other excellent reviews for discussions of the pharmacotherapeutic potential of d3r - selective compounds for substance use disorders associated with other classes of addictive drugs, including ethanol and opiates. since the cloning of the d3r gene in 1990, several key differences have been identified between d3r and the closely related d2r (sharing 78% sequence identity in the transmembrane and binding domains). d2r expression in the human brain is widespread but most prominent in the dorsal striatum ; d3r expression is overall lower than d2r, and the distribution of striatal d3r is more limited to ventral regions, particularly the nucleus accumbens. a recent autoradiography study in postmortem human brain tissue reported that d3r expression is greater and more widespread than has previously been appreciated, including moderate dorsal striatal expression (although still lower density than d2r) and appreciable expression in extrastriatal regions such as the thalamus. the recent development of newer radioligands, such as 5 ([h]ls-3 - 134 in chart 1), with greater d3r selectivity over d2r may further resolve the longstanding difficulty in differentiating d2r and d3r receptor densities in neuronal tissues. the localization of d3rs within the ventral striatum suggests the receptor may play an important role in the rewarding effects of drugs and control of motivational behaviors. the relatively low d3r density in the dorsal striatum, compared to d2r, suggests that ligands sufficiently selective for d3r over d2r may also avoid the undesirable motor coordination and extrapyramidal side effects associated with nonselective d2-like antagonists (e.g., antipsychotics), commonly attributed to d2r blockade. moreover, a recent meta - analysis of randomized placebo - controlled trials with a variety of neuroleptics showed that these clinically available d2r / d3r antagonists were no more efficacious than placebo in improving abstinence or reducing craving for cocaine or meth, further suggesting that nonselective d2-like antagonists do not have clinical utility in this patient population. however, cariprazine (6, rgh-188, chart 1), a partial agonist approximately 10-fold d3r - preferential, is in clinical development as an antipsychotic agent (for review, see ref (59)) and has shown potential utility in preclinical models of cocaine abuse. while the major goal of addiction pharmacotherapy development is to block drug seeking, there has been recent interest in also focusing on cognitive deficits induced by long - term drug abuse. in one of the earlier studies, laszy. used a water labyrinth test to assess spatial memory in rats and found that cognitive impairments induced by scopolamine were reversed by d3r antagonists. more recently, mugnaini. reported that the d3r antagonist 7 (gsk598,809 in chart 1) partially attenuated an attentional bias, as assessed with a stroop task, in abstinent smokers. in a review on d3r and cognition, nakajima. concluded that d3r blockade enhances cognitive function, whereas agonists at d3r appear to impair cognition. recent preclinical studies suggest that the mechanism by which d3r blockade improves cognition involves the facilitation of both cholinergic and da transmission in the frontal cortex. d3r may also influence cognition by modulating creb signaling in the hippocampus as well as through glutamatergic the relationship between d3r and cognition is an area of research that requires much additional work. while nakajima. reported reversal in compromised animals, it remains to be determined whether selective d3r antagonists and/or partial agonists can improve cognition in subjects with cocaine- or meth - induced cognitive disruptions. the initial success of the partial agonist 8 (bp897 in chart 1) in a rodent model of cocaine addiction spurred many laboratories in both academia and pharma to develop new drug - like molecules with d3r selectivity. recently reported analogues in the 4-phenylpiperazine template include functionalization of the butyl linking chain and elaboration of both the terminal aryl amide and phenylpiperazine or head group. these compounds typically follow and expand upon the sar already established for d3r - selective antagonists or partial agonists and, for those that have been tested in vivo, have similar behavioral profiles in animal models of psychostimulant abuse. a novel approach that inserted a cyclobutyl group in the linking chain and replaced the phenylpiperazine head group with tranylcypromine resulted in a recently reported novel series of d3r - selective antagonists. many of the newer analogues have capitalized on variations of the 4-phenylpiperazine template, but some interesting and diverse molecules have also been discovered. notably, the evolution of sar in the glaxo program, led by micheli and colleagues, abandoned the arylamide terminus for a heteroaryltriazole, as seen in 7. this modification resulted in high affinity and selectivity for the d3r target but also metabolic stability and low herg channel activity, predicting that this compound would be bioavailable and safe in humans. other groups have replaced the 4-phenylpiperazine with the tetrahydrobenzothiazole of pramipexole to give full or partial d3r agonists with both high affinity and subtype selectivity. of note, the groups led by dutta and reith have also reported tetrahydrobenzothiazole analogues, but they have replaced the arylamide of the more classic d3r ligands with this functional group and retained an arylpiperazine or an arylamide piperazine to give a novel set of d3r - selective agonists. in this series, it is interesting to contemplate which end of these molecules binds to the orthosteric (da) binding site and whether the other end binds to the secondary binding pocket believed to be occupied by the aryl amide of (r)-1 or a different site within the d3r protein to produce a full agonist profile. a similar hybridization approach that replaced the 4-phenylpiperazine with a 5-aminohydropyrazolopyridine group also resulted in a very interesting series of highly potent d3r agonists that were reported to demonstrate functional bias, a hot topic in the d2-like receptor drug discovery arena (for review, see refs (84 and 85)) but beyond the scope of the present review. gmeiner and colleagues are avidly following these leads and discovering other templates that display functionally biased profiles. the field is still in its infancy in terms of understanding the pharmacological and behavioral consequences of d3r - mediated biased agonism. nevertheless, the combination of small molecule sar and the d3r crystal structure with computational modeling will undoubtedly produce structurally and pharmacologically variant tools in the near future with which to probe these questions. to date, very few d3r - selective or preferential antagonists or partial agonists, 7 appears to be the most clinically investigated compound based on publications in the literature and perusal of clinicaltrials.gov (e.g., clinicaltrials.gov identifiers nct00437632, nct01188967, nct00793468, nct00605241, and nct01039454). briefly, after validating d3r occupancy in a pet study using [c]phno and evaluating its pharmacokinetics,7 was tested in several clinical studies. in overweight and obese subjects, 7 reduced approach bias and attentional bias to palatable food cues but did not alter fmri brain responses to food images. in tobacco smokers, although 7 had some qualified successes in these clinical populations, and many other d3r - selective antagonists and partial agonists have been successful in preclinical models of cocaine or meth abuse, it is unclear at this time whether or not 7 will be evaluated in this patient population. one concern that has emerged is the potential for 7 and other d3r antagonists to increase blood pressure, especially if taken in combination with stimulants (nate appel and jane acri, national institute on drug abuse, personal communication), as d3rs reside in the kidney and contribute to da - mediated regulation of blood pressure. cocaine s cardiovascular effects have been investigated, and increases in both blood pressure and heart rate have been well - documented. indeed, both central and peripheral mediation of increased blood pressure upon acute and chronic administration of cocaine have been investigated with varying results depending on species, dose of cocaine, and acute versus chronic administration. the concern, of course, is that if subjects relapse to cocaine taking, blood pressure elevations due to cocaine exposure could be exacerbated in the presence of a d3r antagonist. human studies investigating the effects of the d2r / d3r partial agonist aripiprazole on cocaine self - administration, drug discrimination, and cardiovascular effects reported a small, acute increase in blood pressure that was not sustained after repeated cocaine administration. further investigation with d3r - selective drugs is required in order to clearly determine whether d3r - selective partial agonists or antagonists are likely to exhibit this potential side effect. for example, in ongoing preclinical studies using nonhuman primates implanted with indwelling telemetry devices, administration of cocaine increases blood pressure and heart rate ; these effects are either unaltered or enhanced by d3r antagonists, but they appear to be attenuated by d3r partial agonists (m.a. newman, unpublished results). also, it is critically important to determine whether this potential cardiovascular risk is mediated via d3r or is an off - target effect of the drug molecule : the latter might be addressed with appropriate structural modifications of the lead compound(s). hence, continued evaluation of structurally diverse molecules with differing off - target profiles remains a priority to further validate the d3r as a target for medication development, especially for psychostimulant abuse. of course, substance use disorders that by themselves do not alter blood pressure or heart rate may be completely treatable with d3r antagonists or partial agonists, without concern for cardiovascular safety. in addition, increases in blood pressure can be monitored and treated, if necessary, with clinically available drugs. in response to the interest in investigating d3r antagonists as potential pharmacotherapies for cocaine abuse, the clinically available anxiolytic buspirone was evaluated in several preclinical addiction models and in a clinical trial with cocaine abusers. while by no means d3r - selective, bergman. reported that buspirone had higher affinity at the d3r compared to the d2r. when tested in monkeys self - administering cocaine, they found that buspirone decreased cocaine - maintained behavior to a larger degree than food - reinforced behavior. mello., using rhesus monkeys that self - administered cocaine 4 times per day, found that continuous infusions of buspirone also decreased cocaine self - administration to a greater degree than food - reinforced responding. unfortunately, the positive results from these preclinical studies did not extend to clinical trials. reported negative effects of buspirone in cocaine abusers and, in fact, found that buspirone increased cocaine use in women. however, it should be noted that this study had a remarkable placebo effect : subjects that were not administered buspirone dramatically reduced their cocaine use. further, it is not clear that the single tested dose (60 mg) of buspirone was adequate to achieve high occupancy of d3r for a sufficient duration of time to be effective. supporting this assumption is a recent study in primates measuring d3/d2r occupancy of buspirone using pet imaging. it was found that the low dose of oral buspirone (1.0 mg / kg) tested, approximately the same dose tested in the winhusen. in contrast, 80% sustained d3r occupancy was achieved by a dose that was 3-fold higher (3.0 mg / kg) and was well - tolerated. these data indicate the importance of using receptor occupancy as a guideline for therapeutic efficacy in that high and sustained levels are most likely needed for the successful treatment of addiction. on the basis of these clinical findings with buspirone, one may (prematurely) conclude that the d3r and, more specifically, d3r antagonists are not viable targets for psychostimulant addiction. we would caution against such conclusions for two reasons : first, buspirone has very complex pharmacology, with known effects at 5-ht1a, d2r, d3r, and d4rs, as well as active metabolites that interact with 2-adrenergic receptors. second, the choice of drug reinforcement schedules used in the preclinical evaluations of buspirone may not have been ideal to detect d3r - mediated antiaddiction effects. one of the interesting aspects of the behavioral pharmacology of d3r compounds is the importance of the schedules of reinforcement used in self - administration studies (see refs (1 and 48) for review). in the preclinical models used by bergman. and, there were no competing reinforcers when cocaine self - administration was studied, and this may be critical to understanding how medications, such as buspirone, can decrease one type of behavior (in their case, food - maintained responding) to a smaller degree than cocaine self - administration. more complex models, such as food drug choice paradigms, measure reinforcing strength (efficacy) with the goal of examining treatments that decrease cocaine self - administration and reallocate responding from cocaine to food. in a recent study, john. found that 5 day treatment with buspirone increased cocaine choice, a finding consistent with the winhusen. clinical study. clearly, the animal models used to evaluate d3r compounds on cocaine self - administration need to be more thoughtfully analyzed in order to achieve translation of preclinical findings to clinical success. for example, in a study using socially housed monkeys, acute buspirone administration decreased cocaine choice relative to food in dominant monkeys but not subordinate animals, suggesting a possible interaction between environmental variables and efficacy of buspirone. importantly, though, it is our premise that using food drug choice paradigms and the study of a range of d3r compounds (see below) will allow for (1) a better understanding of the role of d3rs in cocaine abuse and (2) the identification of potential pharmacotherapies based on the d3r. thus, buspirone should not be considered a representative d3r antagonist, and it is our perspective that this single clinical trial should not deter further research toward developing a d3r - selective antagonist or partial agonist for substance use disorders. it should, however, be noted that treatment - emergent adverse events reported in this study did not include increases in blood pressure in the subjects receiving buspirone. on the basis of the preclinical promise of early leads, our group and others have focused efforts on optimizing d3r affinity and selectivity as well as physical properties (e.g., clogp, tpsa, metabolic stability, etc.) to improve their utility as in vivo tools. as drug abuse is a human behavior, we ultimately must develop tools that are stable in vivo, penetrate the blood brain barrier, and selectively engage d3r. we must also be able to readily scale up the synthesis of these molecules for behavioral studies in rodent and nonhuman primate models, the most translational of which require chronic dosing. like other medicinal chemists, we have optimized lead compounds, using structure activity relationships and, more recently, the d3r crystal structure, to design novel molecules. these efforts have led to the identification of several new lead molecules, including ()-, r-, and s-1, 3, and others including 17 (cjb090),18 (pg619),19 (gcc3 - 09), and 20 (bak2 - 66), to highlight a few (chart 3). these compounds have been evaluated in cell - based binding assays along with compounds reported in the literature to obtain side - by - side data comparisons across the d2-like family of receptors. the binding affinities of representative d2-like compounds, in table 1, were obtained from human d2rs, d3rs, and d4rs expressed in hek293 cells. using membranes from these cells and the antagonist radioligand [h]n - methylspiperone, direct comparisons can be made of these structurally divergent compounds under identical experimental conditions (see supporting information for methods). ki values determined by competitive inhibition of [h]n - methylpsiperone binding in membranes harvested from hek 293 cells stably expressing hd2r, hd3r, or hd4r. table 1 shows the experimentally derived binding dissociation constants (ki) for a number of dopaminergic compounds with varying selectivities for d3r over d2r and d4r. of note, n - methylspiperone, eticlopride, raclopride, and butaclamol are well - known d2-like antagonists, demonstrating high binding affinities across the d2-family of receptors, with the notable exceptions of butaclamol and especially raclopride at d4r. aripiprazole is a clinically available d2-like partial agonist, marketed as abilify, used in the treatment of schizophrenia and mood disorders. it binds with high affinity to both d2r and d3r and has been shown to decrease cocaine self - administration and attenuate reinstatement in laboratory animals. although mixed results have emerged from human studies, a recent phase ii study using chronic treatment with lower doses reported that aripiprazole decreased cocaine craving. because identical binding conditions were used, the data presented in table 1 permit direct binding affinity comparisons across compound classes. compound 7 was the most subtype - selective compound in this set, with 670-fold d3r selectivity over d2r. notably, binding for 21 (yqa14) in this system did not show two - site binding kinetics at d3r as reported previously. the ki determined in this analysis (3.04 nm) is consistent with the ki - low reported previously (2.11 nm) ; coupled with a higher d2r affinity in this analysis than in the previous report (46.9 vs 335 nm), compound 21 appears to be only moderately preferential for d3r binding over d2r when compared to others in this class of d3r ligands and points to the limitations of using cell - based binding affinities to predict d3r selectivities and potencies in vivo. nevertheless, radioligand binding is the first line of testing in most drug discovery programs and is useful as long as compounds are evaluated side - by side under the same assay conditions. as noted in our previous perspective, cell - based functional assay data remain difficult to interpret from an sar standpoint ; hence, binding data remain our primary source of sar, despite these limitations. compound 2 is one of the first d3r - selective antagonists to be reported and has served as an important preclinical tool. early reports demonstrated that 2 significantly lowered the break point in rats trained to self - administer cocaine under a progressive - ratio (pr) schedule of reinforcement. pr schedules require an increase in number of responses following injections of cocaine and the last ratio completed (the break point) is thought to be a measure of reinforcing strength. compound 2 also inhibited cocaine - induced enhancement of brain stimulation reward threshold while neither maintaining self - administration nor altering brain - reward thresholds on its own. in addition, 2 blocked both cue- and cocaine - induced reinstatement of cocaine seeking, a preclinical model of relapse. compound 2, along with 4 and 8, was also evaluated in meth - enhanced brain stimulation reward in rats. notably, the two antagonists (2 and 4) effectively attenuated meth - enhanced brain stimulation reward and did not affect brain stimulation reward on their own ; the partial agonist 8 attenuated meth - enhanced brain stimulation reward, but a high dose inhibited brain stimulation reward itself. hence, 2, along with 4 and 8, demonstrated potential for this class of ligands to be developed toward medications to treat cocaine and meth abuse. although none of these agents would be translated to the clinic, all of them served as templates for the design of new analogues. while 4 was the precursor to 7,2 served as the starting point from which our early lead compounds 3 and 17 were derived. compound 17 was the first partial agonist in our series that was evaluated in nonhuman primates and compared to 2 in two models of cocaine abuse. interestingly, 17, but not 2, attenuated cocaine s discriminative stimulus effects and decreased both cocaine- and food - maintained responding in monkeys that were trained on a second - order schedule of reinforcement. however, in a separate study with squirrel monkeys, 17 failed to attenuate cocaine self - administration or cocaine - induced reinstatement of extinguished cocaine - seeking behavior. as elaborated by achat - mendes., there are several possible reasons for these discrepant results including the different schedule parameters, the different frequencies of cocaine injection per session (maximally 10 in the achat - mendes. study), and the different species used (squirrel monkeys vs rhesus monkeys). another possible explanation could be the cocaine history of the subjects, which has been shown to influence the behavioral effects of d3r compounds. both 17 and the structurally related but more d3r - selective antagonist 3 reduced pr meth self - administration in rats with a history of long access (6 h per day, 6 days per week) to meth.17, in contrast to 3, also reduced pr meth self - administration in rats with a history of short - access to meth (1 h per day, 3 days per week). it is unclear at this time whether or not differences in pharmacokinetics or pharmacodynamics can explain these subtle differences in efficacy across species and models, although pharmacokinetic and metabolism data suggested that 3, like 2, had suboptimal bioavailability. overall, coupled with many other reports in the literature of similar findings with compounds such as 4, 7, and newer generation analogues, these data supported further optimization and development of d3r - selective antagonists or partial agonists. recent reports from the neisewander lab highlight novel 4-phenylpiperazines, exemplified by 22 (chart 3), each varying in their subtype selectivity and degree of partial agonism at d3r, that reduce cocaine self - administration. compound 17 and a newer - generation partial agonist, 18, exhibited interesting effects in rhesus monkeys with a history of cocaine self - administration in comparison to their drug - nave counterparts. as part of our efforts to identify d3r - based behaviors in vivo, a model of d3r - induced yawning in rats was modified, in which yawning could be produced in rhesus monkeys upon administration of the d3/d2r agonist quinpirole. in cocaine - nave animals, quinpirole induces robust yawning, but the partial agonists 17 and 18 failed to do so. however, in monkeys with a history of cocaine self - administration, both 17 and 18 induced yawning similar in magnitude to quinpirole. we reasoned that increased sensitivity to yawning might occur in the monkeys with a cocaine history, as increases in d3r densities had been reported in both human cocaine fatalities as well as in cocaine - exposed rodents. nevertheless, in that study, 18 was unable to attenuate cocaine self - administration under a fixed - ratio (fr) 30 schedule of reinforcement. unlike quinpirole, however, 18 did not reinstate cocaine seeking in these monkeys but rather attenuated cocaine - induced reinstatement. in an effort to continue to optimize d3r affinity, selectivity, and bioavailability, modifications of these lead molecules have led to newer generation compounds such as 19 and 1 (chart 1). both of these compounds have high affinity (ki 12 nm) for d3r and are highly selective over d2r and other off - target receptors. of note, ()-1 was evaluated in 64 radioligand / enzyme assays through the nida addiction treatment discovery program. other than the reported binding affinities at d1r (ki = 4630 nm) and 5-ht1a receptors (ki = 104 nm), ()-1 did not produce > 50% inhibition of binding at any of the receptors evaluated at a concentration of 100 nm and only hit a few at 10 m [e.g., 1 adrenergic, 2 adrenergic, histamine h1, sodium channel site 2, cholecystokinin 1 (cck1), and neurokinin 2 (nk2) ]. in addition, the ic50 for (r)-1 at the herg channel was 0.38 m, as determined using the patchxpress assay through the nimh psychoactive drug screening program (pdsp ; http://pdsp.med.unc.edu). considering its ki at d3r is 2 nm, ()-1 was considered to be highly selective for the d3r, and, despite its marginally acceptable clogp value of 4.8, we chose it as our lead candidate for further development. the addition of a 3-oh group in the linking chain of these molecules creates a chiral center ; therefore, we synthesized the r- and s - enantiomers of 1. in radioligand binding competition studies, we observed a small but significant enantioselectivity at d3r, but not at d2r, with the r - enantiomer having higher d3r affinity. from a structural point of view, this was significant and gave insight on differential binding interactions at the receptor protein level that were further explored. to determine if behavioral effects were also enantioselective, we improved the enantioselective synthesis, evaluated the microsomal metabolism and pharmacokinetics of the racemate, and then tested the racemate and the r- and s - enantiomers in behavioral models of cocaine and meth abuse. absorption for oral ()-1 was slightly delayed, peaking 2 h after administration. the peak plasma concentration (cmax) for ()-1 was 7369 ng / ml following i.v. dosing compared to 522 ng / ml following p.o. dosing ; comparing oral versus intravenous auc parameters gives a low / moderate average absolute bioavailability fraction (f%) of 16.2%. the brain - to - plasma ratio ranged from 6- to 20-fold for ()-1. we investigated the metabolism of ()-1, and these data are presented in table 4. ()-1 was found to be very stable in mouse plasma over a period of 60 min, with 90% of the parent compound remaining. ()-1 was, however, susceptible to phase i and phase ii hepatic metabolism. in mouse liver microsomal incubations in the presence of nadph, which measures phase i metabolism, 21% of the parent ()-1 remained after 60 min. notably, microsomal stability in rats was increased to 37% of the parent ()-1 remaining after 60 min, under the same assay conditions, suggesting higher stability in this species. in mouse microsomes fortified with udpga, which measures phase ii metabolism, 55% of the parent ()-1 remained after 60 min of incubation. no metabolism was observed in microsomes without the cofactors, showing their specificity to cyp- and ugt - dependent instability, respectively. concentration profiles for 10 mg / kg ()-1 in mouse plasma following i.v. data are presented as mean sem ; n = 3 mice per time point. concurrent with the pharmacokinetic studies described above, ()-, (r)-, and (s)-1 were evaluated in a rat yawning model to verify target engagement and determine an effective dose range for further behavioral evaluation. although mouse yawning studies were initially attempted, it was discovered that d3r agonists do not induce yawning in mice, so that approach was abandoned. ()-, (r)-, and (s)-1 each attenuated 7-oh dpat - induced yawning in rats, as demonstrated by a rightward shift in the ascending limb of the dose response curve ; however, no clear evidence of enantioselectivity was observed (j.l. further, this model proved to be difficult to use as an in vivo screen for numerous reasons, including tolerance to the d3r - agonist effect on yawning and significant variation across subjects. it also required a large quantity of test drug to obtain full dose response curves. hence, we discontinued testing compounds in the rat yawning model as an in vivo diagnostic and instead used binding affinities as a guide to dose ranges evaluated in the subsequent rodent studies. using methods described previously,1 was evaluated for its effects in two rat models of drug - taking and drug - seeking behavior. figure 2a shows meth self - administration in rats responding under a pr schedule of reinforcement, comparing baseline performance against pretreatments with vehicle or the enantiomers of 1 ; figure 2b presents these data normalized to each individual animal s baseline performance. separate one - way repeated - measures anovas were conducted on number of injections, which represents pr break points (i.e., the final ratio completed). compound 1 significantly and dose - dependently reduced meth injections [()-1 : f2,18 : 19.36, p 0.05, vehicle vs 10 mg / kg p 0.05, vehicle vs 10 mg / kg p 0.05, vehicle vs 10 mg / kg p 0.05, vehicle vs 10 mg / kg p 0.13, dunnett s test : vehicle vs 3.0 mg / kg p > 0.05, vehicle vs 10 mg / kg p > 0.05 ]. (a) acquisition of meth self - administration and extinction of self - administration. (b) effects of i.p. vehicle or ()-1 on meth - primed reinstatement of meth - seeking behavior. data are presented as mean sem ; p < 0.05 compared to vehicle. reinstatement of drug - seeking behavior, via stressors, drug - associated cues or contexts, or acute exposure to the self - administered drug or related drugs, is a widely used model of drug craving and relapse (for review, see refs (144146) ; see also ref (147)). as with previous d3r - selective antagonists and partial agonists (e.g., 2,3, and 4(31)), ()-1 showed a dose - dependent reduction in meth - primed reinstatement responding. these results add to the growing evidence that d3r signaling plays an important role in the neurocircuitry that drives relapse to drug taking and that d3r - selective compounds could be useful therapeutics in the prevention of relapse. however, it should be noted here that assessing a medication that may prevent relapse requires that the subject abstain from drug taking for a period of time. if the d3r antagonists do not curb drug taking, then abstinence may be very difficult to achieve and hence clinical assessment for this therapeutic benefit will be challenging. it has been proposed that a rosetta stone approach be taken to developing drugs for addiction, wherein the addiction cycle is taken into account for a pharmacological strategy. in the case of d3r antagonists, additional treatment strategies may need to be in place to help subjects attain abstinence before administering d3r - selective therapeutics to prevent relapse. in addition to the rodent model studies, male cynomolgus monkeys were trained to self - administer cocaine under a concurrent schedule in which food was the alternative reinforcer. under these experimental conditions, complete cocaine self - administration dose response curves were determined each session (see ref (112) and supporting information for methods). as shown in figure 4, in all but one monkey, ()-1 either shifted the cocaine choice curve to the left (4 subjects) or had no effect (2 subjects). in one monkey (c-6529), a decrease in choice of the highest cocaine dose was observed. when group data were analyzed, there was a main effect of cocaine dose (f4,30 = 81.8, p < 0.0001) but no main effect of drug pretreatment and no interaction. posthoc multiple comparisons indicated a significant increase in cocaine choice when 0.01 mg / kg cocaine was available as the alternative to food. there were no significant effects of the highest dose tested (5.6 mg / kg) ()-1 on any of the dependent variables of secondary interest (table 5). although ()-1 failed to reduce cocaine choice and increase food choice, which would be a result more congruent with a potential therapeutic, there are several testable hypotheses generated from these results. both concurrent food drug choice and pr responding are considered to be models of reinforcing strength. however, the effects of 1 are different in these two models, suggesting that they are measuring different aspects of self - administration (see ref (150) for an example). one could hypothesize that drugs that share discriminative stimulus effects with cocaine would shift the cocaine choice dose however, in preliminary findings from monkeys trained to discriminate cocaine, 1 does not substitute for cocaine (m.a. nader and a.h. response curve to the left (e.g., refs (112, 151, and 152)). it is also possible that the differences noted are due to drug and/or species differences (see ref (153)) or to differences in drug histories between the rat pr study and the monkey food cocaine choice study. on the basis of our experience and the literature, d3r antagonists are most effective in models of relapse, and perhaps this is the clinical end point that should be singly targeted with this class of drugs. nevertheless, from a basic research standpoint, additional behavioral models of addiction must be explored to fully elucidate the role of d3r in the development of addiction. further, as described previously, neurobiological changes that occur upon chronic exposure to psychostimulant drugs and also during abstinence must also be quantified to better ascertain optimal timing of administration of d3r antagonists or partial agonists for potential therapeutic benefit. one obvious limitation to the present study is that 5.6 mg / kg was the highest dose that could be solubilized and administered intravenously to these subjects. it is certainly possible that the level of d3r occupancy required to observe behavioral effects was not achieved or sustained and future d3r occupancy determination, potentially using pet imaging or another biomarker, will need to be assessed. vehicle or ()-1 on cocaine choice in seven monkeys individually and as a group (lower right panel). abscissae, dose of cocaine (mg / kg) available as an alternative to a food pellet. data in lower right panel represent mean sem. in order to more fully model the clinical situation in which chronic drug treatments are likely to be employed, a follow - up study was conducted in rhesus monkeys that self - administered meth in which ()-1 was administered for 5 consecutive days. in two of three monkeys, 5 day treatment with 3.0 mg / kg ()-1 (i.v.) effects of acute treatment had a more pronounced effect in one monkey (r-1690), suggesting tolerance developed in this subject. when grouped data were analyzed, there were no statistically significant effects of 3.0 mg / kg ()-1 on any of the dependent variables of secondary interest (table 5). in addition to the possibility of suboptimal d3r occupancy, pharmacokinetic studies were conducted only in mice. these experimental sessions are approximately 2 h in duration, and drug self - administration doses are presented in ascending order. if the half - life of 1 is short in monkeys, then it is possible that there is insufficient drug blocking d3rs later in the session when higher cocaine doses are available. an additional possibility is that the elevated da from cocaine or meth is displacing 1 and that d3r antagonists are less effective in models of self - administration compared to reinstatement models or under conditions in which treatment occurs in the absence of psychostimulant availability. indeed, a pet imaging study in rhesus monkeys with [f]ls-3 - 134 suggested a high level of competition between this d3r - selective pet imaging agent and endogenous dopamine in the absence of a psychostimulant drugs. thus, in the presence of psychostimulants, competing da levels may render d3r antagonists ineffective. future research is clearly needed to better understand the conditions under which d3r - selective compounds are effective in nonhuman primate models of psychostimulant addiction and which of these models is reliably translatable to human psychostimulant abusers. effects of acute and chronic administration of ()-1 (i.v.) on meth choice in three rhesus monkeys (mean sem of days 35 of daily treatment). abscissae, dose of meth (mg / kg) available as an alternative to a food pellet. in radioligand competition binding experiments using [h]n - methylspiperone and membranes from hek293 cells expressing hd2r, hd3r, or hd4r, ()-1 exhibits nearly 400- and 1300-fold d3r selectivity over d2r and d4r, respectively. this is more pronounced when considering the enantioselectivity of d3r for the r - enantiomer. the in vitro d3r selectivity of 1, particularly (r)-1, was an improvement over previous generations of 2,3-dichlorophenylpiperazinebutylarylcarboxamides (e.g., 3). as such, 1 and its enantiomers were some of the most d3r - selective ligands in our library and thus the tools we chose to test in vivo. to evaluate the in vivo stability of 1, plasma pharmacokinetics (pk) was studied in mice, revealing plasma stability 01.5 h following i.v. compound 1 was actively metabolized by liver microsomes, but it showed a terminal half - life of approximately 11.5 h following i.v. administration. it should be noted that metabolism and pk studies were done in mice and thus these parameters may be different across species. clearly, pk studies need to be extended to nonhuman primates and brain imaging needs to be utilized to study pharmacodynamics of these compounds entering the brain and occupying d3r. nevertheless, these pharmacokinetic parameters appeared to be promising for a translational candidate, justifying further exploration in both rat and nonhuman primate models of drug addiction. previous studies have demonstrated a trend in which d3r antagonists and partial agonists typically do not affect psychostimulant self - administration with low fr response requirements but attenuate drug taking in paradigms that increase the response requirement (e.g., high fr or pr models) or second - order schedules, suggesting that d3r antagonism does not affect the primary reinforcing effects of drugs but reduces the motivation to self - administer drugs. consistent with that view is the success of d3r antagonists in rodent models of relapse, such as drug-, cue- and stress - induced reinstatement. compound 1 significantly reduced pr responding for meth, as measured by break point. there was no clear difference in potency seen between the (r)- and (s)-enantiomers ; however, this study did not test a sufficient range of doses to reliably determine whether one enantiomer was more potent than the other in reducing pr break point in rats. overall, ()-1 (3.010 mg / kg, i.p.) was efficacious in two rat models of drug self - administration and drug seeking (i.e., reinstatement). in the food cocaine choice model, cynomolgus monkeys were allowed to choose either a food reward or an i.v. overall, 1 had inconsistent effects ; when all data were combined, the general effect that emerged was that 1 shifted the dose response curve of cocaine to the left, i.e., it potentiated cocaine choice over food. in the food meth choice model using rhesus monkeys, one subject (r-1691) appeared to develop tolerance to the effect of 1. however, it must be noted that the food drug choice model is not directed toward relapse, which the rodent studies with 1 and other d3r antagonists most support. further evaluation of 1 and newer generation d3r antagonists and partial agonists are underway in nonhuman primate reinstatement to drug seeking models to determine if the rodent studies can be replicated with these agents in primates with a history of chronic drug taking. chronic dosing with the treatment agent can also be evaluated in the nonhuman primates, which will also inform human studies. in contrast to d3r antagonists such as ()-1 and 7, d3r partial agonists pose several interesting questions. for example, it is known that chronic administration of receptor agonists commonly results in receptor downregulation, whereas chronic administration of receptor antagonists commonly results in receptor upregulation. with regard to partial agonists, a recent pet imaging study in cynomolgus monkeys found that chronic aripiprazole, a d2r partial agonist, produced different effects on d2r binding potential : it appeared to increase binding when the subject had, on average, low d2r availability and decreased binding potential in monkeys with higher average d2r availability. thus, depending on the long - term consequences of cocaine or meth abuse on receptor availability, the effects of receptor partial agonists may be different than antagonists. recently, it was reported that the effects of aripiprazole on cocaine food choice varied depending on whether monkeys were given access to cocaine during aripiprazole treatment. thus, it remains possible that in nonhuman primates and humans d3r partial agonists would be more effective in a setting in which cocaine or meth were not available (e.g., residential treatment facility), at least in the early stages of treatment. finally, there are ongoing studies to evaluate the effects of antagonists and partial agonists on peripheral d3rs, particularly in regard to potential synergistic effects in combination with cocaine- and meth - induced elevations in heart rate and blood pressure, a key treatment risk that must be thoroughly evaluated and thoughtfully considered when describing potential treatment compounds. the translation between rodent and nonhuman primate addiction models and again between nonhuman primate addiction models and humans in a clinical setting is fraught with pitfalls. predecessors of 1 and 7 have failed to successfully clear the hurdles that lead to successful clinical trials. the partial agonist 8 was efficacious in rodent and primate models of addiction, but it failed in clinical trials for cocaine abuse. compound 4 looked promising in rodent models of drug taking and drug seeking, but poor bioavailability and short half - life in primates led glaxosmithkline to halt further development of the compound. the recent failure of buspirone, as described above, may also bode poorly for continuing to pursue this target. its inconsistent behavioral profile in nonhuman primates thus far may be directly related to a poor pharmacokinetic profile, lack of target engagement at the doses and time points tested, or simply lack of efficacy in these models. future studies to parse out the contributing factors to its demise, before reaching the clinic, will be used for the next generation of drug design and choice of behavioral models. nevertheless, newer generation agents in our laboratory and others are already making their way forward in development and will soon be evaluated in these and other models of drug abuse and neuropsychiatric disorders activity relationships and in vivo data will ultimately lead us to successfully identify d3r - selective compounds that have appropriate drug - like properties. a key concern in the field of addiction medicine and indeed all neuropsychiatric drug discovery is the effectiveness of animal models to evaluate preclinical candidates. in one of the first studies using d3r partial agonists, it was reported that the effects of 8 were different in models of drug seeking compared to drug self - administration. the study of d3r compounds has provided a better understanding of the importance of models that incorporate long - term cocaine and meth self - administration and a clearer perception of the importance of behavioral phenotype. for example, it appears that the behavioral pharmacology of d3r partial agonists are different depending on whether the subjects are drug - nave or have a drug history and, for d3r agonists, whether the drug is cocaine or meth. moreover, individual differences in the efficacy of d3r antagonists to reduce drug self - administration have been found involving acute vs chronic treatment, drug history, and social rank. these findings are reflected in human imaging studies involving the d3r - preferential pet ligand [c]phno in cocaine and meth abusers. there are important temporal differences in psychostimulant - induced d3r dysregulation that should be taken into account for the d3r s therapeutic potential. for instance, studies designed to examine the time course of changes in d3r binding and sensitivity observe d3r upregulation after prolonged abstinence (3045 days after the last cocaine administration) but not during self - administration or within 7 days of abstinence. furthermore, the subjects in some of the recent human imaging studies demonstrating d3r upregulation following chronic cocaine or meth use were scanned on average 18.5 20.5 and 50.1 64.4 days, respectively, after their most recent drug use, which appear to reflect substantial periods of abstinence. therefore, although it is clear that d3r expression is significantly altered in drug abusers, perhaps there is a specific window of time in the addiction cycle in which the therapeutic potential of d3r antagonists and partial agonists could be maximally beneficial. the difficulties of treating drug addiction are compounded by the high incidence of comorbid neuropsychiatric disorders with substance use disorders, including depression, anxiety, adhd, and schizophrenia. further complicating the search for translational medications for addiction is the complexity of d3r receptor signaling and genetics. d3r heteromers in the striatum or whether receptor heteromers are a viable target for drug development. d3r heteromer in drug memory reconsolidation, and future studies in this area are of great interest. additionally, there are a number of studies linking various d3r gene polymorphisms with neuropsychiatric disorders. in particular, rs6280, which encodes the functional missense mutation ser9gly, may enhance reward - related da release. this particular polymorphism has been associated with nicotine dependence, alcohol dependence, and early onset heroin dependence. further study will be necessary to determine the significance of receptor heteromerization and genetic variation at d3r in the development of substance use disorders or in the response to an antiaddiction pharmacotherapeutic. given these considerations, it is important that the preclinical and clinical studies showing negative results with d3r antagonists and partial agonists thus far not be used to prematurely rule out the utilization of these pharmacotherapies for addiction. instead, these studies should be used to guide future efforts in designing more appropriate behavioral models to evaluate d3r compounds and determining the subsets of individuals in which d3r pharmacotherapies can be most effective. to reiterate, assessing a medication that may have its greatest utility in the prevention of relapse, as may be the case for d3r pharmacotherapy, requires that clinical subjects abstain from drug taking for a period of time. on the basis of preclinical results thus far, d3r antagonists do not effectively reduce active drug taking, but they do reduce relapse - like behavior. therefore, if d3r antagonists are best suited for a role as a relapse - prevention treatment, then they will likely only be successful in the framework of a larger treatment program in which subjects have already attained abstinence. trials designed to test d3r antagonists in patients actively using drugs may not see any attenuation in drug taking and would not be well - suited to evaluate the abstinence - maintaining therapeutic potential of this compound class. hence, lessons learned from the buspirone case described in section 2.3 should be used in future psychostimulant medication development. this perspective highlights the importance of developing selective compounds to better understand the role of d3r in addiction. it is our opinion that repurposing drugs that are clinically available has utility in guiding future research. however, the use of drugs with d3r affinity, but also many other targets (e.g., buspirone), should not be used to exemplify the pharmacology, biology, and physiology related to this important target. recently, a new d3r - preferential partial agonist, 23 (bp1.4979 in chart 3, communicated by jean - charles schwartz, dopamine 2013 meeting), was introduced by bioprojet, and a clinical trial on smoking cessation has begun (clinicaltrials.gov identifier nct01785147). although data remain unpublished, presumably this compound has been vetted through all of the in vitro and in vivo assays that pharma has at its disposal (as compared to academic drug discovery programs such as ours) to be considered for translation to human studies. the fact that 23 is a partial agonist is of further interest and will provide answers to some of the questions regarding d3r efficacy posed above. results of these clinical investigations will be of great interest to our community of addiction researchers and will very likely determine if the d3r remains a target to pursue for smoking cessation as well as other substance use disorders. one final thought is that d3r antagonists and partial agonists may not prove to be efficacious or safe for treatment of cocaine or meth addiction but may still hold promise for addictions to other substances, such as nicotine, opiates (including prescription pain killers, e.g., oxycodone), alcohol, food and -tetrahydrocannabinol (thc), the active ingredient in marijuana. hence, it is our perspective that the development of selective d3r ligands, with varying efficacies, as research tools that are active and metabolically stable in vivo, can help to elucidate underpinnings of addiction and other neuropsychiatric disorders. hence, future research and development of these agents toward these therapeutic end points remains vitally important. further, developing animal models that can translate preclinical research is challenging and yet key to finding pharmacotherapeutic treatments for this underserved patient population.	the dopamine d3 receptor (d3r) is a target for developing medications to treat substance use disorders. d3r - selective compounds with high affinity and varying efficacies have been discovered, providing critical research tools for cell - based studies that have been translated to in vivo models of drug abuse. d3r antagonists and partial agonists have shown especially promising results in rodent models of relapse - like behavior, including stress-, drug-, and cue - induced reinstatement of drug seeking. however, to date, translation to human studies has been limited. herein, we present an overview and illustrate some of the pitfalls and challenges of developing novel d3r - selective compounds toward clinical utility, especially for treatment of cocaine abuse. future research and development of d3r - selective antagonists and partial agonists for substance abuse remains critically important but will also require further evaluation and development of translational animal models to determine the best time in the addiction cycle to target d3rs for optimal therapeutic efficacy.
otitis media (om) is an inflammatory response of the middle ear (me) caused by multiple factors such as infection and eustachian tube dysfunction. acute otitis media (aom) is one of the commonest pediatric diseases [24 ]. streptococcus pneumonia and haemophilus influenzae are the most common causative microorganisms for aom [57 ]. patients with aom present with 2 important problems infection and effusion. aom is a self - limiting disease, and it has been argued that untreated infections might promote the production of antibodies and thus strengthen the immune response and improve resistance in cases of repeated exposure. furthermore, development of bacterial resistance to common antibiotics is an increasing problem in some parts of the world, which could be limited by strict indications and prescription of narrow - spectrum antibiotics. however, risk of potentially life - threatening complications clearly justifies the use of antibiotics. earlier studies of pneumococcal otitis media in the rat demonstrated that the clinical course of pneumococcal aom in the rat resembles that in human beings. mucosal changes were shown to be present during the acute phase of infection, but these changes persisted throughout the study period. mucosal alterations have been suggested to be of importance for the subsequent development of secretory otitis media, and of recurrent episodes of aom. viral, bacterial, and/or inert respiratory antigens stimulate a complicated interaction between macrophages, mast cells, eosinophils, and neutrophils. the cells produce a cascade of many types of inflammatory mediators, which result in damage by causing epithelial shedding, plasma leakage, edema, and mucus formation. of the numerous inflammatory mediators found in a mee, leukotrienes have been shown to be a major cause of edema, mucus production, and eustachian tube dysfunction [1012 ]. lt c4 and ltd4 stimulate mucus production in human airway cells in vitro and have been shown to depressive activity of human nasal cilia. cysteinyl leukotrienes are products of arachidonic acid metabolism and are released by various cells that are involved in the inflammatory cascade. arachidonic acid metabolites are important in the pathogenesis of middle ear effusion and may even be responsible for the sensorineural hearing loss that is occasionally observed with ome. it seemed appropriate to investigate the possibility that mk might be beneficial in an experimental rat model. although montelukast has long been used in clinical practice, this drug has rarely been of interest in terms of experimental rat models seeking effectiveness for aom therapy. the aim of this study was to compare the effects of penicillin and montelukast in treatment of acute and chronic acute otitis. the animals used in the present study were supervised and handled according to the dokuz eyll university medical school guidelines for care and use of laboratory animals. the experimental animals were housed in plastic cages under laboratory conditions of 28c temperature with 75% relative humidity and a photoperiod of 12-h light / dark cycle. the animals were anesthetized with intraperitoneal injections of 50 mg / kg ketamine hydrochloride (ketalar, park - davis, turkey) and xylazine hydrocloride combination. all procedures were performed under sterile conditions. in all rats, 2 ears and tympanic membranes were examined under an otomicroscope and the ears in which otitis media with effusion was observed were excluded. we administered 0.03 ml suspension of type 3 pneumococci (atcc 49619), at a concentration of 10 cfu / ml) via 26-gauge needle through the healthy tympanic membranes into the middle ear cavities of all animals. all tympanic membranes and middle ears were examined by otomicroscopic examination 48 hours after pneumococci suspension injection. the animals with aom were divided into 4 groups with 14 rats in each group. in group a (antibiotic - treated group, n=20), 14 animals (20 ears) were treated with intramuscular penicillin g (penicillin - g, pfizer warner lambert, abd) 160.000 u / kg once daily for 5 days. in group b (antibiotic and montelukast co - treated group n=23), 14 animals (23 ears) were treated with penicillin g 160.000 u / kg once daily for 5 days and montelukast na (singulair, merck sharp dohme, abd) (10 mg / kg / day) for 21 days intraperitoneally. in group c (montelukast - treated group, n=21), 14 animals (21 ears), were treated with montelukast na (10 mg / kg / day) for 21 days intraperitoneally. in group d (placebo group, n=20), 14 rats (20 ears), were not given any medication, only 2 cc phosphate - buffered saline intraperitoneally. two rats died 1 in the antibiotic and montelukast co - treatment group and 1 infected control and were excluded from the study. on days 7 and 21, 7 rats from each group were otomicroscopically examined under general anesthesia with an intramuscular injection of 50 mg / kg of ketamine hydrochloride and 50 mg / kg of xylazine. their temporal bones were removed, fixed in 10% formalin for 1 day, and decalcified with formic acid for 2 days. specimens of middle ear, including the promontory, bulla, and tympanic membrane, were divided into 2 equal parts. incisions passed through the tympanic membrane and bull and specimens were embedded in paraffin following a dehydration procedure and sectioned serially by a microtome for light microscopy (0.5 m). seven measurements were performed for each specimen : tympanic membrane parameters (thickness, vascularization, and inflammation) and mucosal parameters of tympanic bulla (thickness, vascularization, inflammation, and goblet cell metaplasia) were evaluated separately (olympus bx50, olympus optical co, tokyo, japan). the average value was recorded to increase reproducibility so that changes were graded as 0 if unchanged (as compared to the controls), 1 when mild, 2 when moderate, and 3 when severe for the following variables : mucosa and tympanic membrane thickness was graded as 1 when it had increased by > 10% but 25% but 75%. vascular changes of mucosa and tympanic membrane were graded as 1 when the vascular lumen seen in 1 field had increased by > 10% but 25% but 50%. an inflammatory cell count of mucosa and tympanic membrane 5 cells / field but 10 cells / field was designated as 3. goblet cell metaplasia was defined as the transformation of the normally flat epithelium to goblet cells. the changes are graded as 1 when the transformed cells accounted for 50% of the cells observed. bonferroni correction was used to calculate p values and a p value less than 0.01 was considered statistically significant. comparisons of 2 different data were conducted using the mann - whitney u test and a p value less than 0.05 was considered statistically significant. on days 7 and 21, 7 rats from each group were otomicroscopically examined under general anesthesia with an intramuscular injection of 50 mg / kg of ketamine hydrochloride and 50 mg / kg of xylazine. their temporal bones were removed, fixed in 10% formalin for 1 day, and decalcified with formic acid for 2 days. specimens of middle ear, including the promontory, bulla, and tympanic membrane, were divided into 2 equal parts. incisions passed through the tympanic membrane and bull and specimens were embedded in paraffin following a dehydration procedure and sectioned serially by a microtome for light microscopy (0.5 m). seven measurements were performed for each specimen : tympanic membrane parameters (thickness, vascularization, and inflammation) and mucosal parameters of tympanic bulla (thickness, vascularization, inflammation, and goblet cell metaplasia) were evaluated separately (olympus bx50, olympus optical co, tokyo, japan). the average value was recorded to increase reproducibility so that changes were graded as 0 if unchanged (as compared to the controls), 1 when mild, 2 when moderate, and 3 when severe for the following variables : mucosa and tympanic membrane thickness was graded as 1 when it had increased by > 10% but 25% but 75%. vascular changes of mucosa and tympanic membrane were graded as 1 when the vascular lumen seen in 1 field had increased by > 10% but 25% but 50%. an inflammatory cell count of mucosa and tympanic membrane 5 cells / field but 10 cells / field was designated as 3. goblet cell metaplasia was defined as the transformation of the normally flat epithelium to goblet cells. the changes are graded as 1 when the transformed cells accounted for 50% of the cells observed. bonferroni correction was used to calculate p values and a p value less than 0.01 was considered statistically significant. comparisons of 2 different data were conducted using the mann - whitney u test and a p value less than 0.05 was considered statistically significant. on day 7, in all groups, inflammatory response was as severe as in the control group, drum vessels were dilated, and tympanic membranes were thick and inflamed. on day 21, in the antibiotic - treated group, in 8 ears of 7 rats, inflammatory response was less severe than in the infected groups, but more severe than in the antibiotic - montelukast co - treated and montelukast - treated groups. in the antibiotic - montelukast co - treated group, in 9 ears of 6 rats, inflammatory response was least severe than in the infected groups and the antibiotic - only treated group. six ears showed no inflammatory sign on their tympanic membrane and 1 showed myringosclerosis. in the montelukast - treated group, in 9 ears of 7 rats, inflammatory response was less severe than in the infected groups and the antibiotic - only treated group. six ears showed no inflammatory sign on their tympanic membrane and 3 ears showed effusion. in the infected controls, in 9 ears of 6 rats, inflammatory response was as severe as on day 7. tympanic membranes of 6 rats showed effusion, 2 of them showed retraction of tm, and only 1 was normal. in several areas of the middle ear and bulla, the inflammatory response of the mucosa showed a similar pattern and the degree of inflammatory response were recorded. on day 7, 43 ears of 28 animals were histopathologically examined for 7 parameters : tympanic membrane thickness, vascularization, inflammation, mucosal thickness, vascularization, inflammation and secretory metaplasia. all groups had decreased mucosal vascularization when compared with the placebo group, but in other parameters, all groups showed similar changes (tables 1 and 2). when treated groups were compared with each other according to mucosal vascularization, there was not statistically significant difference between them (table 3). in the early phase of inflammation, polymorphonuclear leucocytes were the dominant cells of inflammation. on day 21, 38 ears of 26 animals were histopathologically examined for 7 parameters ; tympanic membrane thickness, vascularization, inflammation, mucosal thickness, vascularization, inflammation, and secretory metaplasia (table 4). in all groups, no significant difference was found in tympanic membrane inflammation, tympanic membrane vascularization, and mucosal secretory metaplasia data (table 5). in all groups, chronic inflammatory cells in the middle ear and fibrosis of tympanic membrane were observed, but in the control group, acute inflammatory cells were still observed in submucosal sections. mucosal and tympanic membrane changes were found on day 21 in all groups (figure 1). all groups showed significant decrease in tympanic membrane thickness when compared with the control group (group a but when antibiotic - treated, antibiotic and montelukast co - treated, and montelukast - treated groups were compared with each other, no significant difference was found (table 6). the antibiotic and montelukast co - treated group and the montelukast - treated group showed significant decreases in mucosal thickness when compared with the control group (p=0.007, p=0.002). in antibiotic - treated group, no significant difference was found when compared with the control group (p=0.361) (table 6). but when all 3 treated groups were compared with each other, no significant differences were found in mucosal thickness. in mucosal vascularization, there was a significant decrease in the montelukast - only treated group when compared with the antibiotic - only treated group and the control group (p=0.010 and p=0.005, respectively). no significant difference in mucosal vascularization was found between the antibiotic - only treatment and the antibiotic - montelukast co - treatment groups, between the antibiotic - montelukast co - treatment and montelukast treatment groups, and between the antibiotic - montelukast co - treatment group and the control group. in mucosal inflammation, there was a significant decrease in antibiotic - montelukast co - treatment and montelukast treatment group when compared with the control group (p=0.025 and p=0.009, respectively). there was no significant difference in the antibiotic - only treatment group when compared with the control group (p=0.358) (table 6). when the 3 treatment groups were compared with each other, when all groups were compared with each other and with placebo on day 7 and 21, no significant difference was found in goblet cell metaplasia. on day 7, in all groups, inflammatory response was as severe as in the control group, drum vessels were dilated, and tympanic membranes were thick and inflamed. on day 21, in the antibiotic - treated group, in 8 ears of 7 rats, inflammatory response was less severe than in the infected groups, but more severe than in the antibiotic - montelukast co - treated and montelukast - treated groups. in the antibiotic - montelukast co - treated group, in 9 ears of 6 rats, inflammatory response was least severe than in the infected groups and the antibiotic - only treated group. six ears showed no inflammatory sign on their tympanic membrane and 1 showed myringosclerosis. in the montelukast - treated group, in 9 ears of 7 rats, inflammatory response was less severe than in the infected groups and the antibiotic - only treated group. six ears showed no inflammatory sign on their tympanic membrane and 3 ears showed effusion. in the infected controls, in 9 ears of 6 rats, inflammatory response was as severe as on day 7. tympanic membranes of 6 rats showed effusion, 2 of them showed retraction of tm, and only 1 was normal. in several areas of the middle ear and bulla, the inflammatory response of the mucosa showed a similar pattern and the degree of inflammatory response were recorded. on day 7, 43 ears of 28 animals were histopathologically examined for 7 parameters : tympanic membrane thickness, vascularization, inflammation, mucosal thickness, vascularization, inflammation and secretory metaplasia. all groups had decreased mucosal vascularization when compared with the placebo group, but in other parameters, all groups showed similar changes (tables 1 and 2). when treated groups were compared with each other according to mucosal vascularization, there was not statistically significant difference between them (table 3). in the early phase of inflammation,, 38 ears of 26 animals were histopathologically examined for 7 parameters ; tympanic membrane thickness, vascularization, inflammation, mucosal thickness, vascularization, inflammation, and secretory metaplasia (table 4). in all groups, no significant difference was found in tympanic membrane inflammation, tympanic membrane vascularization, and mucosal secretory metaplasia data (table 5). in all groups, chronic inflammatory cells in the middle ear and fibrosis of tympanic membrane were observed, but in the control group, acute inflammatory cells were still observed in submucosal sections. mucosal and tympanic membrane changes were found on day 21 in all groups (figure 1). all groups showed significant decrease in tympanic membrane thickness when compared with the control group (group a but when antibiotic - treated, antibiotic and montelukast co - treated, and montelukast - treated groups were compared with each other, no significant difference was found (table 6). the antibiotic and montelukast co - treated group and the montelukast - treated group showed significant decreases in mucosal thickness when compared with the control group (p=0.007, p=0.002). in antibiotic - treated group, no significant difference was found when compared with the control group (p=0.361) (table 6). but when all 3 treated groups were compared with each other, no significant differences were found in mucosal thickness. in mucosal vascularization, there was a significant decrease in the montelukast - only treated group when compared with the antibiotic - only treated group and the control group (p=0.010 and p=0.005, respectively). no significant difference in mucosal vascularization was found between the antibiotic - only treatment and the antibiotic - montelukast co - treatment groups, between the antibiotic - montelukast co - treatment and montelukast treatment groups, and between the antibiotic - montelukast co - treatment group and the control group. in mucosal inflammation, there was a significant decrease in antibiotic - montelukast co - treatment and montelukast treatment group when compared with the control group (p=0.025 and p=0.009, respectively). there was no significant difference in the antibiotic - only treatment group when compared with the control group (p=0.358) (table 6). when the 3 treatment groups were compared with each other, when all groups were compared with each other and with placebo on day 7 and 21, no significant difference was found in goblet cell metaplasia. as reported previously, untreated experimental pneumococcal aom in rats causes changes in the middle ear mucosa that persist for at least 6 months. the histological changes include a thickened mucosa, an increased number of glands, and the occurrence of ciliated cells. beneficial effects on the mucosal changes were reported in experimental pneumococcal aom when penicillin v was given prophylactically or used as an early treatment. the increased goblet cell density is conceivably a result of both hyperplasia (involving cell division) and metaplasia (involving cell differentiation). to our knowledge, few middle ear data exist on these aspects, but when the tracheobronchial epithelium is exposed to various noxious stimuli, new and perhaps pre - existing goblet cells proliferate by division, and basal and indeterminate epithelial cells differentiate into goblet cells. the mechanisms leading to goblet cell hyperplasia, an epithelial cell metaplasia, are comparatively unknown, although several strong indications exist. loss of intracellular mucus to a critical level stimulates goblet cells to enter the cell cycle. mediators of the inflammatory response to bacterial colonization also play a role, as elucidated by stimulation of goblet cell hyperplasia by neutrophil lysates, inhibition by corticosteroids and nsaids, and administration of antibiotics during middle ear infection. specific mechanisms underlying these findings are partly due to products and metabolites of the cyclooxygenase and lipoxygenase pathways of arachidonic acid. these are activated through cell membrane release, induced by noxious and other agents released during inflammation. prostaglandin e and eicosanoids are examples of such products, which have been shown to induce differentiation of epithelial cells to goblet cells and the release of mucous glycoproteins, possibly through a regulatory mechanism mediated by cyclic adenosine monophosphate. but in our study, goblet cell metaplasia in mucosa was not statically significant in any group compared with infected controls. although clinical trials proving the efficiency of this combination therapy in inflammatory diseases have been reported in the literature, the effect of combination therapy with antibiotics and montelukast in experimental aom has not yet been investigated. cumbs. demonstrated that combination treatment with antibiotics and montelukast in clinical treatment showed a significantly beneficial effect on acute otitis media, compared with an antibiotic - treated group. cysteinyl leukotrienes (cyslts) are being increasingly implicated in the etiology of acute and chronic inflammatory diseases of nonallergic origin, including cardiovascular diseases, autoimmune diseases, and certain malignancies. the spectrum of proinflammatory activities of cyslts may therefore extend beyond eosinophils, monocytes / macrophages, type 2 helper t (th2) lymphocytes, and airway smooth muscle cells, to other types of inflammatory cells such as neutrophils. it also exerts a substantial and apparently direct inhibitory effect on 5-lipoxygenase activity in vitro. thus, montelukast decreases vascular permeability, inhibits inflammatory cell activation, smooth muscle proliferation, and bronchoconstriction and activates mucociliary clearance.. showed that in guinea pigs, ltd4 progressively inhibits ciliary activity, but pge2 promoted it. this may be another means by which montelukast decreases the duration of middle ear effusion after aom. the results of this study indicate that montelukast may have a place in the medical management of aom. acute otitis media commonly presents with only a partial effusion, and spontaneous resolution of the effusion generally occurs in about 50% of ears within 1 month. a full effusion is not only more durable than a partial effusion ; it is also more predictive of a clinically important conductive hearing impairment. montelukast may prove to be most appropriate and cost effective for a small subset of aom patients with bilateral disease and full effusions. however, it is obvious that earlier studies did not show effects of penicillin or montelukast different from placebo. this is probably related to the fact that the accumulating effects of the antibiotic, as well as the leukotriene antagonist (montelukast sodium), become beneficial 7 to 10 days after the inoculation (inflammation). however, the results of this study clearly show that the beneficial effects of the antibiotic (penicillin), as well as the leukotriene antagonist (montelukast), are statistically different from those of placebo in aom in rats. when the parameters of inflammation in the rat middle ear were compared with each other, most of these parameters did not show any statistically different beneficial effects in montelukast and penicillin groups.	backgroundleukotrienes are the major factors in the formation of edema and mucus, as well as development of tuba eustachii dysfunction in acute otitis media. we developed an experimental acute suppurative otitis media model and compared the responses of rats to penicillin and combinations of leukotriene antagonist with respect to histopathological observations conducted in early and late phases.material/methodsa total of 83 ears from 56 wistar rats were used in this study. pneumococcus suspension was injected trans - tympanically into all rats. subjects were classified into 4 different groups with 14 rats in each. in group a, intramuscular penicillin g was injected for a period of 5 days. in group b, intraperitoneal montelukast was injected for 21 days in addition to penicillin. in group c, intraperitoneal montelukast isotonic nacl in group d was injected into rats for 21 days.resultsno significant difference was found between the groups, except for mucosal vascularization with respect to mucosal and tm parameters in early phases. furthermore, considerable deviations were observed for the recuperation of tm and mucosal inflammation for groups in which subjects were injected with montelukast as compared to other groups of the study in the late phases.conclusionswhen the parameters of inflammation in the rat middle ear were compared with each other, most of these parameters did not show any statistically significant beneficial effects in montelukast and penicillin groups.

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