article
stringlengths 0
682k
| abstract
stringlengths 146
3.69k
|
|---|---|
] the atlantic meridional overturning circulation (amoc) is part of the ocean 's global overturning circulation, and amoc variability is linked to climate variability on seasonal to multicentennial timescales [delworth and mann, 2000 ]. the amoc is a meridionally coherent two - layer circulation with warm water flowing northward in the upper 1.1 km compensated by colder water flowing southward at depths between 1.1 and 5 km [kuhlbrodt., 2007 ]. typically these opposing flows each transport an annual average of 18 sv [rayner., 2011 ], resulting in a meridional heat transport (mht) through the south atlantic and across the equator with a maximum of 1.3 pw in the subtropical north atlantic [johns., 2011 ]. the amoc is a major component of earth 's climate system and is responsible for up to 25% of the global ocean - atmosphere meridional heat flux [bryden and imawaki, 2001 ]. much of this heat is released to the atmosphere in the subtropical north atlantic and combined with the release of locally stored summer heat, maintains the maritime climate of the uk and western europe some 510c above the zonal average for these latitudes [rahmstorf and ganopolski, 1999 ]. model simulations have linked amoc variability to climate impacts on decadal timescales [knight, 2005 ; latif., 2004 ], and observations suggest that the amoc is a fundamental component of the seasonal ocean - atmosphere heat budget [rhines., however, until now, direct observational evidence for a link between the amoc and climate has been lacking. in this study, we exploit recent improvements to the ocean observing system in the north atlantic to quantify the anomalous ocean mht associated with a transient weakening of the amoc and the associated impact on subtropical ocean heat content (ohc)., we use estimates of ocean and ocean - atmosphere heat fluxes into the subtropical atlantic ocean between 26.5 and 41n to determine the subtropical atlantic ohc budget. we estimate ocean mht divergence using the rapid - meridional overturning circulation and heat flux array (rapid - mocha) heat transport array at 26.5n [baringer and larsen, 2001 ; cunningham., 2007 ; johns., 2011 ; rayner., 2011 ] and heat transports at 41n derived from available argo floats and satellite altimetry [hobbs and willis, 2012 ; willis, 2010 ] (see section s1 in the supporting information). air - sea surface fluxes are obtained from the era - interim atmospheric reanalysis [dee., 2011 ], and we assume that ohc anomalies at depths greater than 2000 m are negligible. to examine the contributions of meridional heat transport and air - sea fluxes, we define an ohc budget as follows (section s2) : where ohc [j ] is the ocean heat content, t is time and t0 is an initial reference time. f is the air - sea heat flux across the sea surface, s and n are ocean mhts through the southern (26.5n) and northern (41n) boundaries, respectively [j s = w ], and primes denote anomalies relative to a seasonal climatology. to estimate the seasonal cycle in s, we use the first 5 years of the rapid - mocha observations from 1 april 2004 to 31 march 2009 (the reference period) when a seasonal cycle was robustly present, as shown by kanzow. (section s1), but not using data after april 2009, which include the anomalous variability being investigated. this approach was also taken for n. however, mhts and mht divergence is not sensitive to a reference period spanning the anomalous period (section s1). for f, a much longer reanalysis period from 1991 to 2010 was used to calculate a representative seasonal cycle that was insensitive to the inclusion of data after 2009. f is also insensitive to a choice of reference period matching the period of s and n (section s2). for s and n, removal of a seasonal cycle estimated during the reference period (by definition) results in a net zero mean for the integrated mht during the reference period. for f, subsequent to the removal of the seasonal cycle, we adjust the integrated anomalies to have zero mean during the reference period. [5. ] the purposefully designed rapid - mocha array has been measuring the strength and structure of the amoc at 26.5n since april 2004 [cunningham. these measurements recently revealed that the annual average amoc decreased by 30% over a 12 month period beginning in april 2009 [mccarthy., associated with this transport anomaly, there is a sustained cooling of the upper 2000 m of the subtropical atlantic ocean (defined here as the region between 26.5n and 41n) (figure 1a). the most intense cooling was in the mixed layer during the winter months of 2009/2010 and 2010/2011, with temperature anomalies in excess of 0.5c (figure 1b). during the summers of 2010 and 2011, warm anomalies were present at the ocean surface, whereas cold temperature anomalies were sustained below the mixed layer depth. from late 2009, subtropical atlantic ohc decreased over a 12 month (figure 1c) period to a minimum value of 1.3 10 j, then gradually increased during 2011 to 0.5 10 j. these ohc anomalies are robust to incomplete spatial sampling and are not driven by a systematic change to the observing system (section s3). with the exception of a brief 2 month cool period at the end of 2006 that appears localized to the gulf stream location along 41n (section s4), ohc anomalies do not exceed our empirically derived 95% confidence limits until the persistent cooling from 2010. (a) measurement locations and region of interest. bathymetry (0 and 2000 m contours) and topography (grey) of the north atlantic region with the northern and southern limits of the volume shown by black lines at 26.5n and 41n. at 26.5n, the florida cable is given by the short segment between florida and the bahamas, and the principal rapid 26.5n array mooring positions are indicated by dots. (b) area - averaged subsurface temperature anomalies in the subtropical north atlantic (582w, 26.541n) calculated relative to the 19912010 seasonal cycle using monthly means from the en3 v2a gridded objective analysis of quality - controlled subsurface temperature observations [ingleby and huddleston, 2007 ], (http://www.metoffice.gov.uk/hadobs/en3/). black lines indicate the area - averaged depth of selected isotherms across the same region. mean mixed layer depth (white) and the 95th percentile (grey) for which only 5% of grid boxes have a deeper mixed layer defined by kara.. the mld is the depth over which air - sea exchanges drive turbulent mixing of the ocean. (c) six month low - pass filtered ocean heat content anomalies relative to 19912010 above 2000 m (solid black), above the 4c isotherm (red) and above the 14c isotherm (dashed black). ohc uncertainties (grey) are generated by model - based estimates associated with changes in sampling density and locations and are for 10 day values. they are approximated by the ohc uncertainties shallower than 2000 m and 500 m respectively (section s3). the spatial pattern of the ohc decrease is coherent across much of the subtropical atlantic indicating a large - scale forcing, and is not due simply to a shift in the position of the gulf stream (section s5). by july 2010 the negative ohc anomaly extends in a broadband across the subtropical gyre, extending southwestward to 11n in the western atlantic. the total ohc change in the upper 2000 m can be divided into two approximately equal components : (1) changes in the seasonally mixed layer above the 14c isotherm where ocean advection and air - sea heat fluxes contribute and (2) changes between 4c and 14c that are outside the influence of seasonally mixed layer and air - sea fluxes, indicating a role for advection in driving the observed cooling at depth. the observed mhts at 26.5n and 41n are 1.26 0.11 pw and 0.48 0.11 pw, respectively, for the reference period 1 april 2004 to 31 march 2009 (mean sd) (figure 2a and table 1). for both latitudes, the amoc is the dominant mechanism carrying the mht with the horizontal gyre accounting for 10% and 20% (26.5n and 41n respectively) of the total mht [johns., mht variability associated with the amoc is determined almost entirely by circulation changes [jayne and marotzke, 2001 ; johns., for example, at 26.5n the correlation between the amoc and mht variability is r = 0.94 [johns., (a) deseasonalized 6 month low - pass time series of the meridional heat transports [pw ] from the rapid / mocha array at 26.5n (black) [johns., 2011 ] and from the argo / altimetric estimate at 41n (black dashed) [hobbs and willis, 2012 ]. the rapid / mocha at 26.5n data have a temporal resolution of 0.5 days, and 41n data are 3 monthly. the error (grey) for daily values of heat transport at 26.5n is 0.2 pw and at 41n for monthly values is 0.22 pw. horizontal black lines are the mean of the time series from 1 april 2004 to 31 march 2009. both time series are deseasonalized by calculating a mean seasonal cycle for the period 1 of april 2004 to 31 march 2009 (section s1). (b) heat transport divergence (4126.5n) set to zero mean in the period 1 april 2004 to 31 march 2009, where values above zero indicate cooling of the subtropical atlantic. the uncertainty (grey) is the root - sum - square error from 26.5n and 41n. deseasonalized mean and standard deviation of the meridional heat transport at 26.5n and 41n and associated divergence [pw ] [8. ] the most striking feature of the observed records of mht is the large decrease across 26.5n beginning in april 2009 and continuing through spring 2010. this anomaly is only weakly compensated by reduced export of heat at 41n (table 1) and results in a maximum of anomalous mht divergence during the same time period (figure 2b). overall, the interannual variability of mht to the subtropical atlantic is dominated by the amoc strength at 26.5n. while part of the reduction in 2009/2010 is due to a reversal in ekman transports caused by the negative nao in winter 2009/2010, the longer term reduction was driven by an increase in the southward geostrophic transport in the gyre interior [mccarthy., 2012 ]. [9. ] to calculate the ohc change in the subtropical atlantic due to changes in ocean mht and atmospheric heat fluxes, we integrate the ocean mht divergence between 26.5n and 41n and era - interim air - sea heat fluxes through time (section s7). ohc decreases abruptly in the last months of 2009 and early 2010 (figures 3b and 3c), due to mht divergence and reduced surface fluxes into the subtropical north atlantic. the relatively abrupt increases in heat loss due to air - sea fluxes in djf 2009/2010 and december 2010 coincide with two exceptionally negative phases of the nao, such that increased wind speeds over the subtropical gyre result in large anomalies in latent heat loss to the atmosphere (section s8). in contrast, the mht divergence anomaly, that we have shown to be dominated by the amoc at 26.5n, is more sustained and acts to cool the subtropical atlantic through 2010. (a) time series of observed relative heat content above the 4c isotherm determined from the en3 v2a argo data set 6 month low - pass filtered. uncertainties for ohc above the 4c isotherm are approximated by the uncertainty in ohc above 2000 m. (b) integrated ocean heat transport divergence between 26.5n and 41n. (d) ohc budget residual (figures 3a (3b + 3c)). there is also a positive mht divergence in 2005 caused by a decrease in mht at 26.5n and an increase in mht at 41n. while the mht changes at the individual latitudes are not significant, their combined effect produces a positive mht divergence of nearly the same magnitude, but shorter duration than in 2009/2010 (figure 2b). this effect is apparent in the integrated mht divergence (figure 3b), which indicates a cooling between 2005 and 2006. however, although there is a corresponding decrease in ohc (figure 3a), this decrease is not significantly different from zero relative to the estimated error for ohc. [11. ] within errors, the heat budget is balanced, both during the reference period between april 2004 and march 2009 and after april 2009 (figure 3d). the magnitudes of the energy fluxes quantified indicate that the recent cooling in the subtropical atlantic is primarily due to a reduction in the amoc at 26.5n. the mean mht divergence over the subtropical atlantic derived from the mht estimates of figure 2 (0.20 pw) implies a surface heat loss over the subtropical atlantic of 74 19 w m. this is inconsistent with the time - mean values for era - interim air - sea heat fluxes of 19.4 w m an imbalance of 56.4 wm between mht and air - sea fluxes would imply a net ohc change of 12 10 j, which is not observed in figure 3a. however, our analysis is not sensitive to the choice of atmospheric reanalysis as anomalies in era - interim and ncep [kalnay., 1996 ] air - sea heat flux anomalies agree closely, despite a mean difference of 17 w m. this is evidence that air - sea heat flux anomalies are likely better constrained than climatological values (section s9). errors in the climatological values for ocean mht divergence or atmospheric fluxes would result in spurious linear trends after the time integration of anomalies (section s2). we are seeking to explain interannual anomalies in subtropical ohc and not trends, and the integrated ocean mht divergence and atmospheric fluxes are defined to have zero mean for the reference period 20042009 (figures 3b and 3c). extending the reference period to the end of the mht time series by definition results in no net change in ohc due to the integrated mht divergence and atmospheric fluxes but forces a significant trend in ohc throughout the time series (section s2). since within errors no trends are observed in subtropical ohc during the reference period (figure 3a), we conclude that the mean fluxes are nearly balanced and that our assumption of zero net heat divergence for the reference period 20042009 is justified. previous studies argue that, in the midlatitudes, the ocean plays a largely passive role in the generation of upper ocean temperature anomalies on short (monthly - to - interannual) timescales [frankignoul, 1985 ]. in this paradigm, temperature anomalies in the surface mixed layer are generated by the integrated ocean response to stochastic atmospheric forcing of surface fluxes combined with stabilizing negative feedbacks that act to dampen anomalies on timescales of 6 months. in certain regions of the ocean (including the midlatitude north atlantic), it has also been shown that thermal anomalies in the winter mixed layer can persist below the seasonal thermocline and reemerge the following year as the mixed layer deepens [alexander and deser, 1995 ; hanawa and sugimoto, 2004 ]. other studies have suggested that ocean dynamics could play a more active role, with changes in the atmosphere leading to ocean mht anomalies and ocean - atmosphere feedbacks [marshall., [14. ] to examine the processes responsible for ohc changes in the seasonal mixed layer, we define relative heat content change (rhc) following [palmer and haines, 2009 ] (section s10), partitioning ohc anomalies into contributions from changes in the mean temperature above the 14c isotherm (rhct) and changes in the volume of water above the 14c isotherm (rhcv). the advantage of this approach is the ability to separate air - sea heat flux driven changes (rhct) from those arising from changes in ocean circulation (rhcv). the separation into volume and temperature relative ohc contributions presented by palmer and haines is supported here by the close agreement between rhct above 14c and the time integrated era - interim air - sea fluxes (figure 4c). during the winter months of 2009/2010 and 2010/2011, the mean winter mixed layer depth corresponded to the average depth of the 18c isotherm (200 m), but in 5% of the subtropical gyre, minimum mixed layer temperatures were as cold as 14 c (figure 1b). the overall agreement between the surface fluxes and rhct is consistent with our observation that the winter mixed layer depth, and thus the influence of air - sea fluxes, is largely contained above the 14c isotherm. (c) heat content change due to temperature changes rhct and integrated surface heat fluxes from era - interim (red). rhc uncertainties (grey) are generated by model - based estimates associated with changes in sampling density and locations. for rhc warmer than 14c, the rhc error is approximated by the ohcu uncertainty shallower than 500 m (section s3). the total fractional uncertainties in rhcv and rhct are 0.83 ohcu and 0.55 ohcu according to palmer and brohan. rhcv above the 14c isotherm declines throughout 2010, suggesting a reduction in volume due to reduced convergence in this layer. the total rhc change above 14c declines by 0.5 10 j throughout 2010 and 2011 (figure 4a), of which rhct = 0.3 10 j is due to changes in the average temperature (figure 4c) and rhcv = 0.2 10 j due to changes in the volume (figure 4b) : a 60:40 split in heat content changes due to air - sea fluxes and circulation changes, respectively. [17. ] the winter (djf) of 2009/2010 and december 2010 were both characterized by extremely negative phases of the north atlantic oscillation. modeling studies using seasonal forecast systems have suggested that the negative north atlantic oscillation (nao) during winter 2009/2010 was driven by dynamic processes internal to the atmosphere [fereday., 2012 ; in contrast, it has been suggested that the return to a negative nao phase during december 2010 may have been driven by the reemergence of existing subsurface temperature anomalies in the north atlantic [taws.,, demonstrate that anomalous ohc and associated sea surface temperature anomalies in the north atlantic are indeed responsible for successful forecasts of the negative nao state in december 2010. this hypothesis is consistent with modeling and observational studies that have suggested predictability of the nao based on knowledge of preceding atlantic sea surface temperature (sst) anomalies [cassou. our analysis suggests that ocean advection played a significant role in driving the negative temperature anomalies of the seasonally mixed layer in the subtropical atlantic during 2010. building on earlier work linking subsurface temperature anomalies and predictability of the nao [cassou., 2007 ], these results suggest that the ocean mht may be active in the events leading to the extreme negative phase of the nao during december 2010. [19. ] observations show that there has been an abrupt and sustained cooling in the upper 2 km of the subtropical atlantic between 2010 and 2012. the associated reduction in ohc is partitioned equally between the seasonally active mixed layer and the deeper ocean (below the 14 c isotherm). we find that a reduction of the amoc at 26.5n is the largest contributor to the observed cooling in subtropical atlantic. in the seasonally mixed layer, temperature anomalies are a result of both heat loss to the atmosphere and reduced mht associated with a reduction in the amoc. our results emphasize the role for the ocean in the north atlantic climate system on seasonal - to - interannual timescales and suggest a role for the amoc in setting subsurface temperature anomalies that have previously been linked to reemerging sst patterns and subsequent anomalies in the nao [taws., 2011 ] and predictability of december 2010 nao negative state [maidens., 2013 ]. finally, 50 % of the observed ohc anomaly is outside the influence of the surface mixed layer and essentially isolated from the atmosphere on seasonal timescales. we speculate that these temperature anomalies may become climatically relevant on longer (e.g., decadal) timescales | [1 ] observations show that the upper 2 km of the subtropical north atlantic ocean cooled throughout 2010 and remained cold until at least december 2011. we show that these cold anomalies are partly driven by anomalous air - sea exchange during the cold winters of 2009/2010 and 2010/2011 and, more surprisingly, by extreme interannual variability in the ocean 's northward heat transport at 26.5n. this cooling driven by the ocean 's meridional heat transport affects deeper layers isolated from the atmosphere on annual timescales and water that is entrained into the winter mixed layer thus lowering winter sea surface temperatures. here we connect, for the first time, variability in the northward heat transport carried by the atlantic meridional overturning circulation to widespread sustained cooling of the subtropical north atlantic, challenging the prevailing view that the ocean plays a passive role in the coupled ocean - atmosphere system on monthly - to - seasonal timescales. |
taste recognition and memory are linked to hedonic values such as " attractive ", " palatable " or " repulsive ", and also to degree of familiarity, and nutritive or toxic characteristics. the most commonly used taste - memory models are taste preference, conditioned taste aversion (cta) and latent inhibition of conditioned taste aversion (li - cta). the consumption of a novel taste that is not associated with negative visceral consequences leads to the formation of a non - associative form of sensory memory, known as incidental taste memory. habituation followed by exposure to a novel taste, choice preference, and attenuation of neophobia are the models that are most widely used to decipher incidental taste learning and memory. cta is an associative learning paradigm ; by means of cta, organisms learn to avoid a novel food substance (conditioned stimulus, cs) associated with delayed poisoning (unconditioned stimulus, us) [1 - 3 ]. cta can be acquired in a single trial and it differs from other associative paradigms in the long delay between cs and us. in li - cta, a novel taste (cs) is learned days before a cta trial, and it results in retarded performance of a cta task ; a positive tag of the learned taste is established and, consequently, weaker aversion is expressed [4, 5 ]. experimentally, following 3 days of restricted access to water, animals sample novel taste solution. since animals learn novel taste without any negative visceral consequence, they both remember the taste as incidental memory (i.e. no ucs) but also learn that the taste is safe. whereas in cta, lithium chloride (0.05~0.15 m) is injected 40 minutes after the drinking session (ucs), which elicits gastric pain and incomfort and therefore animals learn the given taste as an aversive one. the most commonly used novel taste is saccharin (0.1~0.5% w / v in plain tap water) and sodium chloride (0.1~0.5% w / v also in plain tap water) [4, 5 ]. there is extensive overlapping between the gustatory neural circuits associated with the acquisition and expression of novel - taste and aversive - taste memories. the neural circuitry of taste learning includes the nucleus of the solitary tract (nst, also known as nucleus of tractus solitarius - nts), the parabrachial nucleus (pbn), the parvocellular part of the ventralis postmedial thalamic nucleus of the thalamus (pvpmpc), the nucleus basalis magnocellularis (nbm), the amygdala (amy) and the insular cortex (ic) [5 - 7 ]. recent findings indicate that the nucleus accumbens (nacc), prefrontal cortex (pfc) and perirhinal cortex (pc) also participate in taste - memory formation [8 - 16 ]. although the hippocampus (hip) has been shown to be involved in several forms of learning, its involvement in taste learning is unclear. hippocampal lesion studies in the past decade have yielded inconclusive results regarding the role of the hip in taste learning and cta [17 - 23 ]. recent studies demonstrated that the hip is necessary for the consolidation of attenuation of neophobia, and inhibition of protein synthesis in the hip impairs consolidation of cta extinction. interestingly, another study indicated thatvarious molecular pathways were activated in the hip and in the ic, during different temporal windows following novel taste learning. clearly, further studies are required to identify and define the role of the hip in taste learning. many studies have shown that the ic and amy are highly involved in taste - memory formation, consolidation and retrieval, therefore, in this review we will focus on delineating the prominent role of protein phosphorylation in taste learning in these two temporal lobe structures. memory consolidation, a progressive process of post - acquisition stabilization that facilitates the permanent storage of memory, is dependent on the gene expression and synthesis of new proteins within the relevant brain structures. recent studies elucidated the mechanisms of gene expression and the regulation of translation that underlies taste memory consolidation. phosphorylation of the transcription factor, camp - response element - binding protein (creb) on serine 133 causes it to bind the camp response element (cre), which is then bound to a creb - binding protein (cbp). this complex, in turn, leads the creb to turn the transcription of certain geneson oroff, and creb has been shown to be phosphorylated directly by camp - dependent protein kinase (pka) and ca / calmodulin - dependent protein kinase iv, and indirectly by the extracellular signal - regulated kinase (erki / ii) cascade through activation of rsk2, which is a member of the pp90rsk family of s6 kinases [27, 28 ]. novel - taste learning increased creb phosphorylation in the hip but, interestingly, not in the ic, whereas intraperitoneal (i.p.) moreover, cta conditioning increased creb activation in the lateral septum, lateral amygdala (la) and ic [29, 30 ]. phosphorylated creb activated the transcription of many genes, including c - fos, brain - derived neurotropic factor (bdnf), and ccaat / enhancer binding protein (c / ebp) [31, 32 ]. bdnf stimulated creb phosphorylation, and this creb phosphorylation played a central role in mediating the bdnf response. cta conditioning was correlated with increased expression of bdnf protein from 4 to 12 h after conditioning in the central nucleus of the amygdala (cea), and from 8 to 12 h after conditioning in the ic. also have shown that association of novel taste and malaise was essential for bdnf signalling to be involved in taste learning. this is consistent with previous studies, which found no changes in creb phosphorylation in the ic following novel - taste learning, and activation of creb in the la only following cta. moreover, novel - taste consumption and association of novel taste with malaise in cta learning elicited increased expression levels of c - fos - an immediate early gene whose expression is mediated by creb - in the cea and ic [34 - 36 ]. furthermore, activation of c - fos in the ic and amy has been found to be required for cta memory formation [15, 37 ]. these particular examples clearly suggest that some proteins, e.g., bdnf, that are implicated in taste memory consolidation are mainly related to the consolidation of cs - us association with cta and not to cs or us exposure alone. antisense oligonucleotide - mediated inhibition of creb in the amy, particularly in the cea, has been shown to impair cta memory, and creb mutant mice have also been found to be impaired in cta memory, which suggests that the activity of creb is vital for cta memory formation. another transcription factor that was induced in correlation with taste learning is c / ebp, which also has been associated with hip - dependent learning processes [40 - 42 ]. have found increased expression levels of c / ebp protein 18 h following novel - taste consumption ; furthermore, it is interesting thatfurther study demonstrated that this correlative induction of c / ebp could be deleted if another novel taste was consumed 4 h after the first one, i.e. retrograde amnesia, which suggests that a long process of biochemical changes leads to the increased expression of the proteins required for taste - memory consolidation. lysine acetylation of histones alters the higher - order chromatin structure and exposes dna to transcription processes. novel - taste consumption has been shown to be associated with increased activity of histone acetyl transferase in the ic. moreover, this increased lysine acetylation of p42ack and p55ack proteins appears to be at least partly downstream of activation of erki / ii in the ic, which suggests that novel - taste consumption enhances the chromatin structural reorganization and thereby leads to increased transcription. memory consolidation is generally considered to be a progressive stabilization process that requires the synthesis of new proteins. indeed, pharmacological studies based on the protein - synthesis inhibitor, anisomycin demonstrated the general requirement for protein synthesis in novel - taste learning and cta memory consolidation [4, 45 - 47 ]. translation is the mechanism by which the relevant neurons fine - tune the levels of proteins in both time and space. the primary target for translation regulation is the initiation phase, therefore, key roles in controlling translation are played by translation initiation factors through phosphorylation of the various initiation factors and ribosomal proteins [48, 49 ]. novel - taste learning enhances the phosphorylation of ribosomal protein s6 kinase1 (s6k1) on threonine 389, whereas this phosphorylation was positively correlated with enhanced initiation of translation. interestingly, within the same temporal window, there was an increase in the phosphorylation of a eukaryotic elongation factor (eef2) - whose phosphorylation correlates with a general reduction of the elongation process in translation - on threonine 56 [52 - 54 ]. these results indicate that although novel - taste consumption increases the general initiation of translation, it decreases the elongation rate. on the assumption that the processes take place in the same cell, it is reasonable to suggest that during taste - memory consolidation, in spite of the general increase in the initiation phase of translation, elongation becomes the rate - limiting step allowingonly of poorly initiated mrnas for further translation. this distinct population of mrnas consists of plasticity - related mrnas, such as c / ebp. another hub molecule in translation control is mammalian target of rapamycin - sensitive kinase (mtor). phosphorylated mtor on serine 2448 can regulate several downstream elements of the translation machinery, including s6k1 and eukaryotic elongation factors 1a and 2 (eef1a and eef2). novel - taste learning induced a correlative activation of mtor in a biphasic pattern, at 15 min and 3h following novel - taste learning. moreover, phosphorylation of s6k1 has also been shown to be increased within the same temporal window. biochemical fractionation analysis revealed that phosphorylation of s6k1 was increased in a synaptoneurosomal preparation at 15 min but not at 3 h following novel - taste learning, indicating the importance of spatial segregation of signaling molecules during taste - memory formation. unlike general protein synthesis inhibitors such as anisomycin and cycloheximide, rapamycin selectively inhibits the translation of certain populations of mrnasthat contain an oligo - pyrimidine tract at their transcriptional start (5'top), most notably mrnas encoding ribosomal proteins and elongation factors [57, 58 ]. inhibition of mtor by microinjection of rapamycin locally into the ic reduced expression of eef1a and post - synaptic density protein 95 (psd-95), reduced s6k1 phosphorylation, and increased eef2 phosphorylation ; at the same time, this inhibition of mtor in the ic attenuated long - term taste memory as analyzed by li - cta paradigm. these results support the notion that there is a selective control of translation of proteins during taste - memory consolidation. behavioral analyses of transgenic mice with specific modifications in the translation machinery revealed clear attenuation of taste memory (table 1) : s6k1 knock - out mice showed clear attenuation of taste memory ; the mutant mice that lacked the translation repressor, eukaryotic initiation factor 4e - binding protein (4e - bp2) exhibited enhanced formation of the eif4f complex, and analysis of taste learning in these mice revealed no change in taste recognition, but enhanced cta learning. similarly, mice with reduced phosphorylation of eukaryotic initiation factor 2 alpha (eif2) exhibited enhanced taste learning, but no effect on taste recognition. protein degradation in parallel with protein synthesis regulates the proper turnover of proteins and the quality of the cellular proteome. the ubiquitin- proteasome system (ups), and lysosome- and proteases - mediated proteolysis are the main routes of protein degradation. whereas the lysosome accounts for protein turnover of ~20% in cells, the ups is highly conserved and serves as a principal means of targeting proteins for degradation [63, 64 ]. pharmacological and genetic studies that demonstrated the relationship between the ups and several forms of learning and memory have been reviewed extensively [63 - 65 ]. briefly, pharmacological inhibition of the ups in the hip blocked long - term memory formation in a one - trial inhibitory avoidance task and in a morris water - maze task, and prevented extinction of contextual fear memory [66 - 68 ]. activity have been observed 4 h following a one - trial inhibitory avoidance task in the hip. similarly, polyubiquitinated postsynaptic proteins such as shank and gkap were increased in the hip 1 h after retrieval of contextual fear memory. taken together, these findings indicate that, in addition to gene expression and protein synthesis, the degradation of proteins may be equally important for long - term memory formation. bilateral microinjection of the ups inhibitor, lactacystin into both the ic and the amy appeared to impair the cta memory ; however, the cellular events and mechanisms, and the temporal activity of the lactacystin underlying this behavioral phenotype have not been clearly described. important kinases that are well documented in the field of learning and memory processes are : erki / ii, ca / phospholipid - dependent protein kinase (pkc), pka, and ca / calmodul - independent protein kinase ii (camkii). within the scope of the present review, we will focus on the rolesof these fourpathways in taste learning and memory. activation of erki / ii - a serine / threonine kinase with the two isoforms erki / ii (p42/44) belonging to the mitogen - activated protein kinase (mapk) family of signal cascades - is necessary for several forms of learning and memory. several lines of evidence showed activation of erki / ii by phosphorylation on threonine 202/ tyrosine 204, following novel taste learning [44, 50, 70, 71 ]. however, the temporal window of the activation is not clear ; for example, several studies have shown activation of erki / ii in the ic a few minutes after novel - taste consumption, but found it undetectable after more than 1 h [25, 50, 70, 71 ]. however, swank and sweatt have found sustained activation of erki / ii lasting between 2 and 6 h, accompanied by a second wave of activation in the ic at 24 and 48 h following novel - taste learning in mice. interestingly, when differences in the activation of erki / ii are observed in different studies, there is a tendency to attribute them to differences among the experimental setups. the studies that demonstrated immediate and transient activation of erki / ii have all used the rat model with water as a control [25, 50, 70, 71 ], whereas swank and sweatt used the mouse model with a time point of zero as a control. it is also important to note that most of the studies, including those that observed transient activation of erki / ii, used novel taste in the form of solutions such as saccharin, nacl, etc., but swank and sweatt used blueberry bar, a solid taste source, thus indicating that the texture and the odor of the food, as well as its taste, also plays a key role in taste learning and memory. further examination revealed phosphorylation of elk-1 - a substrate of activated erki / ii - concomitant with muscarinic and n - methy - d - aspartate receptor (nmdar) activation in the ic, following novel - taste learning. as shown in table 2, novel - taste learning also increased the phosphorylation and activation of several members of the mapk pathway, such as raf on serine 259, mek on serine 217/221, p90rsk on serine 381, and jun n - terminal kinase 1/2 (jnk1/2) [44, 70 ]. it has also been shown that consumption of a given novel taste for the second time, but not for the fourth time increased erki / ii activation. these results indicate that erki / ii activity in the ic is no longer significant once a taste has become familiar [44, 70 ]. as in the ic, cta conditioning was found to increase activation of erki / ii in the la and ic. pharmacological inhibition of mek, an upstream kinase of erki / ii, in the ic or amy attenuated cta memory [70, 72 ]. interestingly, the expression of long - term potentiation (ltp), a neural model of learning and memory, in the ic was found to be erki / ii - dependent. as in other behavioral paradigms and brain regions, erki / ii activation is highly correlated with taste learning, probably mainly during the consolidation phase of learning. as mentioned elsewhere [3, 5 ], the upstream mechanism of erki / ii activation has been extensively studied, but the downstream targets of activated erki / ii during taste learning, and especially during consolidation, remain elusive. one of the major roles of activated erki / ii is its involvement in translation regulation. as shown in fig. 1, activated erki / ii enhances the transcription via creb and translation of synaptic - plasticity - related proteins directly through its downstream kinases during taste learning and memory. pkc comprises a family of serine / threonine kinases that has been reported to be necessary in the ic, amy and pbn for formation of a cta memory [75 - 77 ]. injection of us elicited pkc enzyme activity in the membrane - bound fraction in the pbn and, furthermore, cta conditioning increased the activity of pkc persistently for 48 h in the pbn [78, 79 ]. however, despite the identification and characterization of 11 previously known pkc isoforms, the functional role of each isoform in taste learning is not clear, and the temporal activities of pkc in the ic and amy remain to be identified. a constitutively active pkc isoform m zeta (pkm) has been shown to be important for ltp maintenance and hip dependent long - term memory (sacktor, 2011). pharmacological inhibition or lentivirally mediated overexpression of pkm led, respectively, to erasure or enhancement of long - term cta memory [80 - 83 ]. pkm expression was increased in the ic 2 weeks after cta conditioning - a finding that correlated with the long - term cta memory. interestingly, application of the pkm inhibitor, zip into the amy 15 min before us association, but not 30 min after cta conditioning, attenuated cta memory. taken together, these results reveal the importance of pkm in the early and late stages of consolidation of cta memory in the ic ; its role in the amy may be related to the acquisition or early consolidation phase of cta. pharmacological studies showed that inhibition of pkc in the ic, amy or pbn impaired long - term cta memory formation, without affecting short - term cta memory or attenuation of neophobia in the ic [34, 76, 77 ], which suggests that pkc plays a prominent role in formation of aversive - taste memory, but not of novel - taste memory. pka, a serine / threonine kinase composed of four regulatory and two catalytic subunit isoforms has been shown to be activated by camp. although it has not been investigated whether novel taste and/or cta influence the expression and activation of pka, behavioral analyses that involved the targeted disruption of rii subunits of pka in mice, and pharmacological studies using pka inhibitors have shown that the blockage of pka activity in the ic or the amy impaired long - term but not short - term cta memory [85 - 88 ]. further pharmacological study that microinjected camp analog, 8-bromo - adenosine 3'-5 ' camp, into the ic 30 minutes after novel taste consumption and immediately before us, has shown that increasing camp enhanced cta memory. camkii is a serine / threonine kinase that is a highly abundant protein in the post - synaptic density (psd) ; it has a broad range of postsynaptic, membrane - bound and cytoplasmic substrates. activation of camkii, an isoform of camkii, by ca / cam enables intra - molecular auto - phosphorylation of several sites, including threonine 286, 305 and 306. auto - phosphorylation of camkii on threonine 286 enables the subsequent dissociation of bound ca / cam, thereby prolonging camkii activation, which enables partial activity even after full dissociation of ca / cam. thus, camkii can translate a transient event of elevation of intracellular ca into a prolonged change that is independent of that transient change [90, 91 ]. the roles of camkii in synaptic plasticity and in various learning tasks have been intensively investigated [90, 92 - 96 ], but very little is known regarding the role of camkii in taste learning. biochemically, novel - taste consumption was shown to increase expression of camkii protein in the ic. it is important to note that in vitro studies of the enzyme activity that followed ctahave shown that it attenuated the activity of camkii in the pbn. however, a pharmacological study with a classical camkii inhibitor, kn-62 has shown that inhibition of camkii in the pbn following noveltaste and/or cta elicited cta. clearly, many more studies are needed to determine whether the activation of camkii is necessary for taste learning and memory and, if so, what is the nature of its role(s) in these processes. although the functions of protein phosphorylation by protein kinases in learning and memory have been studied extensively, recent studies also highlighted the importance of dephosphorylation by protein phosphatases. protein phosphatases (pp) can be grouped into three main classes, according to sequence, structure, and catalytic function. phosphoprotein phosphatases constitute the largest family, comprising pp1, pp2a, pp2b (also known as pp3), pp4, pp5, pp6, and pp7 and the mg-/mn - dependent (ppm) family, designated pp2c. the superfamiliesof protein tyr phosphatases and the aspartate - based protein phosphatases, respectively, form the second and third classes. the importance and the functional role of pp1/2a in several forms of learning and memory have been documented and, in addition, recent findings also showed the importance of pp1/2a in ltp and ltd [101 - 108 ]. decreased activity of calcineurin (pp3) was observed selectively in the amy 40 min after second cta extinction, whereas pp1 remained unchanged. conditional transgenic mice with genetically inhibited calcineurin showed improved cta memory and enhanced resistance to cta extinction. conversely, conditional transgenic mice with increased calcineurin activity showed impaired memory and substantially faster cta extinction. however, the inhibition or over - expression of calcineurin that followed the cta training appeared not to affect the cta memory, which suggests that calcineurin plays a major role during the cta acquisition. in support of these transgenic animal findings, a recent pharmacological study using okadaic acid - a potent inhibitor of pp1 and pp2a phosphatases - showed that inhibition of pp1 and pp2a enhanced the cta memory if injected locally into the amy 5 min before, but not after cta acquisition. nmda receptors are composed of glun1, glun2 (glun2a - d), and glun3 (glun3a - b) subunits. although the interpretation is not yet definitive, a large body of evidence supports a tetrameric arrangement of nmda receptors, incorporating two glun1 and two glun2 subunits of the same or different subtypes [111 - 114 ]. glun3 co - assembles with glun1 and glun2 to form ternary glun1/ glun2/ glun3 tetrameric complexes, and glun3 has also been hypothesized to serve as an alternative for one of the glun2 subunits [114 - 117 ]. the available evidence indicates that nmdar modulation is mediated through phosphorylation of intracellular loops of its subunits. activation of pka, pkc, fyn (of the src family) kinase, and camkii has been shown to phosphorylate nmdar subunits. the first indication that the nmdar undergoes phosphorylation following taste learning arose from the finding that the net tyrosine phosphorylation was enhanced in several proteins in the rat ic following novel - taste and cta learning. a subsequent study demonstrated that consumption of a novel taste led to increased tyrosine phosphorylation of the glun2b subunit of the nmda receptor in the ic, and that it returned to baseline levels once the taste became familiar. an important finding was that this tyrosine phosphorylation wasindependent of nmdar activity, as indicated by the observation that local application of the nmdar antagonist,2-amino-5-phosphonovaleric acid (apv), did not affect the tyrosine phosphorylation. however, local injection of the muscarinic acetylcholine receptors (achr) agonist, carbachol into the ic mimicked this tyrosine phosphorylation, and release of ach in the ic following novel - taste consumption has also been observed [121, 122 ]. nevertheless, it remains to be determined whether the prolonged endogenous release of ach is the physiological reason for tyrosine phosphorylation of glun2b. it is noteworthy that the release of dopamine was observed following both novel - taste learningand cta, whereas glutamate release was observed following only cta, but not after novel - taste consumption. more specifically, recent findings have shown a correlation between fyn kinase - dependent increase in tyrosine phosphorylation on residue 1472 of glun2b and novel - taste learning. moreover, this increased tyrosine 1472 phosphorylation of glun2b reinforced the association of this nmdar subunit with psd95, a highly abundant psd scaffolding protein that has crucial importance for learning and memory processes. in addition, local injection of the tyrosine kinase inhibitor, genistein impaired novel - taste memory in the li - cta paradigm, and a correlative increase in psd-95 expression was observed after novel - taste learning. as was observed in connection with the enhanced general tyrosine phosphorylation of glun2b that followed novel - taste learning and cta [118, 119 ], novel - taste learning also has been shown to increase the general serine phosphorylation of glun2b. however, this phosphorylation diminished once the taste became familiar, which was not observed in connection with the tyrosine phosphorylation of this protein. although pkc and camkii were shown to phosphorylate glun2b at different sites on serine residues [126, 127 ], it is not clear whether pkc / camkii mediates this glun2b serine phosphorylation. therefore, it will be intriguing to check which of the serine / threonine protein kinases phosphorylates this observed glun2b phosphorylation that followsnovel - taste learning. studies have shown that relocalization of nmdar followed ltp and inhibitory - avoidance learning [128, 129 ]. novel - taste learning increased glun2a and glun2b in the detergent - insoluble fraction (130), and another study showed that novel - taste learning decreased the expressions of glun1, glun2a, and the glun2b subunits of nmdar in the synaptoneurosomal preparation. these differences in nmdar subunits expression from different studies could be attributed to the differences in the fractionation protocol and the sampling time following novel taste learning. the binding of phosphorylated camkii to the overexpressed cytoplasmic (c-) tail of the glun2b in mutant mice that overexpressed c - tail affected its biding to the endogenous glun2b receptor complex. this camkii-glun2b complex formation also was associated with changes in the phosphorylation of the glua1 subunit of -amino-3-hydroxy-5-methyl - isoxazole-4-propionic acid receptor (ampar), and the spatial performance of these mutant mice was impaired, according to morris water maze analysis, which suggests that localization of glun2b is essential for mediating the downstream signaling cascade that leads to activation of learning - related protein changes. thus, it is very important to determine precisely the movement / localization of nmdar, and whether phosphorylation impacts the movement / localization process. ampar comprises hetero - tetrameric complexes composed of at least two of four subunits designated glutamate receptor glua1 - 4 [131, 132 ]. the cytoplasmic tail of the glua1 subunit undergoes distinct phosphorylation on serine 831 and dephosphorylation on serine 845 following ltp and long - term depression (ltd), respectively. functionally, phosphorylation on serine 831 and serine 845 is involved in regulation of ampar channel properties such as modulation of single - channel conductance and total and surface expressions of glua1. following inhibitory avoidance training - an associative learning paradigm wherein animals learn to associate the dark side of a chamber with a foot - shock and therefore avoid the dark side in successive trials - the increased phosphorylation of serine 831 of glua1 has been observed in the dorsal hip, in addition to increased expression levels of total glua1 and glua2 proteins. moreover, spatial learning is also associated with the phosphorylation of glua1, as demonstrated with serine 831/serine 845 double mutant mice in the water maze, an hip - dependent spatial - learning paradigm. taken together, these findings suggest that phosphorylation of ampar and its synaptic incorporation are involved in these aspects of learned behaviors. the application of the ampar antagonist, nbqx directly into the ic impaired both acquisition and retrieval of taste memory, which suggests that the ampa receptor plays a major role in taste recognition and learning. as discussed above, pkm is essential for cta memory [80 - 82 ] and, interestingly, another independent study based on a fear conditioning paradigm has shown that pkm maintains memories by regulating glua2-dependent ampa receptor trafficking. further studies are required, to elucidate whether pkm also maintains cta memory by regulating the glua2 subunit. currently, very little is known about the regulation of ampar in the context of taste learning and memory, therefore, further studies are needed to understand the regulation of ampar with respect to taste learning. tropomyosin receptor kinase b (trkb) is a membrane receptor tyrosine kinase activated by bdnf and neurotrophin 4. there are two tyrosine phosphorylation residues outside the kinase activation domain of trkb : tyrosine 515 and tyrosine 785. the binding of bdnf to its high - affinity receptor trkb leads to the dimerization and autophosphorylation of trkb on tyrosine residues in the intracellular domains, which then triggers one of three signaling cascades, depending on the site of phosphorylation ; they are mapk, phosphatidylinositol 3-kinase (pi3k), and phospholipase c (plc) (yoshii and constantine - paton). used immunoprecipitation followed by immunoblotting with anti - tyrosine antibody to show that cta conditioning increased the tyrosine phosphorylation of trkb, and that the increase in trkb phosphorylation was prolonged ; it was detected 1 and 8 h following conditioning in both the ic and the cea. have shown that inhibition of trk receptors impaired both short - term and long - term cta memory. bdnf - trkb signaling is involved in transcription, translation and trafficking of proteins during various phases of synaptic development, and has been associated with several forms of synaptic plasticity and memory [138, 139 ]. as discussed above, cta conditioning has been shown to increase the secretion and synthesis of bdnf in cea and ic ; and ma. have observed increased phosphorylation of the bdnf receptor, trkb at the time points before and after the increase of bdnf synthesis in the cea and ic, which suggests that cta learning first induced the rapid release of pre - existing bdnf, and then increased bdnf synthesis. pharmacological analyses that used anti - bdnf antibodies and a bdnf antisense oligonucleotide, which, respectively, neutralize the endogenously released bdnf and block de novo protein synthesis, have found that inhibition of bdnf in the cea significantly impaired cta. moreover, exogenous application of bdnf into the ic has been shown to reverse the cta memory deficits caused by the anisomycin - mediated inhibition of protein synthesis. it is consistent with the previous findings that a recent study has also shown that the injection of bdnf immediately after anisomycin was applied 5 and 7 h following cta conditioning reversed the anisomycin - mediated cta impairment ; micro - infusion of bdnf into the ic was also shown to enhance cta retention. these results are clear evidence that bdnf signaling is necessary for the early and late consolidation stages of cta. importantly, recent findings have shown this bdnf - mediated enhanced cta retention to depend on the activation of mapk - erki / ii and phatidylinositol-3-kinase (pi3k), which suggests that trkb - bdnf signaling following cta acts via both the mapk - erki / ii cascade and the pi3k cascade. in the mammalian cns cells that perform a given function or share common functional properties are oft en clustered together. recent studies based on sophisticated methods of optical imaging of intrinsic signals indicated the presence within the ic of discrete clusters of neurons, which responded selectively to different taste modalities [143 - 145 ]. moreover, a functional magnetic resonance imaging (fmri) study has shown that in humans different taste modalities elicited differing specific patterns, with some overlap. however, neither of these imaging techniques currently has the power to provide high - precision resolutionboth in time and at the cellular spatialscale in intact mammalian neural tissue. nevertheless, it is reasonable to assume that clusters of neurons responding to single taste modalities might contain more narrowly tuned neurons. novel - taste consumption and cta activate many intracellular signaling cascades in the ic and the amy, but it is not known whether these cascades occur within a single cell, within narrowly tuned clusters of cells, or in different populations of cells. therefore, techniques with higher cellular and spatial resolution of single neurons need to be developed, to investigate the molecular signaling cascades that underlie taste learning and memory. the recent development of optogenetic methods has rendered feasible investigations of specific cell types in the nervous system, with unprecedented precision and control [147, 148 ]. thus, combinations of optogenetics with laser capture microdissection and genetic manipulation, followed by use of mrna / protein arrays, liquid chromatography - mass spectroscopy, and bioinformatics tools or with new imaging techniques might provide better tools for such as yet unanswered questions. for several decades it was believed that consolidation of memory, a progressive process of changes and stabilization, was a unitary process. however, findings from the recent decade demonstrated that memories restabilize over time [149 - 151 ] and thatconsolidation of memory evolves over time, which suggests that memories change with time. interestingly, it was shown that, in the absence of negative visceral consequences, the strength of novel - taste memory stabilizes over time. despite the differing taste - learning models, available literature on taste learning appears to support the concept that the post - acquisition phase involves activation of waves of several biochemical pathways, and that the reactivation of particular pathways is required for consolidation. as discussed in the present review, several biochemical changes take place within the first hour after taste learning, and these are followed by other waves of activity during the subsequent few hours, which suggests a temporal segregation of signaling cascades that subservetaste - memory consolidation. novel taste and associations of novel taste with malaise differentially release the aforementioned neurotransmitters in the ic and the amy and, accordingly, waves of kinase activation also have been observed. furthermore, novel - taste learning induces two waves of activation of the mtor and s6k, respectively, which suggests that independent rounds of translation activation that lead to increased expression of proteins are required for taste - memory consolidation. ample evidence indicates that the activation of nmdar, trkb, protein kinases, transcription factors, and translation regulators, all of which play roles in synaptic plasticity are also involved in taste learning and in memory formation. the available information regarding the modulation of neurotransmitter receptor response, pluripotent kinase signaling cascades, gene expression, translation regulation and translation by phosphorylation processes has presented opportunities for understanding taste learning and memory formation. | protein phosphorylation and dephosphorylation form a major post - translation mechanism that enables a given cell to respond to ever - changing internal and external environments. neurons, similarly to any other cells, use protein phosphorylation / dephosphorylation to maintain an internal homeostasis, but they also use it for updating the state of synaptic and intrinsic properties, following activation by neurotransmitters and growth factors. in the present review we focus on the roles of several families of kinases, phosphatases, and other synaptic - plasticity - related proteins, which activate membrane receptors and various intracellular signals to promote transcription, translation and protein degradation, and to regulate the appropriate cellular proteomes required for taste memory acquisition, consolidation and maintenance. attention is especially focused on the protein phosphorylation state in two forebrain areas that are necessary for taste - memory learning and retrieval : the insular cortex and the amygdala. the various temporal phases of taste learning require the activation of appropriate waves of biochemical signals. these include : extracellular signal regulated kinase i and ii (erki / ii) signal transduction pathways ; ca2 + -dependent pathways ; tyrosine kinase / phosphatase - dependent pathways ; brain - derived neurotrophicfactor (bdnf)-dependent pathways ; camp - responsive element bindingprotein (creb) ; and translation - regulation factors, such as initiation and elongation factors, and the mammalian target of rapamycin (mtor). interestingly, coding of hedonic and aversive taste information in the forebrain requires activation of different signal transduction pathways. |
although approximately 90% of them pass spontaneously, they can result in perforation or obstruction in the gastrointestinal (gi) tract. toddlers aged 2 - 3 years are most commonly affected ; as children in this age group are ambulatory and more orally explorative. it normally affects the ileocaecal and rectosigmoid regions. according to goh., the most common site of intra - abdominal perforation as the terminal ileum (approximately 39%). we present a case of young female with ingestion of foreign body which was impacted in a small intestine. a 23-year - old female presented to the emergency of the max super speciality hospital, saket, new delhi, india with a history of ingestion of teaspoon, while she was eating ice cream 1 day before. physical examination revealed soft abdomen with mild tenderness around umbilicus and no signs of peritonitis. a plain x - ray abdomen revealed presence of a radio - opaque foreign body (metallic teaspoon) in the stomach. initially, we decided to observe and closely monitor the patient after admitting her and starting conservative expectant treatment. the next day she was not relived, and the abdominal pain continued. in view of that repeat x - ray abdomen was performed in erect and supine position to locate the position of the teaspoon [figure 1 ]. abdominal x - ray showing spoon in jejunum diagnostic laparoscopy was performed with one 10 mm and two 5 mm midline ports. on initial evaluation there were no bowel adhesions. on the exploration and careful examination of upper gi tract jejunum on tracing the loop of jejunum, the spoon was located at the mid jejunal level. enterotomy was performed on the antimesenteric border, and tablespoon identified and was retrieved [figures 2 and 3 ]. the foreign body (spoon) patient had an uneventful post - operative recovery and was ultimately discharged on post - operative day 4. the management of foreign bodies in gi tract is based on collective anecdotal experience of surgeons. the highest incidence of swallowed foreign bodies occurs in children between 6 months and 3 years, and coins are the most commonly ingested foreign bodies. there is a definite predilection for swallowed foreign bodies to become impacted at the level of cricopharyngeus and just below it or at the oesophagogastric junction. in our patient, a large number of foreign bodies will pass through the entire gi tract once past the gastroesophageal junction ; nevertheless it is preferable to observe them in the hospital for a possible need for immediate abdominal exploration should bleeding, or features of perforation occurs. during the observation period, a daily radiograph should be obtained, and patient should be checked for signs of peritonitis or gi bleeding. all sharp foreign bodies should be removed before they pass from the stomach because 15 - 35% of these will cause intestinal perforation, usually in the area of the ileocecal valve. if a sharp foreign body does not progress for three consecutive days surgical intervention should be considered, and if the patient becomes symptomatic, surgical intervention will be necessary. intraluminal endoscopic techniques are limited by the length of the instrument and the curved path it takes. laparoscopic removal of foreign bodies from the peritoneal cavity (translocated intrauterine contraceptive devices) and a needle from the pelvis have been reported previously. laparoscopically removed a toothpick causing small bowel perforation followed by lavage of the abdominal cavity and laparoscopic closure of the perforation including omentoplasty. accurate localisation of foreign body in the small bowel is the main challenge. in our case for irregular objects such as a safety pin, the site requires localisation and then a small enterotomy. depending on the availability of skill and expertise, laparoscopic removal of the gi foreign bodies should be considered an option. the most optional method of retrieval in the form of endoscopy or surgery should be contemplated at the earliest indication. laparoscopic retrieval may be a better option in comparison with laparotomy where facility and proper expertise are available. | ingestion of foreign body is a serious problem commonly encountered in our clinical practice. most of them pass spontaneously, whereas in others endoscopic or surgical intervention is required because of complications or non - passage from the gastrointestinal tract. we present here a case of teaspoon ingestion, which did not pass spontaneously. laparoscopic retrieval of teaspoon was done from mid jejunum after enterotomy and the patient recovered uneventfully. right intervention at the right time is of paramount importance. |
are intestinal protozoa capable of infecting a range of host species, and are important causes of human morbidity and mortality (hunter and thompson, 2005, kotloff., 2013, mainly c. hominis and c. parvum, have been responsible for large - scale waterborne epidemics in the developed world, and are amongst the top four causes of moderate - to - severe diarrhoea in young children in the developing world (checkley., 2015,, 2013, shirley., 2012, sow., 2016). around 200 million people in asia, africa and latin america are reported to have symptomatic giardiasis (feng and xiao, 2011). entamoeba histolytica, the cause of amoebic colitis and amoebic liver disease, is responsible for up to 100 000 deaths annually (stanley jr, 2003). understanding the epidemiology of these parasites is important, both for public health as well as for the health of the animals they infect this is made difficult by morphologically identical parasites sometimes having separate pathogenicity, host ranges and life cycles. for instance, giardia duodenalis is considered a species complex comprised of at least 8 distinct genetic groups (assemblage a to h), with assemblages a and b found both in humans and a range of animal species (thompson and smith, 2011). cryptosporidiosis, giardiasis and amoebiasis are all important diseases in india where poverty, lack of hygiene, free roaming animals, high population density, and infrastructure inadequacies regarding water supply and sanitation, facilitate infection (kaur., 2002, nath., 1999, nath., 2015b) rhesus macaques (macaca mulatta) are one of the most common primates in india, particularly in human - dominated habitats (kumar., 2013). indeed, in some indian districts, the close contact between rhesus macaques and human activities means that they are regarded as a nuisance, particularly due to crop raiding activities (saraswat., 2015). macaque species have been implicated as wildlife reservoirs for zoonotic pathogens such as kyasanur forest disease, a zoonotic tick - borne viral haemorrhagic fever (singh and gajadhar, 2014). nevertheless, it is unclear whether there is transmission of intestinal protozoans between humans and urban monkeys, and if so, how significant this is for public health and for the conservation of the macaques., and entamoeba spp. in rhesus macaques in four districts of north - west india, with the aim of using molecular characterisation of isolates to provide preliminary information on the potential for transmission of these pathogens between macaques and humans. faecal samples (n = 170) were collected from free - living rhesus macaques in four non - overlapping locations in north - west india.troop 1 : located at punjab university, chandigarh. monkeys move freely throughout the campus, spending large amounts of time feeding, defecating and sleeping near areas used for preparation of human food. estimated troop size, 300 animals.troop 2 : located at jakhoo temple, himachal pradesh. primarily based around a forested hilltop temple, however also move freely into the surrounding city of shimla. estimated troop size, 200 animals.troop 3 : located around a small local temple in the municipality of kurali, punjab. this temple also owns a cattle - breeding facility where the troop spends much of its time. there is direct contact between the cows and the moneys, with macaques eating grain provided to the cattle and picking food off the ground contaminated with cattle faeces. estimated troop size, 100 animals.troop 4 : located on the outskirts of a semi - rural town nada sahib, haryana. monkeys move freely throughout the campus, spending large amounts of time feeding, defecating and sleeping near areas used for preparation of human food. troop 2 : located at jakhoo temple, himachal pradesh. primarily based around a forested hilltop temple, troop 3 : located around a small local temple in the municipality of kurali, punjab. this temple also owns a cattle - breeding facility where the troop spends much of its time. there is direct contact between the cows and the moneys, with macaques eating grain provided to the cattle and picking food off the ground contaminated with cattle faeces. troop 4 : located on the outskirts of a semi - rural town nada sahib, haryana. co - exists with roughly 30 tarai grey langurs (semnopuithecus hector). faecal samples (n = 14) were collected from calves from the breeding facility in kurali with which troop 3 was in close contact. rhesus macaques faecal samples were collected non - invasively, and were identified by being morphologically consistent fresh stools located where these monkeys had been observed immediately preceding collection. calf faecal samples were collected directly from the stool after the animal had been observed to defecate. approximately two grammes of faecal material, collected from the middle of the fecal mass, was placed in an 8 ml aliquot of 2.5% (w / v) potassium dichromate, mixed thoroughly, and transported to the parasitology department, norwegian university of life sciences (nmbu) for analysis. one gram of faecal material was transported to the department of medical parasitology, postgraduate institute of medical education and research (pgimer), chandigarh, and kept unpreserved at 4 c for 2 weeks prior to dna isolation. at nmbu, the samples were washed twice with phosphate buffered saline, and then passed through a faecal parasite concentrator with a pore diameter 425 m (midi parasep, apacor, berkshire, england) and centrifuged to create a pellet. giardia cysts and cryptosporidium oocysts were isolated using an in - house immunomagnetic separation method (ims) using dynabeads (gc - combo, life technologies, carlsbad, ca) as previously published (robertson., 2006). briefly, 10 l anti - giardia beads, 10 l anti - cryptosporidium beads, 100 l sl buffer a and 100 l sl buffer b, were used to generate 55 l of purified sample from approximately 200 mg of the faecal pellet. five l of the resulting purified sample was dried and methanol - fixed to welled slides for detection of giardia cysts and cryptosporidium oocysts using a commercially available cryptosporidium / giardia direct immunofluorescent antibody test (ifat ; aqua - glo, waterborne inc., new orleans), in accordance with manufacturer 's instructions. prior to being screened, dried samples were also counterstained with 46 diamidino-2-phenylindole (dapi), a non - specific fluorescent stain that binds to double - stranded dna. stained samples were screened using a fluorescence microscope equipped with appropriate filters (for fitc and dapi) and nomarksi optics. samples were initially screened at 200, and possible findings examined more closely at x 400 and x 1000. the total number and dapi staining of cysts and oocysts on the slide was recorded. due to the large number of giardia positive samples, only those with either over 100 dapi negative cysts, or over 20 dapi positive cysts at pgimer, dna was isolated using qiaamp fast dna stool mini kit, with an incubation at 70 c for 5 min, in accordance with the manufacturer 's instructions. for giardia / cryptosporidium - positive samples, dna was isolated using the remaining 50 l of purified cysts / oocysts after ims using the qiamp dna mini kit (qiagen gmbh) at nmbu. the protocols followed the manufacturer instructions with slight modifications ; cysts / oocysts were first mixed with 150 l of te buffer (100 mm tris and 100 mm edta) and incubated at 90 c/100 c (giardia / cryptosporidium) for 1 h before an overnight proteinase k lysis step at 56 c and spin column purification. dna was finally eluted in 30 l of pcr grade water and stored at 4 c. in all cases, the primary pcr consisted of 8.3 l pcr water, 1 l forward and 1 l reverse primer (at a final concentration of 0,4 mm), 0.2 l bsa (20 mg / l), 12.5 l of 2 hotstarttaqmaster and 2 l of template dna. pcr products were visualized by electrophoresis on 2% agarose gel with sybr safe stain (life technologies, carlsbad, ca). conventional pcr was performed on giardia positive samples at the glutamate dehydrogenase (gdh), triosephosphate isomerase (tpi), -giardin (bg) and small subunit rrna (ssu rrna) genes (caccio., 2008, hopkins., 1997, lalle., 2005, positive samples were purified using a high pure pcr product purification kit (roche, oslo, norway) and sent to a commercial company (gatc biotech, germany) for sequencing in both directions. sequences from both directions were assembled and manually corrected by analysis of the chromatograms using the program geneious. conventional pcr was performed on cryptosporidium positive samples at the ssu rrna gene (xiao., 1999). an entamoeba genus - specific conventional pcr was performed on rhesus macaque samples as previously published (verweij., 2003). a single round multiplex pcr targeting the ssu rrna gene, and that identifies e. histolytica, e. dispar and e. moshkovskii, was performed on all samples (hamzah., 2006). for samples that tested positive on the genus - specific pcr, a species - specific pcr for e. coli was performed as previously described (tachibana., 2009). four entamoeba genus - specific positive samples were not tested for e. coli due to laboratory error. prevalence of giardia, cryptosporidium and entamoeba were compared for the four different macaque troops using the chi - squared test. proportion of samples that resulted in amplification by pcr was compared using fischers exact test. examination of rhesus macaque faecal samples using immunofluorescent microscopy revealed the presence of giardia cysts in 31% (53/170) of samples. macaques excreted 55 to 6325 cysts per gramme faeces (mean, 555 ; median, 165). there was a significant difference in the prevalence of giardia cysts between troops 1, 2, 3 and 4 ; 45% (25/55), 20% (9/55), 33% (15/46) and 17% (4/24), respectively (p < 0.05). of the twenty - six giardia positive samples selected for molecular characterisation, seventeen tested positive at one or more gene, with the ssu rrna loci being the most sensitive (table 1). amplification by pcr was more likely if more than twenty dapi - positive cysts were used for dna isolation, 80% (12/15), than if 10 or less dapi positive cysts were used, 27% (3/11) (p < 0.05). there was no observed correlation observed between the total number of cysts and the likelihood of a sample being positive by pcr. sequences were submitted to genbank and accession numbers are provided (table 1). multiple alignment of consensus sequences at the tpi, gdh, bg and ssu rrna genes showed giardia excreted by macaques to be very similar to each other, 9899%, with differences primarily due to ambiguous nucleotides. importantly, there was heterozygosity of alleles within the sequences corresponding to the reverse internal primer at the bg gene and the reverse internal primer at the ssu rrna genes. blast results of macaque sequences at the tpi, gdh and bg genes showed 99% identity to giardia isolates from humans, common marmosets and a beaver. two samples, 5 and 8 (table 1), showed 100% identity at the bg gene to a giardia isolate from a sheep and human. examination of rhesus macaque faecal samples using immunofluorescent microscopy revealed the presence of cryptosporidium oocysts in 1 of 170 samples, with this animal from troop 3. this sample contained 50 oocysts per gramme of faeces, all of which stained positively with dapi, however was negative by pcr at the ssu rrna gene. examination of rhesus macaque faecal samples using a genus - specific conventional pcr revealed the presence of entamoeba spp. in 79% (132/168) of samples. there was no significant difference in the prevalence of entamoeba spp. between troops 1, 2, 3 and 4 ; 78% (43/55), 69% (31/45), 83% (19/23) and 87% (39/45) respectively (p = 0.21). multiplex pcr for e. histolytica, e. dispar and e. moshkovskii, did not result in amplification in any of the samples (0/168). species - specific pcr for e. coli resulted in amplification in 49% (63/128) of samples positive at the genus - specific pcr. thus, in the other 51% (65/128), no species of entamoeba was identified. there was a significant difference in the prevalence of e. coli between troops 1, 2, 3 and 4 ; 26% (11/42), 75% (21/28), 56% (10/18) and 45% (21/39), respectively (p < 0.01). examination of faeces from domestic calves living together with troop 3, revealed giardia spp. calves excreted 55 to 19 250 cysts per gramme faeces (mean, 4746 ; median, 302). five positive samples were analysed further by pcr, and all five tested positive at one or more loci. sequencing of pcr products revealed assemblage a (kx787052, kx787054) in two calves, assemblage a1 (kx787067) in one calf, assemblage e (kx787051, kx787063, kx787065) in one calf, and a mixed infection of assemblage a1 (kx787062, kx787053) and e (kx787064, kx787066) in one calf. calves were excreting 100 to 5000 oocysts per gramme faeces (mean ; 1480, median ; 700). this study describes a very high prevalence of entamoeba spp., a moderate prevalence of giardia duodenalis assemblage b, and a very low prevalence of cryptosporidium spp. in wild rhesus macaques in india, some of which have relatively close contact with humans and domestic animals. the high prevalence of entamoeba spp. in the macaques is consistent with results from studies in other closely related nonhuman primates (feng., 2011, e. dispar was not identified in this study, but has been detected in macaques from china and nepal (feng., 2013, tachibana., 2013). macaques were not infected with e. histolytica and e. moshkovskii, consistent with previous reports from other wild urban dwelling macaques (feng., 2013, are commonly reported in humans in india, this suggests that macaques are not a wildlife reservoir for these human pathogens, and that transmission from humans to macaques is not common among the macaque troops investigated (nath., 2015a, parija and khairnar, 2005 : parija., 2014). molecular identification of entamoeba spp. in 520 samples from a range of captive nonhuman primate species revealed e. hartmanni (51.9%), e. polecki - like (42.7%), entamoeba histolytica nonhuman primate variant (36%), e. coli (21.5%), e. dispar (2.4%) and e. moshkovskii (1.9%), as well as unidentified entamoeba spp (18.9%). e. polecki and e. hartmanni were not tested for in our study as they are not considered pathogenic to humans, however they may be responsible for the unidentified entamoeba spp observed. the reason for different prevalences amongst the macaque troops is not clear and could be due to a combination of various factors including diet, water sources, microbiome, genetics, and interactions with other humans or animals. the moderately high prevalence of giardia in rhesus macaques in this study is higher than reported for other macaque species, 2.49%, where ifa / pcr was used for diagnosis (sricharern., 2016, ye., 2012, ye., 2014). as these studies also investigated macaque populations in close contact with humans, the difference in giardia prevalence may be due to innate differences in the study populations, or, alternatively, due to different levels of food, water or environmental contamination where these population live. the study population in our study may have an increased exposure to giardia due to its high prevalence amongst humans, domestic animals, and environmental water sources in india (daniels. giardia infection has been associated with human contact in other primate species (gillespie and chapman, 2008, graczyk., 2002, macaques in china and thailand have been reported to be infected with g. duodenalis assemblage a and b, as seen in other nonhuman primates (levecke., 2009, sricharern., 2016, ye., 2012), while in this study macaques around chandigarh were only found to be infected with assemblage b. although this indicates a zoonotic potential for giardia infections in macaques, the results should be interpreted with caution as most of the samples were only positive at one gene and it has been show that some isolates show certain taxonomic grouping at one gene and a different grouping at another gene (lebbad., 2010, robertson., 2006). furthermore, it is difficult to interpret the zoonotic potential of these isolates, as multi - locus typing data can reveal animal isolates to be distinct from human isolates, despite them appearing similar based on a single locus (ryan and caccio, 2013, sprong., 2009). despite close contact with cattle shedding assemblage a and e cysts there was only a single macaque faecal sample that was positive for cryptosporidium, suggesting that this protozoan in not an important parasite in rhesus macaques in this region of india. since this positive sample contained few oocysts and was from the troop that had intimate contact with the calves shedding cryptosporidium oocysts, it is possible that this sample represents carriage, and not a true infection. cryptosporidium may be more common in very young macaques that are likely under - represented in this study due to the sampling technique relying on stool morphology. in this study, using ifa as the gold standard, then pcr at different gene loci had the following sensitivities ; ssu rrna (58%), bg (15%), gdh (12%) and tpi (8%). overall sensitivity of pcr, using all loci, was 65% (17/26) in macaques, and 100% (6/6) in the calves. pcr sensitivity may have been limited by the low number of dapi positive cysts available for dna isolation. alternatively, the allelic sequence heterozygosity observed at the primer binding sites would suggest that the primers used in this study are not optimal for the giardia isolates found in the study population. similar limitations of pcr have been observed in genotyping canine giardia isolates (sommer., 2015). not surprisingly, positive dapi staining of cysts, indicating the presence of nuclear dna, was associated with increased likelihood of a positive pcr result. entamoeba coli, unknown entamoeba spp. and g. duodenalis assemblage b were common in urban dwelling rhesus macaques around chandigarh, india., e. histolytica and e. moshkovskii do not appear to be important pathogens in this population. further molecular investigation is needed to firmly establish the zoonotic potential of giardia infections in macaques. | giardia duodenalis, cryptosporidium spp., and entamoeba spp. are intestinal protozoa capable of infecting a range of host species, and are important causes of human morbidity and mortality. understanding their epidemiology is important, both for public health and for the health of the animals they infect. this study investigated the occurrence of these protozoans in rhesus macaques (macaca mulatta) in india, with the aim of providing preliminary information on the potential for transmission of these pathogens between macaques and humans. faecal samples (n = 170) were collected from rhesus macaques from four districts of north - west india. samples were analysed for giardia / cryptosporidium using a commercially available direct immunofluorescent antibody test after purification via immunomagnetic separation. positive samples were characterised by sequencing of pcr products. occurrence of entamoeba was investigated first by using a genus - specific pcr, and positive samples further investigated via species - specific pcrs for entamoeba coli, entamoeba histolytica, entamoeba dispar and entamoeba moshkovskii. giardia cysts were found in 31% of macaque samples, with all isolates belonging to assemblage b. cryptosporidium oocysts were found in 1 sample, however this sample did not result in amplification by pcr. entamoeba spp. were found in 79% of samples, 49% of which were positive for e. coli. multiplex pcr for e. histolytica, e. dispar and e. moshkovskii, did not result in amplification in any of the samples. thus in 51% of the samples positive at the genus specific pcr, the entamoeba species was not identified. this study provides baseline information on the potential for transmission of these zoonotic parasites at the wildlife - human interface. |
questing nymphal and adult black - legged ticks (i. scapularis) collected from four sites in south kingston, rhode island, and one site in bridgeport, connecticut, were analyzed for the presence of granulocytic ehrlichiae (figure). collections of adult- or nymphal - stage ticks or both were available from ongoing tick surveillance conducted in each region from 1996 to 1999. questing nymphal ticks collected from bluff point in southeastern connecticut in 1997 were also available. the rhode island sites are all located in the state s zone of highest i. scapularis density (17). small rodents, including white - footed mice (peromyscus leucopus) and chipmunks (tamias striatus) live - trapped at the same locations were bled following procedures approved by the institutional animal care and use committees of each institution. briefly, animals were trapped from july to september along transect lines or in trapping grids. blood was stored in edta at -80c until tested for ehrlichiae by polymerase chain reaction (pcr) techniques and dna sequencing. dna was extracted directly from blood samples by using a qiaamp blood extraction kit (qiagen, chatsworth, ca), according to manufacturer s instructions. briefly, detergent lysis was done in the presence of proteinase k for 10 min at 70c. the lysed material was applied to a spin column containing a silica gel - based membrane and then washed twice. purified dna was eluted from the columns in 200 l tris - hcl (10 mm, ph 8.0) and stored at 4c until used as template for pcr amplification. dna was extracted from i. scapularis ticks by a modification of the manufacturer s protocol for the qiaamp tissue kit (qiagen) as described (13). a nested pcr that amplified a 546-bp portion from the 5 region of the 16s rrna gene was used to identify granulocytic ehrlichiae in tick and wildlife samples (13). briefly, primary amplifications consisted of 40 cycles in a perkin - elmer 9600 thermal cycler (perkin - elmer, applied biosystems division, foster city, ca), with each cycle consisting of a 30-sec denaturation at 94c, 30-sec annealing at 55c, and a 1-min extension at 72c. the 40 cycles were preceded by a 2-minute denaturation at 95c and followed by a 5-minute extension at 72c. primary amplifications used primers ge3a and ge10r and reagents from the geneamp pcr kit with amplitaq dna polymerase (perkin - elmer). each reaction contained 5 l of purified dna as template in a total volume of 50 l, as well as 200 m each deoxynucleoside triphosphate (dntp) (datp, dctp, dgtp, and dttp), 1.25 units taq polymerase, and 0.5 m each of primer. reaction products were subsequently maintained at 4c until analyzed by agarose gel electrophoresis or used as template for nested reactions. nested amplifications used primers ge9f and ge2 and 1 l of the primary pcr product as template in a total volume of 50 l. each nested amplification contained 200 m each dntp (datp, dctp, dgtp, and dttp), 1.25 units taq polymerase, and 0.2 m each of primer. nested cycling conditions were as described for the primary amplification, except 30 cycles were used. reactions were subsequently maintained at 4c until analyzed by agarose gel electrophoresis or purified for dna sequencing (13). all samples producing positive pcr products were subjected to dna sequencing reactions with fluorescent - labeled dideoxynucleotide technology (dye terminator cycle sequencing ready reaction kit, perkin - elmer, applied biosystems division). sequencing reaction products were separated, and data were collected with an abi 377 automated dna sequencer (perkin - elmer, applied biosystems division). the full sequence was determined for both strands of each dna template to ensure maximum accuracy of the data. sequences were edited and assembled with the staden software programs (20) and analyzed with the wisconsin sequence analysis package (genetics computer group, madison, wi) (21). questing nymphal and adult black - legged ticks (i. scapularis) collected from four sites in south kingston, rhode island, and one site in bridgeport, connecticut, were analyzed for the presence of granulocytic ehrlichiae (figure). collections of adult- or nymphal - stage ticks or both were available from ongoing tick surveillance conducted in each region from 1996 to 1999. questing nymphal ticks collected from bluff point in southeastern connecticut in 1997 were also available. the rhode island sites are all located in the state s zone of highest i. scapularis density (17). small rodents, including white - footed mice (peromyscus leucopus) and chipmunks (tamias striatus) live - trapped at the same locations were bled following procedures approved by the institutional animal care and use committees of each institution. briefly, animals were trapped from july to september along transect lines or in trapping grids. blood was stored in edta at -80c until tested for ehrlichiae by polymerase chain reaction (pcr) techniques and dna sequencing. dna was extracted directly from blood samples by using a qiaamp blood extraction kit (qiagen, chatsworth, ca), according to manufacturer s instructions. briefly, detergent lysis was done in the presence of proteinase k for 10 min at 70c. the lysed material was applied to a spin column containing a silica gel - based membrane and then washed twice. purified dna was eluted from the columns in 200 l tris - hcl (10 mm, ph 8.0) and stored at 4c until used as template for pcr amplification. dna was extracted from i. scapularis ticks by a modification of the manufacturer s protocol for the qiaamp tissue kit (qiagen) as described (13). a nested pcr that amplified a 546-bp portion from the 5 region of the 16s rrna gene was used to identify granulocytic ehrlichiae in tick and wildlife samples (13). briefly, primary amplifications consisted of 40 cycles in a perkin - elmer 9600 thermal cycler (perkin - elmer, applied biosystems division, foster city, ca), with each cycle consisting of a 30-sec denaturation at 94c, 30-sec annealing at 55c, and a 1-min extension at 72c. the 40 cycles were preceded by a 2-minute denaturation at 95c and followed by a 5-minute extension at 72c. primary amplifications used primers ge3a and ge10r and reagents from the geneamp pcr kit with amplitaq dna polymerase (perkin - elmer). each reaction contained 5 l of purified dna as template in a total volume of 50 l, as well as 200 m each deoxynucleoside triphosphate (dntp) (datp, dctp, dgtp, and dttp), 1.25 units taq polymerase, and 0.5 m each of primer. reaction products were subsequently maintained at 4c until analyzed by agarose gel electrophoresis or used as template for nested reactions. nested amplifications used primers ge9f and ge2 and 1 l of the primary pcr product as template in a total volume of 50 l. each nested amplification contained 200 m each dntp (datp, dctp, dgtp, and dttp), 1.25 units taq polymerase, and 0.2 m each of primer. nested cycling conditions were as described for the primary amplification, except 30 cycles were used. reactions were subsequently maintained at 4c until analyzed by agarose gel electrophoresis or purified for dna sequencing (13). all samples producing positive pcr products were subjected to dna sequencing reactions with fluorescent - labeled dideoxynucleotide technology (dye terminator cycle sequencing ready reaction kit, perkin - elmer, applied biosystems division). sequencing reaction products were separated, and data were collected with an abi 377 automated dna sequencer (perkin - elmer, applied biosystems division). the full sequence was determined for both strands of each dna template to ensure maximum accuracy of the data. sequences were edited and assembled with the staden software programs (20) and analyzed with the wisconsin sequence analysis package (genetics computer group, madison, wi) (21). the percentage positive in each of the 4 years ranged from 6.1% in nymphs in 1998 to 23.3% in adults in 1996. less year - to - year variation was noted in adult ticks, in which infection prevalence ranged from 11.7% (1997) to 23.3% (1996). pcr analysis of edta blood samples from white - footed mice collected in connecticut during summer and fall 1997 and spring 1998 showed that 17 (36.2%) of 47 in 1997 and 3 (60%) of 5 in 1998 were positive (22). all products from samples collected in the bridgeport, conn., area from 1996 through 1998 had sequences identical to the 16s rrna gene (ep - ha) previously amplified and sequenced from documented human infections in the northeast and upper midwest united states and in europe (4). the 16s rrna sequence determined from adult ticks collected from bridgeport in 1999 showed that all 12 positive samples also contained the human agent (ep - ha), although one of the ticks produced a mixed sequence, suggesting the presence of more than one agent. the pcr products from this tick were cloned, and individual clones were purified and sequenced. these data confirmed the presence of a mixed population of ehrlichiae containing some 16s rrna sequences that matched ep - ha and some that differed from ep - ha by two nucleotides. the latter sequence was identical to a variant (called variant 1) previously described in ticks in rhode island and deer in maryland and wisconsin (13,14) (table 2). in contrast to ticks and rodents from the bridgeport area, nymphal ticks collected in 1997 from bluff point in southeastern connecticut contained a nearly equal distribution of ep - ha (5 [55.6% ] of 9 positives) and variant 1 (4 [44.4% ] of 9 positives) ehrlichiae. includes one tick that was positive for both the human granulocytic ehrlichiosis agent and variant 1. the number designations for the ep - ha 16s rdna sequence correspond to those reported by chen. variant base paris are shown in bold.- rhode island samples from i. scapularis ticks, white - footed mice, and chipmunks contained e. phagocytophila variants as well as ep - ha. a total of 123 (22.9%) of 538 ticks were positive for e. phagocytophila by pcr, including 61 (26.3%) of 232 adults and 62 (20.3%) of 306 nymphs. dna sequencing was performed on 92 of these pcr products, and overall, only 24 (26.1%) showed sequences identical to those of ep - ha. the rest of the ticks (53 [57.6% ]) had a novel sequence differing from ep - ha by 2 nucleotides and from variant 1 by 4 nucleotides (hereafter called variant 2) (table 2). pcr testing of blood samples from 19 rhode island chipmunks in 1996 detected 11 (57.9%) positives. dna sequencing of these pcr products showed that nine were identical to the sequence of ep - ha ; the remaining two represented novel variant sequences, each differing from ep - ha by a single nucleotide (variants 3 and 4 ; table 2). although both the white - tailed deer agent and the e. equi / ca human sequence variant (table 2) are amplified by the pcr assay used in this study, neither agent has been detected in potential rodent reservoir populations in connecticut or rhode island. host and vector associations of ep - ha and the four e. phagocytophila variants found in rhode island are shown in table 3. variant 1 detected only in ticks in rhode island and connecticut ; positive deer samples were collected in maryland and wisconsin (13,14). based on samples from 35 the prevalence of e. phagocytophila in i. scapularis ticks (adults and nymphs combined, years 1996 - 1999) was higher in rhode island (22.8%) than in bridgeport (13.3%) (p<0.001 ; fisher s exact test). this finding was also true for adult ticks : 26.3% were infected in rhode island compared with 15.4% in bridgeport (p=0.002). using either the total number of ticks tested or adult ticks only, the prevalence of e. phagocytophila in rhode island compared with bridgeport was 1.7. however, if the 1997 rhode island data, which were skewed by an unusually large number of variants, are removed from the calculations, the percentage of e. phagocytophila - positive ticks (adults and nymphs) was significantly higher in bridgeport (13.3%) than in rhode island (8.2%) (p=0.03). the same analysis, when restricted to the adult tick population, showed no significant difference between connecticut (15.4%) and rhode island (13.4%) ep - positive ticks (p=0.6). if the 1997 rhode island data are excluded, the prevalence of e. phagocytophila in that state compared with bridgeport was 0.6 for the total number of ticks tested and 0.8 for adult ticks only. infection prevalence data were available for adult and nymphal ticks from the same site for 3 years : rhode island in 1997 and 1999 and bridgeport in 1998 (table 1). for two of these, rhode island in 1999 and bridgeport in 1998, the prevalence of e. phagocytophila was significantly higher (rhode island 1999 ; p=0.01) or borderline higher (connecticut 1998 ; p=0.065) in adults than in nymphs. e. phagocytophila infection rates in nymphal and adult ticks from rhode island in 1997 did not differ significantly (p=0.21). temporal trends in tick infection rates showed that the prevalence of e. phagocytophila in rhode island nymphs was highest in 1997 and then declined in 1998 and 1999 (p<0.001 ; chi - square test for trend). however, the high number of variants found in both adult and nymphal ticks from rhode island in 1997 influenced this analysis, as e. phagocytophila prevalence in rhode island in 1997 was significantly higher than in all other years combined (p<0.001). in bridgeport, no significant temporal trends were noted in e. phagocytophila infection rates in adult ticks (p=0.3), nor were significant prevalence or temporal trends noted in the rodents tested from any of the sites. analysis of the proportion of e. phagocytophila - positives that were variants showed that prevalence of the variants in rhode island (73.9% variants) was significantly higher than in bridgeport (0.02% variants) (p<0.001). when the proportion of e. phagocytophila - positives that were variants was compared with the total number of positives for the two connecticut sites, the ticks from bluff point (44.4% variants) showed significantly higher rates than ticks from bridgeport (0.02% variants) (p<0.001). strains of e. phagocytophila found in nature are capable of causing disease in sheep, cattle, horses, dogs, cats, and humans. these strains, including the species previously known as the hge agent and e. equi, are grouped as a single species on the basis of their close relationship at the genetic and antigenic levels. however, biological and ecological differences clearly exist between strains of e. phagocytophila, including varying host pathogenicity, vectors, dna sequence, and geographic distribution. small ribosomal subunit (16s) dna sequences are highly conserved in bacteria and are often used to identify and differentiate bacterial species. the 16s rrna gene sequences amplified from every confirmed human case, except for two isolated cases in northern california, have been identical to the e. phagocytophila - human agent (ep - ha) sequence determined by chen. pcr - positive white - footed mice (n=20) and i. scapularis ticks (n=38) collected in bridgeport from 1996 through 1998 also harbored only e. phagocytophila identical in sequence to ep - ha for a 546-bp region of the 16s rrna gene (4). sequence analysis of pcr products from two connecticut deer blood samples showed dna identical to the ep - ha p44 gene sequence (23). however, an ehrlichia organism with a 16s rrna gene sequence differing from ep - ha by a single nucleotide has been identified in white - tailed deer from maryland and wisconsin and in i. scapularis from rhode island (13,14). in contrast to our results from bridgeport, where we consistently found ep - ha, mice and ticks from rhode island had a significantly lower percentage of isolates identical to ep - ha, but several e. phagocytophila variants with novel 16s rrna gene sequences. these data indicate that variant forms of e. phagocytophila, not yet associated with human or veterinary disease, frequently occur in rhode island. the same or additional e. phagocytophila variants may also occur in other regions of the united states, but this concept remains to be investigated. most pcr assays amplify products from the variant agents that are the same size as the ep - ha pcr product, so that variants are indistinguishable when the products are analyzed only by agarose gel electrophoresis. therefore, results from other human - infection prevalence surveillance studies in ticks and rodents that have not included pcr product sequencing may be misleading. for example, if we had not sequenced our pcr products for 1997, we would have concluded that 46.4% of nymphal and 38.3% of adult i. scapularis ticks collected in southern rhode island were positive for ep - ha. actually, only 11.9% of the positives that were sequenced and an estimated 5.0% of the total ticks tested were ep - ha positive, with the rest of the 1997 rhode island positives consisting of genetic variants not yet associated with human disease. the 16s rrna sequences obtained from tick and rodent samples collected from bridgeport from 1996 through 1998 were identical to ep - ha. however, in 1999, one tick collected in that site was positive for both ep - ha and a variant (variant 1) previously found in rhode island. in a retrospective analysis of ticks collected at another eastern connecticut site (bluff point) close to the rhode island border, variant 1 was found in 1997. our inability to detect variant 1 despite extensive testing of samples collected in bridgeport from 1996 to 1998 and additional studies with larger sample sizes of ticks and rodents from bridgeport and other locations in connecticut are needed to assess the prevalence of ep - ha and the variants, as our bridgeport results may not be representative of the entire state. in fact, the bluff point data suggest that other areas of connecticut may have ep - ha / variant populations quite different from those in bridgeport and more similar to the distribution noted in rhode island. the identification of the coinfected tick in connecticut represents the first detection of more than one strain of e. phagocytophila in a single tick vector in the united states, although the coinfection of ixodes ricinus ticks by 2 e. phagocytophila strains has been reported in europe (24). these data indicate that two strains of the agent are capable of coexisting in a single tick, at least transiently, and that they can survive the molting process, since the coinfection was found in an unfed, host - seeking adult tick. first, the rate of e. phagocytophila - positive ticks (42.2% nymphs and adults) was very high relative to all other tick populations sampled from 1996 through 1999, and many of the positives were variant 2 (79.1% of pcr - positives sequenced). second, the 1997 rhode island ticks represent the only population in which e. phagocytophila prevalence was higher in nymphs than adults (46.4% nymphs and 38.3% adults). finally, variant 2 sequences were also seen in samples collected in 1997 from white - footed mice and chipmunks but were not detected before and have not been detected after 1997. the fact that both nymphal and adult questing ticks were positive for variant 2 suggests that the variant was present in reservoir species during both larval and nymphal feedings and may have been present in reservoirs from late summer 1996 through summer 1997. why this variant appeared only in rhode island during 1997, was the most prevalent strain infecting ticks that year, and then completely disappeared are matters of speculation. variant 2 may be a more common infection in a reservoir species that we did not examine, which may be less commonly targeted by host - seeking ticks. expression of variant 2 in the tick population could have resulted if, during 1996 - 1997, host populations preferred by immature i. scapularis (i.e., white - footed mice and chipmunks) were lower than normal, resulting in a higher proportion of ticks feeding on such atypical hosts harboring variant 2. after molting, nymphs infected as larvae the previous year could have transmitted variant 2 to the more preferred hosts of immature ticks, resulting in the variant 2-positive mice found in 1997. subsequent reestablishment of normal host populations may then have diluted the prevalence of variant 2, as immature tick feeding reverted to the preferred hosts. although the function and biological importance of the genetic differences in e. phagocytophila strains are unknown, we hypothesize that the variants may be interfering with maintenance and transmission of the human disease - causing agent (ep - ha). even if an increased human ehrlichiosis case surveillance effort in connecticut is taken into account, the number of confirmed and suspected cases differs dramatically between the two neighboring states : several hundred cases were reported in connecticut compared with fewer than 25 cases in rhode island during the same time period. from 1995 to 1997, 178 cases were confirmed or suspected (25), and case reports in connecticut increased substantially in 1998 (228 provisional, 104 confirmed, connecticut dept. of public health). these adjacent states share many of the ecologic factors that support natural maintenance of both borrelia burgdorferi and granulocytic ehrlichiae, such as populations of the tick vector i. scapularis and reservoir rodents, including white - footed mice (p. leucopus) (19,22). the incidence of lyme disease in connecticut and rhode island has been the highest in the nation for several years, with connecticut having a reported incidence only approximately 1.3 - 1.5 times higher than rhode island s (26). in contrast, through 1997 there was a 24-fold difference in the incidence of reported hge cases in the two states (connecticut 15.9 ; rhode island 0.67). therefore, the e. phagocytophila variants may have a competitive advantage over the ep - ha, possibly in infecting certain reservoir or vector populations. a lower incidence of ep - ha and less human disease would therefore be expected in areas where the variants predominate, since a lower proportion of ticks would harbor ep - ha. rickettsia rickettsii, the etiologic agent of rocky mountain spotted fever, was first identified in the early 1900s on the basis of its association with human disease (27). subsequent studies of veterinary infections and tick populations identified numerous rickettsia species closely related to r. rickettsii, all clearly members of the spotted fever group but not associated with human disease. these species include r. montana, r. rhiphicephali, r. parkeri, r. bellii, and the east side agent r. peacockii (28,29). nonpathogenic rickettsiae are thought to interfere with the development of more virulent r. rickettsii in dermacentor ticks and may be found more often in ticks than are the more virulent species (30,31). our data suggest that a similar situation may exist among the granulocytic ehrlichiae, with both pathogenic and nonpathogenic genetic variants coexisting in nature. isolation of the new variants will allow us to address this competitive - advantage hypothesis experimentally in both ticks and mice through the use of mixed infections in the laboratory. identification and use of novel gene targets more variable than the 16s rrna gene will eventually permit better assessment of variability between strains of e. phagocytophila (3234). future studies should include e. phagocytophila from additional geographic areas where a substantial number of human cases of granulocytic ehrlichiosis are reported (e.g., new york, wisconsin, minnesota) compared with areas (e.g., new jersey, pennsylvania, delaware, maryland, california) with similar vector densities but with little or no human disease. | primers were used to amplify a 561-bp region of the 16s rrna gene of ehrlichia phagocytophila from ixodes scapularis ticks and small mammals collected in rhode island and connecticut. dna sequences for all 50 e. phagocytophila - positive samples collected from 1996 through 1998 in southwestern connecticut were identical to the sequence reported for e. phagocytophila dna from confirmed human cases. in contrast, the sequences from 92 of 123 e. phagocytophila - positive rhode island samples collected from 1996 through 1999 included several variants differing by 1 - 2 nucleotides from that in the agent infecting humans. while 11.9% of 67 e. phagocytophila - positive ticks collected during 1997 in rhode island harbored ehrlichiae with sequences identical to that of the human agent, 79.1% had a variant sequence not previously described. the low incidence of human ehrlichiosis in rhode island may in part result from interference by these variant ehrlichiae with maintenance and transmission of the true agent of human disease. |
patent ductus arteriosus (pda) has been associated with significant morbidity in preterm infants, especially in very low - birth - weight infants (vlbw). in a hemodynamically significant pda (hspda), there is significant shunting of blood from the systemic to the pulmonary circuit resulting in increased pulmonary blood flow with reduction in effective systemic blood flow. the diastolic runoff from the systemic circulation may lead to major complications like necrotizing enterocolitis (nec), intraventricular hemorrhage (ivh), and renal perfusion abnormalities. the increased pulmonary blood flow contributes to the acute worsening of rds and chronic lung disease later. early treatment of an hspda has been shown to improve symptoms, the need for surgical ligation, duration of ventilator support and hospitalization in premature infants. currently clinical, radiological and echocardiographic assessments used to diagnose a hemodynamically significant pda have substantial limitations. clinical findings like a continuous murmur and bounding pulses are not always present especially in vlbw infants on high ventilator support where the pda could be silent. although echocardiogram has been considered as the gold standard for the diagnosis, it provides assessment at only one point of time and may not give a picture of the rapidly changing dynamic circulation especially in vlbw infants. there is also a lack of consensus in defining an hspda by echocardiographic criteria and size does not always correlate with the degree of shunting. (bnp) have been used as indirect indicators of pulmonary blood flow and left ventricular volume overload. the inactive fragment of bnp, nt - probnp with longer half - life and no circadian variation may serve as a better marker than bnp for assessment of ventricular dysfunction and volume overload. for assessing a reduction in the effective cardiac output, the resistive indices of the superior mesenteric artery (sma) measured as pulsatility index (pi) have been used to indirectly assess the diastolic steal associated with an hspda. this study was undertaken to assess the sensitivity and specificity of nt - probnp in predicting an hspda diagnosed by clinical, traditional and functional echocardiographic criteria including a measure of the sma blood flow pi in vlbw preterm infants. this study was undertaken to assess the sensitivity and specificity of nt - probnp in predicting an hspda diagnosed by clinical, traditional and functional echocardiographic criteria including a measure of the sma blood flow pi in vlbw preterm infants. this was a prospective study conducted at the brookdale university hospital and medical center from january 2008 to september 2009. vlbw infants (less than 1500 g birth weight) were eligible for entry into the study. infants with congenital heart defects, major congenital anomalies, confirmed sepsis, renal failure, persistent pulmonary hypertension and death within 3 days of admission to nicu were excluded. the study was approved by the institutional review board and consent was taken from parents before enrollment of the babies into the study. all babies enrolled in the study were evaluated with echocardiograms on the 3 5 day of life and then every week until the echo showed either a closed pda or non - hspda. a phillips 5500 ultrasonography machine (phillips medical systems, na, bothell, wa) incorporating 2-dimensional, color flow, pulsed and continuous wave doppler was used. if a pda was diagnosed the point of maximal constriction of the color flow jet was measured in millimeters by frame by frame analysis to assess ductal size. the left atrium and aortic root dimensions were measured in the parasternal short axis view at the level of the aortic valve and the la : ao (left atrial : aortic) ratio was calculated. in addition left ventricular dimensions in systole and diastole, tricuspid regurgitant jet gradients to assess systolic pulmonary artery pressures and systolic function by % shortening fraction (sf) were measured. a sagittal subcostal view was used to image the superior mesenteric artery (sma). the pi of the sma calculated as peak systolic velocity - end diastolic velocity / time averaged mean velocity (vti) was measured as an index of change in the local vascular resistance and reduction in effective cardiac output. all measures were performed based on the american society of echocardiography guidelines and an average of two readings were taken and reviewed by a single cardiologist blinded to the clinical status or laboratory data of the patient. hspda was defined as pda size of > 1 mm with at least two additional features of pda (continuous murmur, pulse pressure > 25 mmhg, worsening respiratory status, la / ao ratio > 1.4, pi of sma > 6, base excess > -5). infants with hspda were treated with indomethacin, ibuprofen or surgery depending on the clinical status of the infant and discretion of the neonatologist. blood samples (0.5 ml) for plasma nt - probnp were collected by arterial or venous catheter aspiration or by venous blood sampling along with other routine blood sampling to avoid extra needle sticks and excessive blood sampling. it was collected on the same day that the echocardiogram was done and analyzed immediately. plasma nt - probnp was measured with vitros nt - probnp reagent pack using intellicheck technology (ortho clinical diagnostics, a johnson and johnson company). data including body weight changes, fluid intake and output, respiratory and cardiovascular status were collected every week and data on the length of stay, prevalence of nec, bronchopulmonary dysplasia (bpd as defined by need for supplemental oxygenation at 36 weeks ' postmenstrual age), ivh, and mortality were collected from the charts and neonatal database. analysis was performed using spss, version 15 (spss inc, chicago, il). demographics and clinical characteristics were compared between hspda and non - hspda groups using fishers exact and chi - square tests. t - test and mann - whitney u test were used to compare continuous variables. pearsons correlation coefficient was used to test for correlations between plasma nt - probnp levels and other continuous clinical and echocardiographic variables. the receiver operator characteristic (roc) curve was created to select the best cut - off point of nt - probnp for detection of hspda. analysis was performed using spss, version 15 (spss inc, chicago, il). demographics and clinical characteristics were compared between hspda and non - hspda groups using fishers exact and chi - square tests. t - test and mann - whitney u test were used to compare continuous variables. pearsons correlation coefficient was used to test for correlations between plasma nt - probnp levels and other continuous clinical and echocardiographic variables. the receiver operator characteristic (roc) curve was created to select the best cut - off point of nt - probnp for detection of hspda. sixty - nine babies with mean gestational age (ga) of 272.6 weeks were included in the study for whom a total of 121 echocardiographic studies were done. the hspda group had 22 patients while the non - hspda group had 47 patients. the hspda group had a significantly lower gestational age and birth weight compared to the non - hspda group [table 1 ]. the hspda group had significantly higher nt - probnp levels [figure 1 ], lower base excess, wider pulse pressure and lower mean and diastolic blood pressures. weekly nt - probnp levels demographic and clinical parameters the hspda group had significantly higher estimated pulmonary artery pressures measured by the tr jet gradient and lower pda systolic and diastolic pressure gradients indicating higher pulmonary artery pressures [table 2 ]. echocardiogram parameters there were significant positive correlations of nt - probnp with echo parameters of pda size, la / ao ratio, the resistive index of sma blood flow and higher estimated pa pressures. significant negative correlations were found with low birth weight, gestational age, diastolic blood pressure and base excess [table 3 ]. correlations with nt - probnp in the hspda group, 45% of the infants received intervention ; 6 received ibuprofen, one received indomethacin and 3 underwent surgical ligation. in the hspda group, 6 infants (27%) died and the remaining 16 infants (73%) had pda closure at a mean age of 2.7 weeks. in the non - hspda group, 13 infants (28%) had pda of whom 2 infants (3%) died and the remaining had pda closure at mean age of 2.1 weeks. the hspda group had a higher, but not statistically significant incidence of ivh (27% in hspda group vs. 15% in the non - hspda group, p=0.2), chronic lung disease (36% in hspda group vs. 17% in the non - hspda group, p=0.1), and a significant increase in mortality within 2 weeks (27% in hspda group vs. 4% in the non - hspda group, p=0.01). the nt - probnp levels continued to decline over time in both groups although the mean values remained higher at all times in the hspda group [figure 1 ]. using the receiver operating characteristic curve (roc), nt - probnp levels were a good predictor of an hspda with the area under the curve of 0.981 (p 80% in predicting an hspda. including this as a feature for diagnosing an hspda added a measure of the effective systemic output and ductal steal in addition to the other echocardiographic and clinical parameters. the limitations of this study were a small sample size and no standard protocol for management. also there are a few confounding factors that can influence the validity of nt - probnp levels such as increased pulmonary vascular resistance, concomitant renal impairment, hydration status and sepsis. the strengths of the study were that this was a prospective study performed on all premature infants less than 1500 grams in a single medical center, at preset time intervals thus reducing selection bias and one of the only studies to our knowledge that has used a measure of systemic blood flow and pulmonary blood flow to gauge an hspda. until further randomized control trials are done in vlbw infants to assess the effect of medical treatment and/ or surgical ligation vs. conservative management on short - term and long - term morbidity and mortality, frequent clinical assessments, directed functional echocardiography utilizing measures to assess both pulmonary and effective systemic blood flow and biomarkers like bnp or nt - probnp together may help direct timing and treatment strategies for those patients with an hspda. use of biomarkers could also help determine the relative contribution of filling pressures to worsening respiratory status associated with intermittent reactive pulmonary hypertension in severe rds. this marker may help direct management towards improving ventricular function and reducing the volume overload with the judicious use of inotropes, fluid restriction, diuretics and interventions to close the pda. the pda and premature transitional circulation is dynamic and can lead to rapid and frequent changes in hemodynamic status. serial nt - probnp levels can be used as a simple screening test which is cost effective, easily available and can supplement the information obtained from functional echocardiography to aid in the diagnosis and management of an hspda. | purpose : a hemodynamically significant patent ductus arteriosus (hspda) in premature infants is known to be associated with significant morbidity. recently brain natriuretic peptides and superior mesenteric artery (sma)-resistive indices have been used to effectively diagnose hspda.objective:to assess the sensitivity and specificity of n - terminal probnp (nt - probnp) in predicting an hspda diagnosed by clinical and echocardiographic criteria including pulsatility index (pi) of sma.materials and methods : all preterm neonates < 1500 g were evaluated with echocardiograms and nt - probnp levels on the 3rd to 5th day of life and then every week until the echo showed either a closed pda or non-hspda.results:sixty-nine babies with mean gestational age of 27 weeks were included in the study. nt - probnp levels were significantly higher in the hspda group (n=22) with a meansem of 244203190 compared to 3072332 in the non - hspda group (n=47) (p<0.001). nt - pro bnp level of 5900 pg / ml had 96% sensitivity and 90% specificity of predicting hspda.conclusions:with frequently changing hemodynamics in low - birth weight infants, including nt - probnp and pi of sma improve the ability of assessing the effects of a hspda and will help timing of intervention. |
rupture of the anterior cruciate ligament (acl) is a common knee injury, and subsequently functional instability is often noted [1, 2, 4, 17, 29 ]. acl - reconstruction with bone - patellar - tendon - bone (bptb) autograft used to be the method of choice. this graft is found to give good long - term clinical results [13, 15, 37 ]. metallic interference screws have been the standard graft fixation method in acl - reconstructions with bptb - grafts. these screws give a solid fixation and are well tolerated by the body [11, 19 ]. however, there are some potential disadvantages. the presence of metallic interference screws can complicate revision surgery and may require a second operation for removal. bioabsorbable cannulated interference screws were introduced for arthroscopic acl - reconstruction in the early 1990s [6, 7 ]. different bioabsorbable screws are available, e.g. polyglycolic acid (pga), polylactic acid (pla), poly - l - lactic acid (plla), poly - d - lactic acid (pdlla), and polyparadioxanone (pds). if the bioabsorbable screw is to be an alternative to metal screws, it has to show adequate fixation and remain secure for at least 68 weeks until the bone block has been incorporated and biological fixation is achieved [6, 26, 28 ]. with regard to revision surgery, it is also important that the screw eventually will degrade, and that new bone formation will take place at the site of the implant. studies have revealed that pla screw resorption can take several years [3, 7, 8, 34, 44 ]. they support the conclusion that bioabsorbable screws are a reasonable alternative to metal interference screws [9, 16, 18, 19, 25, 31, 35, 38 ]. bioabsorbable screws are also found to provide as good fixation as metal screws [18, 21 ]. the aim of this study was to evaluate the clinical outcome 7 years following acl - reconstruction with bone - patellar - tendon - bone (bptb) grafts in patients with bioabsorbable plla interference screws and metal interference screws. the postoperative resorption and the bony integration of the bioabsorbable screws evaluated by mri were analyzed. the hypotheses were that there is no difference in the clinical outcome between the groups and that the bioabsorbable interference screws are fully resorbed. this study is a follow - up of patients included and randomized between 2000 and 2001. a total of 41 patients were included in the study, 22 women and 19 men. the included patients had isolated acl ruptures or acl - ruptures with additional minor meniscal lesions and/or minor cartilage lesions (outerbridge grade i and ii). after a diagnostic arthroscopy, the patients were randomized according to the envelope method for the use of either metal interference screws (linvatec, largo, fl, usa) or the bioscrews (linvatec, largo, fl, usa). a total of 21 patients were randomized to reconstruction with bioabsorbable screws and 20 patients to metal screws. there were no statistically significant differences between the two groups in any measured parameter preoperatively. of the 41 patients, 34 (83%) were examined (17 in metal screw group (m), 17 in bioscrew group (b)). one patient did not want to participate (b), one patient did not want to travel (m), and three of the patients were impossible to get in touch with (one in b, two in m). two patients in the bioscrew group were excluded because of graft failure and revision acl - surgery during the follow - up period. three patients were partially excluded from clinical comparisons due to a rupture of the contralateral acl during follow - up (two in b, one in m). these patients were only included in the evaluation of the subjective function, clinical assessment of swelling and lysholm score. the 31 remaining patients (16 in m, 15 in b) underwent all the clinical evaluations. a total of 16 of the 17 patients in the bioscrew group available for the 7 year follow - up had an mri examination performed. the mri was obtained at an average of 7.8 years (7.38.3) after surgery. the follow - up examination included lysholm function score, tegner activity score, knee injury and osteoarthritis outcome score (koos), lachman test, pivot - shift test, kt-1000 arthrometer examination and a clinical assessment of swelling. the patients also graded their subjective knee function as excellent, good, fair or poor. a dedicated knee coil was used to obtain the following image sequences : sagittal turbo spin echo (tse), 4000/16 and coronal fat saturated tse 2600/1416 ; 4-mm slice thickness, and image matrix, 256 384. two oblique coronal tse 2840/14 imaging sequences with slice thickness 3 mm and image matrix 256 384 were added in all patients. the number of excitations was 2 for the fat saturated sequence and 1 for the others. all the mri examinations were analyzed by the same radiologist as in the 2 year follow - up. the integration of the bone block was considered good if no border between the bone block and the bone was visible on mri. widening of the tibial tunnel was estimated at a distance of 2 cm from the joint line. the measurement of the screw volume preoperatively (v1) was calculated from the formula v1 = r h, where r = radius of the screw and h = length of the screw. measurements of volume of possible remnants of screws was calculated from the formula v = (1/2(r1 + r2)) h, where r1 = largest radius and r2 = smallest radius. the femoral tunnels were located at 11 oclock in the right knee and at 1 oclock in the left knee. the femoral bone block was fixed from the inside with a cannulated plla or metal interference screw of 7 25 mm. the bone block in the tibial tunnel was fixed with an interference screw of 8 or 9 25 mm with the knee in extension. the rehabilitation program was the same in both groups and supervised by the same two physiotherapists. a comparison of the differences between the groups was made with the student s t test for continuous variables and with the chi - square test and fisher exact test for categorical variables. in all tests, an alpha level of 0.05 a comparison of the differences between the groups was made with the student s t test for continuous variables and with the chi - square test and fisher exact test for categorical variables. in all tests, an alpha level of 0.05 there was no statistically significant difference between the two groups in any of the five subscales in the koos score (fig. 1), in subjective assessments of knee function (table 1), or in reported pain during the clinical examination. the median tegner activity score had decreased in both groups during the follow - up time from 2 to 7 years (p 5 mm300.02kt-1000 arthrometer (max. force) regrouped <3 mm129 3 mm46n.s.kt-1000 arthrometer (max. man. assessment of laxity in the two groups the mean volume of the screws preoperatively was 1,088 mm (9621,256) for the femur screws and 1,269 mm for the tibia screws. at the 7 year follow - up we could not detect intact screws in the tibia or the femur in any of the 16 patients. a residual screw tract these tracts were surrounded by a hypointense rim and were filled with amorphous soft tissue - like material (fig. the mean volume of this delineated screw - ghost was 406 mm (191678). in five of the patients, the residual screw tract was not clearly threaded and the surrounding rim was more indistinct (fig. there was no sign of a significant bony ingrowth in the screw sites in the tibia in any of the patients.fig. 4comparison of threaded tibial residual tract (a) and a not threaded, more indistinct tibial residual tract (b). both images show reabsorbed screw comparison of threaded tibial residual tract (a) and a not threaded, more indistinct tibial residual tract (b). both images show reabsorbed screw in the femur the residual screw tracts appeared different from the tibial tracts. the tract did not appear threaded in any of the patients, and the surrounding hypointense rim was in general thinner and less distinct than the rim in the tibia. these residual tracts were also filled with a material consistent with soft tissue, and there were no signs of significant bony replacement in the screw sites (fig. 5).fig. 5an example of a not threaded residual screw tract in femur filled with material consistent with soft tissue. it is lined with a thin rim interpreted as sclerotic bone an example of a not threaded residual screw tract in femur filled with material consistent with soft tissue. it is lined with a thin rim interpreted as sclerotic bone the mri did not reveal tibial tunnel widening in any of the 16 patients, and the integration of the bone block in both the femur and the tibia was considered good in all patients. there was no change in bone plug position, visible osteolysis, inflammatory bone marrow edema or other complications in any of the patients. the mean volume of the screws preoperatively was 1,088 mm (9621,256) for the femur screws and 1,269 mm for the tibia screws. at the 7 year follow - up we could not detect intact screws in the tibia or the femur in any of the 16 patients. a residual screw tract these tracts were surrounded by a hypointense rim and were filled with amorphous soft tissue - like material (fig. the mean volume of this delineated screw - ghost was 406 mm (191678). in five of the patients, the residual screw tract was not clearly threaded and the surrounding rim was more indistinct (fig. there was no sign of a significant bony ingrowth in the screw sites in the tibia in any of the patients.fig. 4comparison of threaded tibial residual tract (a) and a not threaded, more indistinct tibial residual tract (b). both images show reabsorbed screw comparison of threaded tibial residual tract (a) and a not threaded, more indistinct tibial residual tract (b). both images show reabsorbed screw in the femur the residual screw tracts appeared different from the tibial tracts. the tract did not appear threaded in any of the patients, and the surrounding hypointense rim was in general thinner and less distinct than the rim in the tibia. these residual tracts were also filled with a material consistent with soft tissue, and there were no signs of significant bony replacement in the screw sites (fig. 5an example of a not threaded residual screw tract in femur filled with material consistent with soft tissue. it is lined with a thin rim interpreted as sclerotic bone an example of a not threaded residual screw tract in femur filled with material consistent with soft tissue. it is lined with a thin rim interpreted as sclerotic bone the mri did not reveal tibial tunnel widening in any of the 16 patients, and the integration of the bone block in both the femur and the tibia was considered good in all patients. there was no change in bone plug position, visible osteolysis, inflammatory bone marrow edema or other complications in any of the patients. the most important finding of the present study was that the bioscrews had degraded 7 years after surgery ; however, the screws were not replaced by bone. the screws used in our study were made of 100% poly - l - lactic acid, a widely used material for fixations. studies have revealed that screw resorption can take several years [3, 34, 44 ]. lajtai. showed that bioabsorbable copolymer (85/15 d, l lactide / glycolide) screws remain intact for 4 months and disappear in 6 months. isomer in a poly - d - l - lactic interference fixation screw speeds the degradations process compared to the plla. mri of the patients in the present study at 2 years showed a mean reduction in screw volume of 63% for the tibia screws and 64% for the femur screws. at the 7 year follow - up, mri showed no remaining bioabsorbable interference screws.. also found no screws apparent on computer tomography (ct) 7 years postoperatively. subtle lamellar bony elements are most often difficult to detect with mri, and can occur even if it is not recognized with the mri imaging sequences chosen in our study. the findings in the present study, however, are in accordance with other studies showing that bony replacement of the screw does not always occur in humans [7, 12, 42, 43 ]. this is compatible with sclerotic bone, confirmed by the radiographic and ct findings of barber.. composite interference screws have recently been introduced to enhance bony integration, and studies show significantly better results compared to pure plla - screws [6, 23 ]. at the 7 years follow - up the tibial tract looked threaded in most of the patients, lined with the rim of sclerotic bone and filled with an amorphous soft tissue material. the tibial tract resembled a screw surface, like a screw - ghost, but the appearance was more indistinct than of an intact screw and the threads differed in size and thickness. the mean volume of these screw - ghost was estimated to 406 mm which was larger than the estimated mean volume of the tibial screws at 2 years postoperatively (403 mm). in six of the patients such threaded tracts were not found in the femur where the tracts appeared more diffuse and indistinct with a thinner rim. the difference between the tract appearance in tibia and femur was obvious in almost all the patients. possible explanations for this could be that the femur screw is positioned closer to the joint line and closer to cortical bone than the tibia screw. this may have consequence for the supply of water, blood and enzymes which are theorized to effect hydrolysis. in studies on bioabsorbable interference screws the presence of screw threads is often used to determine if the screw has degraded., however, found that a plla interference screw revealed clearly delineated on mri after 30 months in vivo actually was undergoing degradation. the explants were seen to be composed of screw remnants, fibrous connective tissue, cortical - like bone and cancellous bone. similarly, park and tibone presented a case study with a late inflammatory reaction 4 years after acl surgery. mri clearly showed the persistence of a tibial pla interference screw, but under the irrigation and debridement they could not find any screw or pla remnants, and the screw observed on preoperative mri was interpreted as a false screw a revealed persistent screw that is actually not there. in our study the so - called screw - ghost could in some patients be misinterpreted as persistent screws which could have a consequence when planning revision surgery after primary acl - reconstruction. drogset. observed osteolysis around the screw in three patients (16%) in the femur and none in the tibia at the 2 year follow - up. the bone block integration was considered good in 17 patients and fair in 10 patients. at the 7 years follow - up, no osteolysis in any of the 16 patients was detected, and integration of all the bone blocks was considered good. studies show that the rate of osteolytic changes after acl surgery with plla bioabsorbable screws is low, and that the incorporation of bone blocks is considered satisfactory compared to metal screws [8, 9, 19, 27 ]. studies on other bioabsorbable fixation materials also show promising results regarding osteolysis and osseous incorporation of bone blocks [10, 23, 25, 33 ]. tunnel enlargement may be a potential problem after primary acl - reconstructions and revision surgery [39, 45 ]. in the present study, bptb - grafts result in less tunnel widening than hamstring grafts. similarly, barber. did not find any difference in tunnel widening between plla - screws and metal interference screws. the majority of reports on bioabsorbable- and metal screws have shown comparable results in terms of stability, functional and clinical outcome [7, 9, 16, 18, 19, 24, 25, 35, 40 ]. the present study did not show any statistically significant differences between the groups concerning subjective knee function, koos score, lysholm score, tegner score, lachman test or assessment of swelling. in the authors opinion, however, the potential advantages of using plla - screws compared to metal screws are not sufficient to warrant the routine use of plla - screws in acl - reconstructions. the limitation of this study is the relatively small number of patients in each group. mri at 7 years showed that the bioabsorbable screws were absorbed ; however, they were not replaced by bone. | abstractintroductionbioabsorbable screws are, at the expense of metal screws, increasingly used as fixation device in acl - reconstructions. the possible advantages with bioabsorbable screws are better postoperative mri evaluations and easier revision surgery.purposethe aim of this study was to compare the clinical outcome after acl - reconstructions with bptb - grafts fixed with metal interference screws or bioabsorbable screws 7 years postoperatively. the resorption of the bioabsorbable screws was also analyzed.methodsbetween 2000 and 2001, 41 patients with acl deficient knees were randomized for the use of bptb - graft fixed with either metal interference screws or bioabsorbable poly - l - lactic acid screws. this is a 7-year follow - up with clinical examinations of both groups and, mri of the patients with bioabsorbable screws.resultsthe clinical and functional results were satisfactory in both groups. there were no significant differences between the groups in any parameter measured, except for better pivot shift results in the bioscrew group (p = 0.04). the mri evaluation showed degradation of the bioscrews. a residual screw tract which appeared threaded was seen in the tibia in 11 of the 16 patients. there was no sign of bony ingrowth in the previous screw site in the tibia in any of the patients.conclusionthe potential advantages of using plla - screws compared to metal screws are not sufficient to warrant the routine use of plla - screws in acl-reconstructions.level of evidencei. |
appropriate management of chronic back pain has been reported to have a positive effect on patients1. its aim is to strengthen the muscles around the spine and to improve motor control ability2. according to panjabi3, the spine is unstable inherently, and three subsystems the spinal column (passive subsystem), the neuromuscular control unit (neural subsystem), and the spinal muscles (active subsystem)have to be balanced to maintain spinal stabilization. panjabi also reported that back pain was the result of deformation of the neuromuscular control unit (neural subsystem) and spinal muscles (active subsystem) due to structural changes in the spinal column, which is a passive structure. this deformation normally occurs in the transversus abdominis (tra) and muldifidus (mf) deep trunk muscles. when functioning as a stabilization mechanism, these two muscles contract simultaneously. a study of the neuromuscular control unit (neural subsystem) and the spinal muscles (active subsystem) in patients with back pain showed delayed recruitment of the mf and reduced cross - sectional area of this muscle compared to healthy individuals4, 5. another study compared the neuromuscular responses of the tra in patients with back pain with those of healthy individuals6. it showed that the patients with back pain had delayed neuromuscular responses, which caused instability of the spine. other than the study by panjabi, who reported that deformed neuromuscular control influences the spinal muscles3, few studies have evaluated the active subsystem of the tra, which plays an important role in lumbar stabilization. abdominal hollowing has been suggested to reduce instability of the trunk, increase the active muscle response, and enhancing the activity of the tra7, 8. abdominal hollowing is a method for inducing voluntary local muscle contraction without global muscle contraction8. one study showed that the activity of the tra was significantly increased following abdominal hollowing, whereas another study found the opposite result7, 9. these conflicting results may be due to the difficulty in voluntarily performing abdominal hollowing, and increase in the activity of the local muscles without recruitment of the global muscles, as well as ambiguous criteria for minimum global muscle recruitment. recently, a maximum expiration exercise was introduced as a training method for maintaining trunk stability while increasing the activity of the expiratory muscles of the tra10. the exercise is aimed at increasing tra activity more than that of the internal oblique (io), and external oblique (eo), global trunk muscles, and it is easier to perform than the abdominal hollowing method10, 11. the aim of the present study was to compare the contraction ability at maximum expiration of the tra in patients with back pain with that of healthy individuals. the study subjects were 15 patients with back pain from the j oriental hospital located in busan, korea, and 15 healthy subjects. the patient group was comprised of individuals with back pain of more than three months ' duration. the control group consisted of subjects who had not experienced back pain during the previous three months. patients who had received lumbar operations or who had back pain due to systematic diseases such as cancer, a severe musculoskeletal disorder, or central nervous system disorders such as stroke were excluded. the subjects were informed of the study 's aim and methods, and the experiment was performed after obtaining the consent of the subjects. to ensure maximum expiration, the subjects were instructed to breathe out maximally, hold their breath, and not to use the brace pattern, which was designed by ishida.10. a logiq book xp (ge healthcare products, milwaukee, wi, usa) system with an 8 mhz linear transducer was used to measure the thickness of the abdominal muscles. the linear transducer was placed at a point 2.5 cm anterior to the center line between the iliac crest and the lower rib. to exclude changes in muscle thickness due to the effect of breathing, the measurements were made when subjects were at rest at the end of natural expiration. the muscle thickness was measured while maximum expiration was maintained for 5 s. the tra muscle was measured three times, and the average value was calculated. the thickness of the muscles was measured using image j software (national institute of health, bethesda, md, usa). the subject group was the independent variable, and the dependent variable was the rate of change in the muscle activity between the healthy subjects and the lbp. the rate of change in muscle thickness was calculated as follows : (thickness of muscle contraction thickness at rest) / thickness at rest 100%. the independent t - test was used to compare the contraction rate and the rate of change (%) in the muscle activity of the tra at rest. as shown in table 1, there was no significant difference in the general characteristics between the groups (p > 0.05). as shown in table 2, there was statistically significant difference between the healthy subjects and the back pain subjects with regard to the thickness of the tra at rest and the thickness of the muscle during contraction (p 0.05). the purpose of this study was to determine whether there is a difference between normal and back pain subjects in the activity of the spinal muscles at maximum expiration. the spinal muscles form an active system and in the spinal stabilizing system proposed by panjabi3. ultrasound is a suitable apparatus for measuring the activity of the tra and the change in the muscle thickness of the tra. electromyographic (emg) activity shows a linear correlation with muscle thickness up to 80% of maximal voluntary contraction (mvc)12. previous studies have reported that the rate of change in the activity of the tra in healthy subjects was 8689% during maximum expiration10, 13. this study showed that the two groups had a significant difference in the thickness of the tra and a significant difference in the thickness of the muscle during contraction, but showed no significant difference in the contraction rate. according to a study by ota and kaneoka14, patients with back pain had a thinner tra at rest than normal subjects, suggesting atrophy of the tra. the present study also showed atrophy of the tra, but voluntary tra muscle activation was similar to that of the normal subjects. the results demonstrate that core strengthening exercises, together with continuous voluntary activation training of the tra, would increase neuromuscular activation in patients with chronic back pain, thereby strengthening the tra muscle and preventing muscle atrophy this study had several limitations. second, the use of ultrasound for the measurements means it was not possible to determine the muscle recruitment order, and future studies of the neuromuscular system at maximum expiration will be needed to determine this. third, the change in the rate of activity of the transversus abdominis in the subjects showed a large deviation. as noted previously by ishida.10, this is due to the different muscle strengths of the subjects, with some finding it more difficult to perform maximum expiration than others finally, local muscles, such as the tra, are mainly involved in the stabilization of posture when a load is applied15, and supine positions require less tra activity for spinal stability. thus, changes in the contraction rate of the tra may not be detected. therefore, future research is required to measure low back pain patients in various positions (sitting, standing) using ultrasonography. | the purpose of the present study was to compare the contraction ability at maximum expiration of the transversus abdominis (tra) in patients with chronic low - back pain (clbp) with that of healthy individuals. [subjects ] we studied 15 patients with clbp and 15 healthy subjects. the subjects were informed of the study 's aim and methods, and the experiment was performed after obtaining the consent of the subjects. [methods ] the thickness of the abdominal muscles was measured using a logiq book xp (ge, usa). the main outcome variable was the ratio of tra thickness at maximum expiration versus in the relaxed position (tra activation ratio). [results ] there was a difference between the healthy subjects and the back pain subjects with regard to the thickness of the tra at rest and the thickness of the muscle during contraction. however, there was no difference in the rate of change in the muscle activity. [conclusion ] in conclusion, clbp patients exhibited atrophy of the tra muscle, but voluntary tra muscle activation was similar to that of the normal subjects. |
parahydrogen is demonstrated to efficiently transfer its nuclear spin hyperpolarization to nitrogen-15 in pyridine and nicotinamide (vitamin b3 amide) by conducting signal amplification by reversible exchange (sabre) at microtesla fields within a magnetic shield. following transfer of the sample from the magnetic shield chamber to a conventional nmr spectrometer, the 15n nmr signals for these molecules are enhanced by 30,000- and 20,000-fold at 9.4 t, corresponding to 10% and 7% nuclear spin polarization, respectively. this method, dubbed sabre in shield enables alignment transfer to heteronuclei or sabre - sheath, promises to be a simple, cost - effective way to hyperpolarize heteronuclei. it may be particularly useful for in vivo applications because of longer hyperpolarization lifetimes, lack of background signal, and facile chemical - shift discrimination of different species. |
|
caries, trauma, and cavity preparations reduce tooth structure that per se decreases the fracture resistance of tooth. abrasion, erosion, non - carious lesions, and aging are the other factors that have such an influence on fracture resistance of teeth. among all factors, extensive cavity preparations and endodontic treatment are the most common reasons for tooth fragility. these procedures can decrease fracture resistance of teeth due to the removal of occlusal marginal ridges. access cavity preparation in endodontic treatment compromises the fracture resistance of teeth, because the preparation is associated with reducing the pulp chamber walls and root dentin. also, dental arch position, tooth anatomy, and changes in mechanical and physical properties of dentin can influence the fracture resistance of teeth. several authors claim that adhesive restorative materials such as composite resins can reinforce the remaining tooth structure after endodontic therapy. however, light - polymerizing direct adhesive materials can cause polymerization shrinkage stresses that result in subsequent microleakage and dentin sensitivity. in order to reduce this negative influence, use of laboratory - prepared indirect resin restorations that adhere to the tooth structure is recommended. they can provide a favorable reinforcement for extensively damaged teeth. on the other hand, although dental amalgam is a material with a long clinical life, it does not have such a reinforcing effect, because amalgam does not adhere to the tooth structure and deforms under compressive stresses. the purpose of this study was to evaluate the fracture resistance and mode of fracture of endodontically treated human premolars with different amounts of remaining tooth structure. seventy sound human premolars free of any cracks or defects and indicated for extraction because of orthodontic treatment were selected for this study and were stored for no longer than 6 months. the selection of teeth was based on having similar bucco - lingual (bl) and mesio - distal (md) dimensions. the selected teeth were divided into 7 groups (n = 10) : group 1(st) : the teeth were left intact without any cavity preparation or root canal treatment and used as the negative control. access cavities were prepared using a high - speed bur and water spray, and the canals were instrumented with k files (kerr corporation) to an apical size 35 using the step - back technique. irrigation with 10 ml of 5.25% sodium hypochlorite preceded each file introduced into the canal. following biomechanical preparation, the canals were dried with absorbent paper points (diadent group international inc., cheongju city, korea) and obturated with gutta percha (diadent group international inc.) and ah 26 root canal sealer (silver free ; dentsply detrey, konstanz, germany) using cold lateral condensation. then, class ii mesio - occluso - distal (mod) cavities (groups 3, 5, 6, and 7) and mesio - occlusal (mo) cavities (groups 2 and 4) were prepared with the gingival cavosurface margin located 1.0 mm above the cement enamel junction. the buccolingual width of each cavity, measured with a digital caliper (mitutoyo corp., kawasaki, japan), was half the inter - cuspal distance and extended into the pulp chamber. the depth of the cavities was 4.0 mm, without proximal steps and flat floor. all teeth were prepared as approximate as possible to the same size using a periodontal probe and standard burs (universal set ; intensiv) to measure the depth and width. the facial and lingual walls of the occlusal segment were prepared parallel to each other. group 2 (mo - nf) : this group was kept unrestored after mo cavity preparation and endodontic treatment and was used as the positive control. group 3 (mod - nf) : this group was kept unrestored after mod cavity preparation and endodontic treatment and was used as the positive control. group 4 (mo - f) : after mo cavity preparation as in group 2, the cavities were etched with 32% phosphoric acid (uni - etch bisco inc., usa) for 30 s for enamel margins and 20 s for dentinal margins, respectively, rinsed for 20 s with an air / water spray, and gently air - dried to avoid desiccation. the primer (all bond 3, bisco inc.) was applied with a microbrush to the tooth surface for 20 s and then air - dried for 5 s. light - curing adhesive (all bond 3, bisco inc.) was applied with another microbrush, the excess was gently air - thinned, and the surface was exposed to a light - emitting diode (led)-polymerization unit with an intensity of 800 mw / cm (starlight pro ; mectron spa, carasco, italy) for 40 s. a matrix retainer (tofflemire matrix ; miltex inc., york, pa) was used and changed for each restoration. the matrix was tightened and held by finger pressure against the gingival margin of the cavity, so that the preparations could not be overfilled at the gingival margin. the composite resin (z 250 micro hybrid composite resin, 3 m espe usa) was placed using the oblique incremental technique, and each increment was no more than 1.5-mm thick to ensure adequate polymerization. each increment was polymerized for 40 s (20 s of slow - rise function repeated 2 times) using the led - polymerizing unit with a power light intensity of 800 mw / cm in contact with the occlusal surface of the tooth. the matrix was removed, and, to ensure adequate polymerization of the deepest parts of the interproximal box, each restoration was further light - cured for 60 s from the buccal aspect and 60 s from the lingual aspect of the box. group 5 (mod - f) : mod cavities were prepared as in group 3, and the cavities were filled as in group 4. group 6 (cc - d) : after obtaining impressions of the teeth, the mod cavities were prepared as in group 3 and the cusps were then reduced for 2 mm. group 7 (cc - ind) : in order for the prepared teeth to be restored with indirect composite resin, restorations impressions were made with a condensation silicone - based material (speedex, colten, switzerland) using a custom - made impression tray. the impressions were poured with a type iv stone and separated from the dies after 1 h. after separation, the cast was carefully evaluated to ensure that the finish line was entirely visible and that there were no distortions, air bubbles, or undercuts prior to sending the cast to the dental laboratory. the dies were coated with separating medium (gradia, gc corporation, tokyo, japan) and onlays were fabricated with indirect composite resin (gradia). each layer of onlays was further polymerized in a light - heat polymerization oven (gradia) for 10 s. each restoration was verified for fit accuracy and adjusted accordingly and then finished with a fine diamond rotary cutting instrument (intensiv fg ; intensiv). the internal surfaces of both the onlays and the teeth were airborne - particle abraded with 50-m silica - coated aluminum - oxide particles (special sand, kumapan ; consorzio onda, grugliasco, italy). then, the teeth were treated, as previously described for group 4, by etching and using primer, and bonding agents. the onlays, after the airborne - particle abrasion, were cleaned with ethyl alcohol (95%), and silane and bonding agents were applied. the duo link cement (bisco inc.) was used as a luting agent, according to the manufacturer 's instructions. the cement was then placed on the tooth, the onlay was seated in place, and the excess cement was removed with a brush. cavosurface margins were coated with a glycerine gel (deox ; ultradent products inc.) to permit complete polymerization of the luting agent. each restoration for the first 10 s was held under load and then polymerized with the led - polymerizing unit from the occlusal, facial, and lingual directions for 20 s in each direction, 3 times each (for a total of 1 min in each direction). after complete polymerization, the specimens were finished with carbide finishing burs (dentsply maillefer) to remove the excess cement, followed by repolishing with rubber cups and points (identoflex ; kerrhawe sa). the selected teeth were then stored in distilled water at 25c, and, subsequently, the root surfaces were marked 3 mm below the crown margin to simulate the biological width and covered with 0.3-mm - thick wax (tenatex wax ; kemdent). the specimens were then embedded in autopolymerizing acrylic resin (ortho - jet ; lang dental mfg co, wheeling, ill) surrounded by a cylindrical - shaped plastic mold (ikea ; rome, italy) with the long axis of the tooth parallel to that of the cylinder. after the first signs of polymerization, the teeth were removed from the resin blocks and the wax on the root surfaces was removed using a hand instrument. light - body silicone - based impression material (speedex) was injected into the resin base, and the teeth were reinserted into the resin base. the specimens were then placed into a universal testing machine (zwick, germany) and loaded compressively at 1 mm / min. the bar contacted the occlusal surface of the restoration and the buccal and lingual cusps of the teeth. the force necessary to fracture each tooth was recorded in newton (n), and the data was subjected to kruskal the fractured specimens were then examined under a stereomicroscope (olympus sz4045trpt, tokyo, japan) with 10 magnification to determine the fracture mode. fractures were considered favorable if adhesive fracture occurred above the cervical line (cej) and was restorable. the mean fracture resistance and the standard deviation for each of the 6 experimental groups are presented in table 1. the mean fracture resistance (newton) and standard deviation for each of the 7 experimental groups statistical analysis indicated that the fracture resistance of groups 6 and 7 was significantly higher than that of the other groups (p = 0.01). teeth restored with composite resin, indirect, and direct composite resin in groups 4 - 7, respectively, showed increased fracture resistance as compared with that in the non - restored group (groups 2 and 3) (p = 0.00). no statistically significant differences were found among groups 4, 5, and 1 (p = 0.25), and group 4 had greater fracture resistance than group 5 (p = 0.33). also, group 3 had the least fracture resistance among those receiving a restoration. chi - square test revealed significant differences among all groups with respect to the mode of fracture. with regard to the fracture mode the specimens in the groups 1, 4, 6, and 7 presented less severe fractures [table 2 ]. the fracture resistance of premolars significantly decreased following endodontic treatment and cavity preparation, because of the loss of tooth structure. premolars are more susceptible to cusp fractures because of their unfavorable anatomical shape, crown root ratio, and crown volume. remaining tooth structure after preparation for indirect restorations is lesser than the amount of tooth structure remaining after preparation for direct materials. in our study, the group 1 presented 837 n fracture resistance value, which is different from the values reported by other studies, such as 2483 n by cobankara., 1124.6 n by soares., and 2451.3 n by plotino. such differences among various studies can result from changes in the storage media of the teeth, the cross - head speed, type and design of load application, testing machine, and anatomical variability of teeth. quality and quantity of the remaining tooth structure are the most important factors affecting fracture resistance ; however, intact marginal ridges form a continuous circle of tooth structure in intact teeth that prevents cuspal fracture. dentin is a suitable solid base for dental restorations, and its structural strength depends on the quality and integrity of tooth anatomical form. it means that, reducing the amount of remaining sound dentin reduces the support provided for the restoration. previous researches have shown that fracture resistance of a posterior tooth with access cavity is about one - third of a sound tooth. after mod cavity preparation, deflection and strain of facial cusps are about 3 times greater than that of sound teeth, and stiffness reduces about 20%. in the current study, the authors found statistically significant differences between fracture resistance and fracture mode of non - restored teeth with mo and mod cavity preparations and fracture resistance of sound teeth, which is similar to the findings of other studies. however, studies by re., reported interesting findings as they failed to find statistically significant differences between unrestored teeth with mod preparation and sound teeth. in this study, restoring the mo and mod cavities with direct composite resin improved the fracture resistance and fracture mode of teeth as high as that of sound teeth. others suggested that composite resin has a cusp - reinforcing effect, which increases the fracture resistance of mod cavity preparations. this reinforcing effect of direct materials is related to the controlled polymerization shrinkage of composite resins. joynt., suggested that incremental composite placement and curing can increase the fracture resistance of premolars with mod cavity preparations. in the present study, the greatest amount of fracture resistance was found in cc - d and cc - ind groups, which means that restoration technique and composite type (direct or indirect) do not lead to significant differences in the amount of required load for fracture. the difference in the fracture resistance of cc - d and cc - ind groups is related to the extensive mod cavity preparation to provide divergent cavity walls for indirect restorations that necessitated removal of greater amounts of tooth structure, as mentioned by soares. reduction in the amount of remaining tooth structure causes decreased fracture resistance. in this study, the fracture resistance of teeth restored with cuspal coverage restorations was even greater than that of sound teeth. young teeth (extracted for orthodontic reasons), with a small amount of dentinal bulk and extended pulp chambers may be the cause of reduced fracture resistance of sound teeth. on the other hand, all teeth were extracted by forceps, which might have led to invisible coronal cracks. one of the limitations of this study was that the samples were not thermocycled and were tested with static loading. also, it is recommended to design future studies that use endodontically treated teeth without any cavity preparation as a positive control group. within the limitations of this in vitro study, the following conclusions were drawn : preparation of access cavity and mo / mod cavities can cause a significant decrease in fracture resistance of teethrestoring the cavities with z250 composite and all bond 3 bonding system increases the fracture resistance of teeth as high as that of the non - restored sound teethdirect and indirect cusp coverage restorations cause a significant increase in fracture resistance as compared to the non - restored and conventionally restored teethconservative direct and indirect adhesive restorations can be used to increase the fracture resistance of endodotically treated premolars as high as that of sound teeth. preparation of access cavity and mo / mod cavities can cause a significant decrease in fracture resistance of teeth restoring the cavities with z250 composite and all bond 3 bonding system increases the fracture resistance of teeth as high as that of the non - restored sound teeth direct and indirect cusp coverage restorations cause a significant increase in fracture resistance as compared to the non - restored and conventionally restored teeth conservative direct and indirect adhesive restorations can be used to increase the fracture resistance of endodotically treated premolars as high as that of sound teeth. | aim : endodontic treatment generally reduces the fracture resistance of teeth. the purpose of this study was to evaluate the fracture resistance and the mode of fracture of endodontically treated human premolars with different amounts of remaining tooth structure.materials and methods : seventy non - carious human premolars were randomly assigned into 7 groups. group 1 (st) did not receive any preparation. the teeth in groups 2 - 7 received root canal treatment and different preparations. group 2 (mo - nf) : mesio - occlusal preparation without filling ; group 3 (mod - nf) : mesio - occluso - distal preparation without filling ; group 4 (mo - f) : mesio - occlusal preparation with direct composite restoration (z250) ; group 5 (mod - f) : mesio - occluso - distal preparation with direct composite restoration (z250) ; group 6 (cc - d) : mesio - occluso - distal preparation with cusp reduction and direct composite restoration (z250) ; group 7 (cc - ind) : mesio - occluso - distal preparation with cusp reduction and indirect composite restoration (gradia gc). the fracture resistance (n) was assessed under compressive load in a universal testing machine (zwick) perpendicular to the occlusal surface at a cross - head speed of 1 mm / min, and the mode of fracture was assessed under stereomicroscope.statistical analysis : data was analyzed by kruskal wallis and mann whitney tests and the mode of fracture was analyzed by chi - square test (p < 0.05).results : statistical analysis showed that mo and mod cavity preparations significantly reduced the fracture resistance of sound teeth. direct composite restorations can improve the fracture resistance, and groups 7 and 6 presented the highest fracture resistance values.conclusions:teeth with adhesive restorations showed significantly higher fracture resistance values as compared with the non - restored ones. |
vomiting is one of the most common symptoms in gastroenterology clinics and usually originates from gastrointestinal (gi) disorders. however, various extraintestinal disorders can also cause vomiting.1,2 patients with thyroid disorders may present with a wide range of gi symptoms (eg, diarrhea, frequent defecation, constipation, dyspepsia, nausea, vomiting and abdominal pain).3 some patients with masked thyrotoxicosis who lack the unique clinical feature usually presented as cardiovascular abnormalities such as heart failure, but also occasionally as gi abnormalities including dysphagia,4 vomiting5 and abdominal pain.6 we report a case of an adolescent female with undiagnosed thyrotoxicosis who was initially treated for vomiting under a diagnosis of duodenogastric reflux and reflux esophagitis. a 13-year - old female patient was referred to the gi outpatient department of our hospital from a pediatric clinic to undergo the esophagogastroduodenoscopy for the evaluation of recurrent vomiting that lasted for 8 days. although vomiting was not related to food intake, she had avoided eating because of recurrent vomiting. she also complained of nausea, boring epigastric pain and palpitation, but denied diarrhea or melena. she had lost 8 kg of weight over 4 months, and her weight was 39.4 kg (10 - 25 percentile) and height was 156 cm (50 - 75 percentile) at presentation. her blood pressure was 100/80 mmhg, pulse rate was 110/min, respiratory rate was 20/min and body temperature was 36.6. there was no definite enlargement or palpable mass on the anterior neck, eye abnormality or lymphadenopathy. the abdomen was soft with normal bowel sound and there was no direct or rebound tenderness on the abdomen. plain abdominal x - ray showed nonspecific findings. because she had weight loss and palpitation with anxiety, thyroid stimulating hormone (tsh) esophagogastroduodenoscopy at the first visit of gi outpatient department revealed mild mucosal erythema on z - line and acute erythematous gastritis with large amount of duodenogastric reflux (fig. she was prescribed a proton pump inhibitor, prokinetic and mucosal coating agent (sucralfate) on the basis of her endoscopic diagnosis with gi department follow up after 2 weeks. her vomiting improved during the first 2 days after medication, however, she visited the emergency department (ed) 5 days later because of recurred vomiting and boring epigastric pain. her blood pressure was 140/80 mmhg, pulse rate was 88/min, respiratory rate was 20/min and body temperature was 36.8. physical examination revealed mild epigastric direct tenderness. the physician of the ed found that she already had blood tests including tsh at gi outpatient department and it revealed tsh 8.0 ng / ml (normal range 0.60 - 1.81 ng / ml), free thyroxine (t4) > 12.0 ng / dl (normal range 0.89 - 1.76 ng / dl), tsh receptor antibody 37.4% (normal range 15.0%), anti - thyroid microsomal antibody 6.22 u / ml (normal range 3.0 u / ml) and thyroglobulin antibody 6.91 u / ml (normal range 3.0 u / ml). thyroid ultrasonography revealed diffusely enlarged glands, decreased parenchymal echogenecity and increased vascularity of both glands (fig. 2). treatment was initiated with propylthiouracil (ptu) 3.75mg / kg / day and propranolol 1 mg / kg / day, after 4 days of treatment, vomiting, epigastric pain and palpitation were improved remarkably. she was discharged with a maintenance dose of ptu (3.75 mg / kg / day) without prokinetic or proton pump inhibitor. liver function tests returned to normal limits, but thyroid function tests were still elevated (tsh < 0.01 iu / ml, free t3 6.31 ng / ml and free t4 5.09 ng / dl). we switched treatment from ptu to methimazole 0.4 mg / kg / day and added inorganic iodine (lugol 's solution) for 5 days to prevent thyroid hormone release. after 3 days, there was no further vomiting, and free t3 and free t4 were decreased to 4.46 ng / ml and 2.24 ng / dl, respectively. she was discharged with a maintenance dose of methimazole (0.4 mg / kg / day) and propranolol (1 mg / kg / day). after 2 months with methimazole and propranolol maintenance therapy, her symptoms were stable and free t4 returned to normal limits (1.27 ng / dl) despite still having low level of tsh (< 0.01 iu / ml). over next 16 months with methimazole monotherapy, vomiting has not recurred and she became euthyroid with normalized tsh receptor antibody. gi symptoms such as hyperphagia, diarrhea, and frequent defecation are commonly associated with thyrotoxicosis, whereas vomiting, abdominal pain, and dysphagia are uncommon. although vomiting, nausea and abdominal pain have not been generally included as common presenting symptoms for thyrotoxicosis, a review of 25 newly diagnosed thyrotoxicosis cases reported that the significant number of thyrotoxic patients complained of vomiting (44%), nausea (28%) and abdominal pain (20%).7 one or more of these abdominal symptoms were included as a chief complaint in 36% of cases reviewed.7 interestingly, thyrotoxic vomiting can occur intermittently, and unique clinical features of thyrotoxicosis including goiter, ophthalmopathy such as exophthalmos or lid retraction are rarely associated with cases of thyrotoxic vomiting.5,6,8 our patient was diagnosed as hyperthyroidism caused by graves ' disease based on autoantibodies and sonographic finding. she was 13-year - old child and the characteristics of pediatric graves ' disease also make clinician difficult to diagnose thyrotoxicosis. in pediatric graves ' disease, the size of the thyroid gland is highly variable and the goiter may go unnoticed in patients with a slightly enlarged thyroid gland, and ophthalmic abnormalities are less severe in children than in adults.9 therefore, children with undiagnosed thyrotoxicosis may initially be referred to cardiologists with a heart murmur, gastroenterologists with gi symptoms and failure to thrive, or psychiatrist because of challenging behavior and school refusal before being referred to an endocrinologist.10 when rare gi symptoms like vomiting present as initial symptoms in patients with thyrotoxicosis and there are no typical features of thyrotoxicosis, it can be misdiagnosed as gi disorders. if the physician is misled to another gi disorder and vomiting is prolonged for several weeks under wrong treatment, this patient not only can progress to a life - threatening situation, but also may have many unnecessary tests under suspicion of functional vomiting or even exploratory laparotomy.1,6,11 in cases of thyrotoxicosis that showed only gi symptoms, diagnostic clues might be chronicity of symptoms, prominent weight loss and tachycardia.6,8,12 in our patient, the prominent weight loss over several months, palpitation and anxiety were clues to the suspicion of thyrotoxicosis. however, since these symptoms are not pathognomic signs of thyrotoxicosis and often accompanied by other gi symptoms, proper diagnosis is difficult and delayed considerably. the first proposed mechanism is an increase in -adrenergic activity due to an increased number of -adrenergic receptors.13 there have been several reports of rapid symptomatic improvement after administration of -blockers before the normalization of thyroid hormones.6,8,12 based on these reports, it is conceivable that the vomiting in thyrotoxicosis may be attributed to sympathomimetic effects of thyroid hormones as dysrhythmias, fever, tremors, palpitations and anxiety. the rapid symptomatic improvement may reflect the ability of -blockers to reduce the peripheral conversion of thyroxine to triiodothyronine.12 second, increased thyroid hormones can cause emesis. there is a reported case that symptoms were not improved by the administration of -blockers but improved after thyroid hormone levels normalized.14 in our case, clinical symptoms were temporarily improved by the administration of -blocker with subsequent recurrence within 5 days and were completely improved after thyroid hormone levels decreased to near normal range by lugol 's solution. the mechanism for vomiting in thyrotoxicosis may be similar to that for vomiting in hyperemesis gravidarum. increased -subunit of human chorionic gonadotropin induces secretion of thyroxine through a tsh - like activity.15 however, it is still controversial whether thyroid hormones themselves act as an emetic factor in thyrotoxic vomiting, although it has been speculated that thyroid hormones stimulate the chemical trigger zone.5 third, thyroid hormones can alter gastric motility and decrease gastric emptying secondary to the malfunction of the pyloric sphincter. a plausible explanation for this phenomenon is that thyroid hormones alter magnesium homeostasis, and hypomagnesemia affect smooth muscle directly or autonomic innervations of the upper gi smooth muscle.16,17 the rate of gastric emptying is slightly increased after the restoration of euthyroidism18 and postprandial tachygastrias are significantly reduced after antithyroid therapy.19,20 fourth, the estrogen was suggested an emetic cause in thyrotoxicosis because there are female predominance in thyrotoxic vomiting and estrogen level could be increased in patients of both sexes with thyrotoxicosis.5,21 and then the raised estrogen level may induce nausea and vomiting only in susceptible patients.22 lastly, it was suggested that functional duodenal obstruction in thyrotoxicosis may present much like the obstruction in superior mesenteric artery syndrome.14 possible causes of this obstruction are prominent weight loss, supine position in a patient with prolonged and severe illness and hypermotility induced volvulus of duodenal third portion. thyrotoxic vomiting has a excellent prognosis and usually improved within several days after initiation of antithyroid treatment in the majority of reported cases.5 - 8,12 however, since our patient was pediatric graves ' disease, we should consider genetic background of graves ' disease and higher frequency of relapse than adult.9 in conclusion, the possibility of atypical thyrotoxicosis should be considered in patients with persistent unexplained vomiting. since symptomatic improvement is related to the thyroid hormone level, appropriate treatment strategies and meticulous surveillance for the thyroid hormone level are mandatory. | the symptoms related to gastrointestinal (gi) tract are sometimes chief complaints in patients with endocrine disease. thyrotoxicosis is a rare, but notable cause for unexplained and repeated vomiting. here, we report an adolescent patient with thyrotoxicosis who was initially presented with repeated vomiting and epigastric pain. a 13-year - old female was referred to a gi outpatient department for evaluation of vomiting and abdominal pain from a pediatric clinic. esophagogastroduodenoscopy revealed acute gastritis with duodenogastric reflux and suspicious reflux esophagitis of minimal change, but there was no significant improvement after treatment and as a result she was admitted to the emergency room. she was subsequently diagnosed as graves ' disease because an initial laboratory test at the gi outpatient department revealed thyroid stimulating hormone < 0.01 iu / ml and additional blood tests showed elevated thyroid hormones and positive thyroid stimulating hormone receptor antibody. the vomiting and epigastric pain improved remarkably after treatment with antithyroid drugs. clinicians should consider the possibility of thyrotoxicosis in patient with unexplained and repeated vomiting. |
otodectes cynotis is a mite of the family psoroptidae, which lives predominantly in the external ear canal and occasionally on the adjacent skin of the head in dogs, cats, foxes, ferrets, and even humans. it is a non - burrowing surface - living mite that feeds on tissue fluid and debris. infestation of the mite results in pruritic symptoms and otitis externa which is characterized by ear canal erythema and the presence of dark brown, ceruminous otic exudate. the prevalence of the mite in dogs ranges from 2% to 29% in london and queensland, and that in cats was from 9% to 37% in florida (usa), greece, and japan [6 - 9 ]. in the republic of korea, the prevalence of o. cynotis was reported to be 22.3% among dogs in an animal shelter, and an ear mite treatment case with selamectin in a dog has been reported. however, no studies about the occurrence and treatment of o. cynotis in cats have been available in korea. in this study, we report 3 cats that had pruritic symptoms in gwangju, korea due to the infestation with o. cynotis. using the korean isolate of o. cynotis, we also experimentally infested domestic cats and assessed the efficacy of 10% imidacloprid/1% moxidectin spot on. in april 2010, pruritic symptoms characterized by scratching, rubbing of the ears, and shaking of the head were recognized in 3 privately - owned siamese cats raised in gwangju, korea. two were females and 1 was male, and they were between 8 months and 2 years old. examination of ear canals using an otoscope (piccolight, kawe, berlin, germany) revealed dark brown, ceruminous otic exudates. numerous live mites were collected from the ears of infested cats from which all developmental stages in life cycle (egg, larva, nymph, and adult) were observed (fig. the mites were preserved in 70% ethanol and mounted on slide glass using pva solution. the average length and width of 6 adult females were 448.3290.0 m, while those of 6 adult males were 360.0277.8 m. caruncles were present on legs 1 and 2 in adult females and on legs 1, 2, 3, and 4 in the adult male (fig. the tarsus of leg 3 in both sexes was equipped with 2 long setae (fig.1d). ten mite - free domestic shorthaired cats (5 males and 5 females ; weight range, 2.0 - 4.3 kg ; age range, 1 - 2 years) were purchased from a commercial vender. cats were thoroughly checked for the presence of any ear mite or other health problems. experimental infestation of uninfected cats was performed twice for 2 weeks in which cerumen containing various stages of mites from the ear of the 3 naturally infested cats was collected in a petri dish and was transferred into both ears of o. cynotis - free cats using an ear swab. one month later, the presence of adult mites and eggs in both ears was confirmed in 10 cats by otoscopic and microscopic examinations. cats did not receive any acaricidal drugs for at least 60 days prior to the treatment. animals were housed in an appropriate accommodation that conformed to accepted guidelines for pen design / floor area, lighting, humidity, temperature, and welfare (including environmental enrichment and social interaction), as required by local and national legislations. ten cats were randomly allocated to the test group and 3 to the control group. the test group was treated with 10% imidacloprid/1% moxidectin spot - on (advocate for cats, bayer animal health gmbh, leverkusen, germany, abbreviated as im / mox hereafter), applied at a dose of 0.1 ml / kg body weight on day 0. the im / mox was administered once topically to the skin in a single spot on each animal 's back at the base of the neck in front of the scapulae. the ears of the cats were examined for the presence of ear mites by using the otoscope. ear scrapes and dry cotton swabs were also used to collect debris in the ears on day 0, 9, 16, and 30 post - treatment (pt), and live ear mites were searched under a dissecting microscope (zeiss, stemi 2000-c, jena, germany). the presence of even 1 live mite in any side of ears was considered as a failure of treatment. the number of cats that had no live mites was compared to the number of all cats tested. the percentage of the drug efficacy was calculated as follows : the percentage of efficacy (%) = x = number of cats with live ear mite y = total number of cats treated upon treatment with im / mox, live mites were recovered only from 1 of 10 treated cats on day 9 (table 1). on days 16 and 30 pt, no live ear mites were observed from the ear canals of treated cats. ear canals of 3 cats in the control group, on the other hand, showed various developmental stages of mite from day 0 to 30. the efficacy of im / mox on o. cynotis in cats was, therefore, 90% on day 9 and 100% on days 16 and 30 pt. the 3 cats in the control group were also treated with im / mox after the chemotherapy study which resulted in a successful removal of mites from the cats. ten mite - free domestic shorthaired cats (5 males and 5 females ; weight range, 2.0 - 4.3 kg ; age range, 1 - 2 years) were purchased from a commercial vender. cats were thoroughly checked for the presence of any ear mite or other health problems. experimental infestation of uninfected cats was performed twice for 2 weeks in which cerumen containing various stages of mites from the ear of the 3 naturally infested cats was collected in a petri dish and was transferred into both ears of o. cynotis - free cats using an ear swab. one month later, the presence of adult mites and eggs in both ears was confirmed in 10 cats by otoscopic and microscopic examinations. cats did not receive any acaricidal drugs for at least 60 days prior to the treatment. animals were housed in an appropriate accommodation that conformed to accepted guidelines for pen design / floor area, lighting, humidity, temperature, and welfare (including environmental enrichment and social interaction), as required by local and national legislations. ten cats were randomly allocated to the test group and 3 to the control group. the test group was treated with 10% imidacloprid/1% moxidectin spot - on (advocate for cats, bayer animal health gmbh, leverkusen, germany, abbreviated as im / mox hereafter), applied at a dose of 0.1 ml / kg body weight on day 0. the im / mox was administered once topically to the skin in a single spot on each animal 's back at the base of the neck in front of the scapulae. the ears of the cats were examined for the presence of ear mites by using the otoscope. ear scrapes and dry cotton swabs were also used to collect debris in the ears on day 0, 9, 16, and 30 post - treatment (pt), and live ear mites were searched under a dissecting microscope (zeiss, stemi 2000-c, jena, germany). the presence of even 1 live mite in any side of ears was considered as a failure of treatment. the number of cats that had no live mites was compared to the number of all cats tested. the percentage of the drug efficacy was calculated as follows : the percentage of efficacy (%) = x = number of cats with live ear mite y = total number of cats treated upon treatment with im / mox, live mites were recovered only from 1 of 10 treated cats on day 9 (table 1). on days 16 and 30 pt, no live ear mites were observed from the ear canals of treated cats. ear canals of 3 cats in the control group, on the other hand, showed various developmental stages of mite from day 0 to 30. the efficacy of im / mox on o. cynotis in cats was, therefore, 90% on day 9 and 100% on days 16 and 30 pt. the 3 cats in the control group were also treated with im / mox after the chemotherapy study which resulted in a successful removal of mites from the cats. in this study, we described the first case of otodectosis in 3 cats naturally infested with o. cynotis in gwangju, korea, and successful treatment of experimentally infested ear mites with 10% imidacloprid/1% moxidectin spot - on. in korea, although the prevalence of o. cynotis in dogs was 22.3%, that of cats has not been studied, probably because of severe bias in population of cats and dogs in korea. according to a study in korea, the population of dogs had been up to 97.8% of total cats and dogs in 2006. this phenomenon might have resulted by the common perception on cats by older generation of korea who generally consider cats as unlucky and sneaky animals. treatments of the ear mite include mechanical cleaning of the ear canal followed by topical or systemic drug administration with such drugs as selamectin, ivermectin, and fipronil. in cats, there exist many difficulties to administer the drugs via oral route which can cause esophagitis, esophageal stricture, a longer esophageal transit time, and retention in mid - cervical region of the esophagus. topical spot - on drugs has recently been considered as an ideal administration route because it can solve those problems of oral administration and be used without cleaning the ear before administration of the drug or treating the hair coat and living environment. the topical spot - on medication can also bypass the intestinal and hepatic first - pass effects. for these reasons, we have chosen the topical spot - on medication route to treat feline otocariosis in our study. among many treatment studies using 10% imidacloprid/1% moxidectin in cats, a single treatment with the drug applied at a dose of 0.1 ml / kg body weight resulted in a treatment success rate of 80%, as assessed 50 days after treatment in cats. the efficacy of advantage multi that contains 10% imidacloprid/1% moxidectin against ear mites was 92.5% after the first treatment and 98.1% after the second treatment in client - owned cats. also, the efficacy of 10% imidacloprid/1% moxidectin against o. cynotis in another study was 100% on days 16 and 30 pt in cats. in our study, the efficacy of 10% imidacloprid/1% moxidectin spot - on was 90% on day 9 and 100% on days 16 and 30 pt which was significantly higher than that of day 0 and indicates that the single spot - on medication of the drug provided more than 90% efficacy against o. cynotis - infested cats. | in april 2010, pruritic symptoms were recognized in 3 privately - owned siamese cats raised in gwangju, korea. examination of ear canals revealed dark brown, ceruminous otic exudates that contain numerous live mites at various developmental stages. based on morphological characteristics of adult mites in which caruncles were present on legs 1 and 2 in adult females and on legs 1, 2, 3, and 4 in adult males while the tarsus of leg 3 in both sexes was equipped with 2 long setae, the mite was identified as otodectes cynotis. ten ear mite - free domestic shorthaired cats were experimentally infected with o. cynotis to evaluate the efficacy of 10% imidacloprid/1% moxidectin spot - on. live mites were recovered from 1 of 10 treated cats on day 9 post - treatment (pt) while no live mites were observed from the ear canals of treated cats on days 16 and 30 pt. the efficacy of 10% imidacloprid/1% moxidectin spot - on on o. cynotis in cats was, therefore, 90% on day 9 and 100% on days 16 and 30 pt. this is the first report of otodectosis in 3 cats naturally infested with o. cynotis in gwang - ju, korea. both natural and experimental infestations were successfully treated with 10% imidacloprid/1% moxidectin spot - on. |
histoplasmosis is a fungal disease caused by the dimorphic fungus histoplasma capsulatum (h. capsulatum) the disease is endemic in certain parts of the world including asia (benevides., 2007). spores containing h. capsulatum can be found in soils contaminated by droppings from birds and bats. patients are infected via inhalation of these spores and up to 80 % of them are skin test positive with histoplasmin (jombo., 2010). most infected patients are asymptomatic or have a self - limiting disease (rana., 2011 ; mahajan., 2000). histoplasmosis usually presents in 2 forms ; pulmonary and extrapulmonary, which is also known as disseminated histoplasmosis (dh). disseminated histoplasmosis is uncommon but frequently (80 %) affects the adrenal glands (goodwin., 1980). in such cases, patients often present with bilateral adrenal masses (kumar., 2003). however, the incidence of hypoadrenalism amongst these group of patients is rare and was reported to be ranging between 7 to 20 % (subramanian., 2005 ; rajesh., hence, a high index of suspicion is prudent as the presentation may mimic other chronic infections or malignancy especially in the elderly or the immunosuppressed host (jaiswal., 2011). we report a case of an elderly man with bilateral adrenal histoplasmosis and normal cortisol response who presented with vague symptoms and hypercalcaemia. a 75-year - old man, with a background medical history of diabetes and hypertension for 10 years, presented to a private hospital with a history of prolonged fever, malaise and marked weight lost for a month. computed tomography (ct) scan of the abdomen revealed bilateral adrenal masses measuring 3.4 x 2.9 cm (right) and 3.9 x 3.1 cm (left) respectively. after being treated empirically with oral fluconazole 200 mg daily for 2 months, his symptoms disappeared and he did not attend subsequent clinic appointments. six months later, he presented to our institution with similar symptoms of generalized bodyaches and 4 month 's duration of significant loss of appetite and weight. on further questioning he had normocytic anaemia (hemoglobin 10.6 x 10/l), mild thrombocytopenia (133 x 10/l) and acute kidney injury with urea of 17.8 mmol / l, potassium 4.4 mmol / l, sodium 144 mmol / l and creatinine 365 mmol / l. both corrected serum calcium and alkaline phosphates were elevated with levels of 3.40 mmol / l and 319 u / l respectively. serology for syphilis, hepatitis a, b, c, toxoplasma antibodies and tumour markers (alpha fetoprotein, cea 125, ca 19 - 9) were negative. serum iron and tibc were low with levels of 8.7 umol / l and 35 umol / l respectively. a repeated ct scan of the adrenals showed persistent bilateral adrenal masses measuring 5 x 4 cm on the left and 4 x 3.6 cm on the right respectively (figure 1(fig. fine needle aspiration cytology of the left adrenal gland was consistent with histoplasmosis infection (figure 3(fig. the patient was treated with liposomal form of amphotericin b 180 mg daily for 2 weeks followed by oral itraconazole 200 mg bd. his serum calcium normalised and creatinine level stabilized at 171 mmol / l after adequate rehydration. the patient was discharged home after 2 months of hospital stay with oral itraconazole which was continued for a total of 1 year duration of treament. on his subsequent clinic appointments, he was asymptomatic, gained weight and tolerated his treatment well. the presentation of disseminated histoplasmosis usually mimics other chronic infections or malignancy as illustrated in this case. the differential diagnoses that should be considered include tuberculosis, histoplasmosis, sarcoidosis, adrenal haemorrhage, metastatic carcinoma and lymphoma (mukherjee., 2005 ; narang., 2009). inhalation of h. capsulatum leading to pulmonary infection is usually asymptomatic or without any sequelae. however, previous study had shown that the reactivation of infection can manifest in severe forms and usually occurs in extreme age and in an immunocompromised or immunosuppressed state such as in a diabetic patient (jaiswal., 2011). our patient is an elderly man with underlying diabetes and had history of contact with pigeons. once infected, the period of latency may extend up to 60 years (jaiswal., 2011). kauffman showed only 12 out of 58 elderly patients with histoplasmosis were found to have adrenal involvement and none of them had adrenal failure (kauffman, 2001). typically, patients with adrenal histoplasmosis present with bilateral adrenal enlargement with normal configuration, peripheral enhancement and central hypodensity as a result of necrosis or haemorrhage on the ct scan (mukherjee., however, bilateral adrenal enlargement with similar characteristics may be also be seen in adrenal haemorrhage, lymphoma, metastatic or disseminated infections such as histoplasmosis, tuberculosis, cryptococcosis, blastomycosis, or aspergilosis (mukherjee., 2005). the presence of central necrosis and peripheral rim enhancements of the adrenals would narrow down the above differentials to tuberculosis and histoplasmosis. hence, cytology or histopathological examination of the adrenal mass after a fine needle aspiration or percutaneous biopsy which exhibits typical microscopic features of h. capsulatum would be the best method to differentiate these two diagnoses especially in the absence of conclusive supporting history which would point to either one. h. capsulatum is an intracellular dimorphic fungus which is usually seen in the macrophages cytoplasm and exhibits narrow based budding (fitzhugh., 2010 ; gopal., 2010). histopathological examination from the repeat adrenal biopsy done a year later was consistent with histoplasmosis. the mortality in untreated dh patients was as high as 80 - 100 % but if treated with antifungal, this very high mortality rate is remarkably reduced to less than 25 % (subramanian., 2005). the recommended treatment for adrenal histoplasmosis is similar to the disseminated histoplasmosis by using amphotericin b. previous study recommended a total dose of 3 to 4 g of amphotheracin b (johnston., 1996). once the condition improves, the therapy can be switched to itraconazole 200 mg bd. in our patient, the primary antifungal therapy instituted was liposomal amphoteracin b for 2 weeks. upon discharge, apart from less nephrotoxicity, the liposomal amphothericin b achieves the cumulative target dose faster than regular amphotericin b, resulting in shortened hospital stay. recurrence of disease has been reported after 9 years after cessation of therapy (rana., 2011). a treatment duration of between 1 to 2 years is recommended to reduce recurrence (rana., 2011). when faced with a patient with bilateral adrenal masses, physicians should always be suspicious of adrenal histoplasmosis especially in endemic areas. it is prudent to obtain a cytological or histopathological examination to confirm the diagnosis so that the appropriate treatment can be instituted to avoid fatal complications. | histoplasmosis infection is endemic in asia and disseminated histoplasmosis (dh) is one form of its presentation (benevides., 2007[1 ]). dh commonly affects both adrenal glands. we describe a case of disseminated histoplasmosis complicated with hypercalcaemia in a 75-year - old immunosuppressed patient who presented with bilateral adrenal masses. the fine needle aspiration cytology of the adrenal mass was positive for histoplasma capsulatum. |
tumor necrosis factor- (tnf-) like weak inducer of apoptosis (tweak, also named tnfsf 12) is a member of the tnf - family of cytokines with multifunctional properties. tweak is expressed as a type ii transmembrane protein and then released into the interstitium after proteolysis of an active soluble 18 kd fragment (stweak). tweak mediates its signaling through binding to its receptor, fgf - inducible 14 kd protein (fn14, also tnfrsf12a or tweakr). in addition, tweak has also been reported to bind another membrane protein, the scavenger receptor cd163, although this has been questioned by others. like stweak, cd163 can be released and measured in blood samples as a soluble fragment (scd163). levels of scd163 in the circulation have been related to stweak levels in a number of diseases like peripheral artery disease, diabetes, chronic renal failure, or systemic sclerosis [48 ]. recently, a role for stweak in cardiac pathophysiology has been proposed [5, 6, 911 ]. interestingly and in contrast to other cytokines, stweak serum levels were found to be reduced in patients with chronic heart failure and dilated cardiomyopathy as well as some other chronic diseases with increased inflammatory activity [47 ]. we and others have also reported the potential value of stweak levels for the prediction of all - cause mortality in patients with chronic stable heart failure or dilated cardiomyopathy [12, 13 ]. however, it is still unclear whether stweak has the potential as a biomarker for disease monitoring in patients with heart failure similar to nt - probnp. in addition, why a single determination of stweak predicts long - term outcome after a couple of years in patients with dilated cardiomyopathy remains unresolved. therefore, we conducted a follow - up study in a cohort of stable patients with dilated cardiomyopathy to evaluate the usefulness of serial stweak determinations and their intraindividual variation over time in relation to their symptoms. patients were seen on a yearly basis in our outpatient clinic with serial determination of clinical, functional, and laboratory parameters with follow - up assessment after four years. a cohort of seventy - eight stable patients diagnosed with dilated cardiomyopathy were identified and followed on a yearly basis in the heart failure clinic of our university hospital that serves as a tertiary referral center in southern germany. eligible patients had to have the diagnosis of dilated cardiomyopathy, to be at least 18 years of age, and to have a reduced left ventricular systolic function with an ejection fraction of less than 50% on echocardiographic evaluation. all examinees enrolled in the present study had to have a stable clinical condition and medication for at least 1 month before inclusion. patients with malignant or inflammatory diseases, acutely decompensated heart failure (nyha class iv), a history of organ transplantation, or significant acute / chronic renal failure (serum creatinine > 2 mg / dl) were not included in our study. patients with significant coronary artery disease, considered to be responsible for the reduced left ventricular function, were also excluded from our study. blood samples of these patients were taken on 3 consecutive follow - up visits once a year in addition to clinical and functional evaluation of the patients including a 6-minute walk test (6mwt) and a peak oxygen consumption study (peak vo2max). soluble tweak, scd163, and nt - probnp levels were determined in different aliquots of the same blood sample. all patients included in our study completed a four - year follow - up visit or a telephone call. blood samples were drawn in all patients once a year during a follow - up visit on three consecutive occasions. plasma samples were generated within 30 minutes of collection by centrifugation at 1000 g for 10 minutes at 4c. to avoid repetitive freeze - and - thaw cycles, plasma samples were taken as part of a multiple biomarker registry project in patients with dilated cardiomyopathy and heart failure, for which only serial plasma samples were available. soluble tweak levels were determined with an elisa by use of a commercially available kit tested for determination of human plasma samples (bender medsystems, vienna, austria) according to the manufacturer 's instructions. briefly, a 1 : 2 diluted test sample was incubated for 3 hours at room temperature in wells precoated with an anti - human stweak antibody together with a biotin - conjugated anti - human tweak antibody which binds to human tweak captured by the first antibody. following incubation unbound biotin conjugated anti - human tweak and streptavidin - hrp is removed during a wash step, and substrate solution reactive with hrp is added to the wells and wells are incubated for approximately 10 to 20 minutes. a colored product is formed in proportion to the amount of soluble human tweak present in the sample. the reaction is terminated by addition of acid and absorbance is measured at 450 nm. a standard curve is prepared from 7 human tweak standard dilutions and human tweak sample concentration is determined. absorbance was measured with an automatic elisa reader (tecan spectra, crailsheim, germany). human stweak is detected with this kit at a threshold of 9.7 pg / ml. intra - assay and interassay coefficients of variation were 7.9% and 9.2%, respectively, according to the manufacturer. all measurements were performed in duplicate by an investigator unaware of patients ' characteristics and outcome. soluble cd163 levels were determined with the macrocd163 elisa assay (iq products, trillium diagnostics). soluble cd163 was only determined once at the time point of study initiation from the same sample as the first stweak value was determined. briefly, a polyclonal antibody recognizing cd163 is immobilized on the surface of a microtiter plate. after incubation with the sample or the standard containing recombinant cd163, a second biotinylated monoclonal antibody recognizing cd163 is added. using tmb (3,3,5,5-tetramethylbenzidine) as a substrate for hrp, the amount of cd163 in the sample can be determined. intra - assay variability of the assay is 36% ; interassay variability is 58%, when measuring duplicates according to the manufacturer. as with stweak determination measurements were performed at the clinical core laboratory of the university hospital heidelberg with an elisa (roche diagnostics, mannheim, germany). data are presented as median (interquartile range) or count and percentages. for all continuous variables that were not normally distributed (tested by the kolmogorov - smirnov test), the optimal plasma stweak cutoff values to predict an adverse outcome in the present study population were calculated by a receiver operating characteristic (roc) curve analysis. patients were compared according to the stweak cutoff value with the use of log - rank test for the 4-year survival curves. statistical analyses were performed with prism 5.0 (graphpad software, san diego, california) and medcalc 9.3.0.0 (medcalc, mariakerke, belgium) software. we serially analyzed a cohort of 78 patients diagnosed with dilated cardiomyopathy and evaluated stweak levels, nt - probnp levels, and functional parameters on a yearly outpatient basis in our heart failure clinic. the median ejection fraction (ef) of our patient cohort was 29% (interquartile range (ir) of 20 to 48%) and the median left ventricular end - diastolic diameter (lvedd) was 60 mm with an interquartile range of 52 to 74 mm at the time point of study initiation. median age of our patients was 55 years (ir : 43 to 64 years). median of stweak at study start was 603 pg / ml (ir : 423 to 839 pg / ml) and that of nt - probnp was 482 ng / l (ir : 211 to 1505). twenty - eight patients (35.9%) had atrial fibrillation, but only 4 (5.1%) developed thromboembolic events during follow - up. overall nyha class of the patient cohort was inversely related to peak oxygen consumption (vo2max) and walking distance during a 6mwt (peak oxygen consumption (vo2max) : 22.0 (ir : 16.6 to 27.2) versus 17.7 (ir : 14.3 to 20.0) versus 13.7 (ir : 12.2 to 17.6) ; 6mwt : 587 (ir : 518 to 638) versus 492 (ir : 430 to 559) versus 395 (ir : 311 to 467) ; nyha class i versus ii versus iii each ; p < 0.05 ; figure 1(a)). moreover, nt - probnp levels significantly increased with each nyha class, whereas stweak levels decreased (figure 1(b)). characteristics of our patient cohort are summarized in table 1. among our 78 patients, 4 deaths and soluble tweak levels were lower in patients who died or were transplanted (372 pg / ml (239 to 405) versus 617 pg / ml (434 to 841) ; median (interquartile range) ; p = 0.0049). roc curve analysis of baseline stweak levels in our patient cohort revealed a cutoff value of 423 pg / ml for the prediction of death or heart transplantation (figure 2(a)). kaplan - meier curve analysis demonstrated a significantly reduced survival in patients with a stweak value below 423 pg / ml (figure 2(a)). we further compared those patients who reported later a significant deterioration of their functional capacity (1 nyha class) over time to those with a stable condition on follow - up. in patients with progressive symptoms, stweak levels were already significantly reduced at the first visit with a median of 432 pg / ml (ir : 322 to 568 pg / ml) (n = 41) compared to 833 pg / ml (ir : 674 to 1066 pg / ml) (n = 37) in stable patients (p < 0.0001) (figure 2(b)). roc curve analysis showed that a cutoff value of 588 pg / ml predicted a deterioration of at least 1 nyha class during follow - up (figure 2(b)). serial evaluation of stweak levels allowed us to analyze whether stweak showed intraindividual variations dependent on the actual clinical condition similar to the use of nt - probnp. however, soluble tweak levels showed little variation in most patients throughout the follow - up period, no matter whether they developed progressive worsening of their clinical and functional parameters or not. soluble tweak levels remained significantly lower in patients with deterioration compared to those with stable symptoms, indicating that interindividual rather than intraindividual differences predicted clinical deterioration (figure 3). similarly, stweak levels correlated with maximum oxygen uptake during exercise (vo2max) and the results of the 6mwt, except for the first visit (6mwt) (first visit : vo2max : r = 0.3820 ; p = 0.0026 ; 6mwt : p = 0.1099 ; second visit : 6mwt : r = 0.3539 ; p = 0.0021 ; vo2max : r = 0.4693 ; p = 0.0001 ; third visit : 6mwt : r = 0.4086 ; p = 0.0002 ; vo2max : r = 0.4097 ; p = 0.0006). similarly, when multivariate regression analysis was used to analyze an independent association between stweak levels and vo2max over all visits, stweak levels remained significantly associated (other variables associated with vo2max on univariate analysis : ejection fraction [p = 0.0258 ] ; nt - probnp [p = 0.0018 ] ; retained variables in multivariate analysis : stweak [p < 0.001 ] ; nt - probnp [p = 0.002 ]). recently, the ratio between scd163 and stweak has been proposed to improve the predictive value of stweak levels in patients with various clinical conditions [48 ]. we therefore analyzed scd163 levels in our cohort of patients with dilated cardiomyopathy on their first visit and evaluated their relation to stweak levels in the same sample. no significant association between scd163 levels and stweak levels was found (p = 0.7904). compared to stweak alone, roc curve analysis of the scd163/stweak ratio in patients with dilated cardiomyopathy increased neither sensitivity nor specificity for the prediction of death / heart transplantation or clinical deterioration on follow - up (p = 0.650 for death / heart transplantation and p = 0.099 for clinical deterioration). here, we report for the first time the results of a serial study of stweak levels in a cohort of patients with dilated cardiomyopathy. similar to previous reports, a single stweak determination predicted long - term outcome in stable patients with dilated cardiomyopathy and reduced left ventricular function. in contrast to nt - probnp, we found that stweak showed little variation on serial determination within individual patients, independently of any clinical or functional deterioration. thus, stweak values differed significantly at baseline between individuals with subsequent progressive clinical deterioration and those with a stable clinical course. moreover, scd163 levels did not add additional information for outcome in our patient cohort. finally, scd163 did not correlate to the reduction of stweak levels in patients with dilated cardiomyopathy. the natural disease progression in patients with dilated cardiomyopathy can be quite variable despite similar echocardiographic and hemodynamic parameters. we and others have recently reported that reduced stweak levels predicted long - term outcome in patients with dilated cardiomyopathy [12, 13 ]. however, how a single determination of stweak could predict outcome and whether stweak levels might be helpful for disease monitoring in these patients remained unclear. in addition, the role of scd163, which has been related to stweak levels in other diseases, was unknown for patients with dilated cardiomyopathy. thus, our study provides new information not only about the usefulness of stweak as a biomarker for patients with dilated cardiomyopathy, but also about the role of scd163 in this setting. our study adds additional evidence that stweak levels may not be useful for disease monitoring but may rather be related to an increased risk for disease progression in a subset of patients. this is of potential interest in the setting of dilated cardiomyopathy, since stweak has also been related to several autoimmune disorders and vice versa autoimmunity has been suggested to play a role in the pathogenesis of patients with dilated cardiomyopathy. for example, autoantibodies against several molecular targets in the heart and additional features of immune activation like cytokine activation and intramyocardial inflammation are detectable even in apparently healthy relatives of patients with dilated cardiomyopathy [1416 ]. thus, decreased stweak levels detected in the circulation of patients with dilated cardiomyopathy might represent a risk factor for a progressive course of the disease. whether stweak plays a role in the pathogenesis of dilated cardiomyopathy or whether the association between reduced levels and outcome represents just an epiphenomenon of changes in the regulation of the immune system remains to be determined. jain. have reported the occurrence of dilated cardiomyopathy in transgenic animals overexpressing tweak. in addition, upregulation of fn14 in stressed myocardium can be found in experimental models of heart failure [18, 19 ]. increased clearance of stweak due to chronic fn14 upregulation in the heart however, sfn14 levels in serum or plasma were not determined in our patient cohort, mainly because their role for stweak levels measured in the circulation has not been studied very well so far. in contrast to sfn14, the ratio between stweak and scd163 has been described to be of value in other clinical settings, although a significant interaction between both molecules has been questioned by others [46, 20, 21 ]. our data suggest that scd163 is less likely to be responsible for the reduction of stweak in patients with dilated cardiomyopathy. moreover, the ratio between scd163 and stweak did not increase sensitivity or specificity for the prediction of an adverse outcome in patients with dilated cardiomyopathy. absolute values of stweak levels in healthy control patients have been reported in several studies using different detection assays in the past reviewed in. usually reported serum levels of stweak in a healthy caucasian population range between 300 and 450 pg / ml, if measured with the assay we used in this study. although we did not include a control cohort in our study, the median of stweak levels detected in our patients with dilated cardiomyopathy was slightly higher. one of the reasons for this finding might be the use of plasma instead of serum for serial evaluation of stweak levels, which could have affected absolute levels of stweak. we can not completely rule out all confounders ; nevertheless, our relatively small study cohort consisted of a homogenous patient population : coronary angiography was used in all patients to exclude significant coronary artery disease, which might have affected stweak measurements. all patients included had three consecutive visits with determination of stweak levels and functional testing and all of them completed 4-year follow - up. almost all patients were on -blockers and angiotensin - converting enzyme inhibitors or angiotensin receptor blockers and, therefore, on optimized therapy. all patients had to be clinically stable ; patients with acute decompensation were not included. patients with a history of malignant disease or a chronic inflammatory disease were also not included in our study. in summary, our study is the first to provide data on serial stweak determination in combination with serial clinical and functional assessment to evaluate the potential of stweak as a biomarker in the setting of dilated cardiomyopathy. as a result, we found that the reduction of stweak levels in patients with dilated cardiomyopathy may not be of value for disease monitoring but may represent a risk factor for disease progression and death. further research will be necessary to elucidate the exact role of stweak as a potential modulator of immune response especially in the setting of dilated cardiomyopathy. | background. tnf - like weak inducer of apoptosis (tweak) has been reported to predict mortality in patients with dilated cardiomyopathy. however, whether it can be used as a biomarker for disease monitoring or rather represents a risk factor for disease progression remains unclear. aim of the study. to evaluate the potential of stweak as a biomarker in patients with dilated cardiomyopathy. results. we conducted a serial study of stweak levels in 78 patients with dilated cardiomyopathy. soluble tweak levels predicted not only a combined mortality / heart transplantation endpoint after 4 years (p = 0.0001), but also the risk for clinical deterioration (p = 0.0001). compared to nt - probnp, stweak remained relatively stable in individual patients on follow - up indicating that inter- rather than intraindividual differences in stweak levels predicted outcome. finally, neither did the scavenger receptor scd163 correlate with stweak levels nor did its determination add additional information on outcome in patients with dilated cardiomyopathy. conclusion. soluble tweak levels in patients with dilated cardiomyopathy may not be of value for disease monitoring but may represent a risk factor for disease progression and death. further research will be necessary to elucidate the exact role of stweak as a potential modulator of immune response in the setting of dilated cardiomyopathy. |
rice is one of the most important staple cereal crops in the world, along with wheat and corn. cereal crops, such as rice, wheat, and corn, are particularly susceptible to changes in environmental conditions. the international panel on climate change (ipcc) predicts that average global temperatures will increase by 14c by 2100, and this global warming will cause the yields of major crops to decrease by more than 25% by 2050. previous studies have described rice yield reductions of 7 - 8% for each 1c increase in temperature. moreover, rice kernel dimensions (length, width, and thickness) decrease at day / night temperatures of 39/34c. high temperature stress occurring when rice plants are at the developmental stages from booting to flowering is the most influential factor affecting rice yields. high temperatures lead to rice spikelet sterility at the flowering stage due to a reduced number of pollinations on the stigma as a result of obstruction to pollen shedding and a decline in the growth and development of pollen grains. after high temperature treatment for one or two days at the flowering stage, the fertilization ability of rice pollen grains decreases due to inhibition of thecae dehiscence, and less than 50% of pollen grains germinate on stigmas under high temperature conditions [6, 7 ]. in rice, high temperature stress leading to significant yield loss due to spikelet sterility has been studied in various ways. since resistance to high temperature is a quantitative trait, mapping quantitative trait locus (qtl) using snp markers is fundamental to the study of this characteristic. qtls that conferred protection from heat stress have been detected in the tolerant n 22 variety on chromosome 4 (qhtsf4.1) and chromosome 1 (qhtsf1.1), explaining 12.6% and 17.6% of the total phenotypic variation, respectively. genome expression studies of the molecular response of rice pollen to high temperature conditions have also been reported. fifteen of the genes identified were further analyzed by real - time reverse transcription - pcr and 13 were repressed in response to high temperature. genes repressed at high temperature were expressed specifically in the immature anther, mainly the tapetum, suggesting an important role for the tapetum in pollen development under high temperature stress. recently, a mirna expression study of panicles of the heat tolerant rice cultivar, n 22, under high temperature conditions was reported. a total of 294 known mirnas and 539 novel mirnas were identified from young panicles. among identified mirnas,. gene ontology (go) category analysis of genes targeted by these mirnas revealed that these were related to various biological process categories and that, among these, the category of cell growth was enriched. rice proteome expression patterns in response to heat have also been monitored using 2d - page. proteome expression patterns were compared among three rice varieties, including a japonica variety, moroverekan, which is highly sensitive to high temperature, an indica variety, ir64, which is adequately tolerant to high temperature, and an aus variety, n22, which is highly tolerant to high temperature. of these, 13 were analyzed by maldi - tof and seven were identified as putative cold shock proteins (csps), an inorganic pyrophosphatase, a serine protease (air3), a dirigent - like protein, a ribosomal protein (s19), a small heat shock protein (shsp), an iron deficiency protein (ids3), and six unknown or hypothetical proteins. multidimensional protein identification technology (mudpit), a representative shotgun proteomic method, has been introduced as a complimentary technique to 2d - page. also, shotgun proteomics approaches make large scale, high - throughput protein identification possible. the shotgun proteomic method involves separation of trypsin - digested proteins by liquid chromatography, followed by identification by tandem mass spectrometry and interrogation of protein databases [12, 13 ]. mudpit allows qualitative, rather than quantitative, analysis ; however, relative quantitative analysis is possible by calculating normalized spectral counts. the tiny size of rice pollen grains makes it almost impossible to collect pure samples to monitor proteome responses., we describe quantitative shotgun proteomics analysis, which allowed us to screen a large number of proteins using only a small amount of material from rice anthers. dianxi4, a chinese rice variety, and ilpum, a korean commercial variety, were grown at konkuk university in an experimental rice paddy field in 15 30 cm rows. one day before anthesis, each individual plant including soil was scooped from paddy field with 15 cm diameter and put into a pot. temperature was gradually increased from 27c to 38c until 08:00 am. at 18:30 pm, the relative humidity was 70% with a 12 h day/12 h night cycle. after 5 days of high temperature treatment, plants were moved to a greenhouse and cultivated until the grain fully matured. as a control, plants with tillers at the same stage of development (bearing panicles) were scooped the same as in the method of the high - temperature - treated rice. the rice in a pot was placed in the rice filed by the end of the day of high temperature treatment ; then the control plants were moved to greenhouse with the high - temperature - treated rice plants. three individual plants for each variety were used and the spikelet fertility was measured from 9 panicles (three panicles per an individual plant). for the proteomic analysis, temperature was gradually increased from 27c to 38c until 08:00 ; then the temperature was maintained through day and night. at the next day at 05:00 am, the relative humidity was 70% with a 12 h day/12 h night cycle. for the control plants, the rice in a pot was placed in the rice filed. three individual plants for each variety were used and rice anther for proteome analysis was harvested from one panicle for each plant. anthers of dianxi4 were collected immediately after one day of high temperature treatment. to ensure that all anthers collected were at the same developmental stage, they were taken from upper part of only one panicle ; anthers emerging from the spikelet were not used in this study. only anthers inside in the spikelet and located in the middle of panicles approximately 3 cm in width were collected. harvested anthers were ground in liquid nitrogen using metal balls and protein was extracted from the resultant powder using extraction buffer (100 mm tris - hcl, ph 8.5 ; 8 m urea ; 5 mm dtt ; 1% lds). the suspension was incubated at room temperature for 30 min, followed by centrifugation at 14,000 g for 15 min. the supernatant was retained and filtered through 0.45 m membrane filters (millipore, billerica, ma, usa). protein concentration was assayed using the 2d - protein quant kit (ge healthcare, piscataway, nj, usa). protein samples (50 g) were loaded on to 412% bis - tris acrylamide gradient gels (invitrogen, carlsbad, ca, usa) and stained using a colloidal blue staining kit (invitrogen, carlsbad, ca, usa). each lane containing separated proteins was sliced into seven pieces, which were then cut into smaller cubes (approximate size, 1 mm) and transferred to eppendorf tubes. sliced gels were destained with destaining buffer (50 mm nh4hco3, ph 7.8 in 50% acetonitrile) and dehydrated in a speedvac. dried samples were reduced at 56c for 45 min in 10 mm dtt (in 25 mm nh4hco3) and alkylated with alkylation buffer (55 mm iodoacetamide in 25 mm nh4hco3) for 30 min at 24c in a dark place. reduced and alkylated samples were dried in a speedvac, mixed with digestion buffer (12.5 ng/l trypsin in 50 mm nh4hco3), and incubated at 36c overnight. tryptic peptides were harvested from gel slices using harvest buffer (5% formic acid in 50% acetonitrile) and incubated at rt for 20 min. combined supernatants were dried out in a speedvac, desalted using pierce c18 spin columns (thermo scientific, rockford, il, usa), and analyzed by lc ms / ms. a nanoflow hplc instrument (easy nlc, thermo fisher scientific, san jose, ca, usa) coupled on - line to a q exactive mass spectrometer (thermo fisher scientific, bremen, germany) was used. analytical columns (12 cm ; 75 m inner diameter) were packed in - house with alltima c18-aq 5 m resin. samples were separated with a linear gradient of 360% buffer b at a flow rate of 250 nl / min. ms data was acquired using the data - dependent top8 method, dynamically choosing the most abundant precursor ions from the survey scan (3002,000 da) for higher - energy collisional dissociation (hcd) fragmentation. the dynamic exclusion duration was 60 s, and the isolation window for precursors was set to 4 m / z. survey scans were acquired at a resolution of 70,000 at m / z 200 and the resolution for hcd spectra was set to 17,500 at m / z 200. each peptide from the lc - ms / ms spectra was searched against the tigr rice pseudomolecule protein database release v7.0 (http://rice.plantbiology.msu.edu/annotation_pseudo_current.shtml) using proteome discoverer (thermo fisher scientific, version 1.3) software. false discovery rate (fdr) in peptide identification through spectrum match to peptide was evaluated by using a decoy database which was created by reversing all the rice protein sequences and it was set to a maximum of 0.01. for the peptides shared between multiple proteins, identified proteins were grouped by the algorithm implemented in proteome discoverers (thermo fisher scientific, version 1.3) and the spectral counts (number of peptide spectrum matches) for proteins include those redundantly identified. carbamidomethylation of cysteine was set as a field modification and oxidation of methionine was set as a variable modification for database searching. the output of the proteome discoverer search was exported to microsoft excel to calculate normalized spectral counts (nspc) [1517 ]. the nspc for each protein k is given by(1)nspck = spc / lki=1nspc / li, where the whole number of ms / ms spectra matching peptides from protein k (spc) is divided by the protein 's length (l), then by spc / l for all (n) proteins in the experiment. rice protein molecular weights (mw) and pi values were calculated using emboss pepstats, embl - ebi (http://emboss.sourceforge.net/download/), and the emboss pepstats algorithm (http://emboss.bioinformatics.nl/cgi-bin/emboss/pepstats). go annotations of rice proteins were retrieved from the tigr rice pseudomolecule protein database ; release v7.0. ilpum, a commercial rice variety, was included in our initial experiments to compare the tolerance of the dianxi4. one of the most heat - sensitive stages of rice pollen development is flowering ; thus, high temperature was applied to the plants at the flowering stage. after 5-day heat - treatment both rice varieties were cultivated in a greenhouse and spikelet fertility was measured after the grain reached full maturity (figure 1). for ilpum, fertility was 28.1 10.0 after high temperature treatment and 93.1 1.4 under normal conditions, indicating that the high temperature treatment was effective. for dianxi4, fertility under normal conditions was 88.4 2.5 and, even after high temperature treatment, fertility was maintained at 77.6 4.6. proteins from anthers of dianxi4 rice plants exposed to high temperature treatment and grown under normal conditions were identified by shotgun proteomic analysis ; anthers were used rather than pollen due to the small size of rice pollen grains. individual anthers are also too small to provide sufficient proteins for proteomic analysis ; therefore, groups of anthers were collected from single panicles and the pooled proteins extracted. for biological replication, shotgun proteomics analysis of three replicates each of the control and high - temperature - treated samples identified 3,266 nonredundant proteins (supplementary table 1 ; see supplementary material available online at http://dx.doi.org/10.1155/2015/238704). the number of identified proteins common to all six samples was approximately 2,000 (supplementary table 2), indicating that not all 3,266 proteins were identified in each sample. this is likely due to the phenomenon of analytical incompleteness, where any single analytical run during shotgun proteomic analysis may identify only a fraction of relevant peptides in a very highly complex mixture. the distribution of the physiochemical characteristics of the identified 3,266 nonredundant proteins were compared with those encoded by the entire rice genome (tigr rice pseudomolecule protein database release v7.0) (figure 2). the pi values of the identified proteins ranged from 3.93 (loc_os07g41694.1) to 12.48 (loc_os01g69020.1). the proportion of identified proteins with pi values of 57 was relatively large compared with those encoded by the whole genome ; however, the pi values of the identified proteins were dispersed across a wide range. the mw of the identified proteins also had a broad range, from 5.29 kda (loc_os04g02670.1) to 486 kda (loc_os09g07300.1), although the proportion of proteins of < 20 kda was below that of all rice proteins. this result is due to the fact that low mw proteins produce fewer peptides that can be detected in a mass spectrometer after digestion with trypsin. relative protein quantities can be estimated from the normalized spectral count with a high degree of accuracy. in general, proteome studies of rice leaves have found rubisco protein and photosynthesis proteins to be present in large quantities. interestingly, our analysis of total proteins identified from rice anthers revealed that pollen allergen proteins (loc_os06g451810, loc_os09g23899, loc_os09g23999, and loc_os04g25190), atp synthase, and cupin domain - containing proteins were present in large quantities. pollen allergen proteins are allergenic to humans but are present in high amounts in plants, suggesting that they have physiological roles required for pollen germination and growth. our proteomic analysis confirms that high quantities of these proteins are present in rice anthers. the observed high levels of atp synthase (loc_os01g49190) suggest a role for this protein in the energy - requiring process of pollen tube germination. two cupin domain - containing proteins (loc_os05g0250 and loc_os01g74480) were also present at high levels. previous studies show that cupins belong to some 18 different functional classes and were originally detected during the early phase of germination in wheat embryos. given the high amounts detected in rice anthers, and the previously reported roles of pollen allergens, cupin, and atp synthase, the results presented herein suggest possible function roles for these proteins in pollen germination. for comparative analysis, the relative quantities of the identified proteins were calculated using the label - free spectral count (sc) method. not all 3,266 identified proteins were reproducibly identified in all control or heat - treated samples ; therefore, we included only proteins identified in all three replicates of either category, with at least two scs per replicate. using these criteria, scs of 1,944 reproducible proteins were normalized (nspc) and the logarithmically transformed nspcs (the natural log (ln) of nspc) were compared using a t - test (supplementary table 3). the average coefficient of determination (r2) between nspcs for the biological replicates was 0.84. proteome changes were detected using a t - test (= 0.05) to compare the expression patterns of dianxi4 grown under normal conditions with those after exposure to high temperature. a total of 139 proteins were differentially expressed between control and treated samples (supplementary table 4). among these 139 proteins, all 139 differentially expressed proteins were analyzed by go category enrichment analysis to determine enriched go terms in biological processes, cellular components, or molecular functions categories. interestingly, in the response to high temperature, one molecular function and 19 cellular component go terms were identified (table 1). the fact that the majority of the enriched go terms were cellular components indicates that the response to high temperature stress affects proteins associated with various cellular components. considering that the damage caused to pollen by high temperature stress results in the inability of pollen to be successfully germinated, the dynamic changes in protein expression associated with cellular components (representing changes in expression levels of these proteins) may be not only a consequence of damage resulting from high temperature stress, but also the result of the tolerance response required to maintain proper cellular components for recovery. next, we conducted mapman analyses to identify changes in metabolic pathways and other processes (figure 3). some metabolic pathways were increased after heat treatment, including lipid, starch, and some amino acid metabolic pathways. interestingly, the heat response process was also increased ; among the 12 proteins involved in the heat response, nine were heat shock proteins (hsp), two were dnak family proteins, and one was a chaperone protein. among the 96 proteins showing increased expression in heat - treated anthers relative to control anthers, nine were hsps, and all were expressed at high levels (table 2). several rice proteomic studies report upregulation of hsps in response to high temperatures [11, 24, 25 ]. together, these previous studies and our results strongly suggest that hsps are associated with improved tolerance to high temperature stress. hsps perform molecular chaperone activities, contributing to cellular homeostasis in cells under both normal and adverse growth conditions and can be divided into high (hsp100, hsp90, hsp70, and hsp60) and small molecular mass proteins (hsp20 or small heat shock proteins (shsps)). interestingly, there were seven shsps among the nine hsps showing increased expression after heat treatment. a previous study by chen. also detected upregulation of several shsps in rice anthers under high temperature conditions. the results of this proteomics study confirm a potentially important role for shsps in rice anthers subjected to high temperature stress ; therefore, we conclude that upregulation of shsps is likely to be important in protecting rice pollen from the adverse effects of high temperature exposure. in addition, four proteins functionally associated with the stabilization of protein structure, one chaperone protein, two dnak family proteins, and one late embryogenesis abundant protein, were upregulated after high temperature treatment. thus, the proteins upregulated in rice anthers in response to heat stress are required for maintenance of cellular homeostasis through the stabilization of proteins required for pollen germination. in addition, three glutathione s - transferases and one superoxide dismutase protein were increased after heat shock treatment. although one of the glutathione s - transferases demonstrated highly increased expression, the overall responses of these antioxidant proteins in the rice anther were less pronounced than those of hsps and other proteins with chaperone functions. furthermore, one trehalose synthase protein demonstrated highly increased expression in response to heat treatment. trehalose is a natural alpha - linked disaccharide formed by a,-1,1-glucoside bond between two -glucose units and is regarded as a protective metabolite against various stress conditions during exposure to different environmental stresses. although the molecular mechanism underlying trehalose protection is not yet fully understood, one hypothesis proposes that, especially during desiccation and heat stress, trehalose replaces water and binds via hydrogen bridges to polar protein residues to prevent the denaturation of protein molecules. the proposed role of trehalose is consistent with that of hsps, which were also found to be increased in response to heat - treatment in this study. in addition to those proteins with previously reported roles, eight of unknown functions showed increased expression in response to heat treatment (descriptions of these proteins are provided in the rice genomic database ; tigr rice pseudomolecule protein database, release v7.0). due to our experimental design, it is not clear whether upregulation of these proteins is associated with high temperature tolerance or a consequence of stress induced damage ; thus, further studies are required to confirm their roles. of the proteins showing decreased expression after heat treatment, the majority showed minimal decreases in expression (supplementary table 3). the protein showing the greatest reduction in expression was loc_os03g15320.1 (glyoxal oxidase - related), which is an extracellular h2o2-producing enzyme ; its decrease is likely associated with upregulation of antioxidant proteins ; however, its exact role in response to high temperature is not clear. the previously reported functions of the other downregulated proteins were metabolism and catalytic activity, suggesting that high temperature stress may affect certain metabolic processes. in summary, through quantitative shotgun proteomic analysis of rice anthers, we identified 3,266 nonredundant rice proteins, one of the largest rice anther protein identification experiments to date. atp synthase, cupin domain - containing proteins, and pollen allergens were present in the rice anther in large amounts, suggesting a potentially important physiological role for rice pollen allergens. a comparison of protein expression patterns in rice anthers cultivated under normal conditions or exposed to high temperature treatment at the flowering stage suggested that the tolerance of dianxi4 may be due to highly increased expression of heat shock, dnak family, chaperone, and trehalose synthase proteins, which maintain protein homeostasis in pollen cells in response to high temperatures. | in rice, the stage of development most sensitive to high temperature stress is flowering, and exposure at this stage can result in spikelet sterility, thereby leading to significant yield losses. in this study, protein expression patterns of rice anthers from dianxi4, a high temperature tolerant japonica rice variety, were compared between samples exposed to high temperature and those grown in natural field conditions in korea. shotgun proteomics analysis of three replicate control and high - temperature - treated samples identified 3,266 nonredundant rice anther proteins (false discovery rate < 0.01). we found that high levels of atp synthase, cupin domain - containing proteins, and pollen allergen proteins were present in rice anthers. comparative analyses of 1,944 reproducibly expressed proteins identified 139 differentially expressed proteins, with 95 increased and 44 decreased in response to high temperature conditions. heat shock, dnak family, and chaperone proteins showed highly increased expression, suggesting that the high temperature tolerance of dianxi4 is achieved by stabilization of proteins in pollen cells. trehalose synthase was also highly increased after heat treatment, suggesting a possible role for trehalose in preventing protein denaturation through desiccation. |
in times of increasing environmental and industrial standards for technologies and innovation toward safer substances closely connected to subsequent legal surveillance in most european union (eu) countries, it may strike us as strange to communicate that we are confronted with a group of new problematical substances in the environment. long forgotten are the impressions of great environmental disasters, fish dying in running waters, the disappearance of predatory mammals and birds scenarios such as these belong to bygone days. for quite some time now, in many eu countries water condition maps indicate a mainstream trend : the status of water quality in europe seems to be improving (nixon. 2003)although one should bear in mind that these statements do not refer to the biological status of water quality as required by the newly introduced eu water framework directive (european parliament and the council of the european union 2000). today s problematical pollutants are not characterized by high environmental concentrations but occur in the ultratrace range. in many cases even modern analytical techniques are not well enough developed to detect those substances in the subnanogram per liter range. daughton and ternes (1999) coined the term lingering environmental toxicants, identifying personal care products, pharmaceuticals, and endocrine disruptors as integral parts of this substance group. regarding their common distribution in the environment, optimized biological activity, and effectiveness at comparatively low concentrations, they obviously show some similarities. therefore, the class of substances possessing hormonal activities incorporates diverse groups such as man - made and natural chemicals (e.g., pesticides, plastisizers, alkylphenol ethoxylates as industrial surfactants, organotin compounds, polychlorinated biphenyls, phytoestrogens, natural hormones) and pharmaceuticals (e.g., ethinylestradiol, methyltestosterone, trenbolone). there is a wide range of biophysical properties, from persistent to rapidly degraded, lipophilic to hydrophilic, and nontoxic to very toxic, that can be attributed to endocrine modulators. even from a mechanistic point of view, one can not define a unique mode of action. although some of the substances take effect by binding but not activating the receptor (antagonistic effect), others may mimic biological activity by attaching to the receptor to produce hormonelike action to an inflationary extent (agonistic effect). furthermore, endocrine toxicants may act indirectly in a non - receptor mediated manner when binding to transport proteins (e.g., corticosteroid - binding globulin, steroid hormone binding globulin, thyroid hormone binding globulin, growth hormone binding protein) or by blocking, increasing, and decreasing enzyme activities or other metabolic pathways to affect the synthesis of endogenously produced hormones. there are a number of less well - known endocrine tissues and hormones other than those producing sexual steroid hormones (e.g., those affecting the hypothalamus and pituitary gland, thyroid and parathyroid glands, adrenal glands, and endocrine pancreas). in any case, a particularly large number of publications on the impairment of reproduction, sexual differentiation, and development caused by hormonally active substances (colborn. 2004) drove research toward a strong focus on sex steroids produced by the gonads. endocrine disruption is a global phenomenon. across the world, endocrine - related effects have been reported in wildlife causing reproductive failure and in some cases population declines (candia carnevali. these effects range from feminization of males and virilization of females to suppressed thyroid and immune functions and altered development. nevertheless, the greatest attention to endocrine disruption has been paid to estrogenic effects, although a clear cause effect relationship has been established for the androgenic activities of organotin compounds in mollusks (bettin. 1996 ; horiguchi. 1997 ; oehlmann and schulte - oehlmann 2003). in prosobranch snails, tributyltin and triphenyltin compounds interfere with key enzymes of the sexual steroid metabolism. moreover, these compounds are known also to affect comparable or the same molecular targets in other taxa, including humans (doering. 2002 ; heidrich. therefore, it was self - evident that research within the framework of the eu project comprendo (comparative research on endocrine disrupters phylogenetic approach and common principles focussing on androgenic / antiandrogenic compounds) would offer the unique opportunity to study a real wildlife human connection when zooming in on effects mediated by androgen- and antiandrogen - mimicking compounds. comprendo is one of the four projects that form the core of the credo (coordinating european environmental and human health research into endocrine disruption) cluster and is funded by the european commission s fifth framework programme for research, technological development and demonstration activities in the european community. the overall goal of comprendo is to improve the understanding of the effects of endocrine - disrupting chemicals (edcs) on aquatic wildlife and humans to improve environmental quality standards and public health in europe. to this end the special issue of edcs and their potential to cause serious health problems in wildlife species make it questionable whether drinking water and food are still without impact on normal development and sexual differentiation and also whether they allow a normal reproduction and the aging of individuals without avoidable health effects. a major concern for human health is the suspected intake of edcs via the diet. to give consideration to the latter in comprendo, human exposure to androgenic and antiandrogenic compounds (aacs) is assessed in all participating european partner countries via food basket analyses (identification of background concentrations in human food). in parallel, an exemplary aac residue determination in human samples (blood, urine, placenta) in european areas with higher and lower environmental and industrial standards is accomplished. environmental samples (sediment and biota) are additionally taken and chemically analyzed in these regions. this is to assess the exposure of wildlife populations to aacs as well, and to characterize potential effects in highly contaminated and reference areas. presently, the organisms that are used to assess effects of chemicals and in turn infer potential health and ecosystem - level effects are very limited. as new problems arise, we need to be mindful of what chemical tests are the most appropriate and fit for purposes of environment protection rather than necessarily being constrained by existing test systems. this is why comprendo aims to identify new test species and toxicological end points to help safeguard the protection of organisms living in aquatic ecosystems. to do this a wide range of organisms from invertebrates (echinoderms, crustaceans, mollusks) to vertebrates (fish, amphibians) and human models (human cell lines, tissues, rodents) are under investigation to develop an understanding of the commonalities and differences in responses to aacs across the animal kingdom. initially, the application of positive controls for aacs known to cause direct receptor - mediated or indirect functional effects [e.g., methyltestosterone and mibolerone as androgen receptor agonists ; letrozole as steroidal aromatase inhibitor ; cyproterone acetate, flutamide, and prochloraz as androgen receptor antagonists ; 4,7-dimethyl-4-azacholestan-3-one (mk 386) and finasteride as reductase type 1 and 2 inhibitors ] provides a basis for the assessment of the potential vulnerability of representative aquatic wildlife groups and humans. second, test species are exposed to several compounds [organotins, e.g., mono-, di-, tri - butyltin and triphenyltin compounds, and pesticides such as fenarimol, vinclozolin, linuron, diuron, p, p-dde (dichlorodiphenyl - dichloroethylene) ] at environmentally relevant concentrations to identify sensitive and easily measurable end points (genetic, biochemical, physiological, histological, morphological) and critical stages of exposure (via early - life stage and life - cycle tests). these monosubstance tests are linked to sediment exposure studies (sediments originating from environmental sampling) representing complex environmental mixtures. by means of comparison, the results are used to determine whether pure substances or mixtures adversely affect communities in ecosystems. most of the compounds suspected as endocrine disruptors end up in aquatic ecosystems by sewage effluents. aquatic organisms are almost continually exposed to these chemicals and, as a matter of plausibility, therefore provide sentinel organisms for detecting adverse biological effects. determining whether a chemical, especially at sublethal concentrations, can affect endocrine - mediated functions in wildlife populations is difficult and may require a prolonged and profound understanding of the animal s life history, morphology, and the influence of the local environmental conditions (tagatz 1968). although it is unquestionable that invertebrates are in principle sensitive to edcs, their use as standard biotest organisms has been impeded by the lack of laboratory cultures and detailed insights into invertebrate endocrinology. therefore, the establishment of long - term cultures of selected species of mollusks (marisa cornuarietis, potamopyrgus antipodarum) and crustaceans (hyalella azteca, arcatia tonsa) will be used for studies on endocrine disruption to potentially provide new standard test organisms on the international scale, such as tests mandated by the organisation for economic cooperation and development (2006) inter alia robustness, synchronicity of development, number of generations per year, ease of handling, diet preferences and the possibilities to achieve a standardization of the diet, cost of culture, and sensitivity of species in the exposures have been important evaluation criteria for checking their suitability for the establishment of laboratory cultures. the invertebrate reproductive cycle can be highly complex (sastry 1968, 1970) and is controlled by many environmental stimuli, including light intensity, temperature, desiccation, and diet (ansell and trevallion 1967 ; copeland and bechtel 1974 ; largen 1967 ; tessier. this is why laboratory cultures will finally be optimized in terms of favorable culture conditions (temperate and light / dark regimen, water chemistry, standardized food, animal densities in tanks) and a characterization of the species - specific biology under the chosen culture conditions [reproductive phase(s), time and length of spawning, duration of different development phases, time until puberty, length of sexually active life phase ]. on the level of sexual development, reproduction, and differentiation, endocrine modulators may produce a variety of deviations compared with normogenesis and unaffected progeny. therefore, test species will be checked for pathomorphoses and histopathological aberrations of target organs (e.g., sexual glands, gonads, midgut gland) during exposure. tracing of cell cycle kinetics, apoptosis, protein content, and mitochondrial respiration provides additional information on disturbances of cytologic processes. sex steroid concentrations (androstenedione, testosterone, dihydrotestosterone, 11-ketotestosterone, estrone, and estradiol) are checked in all species under investigation during an unspoiled annual reproductive cycle to be linked with comparative data generated during the exposure s run. the same approach is applied for the determination of phase i and ii metabolism. in this regard, the activities of cytochrome p450 aromatase and 3-, 11-, and 17-hydroxysteroid dehydrogenase are analyzed for phase i. because interferences with hormone phase ii metabolism will alter hormone excretion rates and may lead to hormonal imbalance in exposed organisms, the activities of several enzymes, such as sulfotransferase, acyl - coenzyme a acyltransferase, and sulfotransferases, are analyzed in human cell lines and tissue (placenta, prostate, liver), digestive gland, liver, and gonad of vertebrates and invertebrates in parallel. in the development of new tests for aacs, innovative approaches are being employed, including the development of a multispecies gene array. here, a suite of key genes that control sexual function is being isolated and applied to a matrix, and the array subsequently used to screen for the effects of aacs on target gene expression. it is envisioned that this approach will offer new perspectives in the application of modern molecular techniques in our understanding of endocrine disruption. the comprendo work program reflects a holistic attempt that will have relevance far beyond the chosen group of compounds. environmental and human health aspects are simultaneously addressed by using an interdisciplinary and comparative phylogenetic approach. studies of this nature require a comprehensive understanding of the endocrinology of sentinel organisms and where there are fundamental gaps of knowledge in baseline endocrinology (notably in invertebrates). a common and distinguishing mark of the endocrine system of all animal phyla consists of a widely distributed system of glands, tissues, and diffuse groups of widespread cells that synthesize and release chemical messengers into the circulatory system. doubtless the regulation of many parts of biological processes has been widely conserved, although individual components of the endocrine system have passed through profound evolutionary processes. consequently, remarkable divergences resulting in distinct differences in the endocrine systems of the diverse taxa can be expected (de fur. 1999), but on the other hand, we have reason to suppose that in the animal kingdom many similarities and analogues of metabolic pathways also exist. it is likely that genes, proteins, or receptors differ in structural configuration across taxa, and even if the differences are very small, they may constitute the basis for different effects and reactions in test species when exposed to the same substances and concentrations. for example, conventional radioreceptor assays have provided evidence for androgen- and estrogen - specific binding sides in mollusks, crustaceans, and echinoderms (oehlmann. nevertheless, their binding affinity to direct - acting aacs varies enormously compared with those of vertebrates. although parts of vertebrate - type sex steroid metabolism can be identified in the different invertebrate test species, the conversion rates from androgens to estrogens might be identified to be much lower compared with those of vertebrates. different activity patterns of the enzymes involved (occurrence, function, biosynthesis) or other interspecies differences with respect to the metabolic pathways (e.g., conjugating activities) may turn out to be responsible for this. however, these and other findings make it worthwhile to clone and sequence the androgen and estrogen receptors in the different species under investigation and to focus on deciphering their endocrine systems. finally, this could pave the way toward an extrapolation of data derived from animal experiments to human health based on factual knowledge and improve our knowledge on the extent and nature of endocrine disruption by aacs with a critical evaluation of their role as disruptors of physiological functions in wildlife and human populations (risk assessment). | tens of thousands of man - made chemicals are in regular use and discharged into the environment. many of them are known to interfere with the hormonal systems in humans and wildlife. given the complexity of endocrine systems, there are many ways in which endocrine - disrupting chemicals (edcs) can affect the body s signaling system, and this makes unraveling the mechanisms of action of these chemicals difficult. a major concern is that some of these edcs appear to be biologically active at extremely low concentrations. there is growing evidence to indicate that the guiding principle of traditional toxicology that the dose makes the poison may not always be the case because some edcs do not induce the classical dose response relationships. the european union project comprendo (comparative research on endocrine disrupters phylogenetic approach and common principles focussing on androgenic / antiandrogenic compounds) therefore aims to develop an understanding of potential health problems posed by androgenic and antiandrogenic compounds (aacs) to wildlife and humans by focusing on the commonalities and differences in responses to aacs across the animal kingdom (from invertebrates to vertebrates). |
the cellular signals initiated by reactive electrophilic species, such as reactive oxygen species (ros), reactive nitrogen species (rns), and reactive carbonyl species (rcs) are delivered through redox chemistry. this primarily involves the post - translational modification of specific amino acid residues, especially cysteine sulfhydryls, on signaling proteins (satoh and lipton, 2006). according to the classical view, the reaction of cysteine sulfhydryl groups maintains protein structure through disulfide bond formation and regulates catalytic centers through metal binding (lipton., 2002). however, the paradigms for function of cysteine sulfhydryl reactions have been extended by the discovery of s - nitrosylation in biological systems. the transfer of a nitric oxide (no) group to a key cysteine sulfhydryl represents a new model of cysteine sulfhydryls acting as sensors of redox status to regulate protein functions. the physiological significance of no in signal transduction was established by the demonstration that no generated by endothelial cells relaxes vascular smooth muscle through the activation of guanylate cyclase and cyclic gmp - dependent kinase (murad, 1986). the extensive range of no - based signaling was illustrated by the discovery of no synthases (noss) with well - conserved phylogenetics and pervasive tissue distributions (murad, 2006). in addition to the well - characterized binding of no to the heme iron of guanylate cyclase, s - nitrosylation was suggested to be an alternative physiological no - based protein modification (stamler., 1992a, b) and this theory has been proven by the identification of more than a hundred substrates for s - nitrosylation (stamler., 2001). important examples include s - nitrosylation of the nr2a subunit of n - methyl - d - aspartate (nmda) receptors (lipton., 2002), ryanodine receptors (ryr ; (eu., 2000), gapdh (hara., 2005), and protein - disulfide isomerase (pdi ; uehara., 2006). there is a growing awareness of s - nitrosylation as a post - translational protein modification regulated with precise temporal and spatial characteristics (stamler., 2001 ; barouch. these characteristics confer specificity to no - derived effects, allowing s - nitrosylation to function as a prototype of mechanisms that convey redox - based cellular signals (stamler., 2001). analogous to no, other electrophiles can activate or inhibit specific signal transduction pathways by reacting with cysteine sulfhydryls and thus causing modification of protein structure. ros can directly oxidize cysteine sulfhydryls in vitro and in vivo (berlett and stadtman, 1997). for example, h2o2 oxidizes sulfhydryl groups to sulfenic acid, which is rather unstable and reactive and rapidly forms stable intra- or intermolecular disulfide bonds if other adjacent sulfhydryl groups are present (claiborne., 1999). post - translational modifications include the addition of rcs on proteins, generically termed as protein carbonylation, with the most reactive and common form of these carbonyl groups being aldehydes.,-unsaturated aldehydes, including 15-deoxy--prostaglandin j2 (15d - pgj2), 4-hydroxy-(2e)-non - enal (4-hne), and acrolein are reactive aldehydes generated from polyunsaturated fatty acid oxidation. because of the presence of electron - withdrawing functional groups, the double bond of these compounds serves as a site for michael addition with the sulfur atom of cysteine. through the modification of key cysteine sulfhydryls, these electrophiles can mediate various biological actions including functional regulation of the ib kinase (ikk) subunit, the nuclear factor (nf)-b p65 subunit (rossi., 2000 ; straus., 2000), kelch - like ech - associated protein (keap1 ; eggler., 2005), thioredoxin (shibata., 2003), and peroxisome proliferator activated receptor (ppar) (shiraki., 2005). thus, highly conserved redox reactions of cysteine sulfhydryls can be elementary molecular processes in the same way as phosphorylation reactions of threonine, serine, and tyrosine residues are in cell signaling. studies of transient receptor potential (trp) proteins (trp), which form a variety of ca - permeable cation channels, have significantly extended our knowledge of the molecular basis of sensory biology. trp homologs are grouped into six subfamilies [canonical (c), vanilloid (v), melastatin (m), polycystic kidney disease (p), mucolipin (ml), and ankyrin (a) ] by the homology of their protein sequences (clapham, 2003 ; voets., 2005). because of their distinct activation mechanisms and biophysical properties, trp channels are highly suited to function in sensory receptor cells, either as molecular sensors for environmental or endogenous stimuli or as modulators of signal transduction cascades downstream of metabotropic receptors. in fact, trp channels play crucial roles in many types of senses, including touch, taste, and smell in mammals (clapham, 2003 ; voets., 2005). recently, it has been demonstrated that a group of trp channels are cell sensors for changes in redox status (hara., 2002 ; aarts., 2003 ; the trpm2 channel, the first identified ros - sensitive trp channel, is activated indirectly by h2o2 through the production of nicotinamide adenine dinucleotide and its metabolites, adp - ribose (adpr) and cyclic adpr (hara., 2002 ; accumulated evidence indicates that trpm2 mediates several cellular responses, including the h2o2-activated ca influx that mediates cell death (hara., 2002) and, in pancreatic -cells, the ca or cation influx that drives insulin secretion (togashi., 2006 ; uchida., 2011). recently, through studies using trpm2 knockout (ko) mice, we have demonstrated that h2o2-activated ca influx through trpm2 induces chemokine production in monocytes, which aggravates inflammatory neutrophil infiltration (yamamoto., 2008). in addition to trpm2, certain members of the trpc and trpv subfamily, including trpc5 and trpv1, are activated directly by no, oxidants, and other chemical agents through modification of cysteine free sulfhydryl groups (yoshida., 2006). trpc5 is also activated by reducing substances such as thioredoxin (xu., more recently, trpa1 channel activation has been shown to occur following oxidative cysteine modification by pungent compounds and inflammatory mediators (hinman., 2006 ; macpherson., 2007 ; takahashi., 2008). we focus on the three types of trps : trpc5, trpv1, and trpa1 to extend our understanding of the biological significance of cysteine modifications by oxidants and electrophiles, and the physiological consequences of these chemical reactions in signal transduction pathway and in sensory neuronal responses. trpc5 was cloned from the mouse brain and functionally identified as a receptor - activated ca - permeable cation channel linked to phospholipase c (plc ; okada., 1998 ; although it is still controversial whether depletion of ca stores can activate trpc5, a number of proteins and factors have been shown to act as direct triggers and modulators of trpc5 channel activation. for example, binding of intracellular ca and calmodulin (cam) have been implicated in trpc5 activation and modulation (ordaz., 2005 ; shimizu., 2006 ; blair., 2009 ; gross., 2009), while membrane polyphosphoinositides, such as phosphatidylinositol 4,5-bisphosphate (pip2), exert both stimulatory and inhibitory effects in regulating trpc5 channel activity (trebak., 2009). trpc4 and trpc1, the closest structural homologs of trpc5, interact with the trpc5 protein (lockwich. yuan., 2003 ; obukhov and nowycky, 2004 ; goel., 2005 ; schindl., 2008 ; miehe., 2010). as trpc1 interacts with both trpc5 and caveolin-1 (lockwich., 2000 ; strbing., 2001), it is likely that trpc5 forms protein complexes with caveolin-1 (the importance of which will discussed below). an array of these proteins and factors can cooperatively control the function of trpc5 channelsomes. trpc5 is potently regulated by cysteine modifications and is activated by no via cysteine s - nitrosylation (yoshida., 2006). by performing labeling and functional assays with cysteine mutants, we showed that cysteine residues accessible from the cytoplasm, namely cys553 and nearby cys558 on the n - terminal side of the putative pore - forming region between the fifth and sixth transmembrane domains, are essential for mouse trpc5 activation in response to no (figure 1). the corresponding cysteine sites of trpc1, trpc4, trpv1, trpv3, and trpv4 are potential targets of nitrosylation that leads to channel activation (see also below for nitrosylation of trpv1 ; figure 2). no - activated trpc5 channels were significantly but not entirely suppressed by ascorbate, which reduces s - nitrosothiols to thiols but not disulfides. however, dtt, which reduces both s - nitrosothiols and disulfides to thiols, fully suppressed no - activated trpc5 channel activity. thus, both nitrosylation and disulfide bond formation are very likely to be involved in no - induced trpc5 activation. in an s - nitrosylation assay (jaffrey., 2001), s - nitrosylation was abolished by the mutation of cys553 of trpc5, whereas it was unaffected by mutation of cys558. as proposed for the acid base catalysis of hemoglobin nitrosylation in proteins with high no sensitivity, basic and acidic amino acids surrounding s - nitrosylated cysteines enhance the nucleophilicity of the sulfhydryl and therefore the s - nitrosylation of this group (hess., indeed, in trpc5, charged residues flanking cys553 and cys558 may confer modification susceptibility to no. our data may suggest that the trpc5 channel is opened via the s - nitrosylation of cys553 and a subsequent nucleophilic attack of nitrosylated cys553 by the free sulfhydryl group of cys558 to form a disulfide bond that stabilizes the open state (figure 1). however, the no sensitivity of trpc5 channels has been disputed by several groups (xu. it is possible that no sensitivity of trpc5 is dependent on culturing conditions, application systems of drugs, cell density during measurements, or other experimental conditions, which may affect the modification state of trpc5 proteins, levels of antioxidants, or other molecular and cellular states. no modify the free sulfhydryl group of cys553 accessible from the cytoplasmic side to open the activation gate. the modified cys553 can be further attacked nucleophilically by the free sulfhydryl group of cys558 to form an intramolecular disulfide bond, which may stabilize the channel in activation states. conserved cysteine residues on the n - terminal side of putative pore - forming regions in trps. high extracellular concentrations of thioredoxin are apparent in rheumatoid arthritis, an inflammatory joint disease that disables millions of people worldwide (burke - gaffney., 2005 ; smolen., 2007). trpc5 is also activated by the reducing agent, dtt, and by extracellular reduced thioredoxin, which both cleave a disulfide bridge in the predicted extracellular loop adjacent to the ion - selectivity filter of trpc5 (xu., 2008). blockade of thioredoxin - activated trpc5 enhances secretory activity and prevents the suppression of secretion, suggesting that trpc5 has a protective role against the progression of rheumatoid arthritis. thus, the cysteine residues of trpc5 that are important for the actions of no and oxidants are also functional targets for reducing agents. firstly, as mentioned above, trpc1 has been described to form heterotetrameric channels with trpc5 (strbing., 2001) and a protein complex with caveolin-1 in caveolae / lipid raft domains (lockwich., 2000 2003), which regulate plasma membrane trafficking of trpc1 (brazer., 2003). in fact, we have found an interaction of trpc5 with caveolin-1 and enos by co - immunoprecipitation experiments as well as by co - localization of trpc5 with caveolin-1 (mori., unpublished data). secondly, among numerous caveolin - associated proteins linked to signaling cascades (couet., 1997 ; garca - cardea., 1997 ; sato., 2004 ; quest., 2008), three isoforms of nos, such as enos, have been identified (kone., 2003). the inhibitory association of caveolin is disrupted by the binding of ca cam to enos, leading to enos activation (ju., 1997 ; michel., 1997a, b ; rizzo., 1998 ; bernatchez., 2005). thirdly, enos is known to be activated by different kinases, including akt, protein kinase a, and protein kinase c (garca - cardea., 1996 ; fulton., 1999 based on these papers and our own data, we can propose a plausible model to describe the role of the trpc5 channelsome in regulating receptor - activated no production in vascular endothelial cells (figure 3). in this model, trpc5 proteins form complexes with vasodilator receptors, g - proteins, plcs, and enos, and these complexes are anchored in caveolae by the scaffolding protein, caveolin-1. upon vasodilator receptor stimulation, trpc5 is activated by the plc cascade to induce ca influx, which elevates the intracellular ca concentration ([ca]i) and forms ca cam. this releases enos from the inhibitory control of caveolin-1 and leads to an initial no production, which activates uncomplexed trpc5 channels. ca influx via no - activated trpc5 channels then induces secondary activation of enos to amplify the production of no, resulting in a positive feedback cycle of receptor - activated ca and no signaling. this model has been neatly summarized by stamler and colleagues in a short review (foster., 2006) based on our data (yoshida., 2006). proposed model for trpc5-mediated feedback cycle of receptor - activated ca and no signaling in caveolae of endothelial cells. stimulation of gpcrs (such as the atp - activated p2y receptor) induces ca influx and activation of enos as a consequence of binding of ca cam and release of enos from caveolin-1. trpc5 undergoes enos - dependent s - nitrosylation after gpcr stimulation, resulting in amplified ca entry and secondary activation of enos to amplify production of no. our immunolocalization studies have revealed that trpc5 is distributed on both the apical and basal membrane in the endothelial cell layer of vascular tissue (mori, unpublished data). given that vasodilator receptors are distributed at the apical luminal surface of the endothelial cell layer, an initial burst of no produced there may diffuse across the cytoplasm and activate trpc5 located at the basolateral membrane. this could lead to an efficient propagation of ca signals directed toward the basal membrane. this feedback mechanism might further contribute to global [ca]i elevation / oscillation and full activation of enos at the golgi (fulton., 2002) of endothelial cells, leading to synchronization of neighboring smooth muscle cells during relaxation of vascular tissues. this idea is substantiated by the fact that genetic disruption of trpc4 (the closest relative of trpc5), which we demonstrated to be colocalized with trpc5 in the endothelial cell membrane (yoshida., 2006), impairs agonist - dependent vasorelaxation (freichel., 2001). interestingly, the importance of trpc5 has been demonstrated in neurite extension (greka., 2003). as no signals are reported to regulate neurite extension (zhang., 2005), the feedback mechanism might also be important in growth cone morphology. trpc5 channelsomes might also be involved in the activation of enos by shear stress (fulton., 1999 ; boo., 2002), which is purported to proceed through a ca - independent mechanism because membrane stretch has been reported to activate trpc5 independently of plc function (gomis., 2008). even where initiation of no production is evoked independently of ca, the secondary amplification phase might be mediated by ca influx via no - activated trpc5 channels. thus, the positive feedback regulation of ca signals by no - activated trp channels can be involved in diverse biological systems. trpv1 has been implicated in a wide variety of cellular and physiological processes, including detection of noxious physical and chemical stimuli, making it a promising target for the development of analgesic drugs with ultra - specificity for the origin of the pain. trpv1 is a non - selective cation channel that is activated by noxious stimuli such as heat (> 43c ; caterina., 1997), acidic ph (tominaga., 1998), and environmental irritants and endogenous algesic substances including capsaicin (caterina., 1997), camphor (xu., 2005), allyl isothiocyanate (aitc) from mustard oil and wasabi (everaerts., 2011), 12-hydroperoxyeicosatetraenoic acid (12-hpete ; hwang., 2000 ; shin., 2002), bradykinin (premkumar and ahern, 2000), and anandamide (zygmunt., 1999) although expression of trpv1 was originally reported to be restricted to primary afferent nociceptors of the dorsal root ganglia (drg), trigeminal ganglia, and nodose ganglia (szallasi., 1995 ; caterina., 1997 ; helliwell., 1998 ; tominaga., 1998 ; ward., 2003 ; brierley., 2005 ; christianson., 2006), recent studies have argued for a much wider distribution, both in the central nervous system and in non - neuronal tissues (mezey., 2000 ; roberts., 2004 ; tth., 2005 ; cristino., 2006 ; steenland., 2006 ; analysis of trpv1-deficient mice confirmed the specificity and activation kinetics of various receptor - specific stimuli (caterina. trpv1 is a useful model for studying the biochemical regulation of the multimodal sensitivity of cells to stimuli. for example, the sensitivity of trpv1 to capsaicin and noxious heat can be greatly enhanced by mild acidosis (chuang., 2001 ; ji., 2002 ; amadesi., 2004, 2006 ; inflammatory agents that activate plc signaling pathways (premkumar and ahern, 2000 ; tominaga., 2001). such integration of different signaling pathways allows trpv1 to detect subthreshold stimuli, and provides a mechanism through which tissue injury produces thermal hypersensitivity (julius and basbaum, 2001). cysteine modifications also mediate the trpv1-activating actions of several regulatory factors (yoshida., 2006). the alignment of amino acid sequences surrounding cys553 and cys558 of trpc5 with counterpart sequences shows cysteines conserved on the n - terminal side of the putative pore - forming region, which is located between the fifth and sixth transmembrane domains in trpv1. indeed, trpv1 channels are activated by no by itself, while a trpv1 mutant with substitutions at these conserved cysteines gives significantly suppressed responses to no. no also enhances the sensitivity of trpv1 to h and heat, suggesting that nitrosylation - induced ca entry through trpv1 is involved in heat or pain sensation. sensitizing effects of oxidizing agents such as diamide and chloramine - t support the existence of these counterpart cysteines in trpv1 (susankova., 2006). trpv1 also shows sensitivity to pungent compounds from onion and garlic, such as allicin (macpherson., 2005), through covalent modification of a single cysteine residue located in the n - terminal region (salazar., 2008). trpv1 activation by allicin is of physiological significance, because trpv1 mediates part of the response to this compound in isolated drg neurons and in an in vivo model. recently, c - terminal cytoplasmic cysteine residues that sensitize trpv1 activation upon oxidative challenge have been identified (chuang and lin, 2009). robust oxidative modulation recovers the agonist sensitivity of receptors desensitized by prolonged exposure to capsaicin. moreover, oxidative modulation operates synergistically with kinases and proton modulation. considering that tissue damage and inflammation produce ros, sensitization of trpv1 under oxidative challenge is likely to play a role in nociceptor pain sensation during inflammation, infection, and tissue injury. trpa1 is the only member of the trpa sub - branch of the trp gene superfamily in mammals, characterized by a large number (17) of amino - terminal ankyrin repeats (gaudet, 2008). although trpa1 was first cloned from a fibroblast cell line (jaquemar., 1999), expression of trpa1 is largely restricted to a subset of nociceptive c - fiber nerves, including somatosensory and vagal nerves (story., 2003 ; bandell., 2004 ; jordt., trpa1 was initially identified as a cold - sensitive ion channel in a small subset of cells (story., 2003). pharmacological experiments have revealed that trpa1 is the sensory neuronal receptor for pungent compounds such as aitc (bandell., 2004 ; jordt., 2004), cinnamaldehyde from cinnamon (bandell., 2004), and allicin from onion and garlic (macpherson., 2005). these compounds are potentially susceptible to nucleophilic attack by the sulfhydryl groups of cysteine residues (bautista., 2005 ; macpherson., 2005) similar to capsaicin, mustard oil activates sensory neurons, causing acute pain, thermal and mechanical hyperalgesia, and neurogenic inflammation (jancs., 1967 ; bautista., ; wang., 2008 ; schmidt., 2009), cannabinoids (jordt., 2004), caffeine (nagatomo and kubo, 2008), nicotine (talavera., 2009), and heavy metals including zinc, cadmium, and copper (hu., 2009 ; gu and lin, 2010). recent reports have demonstrated that the biophysical and pharmacological properties of trpa1 expressed in trigeminal and vagal neurons have intriguing parallels with those of the proposed reactive airway irritant receptor (bessac and jordt, 2008 ; taylor - clark and undem, 2011). different approaches have provided strong support for the proposal that the activation mechanism of trpa1 by aitc and cinnamaldehyde is dependent on covalent cysteine modifications (figure 4 ; table 1). systematic mutation of candidate acceptor sites identified three neighboring cysteines within the cytoplasmic n - terminus on human trpa1 (cys621, cys641, and cys665) whose simultaneous mutation negates the channel - activating effects of several cysteine - modifying reagents (hinman., 2006). mass spectrometry independently implicated three cysteines in mouse trpa1 (cys415, cys422, and cys622), which are conserved in the human homolog (as cys414, csy421, and cys621), as the target site for electrophilic agonists (macpherson., 2007). recently, we have shown that a variety of inflammatory mediators [15d - pgj2, no, h2o2, and protons (h) ] activate human trpa1 in heterologous systems and mouse trpa1 in dissociated sensory neurons (takahashi., 2008). this finding suggests that trpa1 channels are targeted by an array of inflammatory mediators to elicit inflammatory pain in the nervous system. functional characterization of site - directed cysteine mutants of trpa1 in combination with labeling experiments using biotinylated 15d - pgj2 demonstrated that modifications of cytoplasmic n - terminal cys421 and cys621 are responsible for the activation of trpa1 by 15d - pgj2. in trpa1 responses to other cysteine - reactive inflammatory mediators, such as no and h2o2, the extent of impairment by respective cysteine mutations differed from those in trpa1 responses to 15d - pgj2. this characteristic can be attributed to a difference in the mechanisms employed by 15d - pgj2 and no / h2o2 to achieve the modification of free sulfhydryl groups of cysteine residues in trpa1 proteins : 15d - pgj2 modifies sulfhydryl groups through the michael addition reaction, while no and h2o2 modify sulfhydryl groups through the redox reaction. cysteine residues such as cys421 can be deprotonated by nearby basic residues to form a thiolate anion in resting states and are protonated by acidic ph in activated states. this is a likely physiological scenario, considering that thiolate anions exert nucleophilic attack on 15d - pgj2, no, and h2o2. according to the structural model, cys421 is spatially located close to the basic residue his418 on the same side of an -helix (gaudet, 2008). notably, trpa1 is activated by external nh4 + -induced intracellular alkalization (fujita., 2008) as well as acidic ph (takahashi., 2008 ; wang., 2010), suggesting that the ph dependency of trpa1 activity has an inverted bell - shape with the minimum around physiological ph of 7.4. interestingly, different sets of cysteine residues have been identified by independent groups (table 1). this can be attributed to differences among mechanisms underlying modification of cysteine residues as described above for 15d - pgj2 and no / h2o2. in ryr1, which is a redox - sensitive ca channel, only 12 of the 100 cysteine residues are redox - modified (hyper - reactive cysteines) and two of the 12 hyper - reactive cysteines are s - nitrosylated but not s - glutathionylated, whereas for a further two, the reverse applies (aracena - parks., 2006). in keap1, which is a molecular sensor for intracellular redox changes, different cysteine residues have been reported to display different preferences for alkylating reagents (dinkova - kostova., 2002 ; eggler., 2005 ; hong., 2005). the discrepancy may be due to the differences among the mutants employed by the groups : hinman. (2006) showed data only for triple trpa1 mutant, which carries mutations of cys621, cys641, and cys665, while others used single mutants. to fully understand cysteine residues responsible for trpa1 activation, analyses of its three dimensional structure is essential. cys415, cys422, and cys622 in mouse trpa1. structural model for trpa1 protein. four identical trpa1 subunits are believed to be combined in the formation a functional channel. each subunit spanning the plasma membrane six times (transmembrane domains s1s6) has a long cytoplasmic n - terminal domain. ovals indicate ankyrin repeats, while filled circles indicate cysteine residues identified as crucial sites for covalent modification of trpa1 (hinman., 2006 ; macpherson., 2007 ; trevisani., 2007 ; these include a variety of rcs such as acrolein (2-propenal ; bautista., 2006), 4-hne (trevisani., 2007), 4-oxonon - enal (taylor - clark., 2008a), and 15d - pgj2 (andersson., 2008 ; maher., 2008 ; takahashi., 2008 ; taylor - clark., 2008b) ; ros such as hypochlorite (bessac., 2008), h2o2 (andersson., 2008 ; bessac., 2008 ;, 2008 ; takahashi., 2008), and o3 (taylor - clark and undem, 2010) ; and rns such as no (sawada., 2008 ; takahashi., 2008), peroxynitrite (sawada., 2008), and nitrooleic acid (taylor - clark., 2009). many trpa1 stimulants activate bronchopulmonary c - fibers of vagal nerves, which innervate the airways and play a critical role in the detection of the airway microenvironment (kubin., 2006). oxidative stress and associated compounds have been shown to activate unmyelinated bronchopulmonary c - fibers, initiating action potentials in these nerves that conduct centrally to evoke unpleasant sensations such as urge to cough, dyspnea, and chest - tightness and to stimulate / modulate reflexes such as cough, bronchoconstriction, respiratory rate, and inspiratory drive (bessac and jordt, 2008). in vivo inhalation of h2o2 and hypochlorite given as an aerosol evokes a decrease in respiratory rate and an increase in end expiratory pause, which was abolished in trpa1 ko mice (bessac., 2008). in addition, o3 evokes robust action potential discharges from cinnamaldehyde - sensitive mouse bronchopulmonary c - fibers, which were reduced by approximately 80% by the generic trp channel blocker, ruthenium red (taylor - clark and undem, 2010). o3 also causes airway nociceptor hyperexcitability (ho and lee, 1998 ; taylor - clark and undem, 2010), although it is unclear if this is due to trpa1 function. recently, our systematic evaluation of the oxidation sensitivity of trp cation channels using reactive disulfides with different electrophilicity reveals the capability of trpa1 to sense o2 in bronchopulmonary c - fibers (takahashi., 2011). the role of trpa1 in airway afferent signaling has been studied in association with cigaret smoke and inflammation. it is well established that cigaret smoke contains nicotine and inflammation induces bradykinin, lipoxygenase products, and cyclooxygenase products, all of which can modulate airway nociceptor function independent of oxidative stress (carr., 2003 ; kwong and lee, 2005 ; lee., 2007 ; taylor - clark., 2008c ; talavera., 2009, 2008, 2009 ; lin., 2010) and inflammation (joseph., 2008 ; kuhad and chopra, 2009 ; kamboj., another important role for trpa1 has been suggested from a mouse model of allergic asthma (caceres., 2009). allergen - induced eosinophilia, mucin production, and airway hyper - reactivity were reduced by a trpa1 inhibitor, hc-030031 in wild - type mice by 35, 90, and 90%, respectively. these markers of allergen - induced airway inflammation were reduced in trpa1 ko mice. because trpa1 is exquisitely sensitive to oxidants, further analyses are necessary to determine whether trpa1 activation induced by smoke and inflammation is independent of ros. transient receptor potential channels can respond to multiple activation triggers and therefore serve as polymodal signal detectors. an important aspect of this multimodal activation of trp channels is its role in signal integration and amplification. when a trp channel participates in a specific signaling cascade and is activated by downstream or upstream constituents (molecules / proteins / enzymes), in addition to the primary activation trigger immediately upstream, the trp channel equips the cascade with positive feedback or feed - forward loops. this mechanism, which is capable of ensuring the fidelity of cellular responses and minimizing variation in their magnitude, may synchronize the responses of neighboring cells that comprise functional domains within tissues. for example, in vascular endothelial cells upon vasodilator receptor stimulation, ca influx via no - activated trpc5 channels can amplify production of no by enos, resulting in the enhancement of no production in nearby endothelial cells and no - dependent relaxation of smooth muscle cells. the intercellular amplification of no production eventually leads to vasodilation synchronized at the vascular tissue level. the studies summarized above clearly indicate that multiple inflammatory signals conveyed by oxidants, lipid products, and protons converge at trpv1 and trpa1 to increase the excitability of sensory and vagal neurons during inflammation. thus, trpv1 and trpa1 are sensors that translate oxidant and electrophilic stimuli into electrical signals in sensory and vagal neurons. however, the role(s) of trpv1 and trpa1 ca permeability in controlling ca signaling pathways is still elusive in neurons. how cellular signals amplified by activation of redox - sensitive trp channels are terminated is also unclear. our study of trp channels is now extending from functional description of single molecules to analysis and integration of molecular systems controlled by trp channels. the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. | proteins are capable of sensing the redox status of cells. cysteine residues, which react with oxidants, reductants, and electrophiles, have been increasingly recognized as the mediators of this redox sensitivity. cation channels encoded by the transient receptor potential (trp) gene superfamily are characterized by a wide variety of activation triggers that act from outside and inside the cell. recent studies have revealed that a class of trp channels is sensitive to changes in redox status and is notably susceptible to modifications of cysteine residues, such as oxidation, electrophilic reaction, and s - nitrosylation of sulfhydryls. in this review, we focus on trp channels, which directly sense redox status, and discuss the biological significance of cysteine modifications and the consequences of this chemical reaction for physiological responses. |
as a result of the development of endoscopic submucosal dissection (esd), the en bloc resection of larger gastrointestinal neoplasms can be performed successfully. however, esd still has many limitations, including its technical difficulty, long procedure times, and risks of perforation and bleeding. because the colorectal wall is much thinner than the gastric wall, the risk of perforation is considerably greater in colorectal esd than in gastric esd. the perforation rates associated with colorectal esd were approximately 10% in several reports1,2 although this rate has been decreasing as the skills, materials, and devices for esd are improved. adequate tissue tension and good visibility of the tissue to be dissected are very important for effective and safe dissections, and they can be achieved by traction of the tissue to be dissected. in this article, we will discuss various techniques for achieving tractions during esd and their advantages and disadvantages. traction decreases the contact area between the tissue and the electrosurgical knife by increasing the tissue tension (fig. 1). the current density increases as the contact area decreases, which enables a more effective cut. secondly, traction can provide better submucosal exposure (fig. the precise dissection of the submucosal layer is the most important step for preventing perforation, and the electrosurgical knife should always aim for the submucosal layer during dissection. therefore, improved submucosal exposure through traction is helpful for more effective and safer dissections surgeons are usually aided by the hands of assistants or by various instruments to maintain the tissue tension and visibility of the tissue to be dissected. in esd, achieving good traction is not easy because only an electrosurgical knife can be passed through the single working channel. the transparent distal attachment is a simple device that provides tissue tension and submucosal exposure. by inserting the transparent hood in between the flap and its base, more submucosa can be exposed and better tissue tension can be achieved (fig.3). this technique can also keep the tip of the endoscope at a suitable distance from the tissue such that the phenomenon of red - out can be avoided. traction force can be simply achieved through gravity.3 the direction of the traction force can be controlled by changing the position of the patient. while the patient 's position during gastric esd is limited to left lateral decubitus, various positions, even including the prone position are allowed during colorectal esd. locating fluid on the opposite side of the lesion, which can cause the flap to be strained to the side of the luminal center, is usually preferred. however, submucosal exposure may not be insufficient in the early stages of the dissection because the flap has not yet been sufficiently prepared. as the dissection progresses, the weight of the flap increases, and thus the traction force increases due to gravity (fig. insufficient submucosal exposure at an early stage can be complemented by the submucosal injection of longlasting materials, such as glycerol or hyaluronic acid4 and the use of a transparent hood. the traction force by gravity can be strengthened by applying a small weight.5 this sinker system consists of a 1-g weight, a clip, and a connecting nylon thread. by attaching the clip to the mucosal edge after a circumferential incision, the clip is attached to the tip of the flap and the end of the thread is pulled though a thin tube by an assistant during the colorectal esd. pushing the endoscope and pulling the thread can together lift the flap.6 various methods using clips and a thread has also been attempted in esophageal or gastric esd.7 - 12 the traction force can be prepared internally using elastic materials. a rubber band between a clip attached to the tip of the flap and another clip attached to normal mucosa can provide a continuous traction force during colorectal esd.13 the method can also be applied to gastric lesions,13 - 15 and a thin spring can replace a rubber band.16,17 the use of an extracorporeal electromagnet was attempted in gastric esd.18 in this trial, a large extracorporeal electromagnet was used to attract a small magnet that was attached to the mucosal edge for traction. external forceps can be used for traction in rectal esd.19 after a pair of bendable forceps is introduced with the help of other grasping forceps, and fixed to the mucosal edge, the bendable forceps are bent or pulled to elevate the lesion. forceps to elevate the mucosa can be introduced through a channel within a specialized transparent hood.20 similar methods have also been used for esophageal21 or gastric esd.22,23 an additional thin endoscope can be introduced for traction during rectal esd,24 and this method has also been attempted during gastric esd.25 - 28 percutaneous tractions,29,30 using a catheter and a double channel endoscope,31 and development of a new endoscope with two movable instrument channels32 have each been tested for gastric esd. the various trials attempting to achieve a proper traction force during esd are listed in table 1. one of the advantages of traction by gravity is that it does not require any additional devices. moreover, the direction of traction force can be easily controlled by changing the patient 's position. as mentioned above, traction force by gravity may not be sufficient at early stages of dissection, in which case the traction force can be complemented by a transparent hood and submucosal injection. in contrast, traction by adjunctive devices can create a good traction force from the early stages. specialized, expensive, or even huge devices may be required.18,32 some methods are more invasive than conventional esd,29,30 and the endoscope should occasionally be withdrawn and reinserted to provide traction.5 - 12,19,22 the movement of the primary endoscope may be hindered by the second endoscope to maintain traction.24 - 27 moreover, majority of the methods has not been tested in colorectal esd (table 1). the advantages and disadvantages of traction by gravity versus traction by adjunctive devices are listed in table 2. the maintenance of tissue tension and good submucosal exposure during dissection is one of the most important factors for an effective and safe dissection. various traction methods using adjunctive devices have been developed and may be useful for difficult cases. traction by gravity is not only a simple method, but also a useful method for most colorectal esd cases and is preferred by majority of experts. traction using adjunctive devices can thus be reserved for extremely difficult cases or for endoscopists in their learning periods of colorectal esd. | colorectal endoscopic submucosal dissection (esd) still remains a technically difficult procedure. the maintenance of tissue tension and good submucosal exposure during dissection is one of the most important factors for an effective and safe dissection. although various traction methods have been developed, traction by gravity is one of the most useful method for colorectal esd. traction using adjunctive devices can thus be reserved for extremely difficult cases or for endoscopists in their learning periods for colorectal esd. |
the efficacy of therapeutic antibodies stems from various natural functions of antibodies neutralization, antibody - dependent cell - mediated cytotoxic (adcc) activity, or complement - dependent cytotoxic (cdc) activity, or the antibody can be utilized as a drug delivery carrier (missile therapy) (fig. neutralization : many therapeutic antibodies utilize neutralization to block the pathophysiological function of their target molecules. in this case, antibodies bind to the ligand or receptor that is expressed on the cell surface and block the target signaling pathway. when the signaling in the tumor through these ligands or receptors is diminished, it can result in cellular activity being lost, proliferation being inhibited, pro - apoptotic programs being activated, or cells being resensitized to cytotoxic agents. adcc : to trigger adcc, the fv binding domain of an antibody binds to a specific antigen expressed on the surface of a target cell. the antibody is then able to recruit immune - effector cells (such as macrophages and nk cells) that express various receptors able to bind to the fc and thus activate the immune - effector cells to lyze the target cell. antigen complex, activates a cascade of complement proteins, and causes a complex to form that attacks the membrane. both adcc and cdc are interactions that involve components of the host immune system and, among the therapeutic antibodies being developed for cancer, there are presumably products that utilize more than one mechanism (adcc, cdc, and neutralizing functions) in their pharmacological actions. drug delivery carrier : antibodies can be applied as drug delivery carriers when conjugated to radioisotopes, toxins, drugs or cytokines. the advantage of these conjugates over conventional drugs is that cytotoxic agents can be delivered directly and at higher local concentrations to tumor tissues, without causing damage to normal cells. antibodies that bind and/or cross - link to target molecules and thus stimulate several signal pathways are also under research. however, these agonistic antibodies have not been placed on the market at this point. below are examples of the histopathological changes induced by therapeutic antibodies in non - clinical studies. as examples of therapeutic antibodies that use neutralization to block the pathophysiological function of their target antigens, we will show the changes caused by an anti - vascular endothelial growth factor (vegf) antibody and by an epidermal growth factor receptor (egfr) antibody. for those that use adcc and cdc, we will give examples of biological reactions to an anti - cd20 antibody. it binds to vegf and blocks vegf from uniting with its receptors (vegfr-1 and -2), which then blocks the signal transduction of vegf. vegf is the main factor that controls angiogenesis, and its expression is increased in most human tumors and is related to tumor proliferation / metastasis. hence, bevacizumab was approved for colorectal cancer, non - small cell lung cancer except squamous cell carcinoma, breast cancer, and so on. because the therapeutic blocks all the signaling transduced by vegf, cynomolgus monkeys treated repeatedly with bevacizumab via intravenous injection exhibited several pathological adverse effects on the epiphyseal growth plate, ovary, and uterus. lesions on the epiphyseal growth plate were characterized by a linear cessation of growth line and chondrocyte hyperplasia. in the ovary, arrested follicular development and absent corpora lutea were shown, and in the uterus, a decrease in endometrial proliferation and in the number of menstrual cycles were also seen. it is well known that vascularization of the epiphyseal growth plate region represents a key mechanism for chondrogenesis (cartilage production) and osteogenesis (bone formation). a small - molecule vegf inhibitor that inhibited angiogenesis in rats showed epiphyseal growth plate lesions it is reported that vascularization is essential for corpus luteum and endometrial formation ; therefore, biological reactions caused by an anti - vegf antibody are considered to be specific reactions by the target molecule in the organs and tissues in which vascularization was constantly maintained. cetuximab (erbitux) is a recombinant human / mouse chimeric anti - egfr monoclonal antibody. cetuximab binds to egfr selectively, blocks egfr from uniting with its ligand, egf, and then blocks the signal transduction of egf. egfr is a transmembrane glycoprotein that is expressed in epithelial tissues and acts as a receptor. binding of egfr to egf induces receptor dimerization and tyrosine autophosphorylation and leads to cell proliferation and differentiation,. egfr is expressed in normal tissues and also in many solid tumors, including colorectal cancer. hence, cetuximab is approved for colorectal cancer and squamous cell carcinoma of the head and neck. in cynomolgus monkeys, cetuximab was given by repeated intravenous injection and it resulted in dermatologic lesions characterized by hyperkeratosis, parakeratosis, abscess, and acantholysis with bullosa at the external integument. similar changes were observed in the epithelial mucosa of the nasal passage, esophagus, and tongue at the highest dose level. in addition, deaths due to sepsis associated with ulcerative dermatitis were observed in the animals at the highest dose level. rituximab (rituxan) is a chimeric murine / human monoclonal antibody targeted against the pan - b - cell marker cd20. rituximab binds to b cells that express cd20 and induces cell death through cdc or adcc. cd20 is expressed in non - neoplastic b cells (pre, immature, mature, and activated) and neoplastic cells derived from b cells. rituximab is indicated for the treatment of patients with non - hodgkin s lymphoma (nhl), chronic lymphocytic leukemia (cll) and rheumatoid arthritis. in a non - clinical study, rituximab was administered to cynomolgus monkeys repeatedly via intravenous injection (1/ week), and changes were found in immune - hematopoietic tissues. the total number of lymphocytes decreased in peripheral blood owing to a decrease of b cells, and atrophy of lymphoid follicles and a decrease of cd20-positive b cells were seen in the spleen and systemic lymph node. all of the cells affected by cytotoxicity were b cells that express cd20, and the reaction is considered to be specific to the target molecule. the changes induced by a therapeutic antibody in non - clinical study are thought of as biological reactions that are dependent on the target molecule. for example, with a blocking antibody the changes occur in the tissues and organs in which the targeted pathway functions. with antibodies that target specific ligands, changes are found in organs and tissues that express the receptor of the targeted ligand, and with antibodies that target specific receptors, changes are found in organs and tissues that express the targeted receptor. with a cytotoxic antibody the changes are found in the tissues and organs that express the target molecule. although the biological reactions induced by a therapeutic antibody are dependent on the target molecule and the target molecules selected in this paper, vegfr and egfr, were expressed broadly in normal tissues, the biological changes were not observed in all the organs and tissues that express the target molecule. with a blocking antibody, differences in the biological reactions may depend not only on expression of the target molecule but also on how the target pathway contributes to maintenance of homeostasis. the existence of alternative systems that compensate for the blocked pathway is thought to be an important factor of toxicologic changes. cytotoxicity antibodies are reported to have biological reactions that are not induced in all the cells in which antigen is expressed. we analyzed cdc induction in a non - clinical in vivo model and demonstrated that the biological response to an antibody with a cdc mechanism is regulated not only by the distribution of the target molecule but also by various other factors, ranging from antibody distribution to the nature of the host immune system and the presence of membrane complement regulatory proteins,. hence, when a therapeutic antibody induces cytotoxic change via the host immune system, cdc, or adcc, the immune regulatory system is an important factor on the occurrence of toxic effects. it binds to vegf and blocks vegf from uniting with its receptors (vegfr-1 and -2), which then blocks the signal transduction of vegf. vegf is the main factor that controls angiogenesis, and its expression is increased in most human tumors and is related to tumor proliferation / metastasis. hence, bevacizumab was approved for colorectal cancer, non - small cell lung cancer except squamous cell carcinoma, breast cancer, and so on. because the therapeutic blocks all the signaling transduced by vegf cynomolgus monkeys treated repeatedly with bevacizumab via intravenous injection exhibited several pathological adverse effects on the epiphyseal growth plate, ovary, and uterus. lesions on the epiphyseal growth plate were characterized by a linear cessation of growth line and chondrocyte hyperplasia. in the ovary, arrested follicular development and absent corpora lutea were shown, and in the uterus, a decrease in endometrial proliferation and in the number of menstrual cycles were also seen. it is well known that vascularization of the epiphyseal growth plate region represents a key mechanism for chondrogenesis (cartilage production) and osteogenesis (bone formation). a small - molecule vegf inhibitor that inhibited angiogenesis in rats showed epiphyseal growth plate lesions it is reported that vascularization is essential for corpus luteum and endometrial formation ; therefore, biological reactions caused by an anti - vegf antibody are considered to be specific reactions by the target molecule in the organs and tissues in which vascularization was constantly maintained. cetuximab (erbitux) is a recombinant human / mouse chimeric anti - egfr monoclonal antibody. cetuximab binds to egfr selectively, blocks egfr from uniting with its ligand, egf, and then blocks the signal transduction of egf. egfr is a transmembrane glycoprotein that is expressed in epithelial tissues and acts as a receptor. binding of egfr to egf induces receptor dimerization and tyrosine autophosphorylation and leads to cell proliferation and differentiation,. egfr is expressed in normal tissues and also in many solid tumors, including colorectal cancer. hence, cetuximab is approved for colorectal cancer and squamous cell carcinoma of the head and neck. in cynomolgus monkeys, cetuximab was given by repeated intravenous injection and it resulted in dermatologic lesions characterized by hyperkeratosis, parakeratosis, abscess, and acantholysis with bullosa at the external integument. similar changes were observed in the epithelial mucosa of the nasal passage, esophagus, and tongue at the highest dose level. in addition, deaths due to sepsis associated with ulcerative dermatitis were observed in the animals at the highest dose level. rituximab (rituxan) is a chimeric murine / human monoclonal antibody targeted against the pan - b - cell marker cd20. rituximab binds to b cells that express cd20 and induces cell death through cdc or adcc. cd20 is expressed in non - neoplastic b cells (pre, immature, mature, and activated) and neoplastic cells derived from b cells. rituximab is indicated for the treatment of patients with non - hodgkin s lymphoma (nhl), chronic lymphocytic leukemia (cll) and rheumatoid arthritis. in a non - clinical study, rituximab was administered to cynomolgus monkeys repeatedly via intravenous injection (1/ week), and changes were found in immune - hematopoietic tissues. the total number of lymphocytes decreased in peripheral blood owing to a decrease of b cells, and atrophy of lymphoid follicles and a decrease of cd20-positive b cells were seen in the spleen and systemic lymph node. all of the cells affected by cytotoxicity were b cells that express cd20, and the reaction is considered to be specific to the target molecule. the changes induced by a therapeutic antibody in non - clinical study are thought of as biological reactions that are dependent on the target molecule. for example, with a blocking antibody the changes occur in the tissues and organs in which the targeted pathway functions. with antibodies that target specific ligands, changes are found in organs and tissues that express the receptor of the targeted ligand, and with antibodies that target specific receptors, changes are found in organs and tissues that express the targeted receptor. with a cytotoxic antibody the changes are found in the tissues and organs that express the target molecule. although the biological reactions induced by a therapeutic antibody are dependent on the target molecule and the target molecules selected in this paper, vegfr and egfr, were expressed broadly in normal tissues, the biological changes were not observed in all the organs and tissues that express the target molecule. with a blocking antibody, differences in the biological reactions may depend not only on expression of the target molecule but also on how the target pathway contributes to maintenance of homeostasis. the existence of alternative systems that compensate for the blocked pathway is thought to be an important factor of toxicologic changes. cytotoxicity antibodies are reported to have biological reactions that are not induced in all the cells in which antigen is expressed. we analyzed cdc induction in a non - clinical in vivo model and demonstrated that the biological response to an antibody with a cdc mechanism is regulated not only by the distribution of the target molecule but also by various other factors, ranging from antibody distribution to the nature of the host immune system and the presence of membrane complement regulatory proteins,. hence, when a therapeutic antibody induces cytotoxic change via the host immune system, cdc, or adcc, the immune regulatory system is an important factor on the occurrence of toxic effects. recently, antibody engineering techniques have progressed and it is now possible to create antibodies with a diverse selection of functions, such as antibodies with more efficient and long - lasting neutralizing effects, agents that cause cytotoxicity at lower molecule expression levels, or bispecific antibodies that can recognize two different molecules simultaneously to induce new biological responses. these recent advances along with the discovery of novel target molecules shed light on the possibility of new therapies. as the functions and target molecules of antibodies become more and more diverse, it becomes increasingly necessary to understand how the target molecule functions biologically and what will be the biological response to the modified functions induced by the antibody. the toxicological pathology associated with these issues will also need to be evaluated and researched most carefully. | antibodies can swiftly provide therapeutics to target disease - related molecules discovered in genomic research. antibody engineering techniques have been actively developed and these technological innovations have intensified the development of therapeutic antibodies. from the mid-1990 s, a series of therapeutic antibodies were launched that are now being used in clinic. the disease areas that therapeutic antibodies can target have subsequently expanded, and antibodies are currently utilized as pharmaceuticals for cancer, inflammatory disease, organ transplantation, cardiovascular disease, infection, respiratory disease, ophthalmologic disease, and so on. this paper briefly describes the modes of action of therapeutic antibodies. several non - clinical study results of the pathological changes induced by therapeutic antibodies are also presented to aid the future assessment of the toxic potential of an antibody developed as a therapeutic. |
inhaled foreign bodies (fbs) are a common paediatric emergency and perhaps among the greatest cause of accidental death at home among nigerian children, although the exact incidence is not known because many are not recognised especially in rural communities.1 it is equally a major source of morbidity and mortality in even the most sophisticated of centres. this is particularly so in the paediatric age group especially the under - fives.2 this is said to be due to their inquisitiveness, adventurous nature, lack of molars for proper mastication, playing and running with food in the mouth with consequent incoordination in swallowing and glottic closure.3 with the advent of the ventilating bronchoscope, skilled personnel and better anaesthetic techniques, the mortality rate is said to have reduced from 24 to 3% or even less in developed countries.4 this, however, is not the case in developing countries especially sub - saharan africa, where the mortality rate is variable but reported cases range from 2.7 to 8.3%567 with majority probably under reported as most die before getting to a definitive point of care. the classical symptoms during fb inhalation are a sudden onset of paroxysms of cough, stridor and dyspnoea and occasionally this history is unavailable. the commoner signs are respiratory difficulty, reduced air entry in an affected lung, wheezing and/or crepitations. different types of foreign bodies (fbs) have been reported from the ordinary to the most bizarre. these range from inorganic materials such as needles, nails, whistles, toy parts and beads / rosary to organic substances such as groundnuts, maize seeds, water melon seeds, beans, variety of grains, etc. nonetheless, one thing is sure they are all a major source of anxiety and fear to the patient, caregiver and perhaps the attending physician as well. this broad classification becomes important when considering obstruction to airflow, amount of mucosal reaction, inflammation / infection and abscess formation akin to organic foreign bodies in the tracheobronchial tree. on the other hand, inorganic fbs may cause total or partial obstruction which may be tolerated for some time with mild signs and symptoms leading to formation of granulation tissue after a period of time.8 the clinical course and outcome of inhaled foreign bodies largely depends on the nature / type of foreign body, the site of arrest or impaction along the tracheobronchial tree and perhaps availability of skilled manpower especially in developing countries. foreign bodies can get impacted at any point from the laryngeal inlet to the terminal bronchioles, but more often these fbs get lodged in the right main bronchus. this is due to the right main stem bronchus being more in line with the trachea, thereby, creating a relatively straight path from the larynx to bronchus. despite this anatomical arrangement some controversies exist to suggest that more fbs are lodged in the left main bronchus compared to the right bronchus. in the same vain, some studies also posit that the site of election for impaction has an almost equal incidence in children, while others claim that this arrangement becomes relevant only after 15 years of age.1910 however, the site of impaction of fbs may ultimately depend on the position of the patient at the time of inhalation. the right main bronchus is commoner in the erect position and right lateral position while fbs which are small enough preferentially lodge in the left main bronchus in the left lateral position. this may also account for the difference in distribution of site of lodgment between adults / adolescent and children.1 this is a 6-year retrospective review of case notes of patients who have had bronchoscopies performed for foreign body inhalation at the otorhinolaryngology department of aminu kano teaching hospital, kano from january 2005 to december 2010. all the cases were referrals from the accidents and emergency unit, emergency paediatric unit and direct referrals from peripheral hospitals as well. the hospital is the main tertiary centre serving a population of over 10 million nigerians with three other states as catchment areas (katsina, jigawa and bauchi states). a total of 39 patient case notes were retrieved for the study and only 35 were included for analysis, one case note had incomplete information / missing pages, two case notes revealed a failed / negative bronchoscopy, with one recorded mortality due to cardiac arrest intra - operatively. basic information such as date of birth, age groups, gender, indication for bronchoscopy, type of fb, sites of fb impaction, and surgical outcome were recorded and analysed. in our centre ibm spss (for windows, version 21) software was used to analyse this data and for ease of interpretation presented in the form of tables and charts. statistically significant association between variables was explored using a two - tailed chi - square test and a confidence interval of 95.0%. ibm spss (for windows, version 21) software was used to analyse this data and for ease of interpretation presented in the form of tables and charts. statistically significant association between variables was explored using a two - tailed chi - square test and a confidence interval of 95.0%. there were 18 (51.4%) males and 17 (48.6%) females with a male : female ratio of 1.1:1. the most affected age group is 3 - 5 years (31.4%), followed by 0 - 2 years (25.7%) old age group [table 1 and figure 1 ]. age group by sex distribution of foreign bodies among our patient groups chart depicting percentage of foreign body inhalation by age groups with highest among the under - five 's groundnuts and whistles were the most frequently inhaled fbs in our series with 9 (25.7%) each [figure 2 and table 2 ]. subdivision of types of fbs into two broad groups revealed organic 18 (51.4%) foreign bodies and 17(48.6%) inorganic foreign bodies [figures 3 and 4, respectively ]. chart to show the frequency of foreign body types as seen in our patient population variety of foreign bodies removed at bronchoscopy during the period under review distribution of organic foreign bodies by (groundnut with highest) percentage distribution of inorganic foreign bodies by (whistles with highest) percentage with regard to the bronchus, overall fbs were more preferentially lodged in the left main bronchus in 8 (22.9%) patient against the right main bronchus in 6 (17.1%) patients. the 9 - 11-year and 3 - 5-year - old age groups were especially noted to have fb impaction involving the left main bronchus 3 (42.9) and right main bronchus 2 (18.2%) (p = 0.592), respectively. however, the majority of extracted fbs from the airways had no label for the specific site of impaction 15 (42.9%). [figures 5 and 6 ] distribution of impaction sites by percentages for foreign bodies in the tracheobronchial tree in our patient population frequency of impaction site by age group cluster bar chart analysis to determine relationship between age groups of these patients and types of inhaled foreign bodies revealed a strong association between these variables [exact test p = 0.001 ; phi and cramer 's v = 1.255 and 0.725, respectively, and contingency coefficient 0.782 ] further attempts to find relationships within variables such as sex of patients and nature of inhaled fbs, age groups and site of impaction, organic and inorganic fbs did not reveal any significant associations (p > 0.05) including the effect of age and gender in this study. our study clearly revealed that children below 5 years of age were most susceptible with a slight male preponderance. this was also the finding by gulshan., and is probably due to the over active nature of male children compared to their female counterparts.11 a higher male preponderance ratio of atleast 2:1 or more, has been reported by several authors this may be due to variations in sample size when compared to ours.6111213 in contrast, from available literature we did not find a higher female preponderance. moreover, with regard to variety of fb 's, groundnuts and whistles were the most common fb 's accidently inhaled with equal frequencies in our series [table 2 ]. to our knowledge, no recent study has reported an equal frequency of these fbs, much less reporting whistles as high as 25.7%. a study in pakistan showed that whistles were the commonest inhaled foreign body with a frequency of 57.7%.14 however, two recent studies show a frequency of 5.26% and 17.3%, respectively,1011 which is still less than findings from our series. children under 5 years are said to be generally more susceptible to fb inhalation due to their oral tendency, playing or running around with food pieces in the oral cavity and also parental nonchalance with regard to close supervision. many studies allude to this same finding.11516 children between the ages of 3 and 5years were the highest in our series [figure 1 ], this was also the finding of onotai ; however, other studies report a higher incidence in children less than or equal to 3 years.2121718 these periods coincide with the periods of inquisitiveness, adventure and exploration in a child 's developmental milestone, and by implication require closer attention and supervision from parents / caregivers. by and large organic fbs are commoner than inorganic fbs ; our study revealed 51.4% and 48.6% respectively and this was the finding by other authors.1920 the commonest organic fb inhaled in children is the groundnut and this concurs with our finding (25.7%) and is corroborated by other studies as well.1011162122 in contrast, a recent study in kenya revealed that the commonest organic fb in their series was beans while the second were peanuts / groundnuts.6 this was closely followed by seeds, although the particular type of seed was not documented in some instances due to its fragmented nature, but in addition to maize seeds, these still made up a large portion of inhaled organic fbs [figure 3].1618 kolanut and vegetable matter were the least (< 10%) but what we found, however, was the feeding, in some cases, of kolanut to children either by older siblings or accidently inhaled by the child while the parents were looking away or were not around. kolanut is a commonly chewed nut by adults in nigeria especially in the north, and requires molars to adequately grind them enough to be swallowed, but how they end up in the mouths of children since they lack molars says a lot about their care, or perhaps due to careless storage by the parents. other studies have revealed fishbone as the commonest type of fb seen especially in south - south nigeria.12 generally, inorganic fbs portend grave danger with acute asphyxiation from total obstruction or respiratory difficulty from partial obstruction. with regard to whistles, in our part of the country during festivities or ceremonies sweet candies are frequently giving to children. however, a variety of sweet candy is now available which is made combined with a whistle embedded within the sweet candy and after sucking out the candy bit, the children then go ahead to indulge in the reward by blowing the whistle as part of the fun. and as such, older children with whistles as fbs (25.7%) accounted for a greater portion of this group and this coincided with the islamic festive periods of eid - el - fitr or -kabir and in the wet season [figure 4 ]., reported the same observation during the wet season in ibadan south - eastern nigeria,1 a similar turkish study also reported similar findings highlighting an association between fbs with seasonal, geographic locality and sociocultural environment of affected patients.23 the seasonal significance of this finding at this time is unclear and may require further observations. the finding of whistles as part of other toys or snacks is, however, not uncommon, as this was also reported in abbottabad, where they mostly accounted for (17.3%) second highest after groundnuts.10 in one study in saudi arabia, the whistle could not be removed transglottically and tracheostomy with tracheal slit had to be performed, in our series all the whistle were successfully extracted transglottically via a rigid bronchoscope. this further emphasises the danger posed by these toys of sweetness. other varieties of fbs are more straightforward in terms of extraction with the appropriate forceps at hand. ball bearings in particular, had to be removed with a ventilating bronchoscope via the tracheostome. this obviously requires cooperation between the anaesthetist and endoscopist in addition to relevant skill and experience. by and large, most studies show organic fbs to be most prevalent in comparison to inorganic fbs116 and this was our finding as well [figures 3 and 4 ]. with regard to site of impaction or lodgment of foreign bodies, most studies have reported the preferential lodgment of fbs in children and adults to the right main bronchus as compared to the left main bronchus.15162425 however, in this review we did not record fb in both bronchi at the same seating as reported by a previous study.16 location of impacted fb is somewhat contentious ; most authors have argued that there is a preferential lodgment of fb in the right main bronchus than to the left main bronchus. this is said to be dependent on the patient 's age and relative physical position at the onset of inhalation. up until 15 years of age the angle made by the bronchi with the trachea is about the same, by implication an equal frequency is thereby recorded. however, after this age with continued growth and development, the left main bronchus assumes a more acute angle than the right and as such, the right bronchus is therefore more in line with the trachea. this may account for the relative higher frequency of fbs in the right main bronchus than in left as alluded to by many authors.41021 conversely, a recent study using tracheobronchial widths and angles from a large database of paediatric radiographs tested this hypothesis using standardised techniques with good reproducibility, and they concluded that an inhaled foreign body is more likely to enter the right bronchial tree than the left in children of all ages. however, in another recent study the authors claim that variability in the position of the carina with respect to the mid - trachea may explain why this right - sided preference is less marked in children compared to adults. this is because, in most children, the carina was positioned to the left of the mid - trachea, but in 34% of cases (40% of infants), it was to the right of the mid - trachea.24 in another related study the authors posit that aspirated foreign bodies are equally distributed between the left and right main bronchus that in children aged 3 and older foreign bodies were more commonly found in the right main bronchus.1826 in our review of these paediatric cases, we found a preferential lodgement of fbs in the left bronchus than the right with 22.9% and 17.1%, respectively [figures 5 and 6 ]., as well.27 although, as previously highlighted, the lack of specific labelling for site of impaction might have affected this result inadvertently. however, it is not uncommon.27 it suffices to say then that the bronchi, irrespective of laterality are the commonest areas of impaction along the tracheobronchial tree. our study showed a combined frequency of 40% (right and left) of bronchi involvement [figure 5 ] and this was the finding of some authors as well.172328 with regard to the rest of the tracheobronchial tree, the larynx and trachea have the lowest prevalence except in children less than 1 year,17 [figure 5 ] this was our finding as well. finally, our finding of left bronchial lodgment of fbs in specific age groups of 9 - 11 and 3 - 5 years was not statistical significant and as such no reliable comment can be made at this time. it suffices to say that proper and/or specific identification of extracted seeds and accurate delineation of impaction sites may have affected significantly the overall outcome measures in this study. possible biases may have existed in the recorded information, and some patient information was missing or not reported by the attending surgeon. we also recognise that perhaps the sample size may be too small to make population - based conclusions. it suffices to say that proper and/or specific identification of extracted seeds and accurate delineation of impaction sites may have affected significantly the overall outcome measures in this study. possible biases may have existed in the recorded information, and some patient information was missing or not reported by the attending surgeon. we also recognise that perhaps the sample size may be too small to make population - based conclusions. children particularly infants will always be curious leading to accidents and as such parents / caregivers have a fundamental role to play by providing constant supervision. the government must organise regular campaigns and proper education of the public both in the news / print media on the risk of foreign body aspiration especially among the under - five 's. furthermore, the left main bronchus is a common area for impaction of fbs as well, although this may ultimately depend on the position of the patient at the time of inhalation. we suggest that a strong legislation be put in place to regulate sweet and/or toy - producing companies on whistles (toys) manufactured with candies / sweets, and if possible be totally withdrawn from the market. | background : inhaled foreign bodies are common paediatric emergencies and a major cause of accidental deaths at home among nigerian children especially among children below the age of 5 years. different types of foreign bodies have been reported from the ordinary to the most bizarre. we evaluated the types of foreign bodies and sites of possible impaction of these foreign bodies along the tracheobronchial tree.materials and methods : a 6-year case record of 35 patients with foreign body inhalation was reviewed from january 2005 to december 2010 at the otorhinolaryngology department of bayero university / aminu kano teaching hospital, kano north - western nigeria. basic information such as date of birth, age groups, gender, indication for bronchoscopy, type of foreign bodies, sites of foreign body impaction and surgical outcome were extracted and analysed.results:thirty-five patients [18 (51.4%) males and 17 (48.6%) females, aged 6 months-10 years ] with a mean age of 5.1 years and median of 5 years (+ /- 3.0 standard deviation). the most affected age group with 31.4% is 3 - 5 years. groundnuts and whistles were the most frequently inhaled foreign bodies with an equal frequency of 25.7%, respectively. foreign bodies were preferentially lodged in the left main bronchus in 8 (22.9%) patients compared to the right main bronchus in 6 (17.1%) patients.conclusion:public enlightenment both in the news / print media on the risk of foreign body aspiration especially among the under - five 's is highly advocated. the left main bronchus may be a common site for impaction as well. |
preoperative radiotherapy, often combined with chemotherapy, followed by a total mesorectal excision (tme), has become the standard of care for patients with locally advanced rectal cancer. the success of radiotherapy is critically dependent on delivering an adequate radiation dose to tumor volumes while maximally sparing normal tissues and organs at risk. this review focuses on the clinical application of multimodality imaging in target delineation and treatment planning for rectal cancer radiotherapy, especially for preoperative radiotherapy. historically, simulation and planning of radiotherapy for rectal cancer was based on orthogonal films. rectal contrast and bony landmarks were used to delineate the treatment volumes, supplemented by clinical examination to aid definition of the inferior extent of the tumor. the availability of computed tomography (ct) and sophisticated treatment planning software has improved target definition, reduced geographic miss, and enabled the design of precision three - dimensional conformal radiotherapy (3dcrt) (fig. studies have shown that ct planning has advantages over orthogonal films in terms of definition of anterior and superior tumor borders and reduced toxicity in comparison with historical controls. however, visualization of pelvic structures using ct is limited by poor soft - tissue contrast between pelvic structures of similar hounsfield units. when contouring target volumes for rectal cancer, it is important to set an image gray scale that maximizes the contrast between soft - tissue infiltration and normal fat. it is also important to adequately visualize air in the bowel (particularly the large intestine) to properly identify normal tissue. it is better to select a window and level that emphasize densities within the mesorectum, and which adequately display the contrast between air in the large bowel and the surrounding fat. myerson and drzymala recommend a level of about 60 hounsfield units and a somewhat larger than routine window (250 hounsfield units) of approximately 600 hounsfield units to help better identify both loops of bowel and perirectal soft - tissue densities. figure 1outline of the clinical target volume (ctv) on the simulation ct scan of a patient with rectal cancer in the prone position. the ctv encompasses the possible regions into which the microscopic disease may extend, or regions with a high risk of involvement based on clinical experience (invisible tumor). outline of the clinical target volume (ctv) on the simulation ct scan of a patient with rectal cancer in the prone position. the ctv encompasses the possible regions into which the microscopic disease may extend, or regions with a high risk of involvement based on clinical experience (invisible tumor). according to previous research and our clinical experience, we generally distend the rectum with 50 ml of air at the time of simulation, so that the imaged boost target volume will incorporate an upper bound on the extent of rectal movement. this maneuver will not be feasible in patients who have substantial tumor - associated pain. however, such patients invariably have very advanced cancers, which are fixed anyway. knowledge of bowel location and dose volume histogram information is important in planning boost portals and interpreting treatment plans and outcome. during the past 2 decades, magnetic resonance imaging (mri) has emerged as the most accurate staging modality for locally advanced rectal cancer. dual phased - array, thin - section mri has been shown to be the gold standard for rectal cancer staging, with sensitivities of 7191% and specificities of 78100% in detecting depth of penetration and lymph node metastasis. mri addresses many of the limitations of ct, such as definition of depth of invasion through the rectal wall into local structures, and extension into presacral space and mesorectal circumference, which are high - risk areas for recurrence (fig. mri - defined tumor volumes for radiotherapy planning of rectal cancer could result in smaller, shorter, more accurate tumor contours, lying further from the anal sphincter. this would facilitate dose sparing to the normal tissues and escalation to the target. figure 2outline of the gross tumor volume (gtv) on the simulation ct scan of a patient with rectal cancer in the supine position (left). the gtv defines the tumor volume determined by clinical examination and imaging modalities (visible, palpable). outline of the gross tumor volume (gtv) on the simulation ct scan of a patient with rectal cancer in the supine position (left). the gtv defines the tumor volume determined by clinical examination and imaging modalities (visible, palpable). oneill. reviewed imaging and planning data for 10 patients with locally advanced low rectal cancer. estimates of tumor volume, tumor length, and height of proximal tumor from the anal verge were larger on planning ct than on mri. tumor volumes defined on mri are smaller, shorter, and more distal from the anal sphincter than ct - based volumes. tan. compared the volumetric and spatial relationships of gross tumor volume (gtv) derived from ct (ct - gtv) and gtv derived from mri (mr - gtv) to determine the utility of multimodality imaging for radiotherapy treatment planning in rectal cancer. tumor volumes were analyzed for 3 anatomical subregions (sigmoid, rectal, and anal). ct - gtv provided adequate spatial coverage of tumor in reference to mr - gtv with an average mean discrepancy of 0.12. ct - gtv coverage was inadequate for tumors with mri evidence of anal and sigmoid invasion. the investigators concluded that conventional simulation ct imaging provided a reasonable estimate of the gtv. multimodality imaging with staging mri can assist target volume definition where there is involvement of the sigmoid and anorectal region, and avoid geographic misses. in the study conducted by seierstad., rectal tumor volumes assessed by mri prior to and after 2 cycles of chemotherapy given in a neoadjuvant setting were used as input for a novel simultaneous integrated boost (sib) strategy in preoperative radiotherapy for locally advanced rectal cancer. sagittal and axial t2-weighted mri of the pelvis was acquired using a fast spin - echo sequence (repetition time range, 40006000 ms ; echo time, 85 ms ; echo - train length, 12 ; number of excitations, 4 ; matrix size, 512 512 ; slice thickness, 5 mm ; slice gap, 0 mm). in all tumor - containing axial t2-weighted mri, gtv was manually delineated by an experienced mr radiologist. planning ct images were rigidly coregistered with pre- and post - chemotherapy mri using the coregistration option mutual information in the treatment planning system. from the image registration, the investigators pointed out that mri facilitated the delineation of boost volume for rectal cancer. compared with ct, mri defines the target volume of rectal cancer more simply and accurately. adding the invasion part on mri to the target volume in addition, mri can be used in determining the target volume for rectal cancer brachytherapy. however, mri also has its disadvantages, including lack of electron density information and potential image distortion. tissue electron densities are required for accurate dose calculations and to account for tissue inhomogeneities within the treatment volume. unlike ct data, whereby electron density can be automatically calibrated from hounsfield units, mri signal intensity has no such correlation. image coregistration has become an integrated tool in radiotherapy planning, and facilitates the use of multimodal imaging approaches for optimal target delineation. mri coregistration accuracy of 4 commercial rigid - body techniques for external - beam radiotherapy treatment planning for rectal patients. seventeen patients with biopsy - proven rectal cancer were scanned with ct and mri in the prone position without the use of fiducial markers. two automated and 2 manual techniques on 2 separate treatment planning systems were compared with a reference coregistration. accuracy and reproducibility were analyzed using a measure of target registration error (tre) that was based on the average distance of the misregistration between vertices of the clinically relevant gtv as delineated on the ct image. an automated technique achieved the greatest accuracy, with a tre of 2.3 mm. both automated techniques demonstrated perfect reproducibility and were significantly faster than their manual counterparts. there was a significant difference in tre between registrations performed on the 2 planning systems, but there were no significant differences between the manual and automated techniques. the automated registration technique offered a fast and accurate solution, with associated uncertainties within acceptable treatment planning limits. more recently, diffusion - weighted mri (dwi) has been documented as a potential tool for evaluation of tumors in the body. dwi is used to measure the brownian motion of water molecules in tissue, which has been shown to be inversely proportional to cellular density, presumably because increased cellular density limits water diffusion in the interstitial space. the apparent diffusion coefficient (adc), a quantitative parameter measured on dwi, has been shown to be useful for evaluating solid tumors in the abdomen and pelvis. dwi evaluates the diffusion capacity of water molecules and obtains information about microscopic structures such as cell density or necrotic cell clusters, and therefore indirectly assesses tumor aggressiveness. rao. found that the addition of dwi to conventional t2-weighted imaging provides better detection of rectal cancer. in another study, gu made a comparison between mri and positron emission tomography (pet) combine with ct (pet / ct) for rectal cancer. they found the significant negative correlations between adc and standardized uptake value (suv) to suggest an association between tumor cellularity and metabolic activity in primary rectal adenocarcinoma. van brussel. investigated the potential role of dwi in the delineation of the target. they used dwi in intensity - modulated radiotherapy (imrt) dose painting for 5 rectal cancer patients. the boost volume was defined as a focal area of high signal detected in the rectum in high b value images (1000 s / mm). photon imrt and an sib were used to treat the whole pelvis with 45 gy, and the region with the maximum diffusion restriction (on dwi with the highest b value) with 52.5 gy in 25 treatments. volume constraints for small bowel were met in all 5 patients with a combination of 6, 7, or 8 beams. as a functional imaging modality, dwi can detect rectal cancer more accurately and provide tumor metabolic information, which facilitated the dose painting of imrt. mri can affect the determination of both gtv and clinical target volume (ctv). the radiation therapy oncology group (rtog) recommended that rectal cancer ctv should include at least the internal iliac, presacral, and perirectal nodal regions. although the contrast agent has not obtained approval by the us food and drug administration, some studies have suggested that lymphotropic nanoparticle mri (lnmri) can improve the ability to characterize lymph nodes as benign or malignant beyond size criteria alone. as a novel imaging technique, lnmri is mostly used in lymph node diagnosis of genitourinary tumor. just as for prostate cancer application of mri in radiotherapy treatment planning for rectal cancer is an emerging research area. we are unable to provide recommendations regarding the optimum protocols for the use of mri in radiotherapy planning given the relatively lower experience and published patients numbers utilising this technique compared to that available for ct. pet has been recognized as an important imaging modality for the diagnosis and staging of many malignant diseases because of its ability to provide metabolic activity information on malignant tumors. for colorectal cancer, pet / ct has an accuracy between 83% and 93% for initial staging, 96% sensitivity and 97% specificity for local recurrence, and 95% sensitivity and 98% specificity for detecting distant metastases. pet / ct is increasingly being applied in oncology to assist with initial staging of tumors as well as monitoring response to therapy in several disease sites. this has translated into increased incorporation of combined - modality pet / ct into radiation oncology for treatment planning (fig. figure 3axial unenhanced ct scan (left) shows ill - defined soft - tissue thickening in the perianal region (arrow) without definite evidence of a nodule / mass. fusion pet / ct image (right) shows an intense focus of hypermetabolism corresponding to the soft - tissue thickening, suggesting rectal cancer, which can help the radiation oncologist to define the gtv. axial unenhanced ct scan (left) shows ill - defined soft - tissue thickening in the perianal region (arrow) without definite evidence of a nodule / mass. fusion pet / ct image (right) shows an intense focus of hypermetabolism corresponding to the soft - tissue thickening, suggesting rectal cancer, which can help the radiation oncologist to define the gtv. buijsen. compared ct-, mri-, and pet / ct - based tumor length measurements in rectal cancer with pathology. twenty - six patients with rectal cancer underwent both mri and pet / ct imaging followed by short - course radiotherapy (5 5 gy) and surgery within 3 days after radiotherapy. tumor length was measured manually and independently by 2 observers on ct, mri, and pet / ct. pet / ct - based tumor length measurements were also generated automatically using the signal - to - background - ratio (sbr) method. the sbr method was used to find for each patient a percentage threshold of the maximal suv within a user - defined volume of interest around the tumor. manual pet / ct measurements reached a good correlation with pathology, but less strong than automatic pet / ct - based measurements, which provided the best correlation. this study demonstrated that automatically generated pet / ct - based contours show the best correlation with the surgical specimen, and thus provide a useful and powerful tool to accurately determine the largest tumor dimension in rectal cancer. bassi. compared ct with pet / ct scans with respect to rectal tumor volumes for 25 patients with rectal cancer who were candidates for preoperative radiotherapy. the gtv and ctv were delineated on the basis of ct and pet / ct. in 24% of the patients, pet / ct affected tumor staging or the treatment purpose ; in 12%, pet / ct showed an uptake in the regional lymph nodes, and in one case also in the liver. the pet / ct gtvs were statistically significantly larger than the ct gtvs, the mean difference being 25%. compared delineation of gtv using pet / ct and mri for a group of patients with rectal cancer. standard gtv was delineated using information from clinical examination, ct, and mri (gtv - mri). thereafter, a gtv - pet was defined in the pet / ct, and the target volume delineations were compared for total volume, overlap, and mismatch. the median volume of gtv - mri was larger than that of gtv - pet : 111 ml versus 87 ml (p < 0.001). in many cases, the gtv - mri contained the gtv defined on the pet / ct images as subvolumes, but when a gtv total was calculated after the addition of gtv - pet to gtv - mri the volume increased, with a median of 11%. another advantage of pet scanning is the ability to use the quantitative information of the tracer uptake within the tumor to automatically create a contour around the tumor. this contouring process significantly reduces the interobserver variability in the interpretation of images, as it eliminates the human factor, increases consistency, and diminishes interobserver variability. analyzed the interobserver variation for target volume delineation in preoperative radiotherapy of rectal cancer, and concluded that pet / ct may allow a reduced interobserver variation in gtv delineation in comparison with ct. analyzed the effect of the use of pet / ct on the interobserver variation in gtv definition in rectal cancer. forty - two patients diagnosed with rectal cancer underwent pet / ct for radiotherapy planning. an automatic contour was created on the pet scan using the source - to - background ratio. the gtv was delineated by 5 observers in 3 rounds : using ct and mri, using ct, mri, and pet, and using ct, mri, and pet auto - contour. cis increased significantly using pet, and the best interobserver agreement was observed using pet auto - contours. pet seems to be promising in target determination, but there are several methodological issues that need to be addressed, including the method for tumor volume segmentation and the selection of optimal tracer for rectal cancer. in the study by day., 3 segmentation methods were evaluated and compared for patients with rectal and anal cancer : percentage of the maximum suv (suv%max), fixed suv cutoff of 2.5 (suv2.5), and mathematical technique based on a confidence - connected region - growing (ccrg) method. the ccrg method is based on a region - growing method using the pixel intensity data of the tumor region in an iterative statistical manner to detect the edge of the tumor mass. pet / ct imaging studies for 18 patients who received radiotherapy were used to evaluate the segmentation methods. a pet - avid (petavid) region was manually segmented for each patient, and the volume was then used to compare the calculated volumes along with the absolute mean difference and range for all methods. for the suv%max method, the volumes were always smaller than the petavid volume by a mean of 56%. the volumes from the suv2.5 method were either smaller or larger than the petavid volume by a mean of 37%. this study showed that the ccrg technique can be used in the segmentation of tumor volumes on pet images for patients with rectal cancer. roels. investigated the use of pet / ct with fluorodeoxyglucose (fdg), fluorothymidine (flt), and fluoromisonidazole (fmiso) for radiotherapy target definition and evolution in rectal cancer. pet / ct was performed before and during preoperative chemoradiotherapy in 15 patients with resectable rectal cancer. pet images were used for treatment target delineation, and ct images on the different time points were nonrigidly registered. mismatch analyses were carried out to quantify the overlap between fdg and flt or fmiso tumor volumes (tv) and between pet tvs over time. ninety sequential pet / ct images were analyzed. on each time point, the mean fdg - pet tv was significantly larger than the fmiso - pet tv but not significantly larger than the mean flt - pet tv. there was a mean 65% mismatch between the fmiso and fdg tvs obtained before and during crt. flt tvs corresponded better with the fdg tvs (25% mismatch before and 56% during chemoradiotherapy). during chemoradiotherapy, on average 61% of the mean fdg tv (7 ml) overlapped with for flt, the tv overlap was 49%, and for fmiso only 20% of the tv during chemoradiotherapy remained inside the contour at baseline. it was concluded that fdg, flt, and fmiso - pet reflect different functional characteristics that change during chemoradiotherapy in rectal cancer. flt and fdg show good spatial correspondence, while fmiso seems less reliable owing to the nonspecific fmiso uptake in normoxic tissue and tracer diffusion through the bowel wall. fdg and flt - pet / ct imaging seem most appropriate for integration in preoperative radiotherapy for rectal cancer. pet / ct affects rectal cancer staging, and increase target definition and contouring consensus. after thorough methodological research and combined use with mri, pet / ct may come to play an essential role in target definition for rectal cancer. ct simulation is a cornerstone of these modern techniques, but has limitations, such as poor soft - tissue contrast between pelvic structures and partial volume effects. mri and pet can overcome these limitations and provide additional information for the planning of rectal cancer treatment. in addition, pet can affect rectal cancer staging, and increase target definition and contouring consensus. however, there is long way to go before these imaging modalities are routinely used in clinical settings. several questions, such as the registration method for mri and ct, the value of dwi in rectal cancer radiotherapy treatment planning, the tumor volume segmentation method of pet imaging, and the optimal tracer for rectal cancer pet imaging, need to be answered by further research. | abstractradiotherapy plays an important role in the treatment of rectal cancer. three - dimensional conformal radiotherapy and intensity - modulated radiotherapy are mainstay techniques of radiotherapy for rectal cancer. however, the success of these techniques is heavily reliant on accurate target delineation and treatment planning. computed tomography simulation is a cornerstone of rectal cancer radiotherapy, but there are limitations, such as poor soft - tissue contrast between pelvic structures and partial volume effects. magnetic resonance imaging and positron emission tomography (pet) can overcome these limitations and provide additional information for rectal cancer treatment planning. pet can also reduce the interobserver variation in the definition of rectal tumor volume. however, there is a long way to go before these image modalities are routinely used in the clinical setting. this review summarizes the most promising studies on clinical applications of multimodality imaging in target delineation and treatment planning for rectal cancer radiotherapy. |
lung cancer is the leading cause of cancer - related deaths. according to the world health organization (who), it is divided into two categories based on its biology, therapy and prognosis as small cell lung cancer and non - small cell lung cancer (nsclc). nsclc, accounting for more than 85% of all lung cancer cases, consists of two major histological subtypes ; squamous and non - squamous (adeno, large cell and other cell types). only about 15.6% of all lung cancer patients are alive 5 years or more after diagnosis. the majority of patients with lung cancer are at the advanced stage at time of diagnosis. therefore, late diagnosis is still a problem in the effort to improve the outcomes and early stage at diagnosis is an important prognostic factor. defining the high risk group patients is important for early diagnosis and chance of cure. new biological markers, which can predict patients with higher risk and poor outcome, can be a new therapeutic approach. the potential uses of the markers include aiding early diagnosis, determining prognosis, prospectively predicting response or resistance to specific therapies, and monitoring therapy in patients with advanced disease. kallikreins, a subgroup of the serine protease enzyme family, are expressed in many tissues, including steroid hormone - producing or hormone - dependent tissues such as the prostate, breast, ovary, and testis. human tissue kallikreins are a family of 15 highly conserved serine proteases encoded by the largest contiguous cluster of protease genes in the human genome. measurement of kallikrein genes and proteins in serum and tumor tissue extracts may be useful for the purpose of disease diagnosis, monitoring, prognosis, or subclassification.. showed that human kallikrein 11 (hk11) positivity is associated with a slower disease progression and hk11 is an independent marker of favorable prognosis in patients with ovarian cancer. in a recent study, we observed that hk11 expression in gastric cancer is associated with a better prognosis. in the current study, we aimed to evaluate the prognostic value of immunohistochemical expression of hk11 in patients with non - metastatic nsclc treated with chemoradiotherapy. a total of 88 eligible patients with non - metastatic nsclc were treated with chemoradiotherapy. patients who were 0.05). the estimation of disease - free and overall survival by kaplan - meier was 11 months and 17 months, respectively (data not shown). fig. 2 shows disease - free survival according to the degree of staining with hk11. although not statistically different, the estimation of disease - free survival by kaplan - meier was higher in patients with hk11 strongly positive (2 +) than in those with hk11 weakly positive (1 +) (12 months vs. 9 months, p=0.113). the estimation of overall survival by kaplan - meier was significantly higher in patients with hk11 strongly positive (2 +) than in those with hk11 weakly positive (1 +) (20 months vs. 11 months, p=0.032). the overall survival rates according to characteristics of the patients are shown in table 2. histology of cancer, t status, response to chemoradiotherapy, and the degree of staining with hk11 were the characteristics that significantly influenced the overall survival (p=0.021, p=0.038, p=0.011, and p=0.032, respectively). the overall survival and p - values adjusted for patient characteristics in patients with hk11 weakly positive (1 +) and strongly positive (2 +) are shown in table 3. a significant difference in the overall survival adjusted for age groups, sex, t status, and n status was observed between the two groups (p=0.043, p=0.026, p=0.041, and p=0.039, respectively). however, no significant difference in the overall survival adjusted for histology and response to chemoradiotherapy was observed between the two groups (p > 0.05). univariate and multivariate analyses were performed to determine the risk factor(s) related to overall survival. table 4 shows the results regarding eight variables examined in univariate analysis as potential risk factors for overall survival. in univariate analysis four of the eight factors (histology of cancer, t status, response to chemoradiotherapy, and the degree of staining with hk11) differed significantly between these groups (p 0.05). interactions occur between serine proteases and substrates of serine proteases and many other substrates, including extracellular matrix proteins. serine proteases play a role in cascade pathways, such as the blood coagulation cascade, the dissolution of blood clots, the intestinal digestive enzymes, and the apoptosis pathway. kallikrein cascade seems to be valuable in elucidation of the various mechanisms underlying cancer invasion and metastasis. prostate - specific antigen (psa ; hk3) is the most used tumor marker. the most successful clinical use of psa is currently in the diagnosis and monitoring of prostate cancer. similarly, kallikreins such as hk4, hk6, hk9, hk10, hk11, and hk12 are emerging new markers for the diagnosis and prognosis of various tumors. yu. reported that over - expression of hk11 was associated with poor prognosis in patients with low rectal carcinoma. diamandis. found that hk11 is an indicator of favorable prognosis in ovarian cancer patients. they observed that patients with hk11-positive tumors had a significantly longer progressionfree survival and overall survival and hk11 was an independent prognostic indicator of progression - free survival. similarly, in another study conducted by these authors in patients with ovarian cancer, it was found that hk11-positive tumors were more frequently associated with early stage disease, pre-/peri - menopausal status and patients who exhibited complete or partial response to chemotherapy and patients with hk11-positive tumors had a significantly decreased risk of relapse and death. in the literature, there are a limited number of studies on the importance of hk11 in lung cancer. planque. examined the clinical value of 11 members of the hk family as potential biomarkers for the diagnosis of lung cancer. they found that patients with nsclc had lower levels of klk5, klk7, klk8, klk10, and klk12, and higher levels of klk11, klk13, and klk14 compared to controls and proposed a multiparametric panel of kallikrein markers for the diagnosis of nsclc with relatively good accuracy. similarly, xu. evaluated the diagnostic and prognostic value of serum human kallikrein related peptidases 11 levels were significantly higher in nsclc compared to that in the controls and nsclc patients with serum high human kallikrein related peptidases 11 had a longer overall survival and progression - free survival than those with low human kallikrein related peptidases 11 might be a useful diagnostic and prognostic test for nsclc patients. in the current study, we observed that the stronger hk11 expression was associated with better survival rates in patients with nsclc. overall survival rates were significantly higher in patients with hk11 strongly positive (2 +) compared to those with hk11 weakly positive (1 +). in addition, although not statistically different, disease - free survival rates were higher in patients with hk11 strongly positive (2 +) compared to those with hk11 weakly positive (1 +). cancer cells and some cells of the immune system produce substances that induce apoptosis in normal tissues. for example, the p53 tumor suppressor gene stimulates apoptosis, whereas the bcl-2 oncogene inhibits it. hk11 may improve the prognosis by enhancing chemoradiotherapy - induced apoptosis in lung cancer. in our recent study on the value of hk11 expression in gastric cancer similarly, in the current study, we found that prognosis worsens as staining with hk11 in lung cancer tissue is decreasing. on the other hand hk11 may be a prognostic biomarker of nsclc, which is usually diagnosed at advanced stage, and new therapeutic and prognostic indicators are needed. the stronger hk11 expression in nsclc appears to be associated with better survival rates. on the other hand, | purposeinvolvement of human kallikreins (hks) in human cancers has been reported and several hks are promising biomarkers of various cancers. the aim of this study was to evaluate the prognostic significance of hk11 expression in patients with non - metastatic non - small cell lung cancer (nsclc).materials and methodsthe study included 44 patients with nsclc. hk11 expression was determined by immunohistochemical staining.resultsthe estimation of disease - free and overall survival by kaplan - meier was 11 months and 17 months, respectively. the estimation of overall survival by kaplan - meier was significantly higher in patients with hk11 strongly positive (2 +) than in those with hk11 weakly positive (1 +) (20 months vs. 11 months, p=0.032). although not statistically different, the estimation of disease - free survival by kaplan - meier was higher in patients with hk11 strongly positive (2 +) than in those with hk11 weakly positive (1 +) (12 months vs. 9 months, p=0.113). multivariate cox regression analysis showed that the overall survival rates were significantly associated with response to chemoradiotherapy and the degree of staining with hk11.conclusionthe stronger hk11 expression in nsclc appears to be associated with better survival rates. hk11 may be a prognostic biomarker of nsclc. |
acinetobacter baumannii is an important nosocomial pathogen that causes pneumonia, bacteremia, meningitis, urinary tract infections, and other inflammation - related diseases [15 ]. it is difficult to treat a. baumannii infections owing to the occurrence of drug resistance and the ability of the pathogen to propagate worldwide. this infection contributes to the high mortality rates of in - patients (23%) and patients in the intensive care unit (icu ; 43%) and represents a major clinical issue. currently, a. baumannii drug resistance is believed to be related to specific antibiotic hydrolases produced by a. baumannii ; these hydrolases could alter drug - binding proteins, bacterial structure, and the number of porins, and could increase the activity of efflux pumps [710 ]. furthermore, a. baumannii can live in the form of a biofilm in the external environment, resistant to disinfectants, ultraviolet light, and host immune defenses. a previous review illustrated several specific genes, including csua / babcde, ompa, abai, and pgaabcd, that may determine the biofilm formation of a. baumannii. furthermore, alternative protein complexes involved in biofilm formation are assembled in different a. baumannii strains and are highly correlated with the uneven distributions of different biofilm - associated protein (bap) types. the relationship between biofilm - related genes and a. baumannii biofilm formation was described in previous studies [1418 ]. for example, breij. suggested that there may be an association between the csua / babcde gene and a. baumannii biofilm formation on abiotic surfaces. furthermore, ompa can be integrated into host epithelial cell and mitochondrial membranes and induce cell death, or participate in the extrusion of compounds from the periplasmic space through the outer membrane and couple with inner membrane efflux systems, which may be associated with drug resistance in a. baumannii infections. the autoinducer synthase abai is necessary for biofilm formation and plays an important role in the late stages of biofilm maturation. moreover, the ability of a. baumannii to adhere to epithelial cells may be enhanced by bla - per1. currently, although several potential relationships have been detected in previous studies, the relationship between biofilm - related genes, biofilm formation, and drug resistance of a. baumannii in china remains unclear. furthermore, the correlations between antibiotic resistance and the four a. baumannii genes related to biofilm formation are still controversial. therefore, we conducted a retrospective study to explore the potential association between the four biofilm - related genes and drug resistance by detecting csua / babcde, ompa, abai, and bla - per1 in a. baumannii isolates from clinical specimens. this study was approved by the ethics committee of longyan first affiliated hospital of fujian medical university, longyan, fujian, china (2012001). the purpose and procedures of the study were carefully explained to all participants and written informed consent was obtained from all participants. all the clinical isolates analyzed in this work were collected as part of routine medical care. one hundred twenty - two patients with lower respiratory tract infection by a. baumannii were enrolled in this study after hospitalization in various departments at fujian longyan first hospital between january 2013 and september 2014. patients primary disease, aggressive treatment, clinical symptoms, temperature, white blood cell count (wbc), and x - ray examination results were recorded by investigators. the patients had clinical symptoms that included cough with purulent sputum or increasing sputum volume, moist crackles, or lung x - ray examination with pulmonary infiltrates or with fuzzy and increased lung markings. furthermore, we also carried out laboratory tests, imaging examinations, and microbiological examinations. using diagnostic criteria for hospital infections as the basis for diagnosis, patients were eligible for inclusion in the study if the following criteria were met : (1) a. baumannii was detected from two consecutive sputum cultures, and (2) a. baumannii 10 cfu / ml was detected from lower respiratory tract secretions that were collected by fiber optic bronchoscopy or artificial airways. a. baumannii bacteria were obtained from sputum specimens from the first deep lung expectorant of patients after waking and rinsing their mouths using normal saline. strains were detected using a bd phoenix100 automated microbial identification system (becton dickinson and company, nj, usa). the reference strains escherichia coli atcc25922 and pseudomonas aeruginosa atcc27853 were used as controls (oxoid company, basingstoke, england). a. baumannii genomic dna was extracted using a tiangen bacterial genomic dna extraction kit (tiangen biotech company, beijing, china) according to the manufacturer s instructions. the various target gene primer sequences [1618 ] and product lengths are shown in table 1. each biofilm - associated gene amplification reaction included 1 l each of p1 and p2 primers (10 nm), 3 l of 200 mm dntps, 3 l of buffer containing 15 mm mgcl2, 2 l dna template, 5 u/l taq enzyme (1 l), and 19 l of double - distilled h2o for a total reaction volume of 30 l. the pcr thermal cycling parameters for abai and csue were an initial denaturation at 95c for 10 minutes ; followed by 35 cycles of 95c for 30 seconds, 63c for 30 seconds, and 72c for 1 minute ; and a final extension at 72c for 10 minutes. the pcr thermal cycling parameters for ompa and bla - per1 were an initial denaturation at 95c for 10 minutes ; followed by 35 cycles of 95c for 30 seconds, 52c for 30 seconds, and 72c for 1 minute ; and a final extension at 72c for 10 minutes. by comparing the resulting bands and a dna base pair marker following 2% agarose gel electrophoresis, products that showed the expected molecular weight were regarded as positive. the microdilution quantitative minimum inhibitory concentration (mic) method was used to determine the antimicrobial susceptibility of each strain. according to the us clsi2013 susceptibility criteria, multidrug resistance was defined as isolates showing drug resistance to three or more of the following five classes of antibiotics : cephalosporins, carbapenems, compounds of -lactamase inhibitors, fluoroquinolones, and aminoglycosides. pan - drug resistance was defined as isolates showing drug resistance to all classes excluding polymyxin.. comparisons between positive and negative genes were made using t - tests and tests. furthermore, drug resistance was investigated by logistic regression for analyzing the odds ratio after adjustment for gender, mean age, department, ventilator, tracheotomy, cardiovascular disease, and diabetes. statistical analyses were performed with two - sided tests. differences with p - values of less than 0.05 the data were analyzed using statistical package for the social sciences version 19.0 (spss 19.0). this study was approved by the ethics committee of longyan first affiliated hospital of fujian medical university, longyan, fujian, china (2012001). the purpose and procedures of the study were carefully explained to all participants and written informed consent was obtained from all participants. all the clinical isolates analyzed in this work were collected as part of routine medical care. one hundred twenty - two patients with lower respiratory tract infection by a. baumannii were enrolled in this study after hospitalization in various departments at fujian longyan first hospital between january 2013 and september 2014. patients primary disease, aggressive treatment, clinical symptoms, temperature, white blood cell count (wbc), and x - ray examination results were recorded by investigators. the patients had clinical symptoms that included cough with purulent sputum or increasing sputum volume, moist crackles, or lung x - ray examination with pulmonary infiltrates or with fuzzy and increased lung markings. furthermore, we also carried out laboratory tests, imaging examinations, and microbiological examinations. using diagnostic criteria for hospital infections as the basis for diagnosis, patients were eligible for inclusion in the study if the following criteria were met : (1) a. baumannii was detected from two consecutive sputum cultures, and (2) a. baumannii 10 cfu / ml was detected from lower respiratory tract secretions that were collected by fiber optic bronchoscopy or artificial airways. a. baumannii bacteria were obtained from sputum specimens from the first deep lung expectorant of patients after waking and rinsing their mouths using normal saline. strains were detected using a bd phoenix100 automated microbial identification system (becton dickinson and company, nj, usa). the reference strains escherichia coli atcc25922 and pseudomonas aeruginosa atcc27853 were used as controls (oxoid company, basingstoke, england). a. baumannii genomic dna was extracted using a tiangen bacterial genomic dna extraction kit (tiangen biotech company, beijing, china) according to the manufacturer s instructions. the various target gene primer sequences [1618 ] and product lengths are shown in table 1. each biofilm - associated gene amplification reaction included 1 l each of p1 and p2 primers (10 nm), 3 l of 200 mm dntps, 3 l of buffer containing 15 mm mgcl2, 2 l dna template, 5 u/l taq enzyme (1 l), and 19 l of double - distilled h2o for a total reaction volume of 30 l. the pcr thermal cycling parameters for abai and csue were an initial denaturation at 95c for 10 minutes ; followed by 35 cycles of 95c for 30 seconds, 63c for 30 seconds, and 72c for 1 minute ; and a final extension at 72c for 10 minutes. the pcr thermal cycling parameters for ompa and bla - per1 were an initial denaturation at 95c for 10 minutes ; followed by 35 cycles of 95c for 30 seconds, 52c for 30 seconds, and 72c for 1 minute ; and a final extension at 72c for 10 minutes. by comparing the resulting bands and a dna base pair marker following 2% agarose gel electrophoresis, products that showed the expected molecular weight the microdilution quantitative minimum inhibitory concentration (mic) method was used to determine the antimicrobial susceptibility of each strain. according to the us clsi2013 susceptibility criteria, multidrug resistance was defined as isolates showing drug resistance to three or more of the following five classes of antibiotics : cephalosporins, carbapenems, compounds of -lactamase inhibitors, fluoroquinolones, and aminoglycosides. pan - drug resistance was defined as isolates showing drug resistance to all classes excluding polymyxin. comparisons between positive and negative genes were made using t - tests and tests. furthermore, drug resistance was investigated by logistic regression for analyzing the odds ratio after adjustment for gender, mean age, department, ventilator, tracheotomy, cardiovascular disease, and diabetes. statistical analyses were performed with two - sided tests. differences with p - values of less than 0.05 the data were analyzed using statistical package for the social sciences version 19.0 (spss 19.0). as shown in table 2, there were 102 men and 20 women enrolled in this study. additionally, 54.9% (67/122) of a. baumannii - infected patients were admitted to the icu, and 33 patients (27%, 33/122) had chronic obstructive pulmonary disease (copd). the long - term invasive treatments to which patients were exposed included tracheotomy, mechanical ventilation, intravenous indwelling catheterization, retention catheterization, and sputum suction. the proportion of infections associated with tracheotomy, mechanical ventilation, and sputum suction were 74.07% (40/54), 71.67% (43/60), and 46.67% (28/60), respectively. patients had cough, sputum, pulmonary moist rales, imaging changes, and other clinical symptoms. in the 122 sputum specimens, the detection rates of abai and csue were both 59.8%, and those of ompa and bla - per1 were 100% and 0%, respectively. the results of our analysis of biofilm - related genes are shown in table 3. abai- and csue - positive and negative specimens showed statistically significant differences in the rates of resistance to amikacin, ampicillin / sulbactam, cefepime, cefoperazone / sulbactam, cefotaxime, ceftazidime, ciprofloxacin, gentamicin, imipenem, meropenem, piperacillin / tazobactam, tetracycline, cotrimoxazole (smzco), and levofloxacin (p0.05). the odds ratios for the association between specific drug resistance rates and positivity for the abai and csue genes are presented in table 5. overall, we noted that abai and csue positivity were associated with a statistically significant impact on amikacin, ampicillin, cefepime, sulbactam, cefotaxime, ceftazidime, ciprofloxacin, gentamicin, imipenem, meropenem, piperacillin, tetracycline, smzco, and levofloxacin resistance after adjustment for gender, mean age, department, ventilator, tracheotomy, cardiovascular disease, and diabetes. as shown in table 2, there were 102 men and 20 women enrolled in this study. additionally, 54.9% (67/122) of a. baumannii - infected patients were admitted to the icu, and 33 patients (27%, 33/122) had chronic obstructive pulmonary disease (copd). the long - term invasive treatments to which patients were exposed included tracheotomy, mechanical ventilation, intravenous indwelling catheterization, retention catheterization, and sputum suction. the proportion of infections associated with tracheotomy, mechanical ventilation, and sputum suction were 74.07% (40/54), 71.67% (43/60), and 46.67% (28/60), respectively. patients had cough, sputum, pulmonary moist rales, imaging changes, and other clinical symptoms. in the 122 sputum specimens, the detection rates of abai and csue were both 59.8%, and those of ompa and bla - per1 were 100% and 0%, respectively. the results of our analysis of biofilm - related genes are shown in table 3. abai- and csue - positive and negative specimens showed statistically significant differences in the rates of resistance to amikacin, ampicillin / sulbactam, cefepime, cefoperazone / sulbactam, cefotaxime, ceftazidime, ciprofloxacin, gentamicin, imipenem, meropenem, piperacillin / tazobactam, tetracycline, cotrimoxazole (smzco), and levofloxacin (p0.05). the odds ratios for the association between specific drug resistance rates and positivity for the abai and csue genes are presented in table 5. overall, we noted that abai and csue positivity were associated with a statistically significant impact on amikacin, ampicillin, cefepime, sulbactam, cefotaxime, ceftazidime, ciprofloxacin, gentamicin, imipenem, meropenem, piperacillin, tetracycline, smzco, and levofloxacin resistance after adjustment for gender, mean age, department, ventilator, tracheotomy, cardiovascular disease, and diabetes. a. baumannii is an opportunistic pathogen that can colonize the skin, conjunctiva, oral cavities, respiratory tract, gastrointestinal tract, and urinary tract. furthermore, a. baumannii infections frequently occur in the intensive care unit and respiratory department. these patients are often in critical condition, have lower immune function, exhibit poor nutritional status, and may also have diabetes, cancer, chronic wasting disease, and other diseases of the brain, heart, kidneys, or lungs. the symptoms caused by lower respiratory tract infections of a. baumannii include cough, sputum, and pulmonary moist rales. in addition, patients often exhibit distinct x - ray images, such as multiple scattered small patchy shadows, large patchy shadows, cystic lung or cylindrical bronchiectasis, and pleural effusions, which lack clinical specificity. therefore, microbiological examinations are needed to confirm a. baumannii infection as soon as possible in order to maximize recovery rates. the findings of our study suggested that abai- and csue - positive strains were associated with 14 types of antimicrobial resistance, as shown in table 4. furthermore, after adjusting for gender, mean age, department, ventilator, tracheotomy, cardiovascular disease, and diabetes, abai and csue were associated with significant effects on amikacin, ampicillin, cefepime, sulbactam, cefotaxime, ceftazidime, ciprofloxacin, gentamicin, imipenem, meropenem, piperacillin, tetracycline, smzco, and levofloxacin resistance. bacterial biofilms are associated with problems in the prevention and treatment of a. baumannii infection. biofilms irreversibly adhere in the host s tissues or on abiotic surfaces and are supported by polymer matrices that are secreted by the microorganisms themselves, facilitating the formation of bacterial communities. this viscous matrix can separate bacteria from harmful external factors, thereby increasing the resistance of the microbial community. these observations may be explained as follows : (1) permeation limitation : bacteria at high density in biofilms can produce an extracellular matrix that impedes antibiotic penetration ; (2) nutrition restrictions : bacteria in biofilms are maintained in a state of low metabolism and a slow growth rate, making them less sensitive to outside stimuli, such as antibiotics ; (3) phenotype inference : biological membranes select or induce strains with resistant phenotypes and increase the number of antibiotic resistance genes and the expression of resistance efflux pumps, leading to drug resistance ; (4) immune suppression : a. baumannii biofilms are a natural physical barrier that limit immune - mediated killing of the organism ; and (5) quorum sensing : following an increase in the number of a. baumannii, some bacteria are detached from the surface of the biofilm by quorum sensing and are transformed into a planktonic growth state, allowing bacteria to adhere to appropriate media and leading to the spread of infection and relapse. the clinical signs of a. baumannii - infected patients and the possible factors that influence drug resistance were also examined in this study. the reasons for this include permeation limitation, nutrition restrictions, phenotype inference, immune suppression, and quorum sensing, as discussed above. therefore, the following prevention and treatment methods are recommended : (1) regular disinfection of wards and medical containers should be conducted ; (2) medical personnel should practice hand hygiene ; (3) the duration of mechanical ventilation and body catheterization should be shortened ; and (4) early diagnosis and drug resistance testing should be a priority in order to enhance preventive measures. first, stratified analyses based on potential confounders were not conducted due to the insufficient sample size. second, patients with different disease statuses and admitted to different clinical centers were enrolled in this study, which may have affected the observed results. thus, an improved understanding of the association between biofilm formation and resistance mechanisms is needed because once drug - resistant strains form biofilms, treatment becomes extremely difficult. abai- and csue - positive strains were the most resistant to the 14 types of antimicrobials ; therefore, detecting these genes can guide antibiotic use in a. baumannii - infected patients. further studies are needed to determine whether strains carrying abai and csue are associated with biofilm formation. a. baumannii infected patients were most commonly admitted to intensive care units and the respiratory department, and the patients frequently had chronic obstructive pulmonary disease. the patients infected with a. baumannii - induced lower respiratory tract infections lacked clinical specificity. the majority of the a. baumannii isolates from mainland china were susceptible to polymyxin e. abai- and csue - positive strains were associated with higher incidence of 14 types of antimicrobial resistance. detecting the abai and csue genes can provide important information for clinical treatment by certain antibiotics. an improved understanding of the association between biofilm formation and resistance mechanisms is needed, and some preventive measures such as shortening the duration of mechanical ventilation should be instituted to avoid biofilm formation. | backgroundacinetobacter baumannii is an important nosocomial pathogen which shows a high level of mortality risk. several papers have reported biofilm formation as a well - known pathogenic mechanism in a. baumannii infections and exceptional antibiotic resistance. the study aims to explore the potential relationships between biofilm - related genes and antimicrobial resistance.material/methodssamples from 122 patients with lower respiratory tract infections of a. baumannii were collected at fujian longyan first hospital from january 2013 to september 2014. a. baumannii was isolated from sputum specimens. biofilm - related genes including abai, csue, ompa, and bla - per1 were analyzed by pcr. the minimum inhibitory concentration method was used to determine the sensitivity of each strain to antibiotics.resultsthe clinical manifestations of a. baumannii - induced lower respiratory tract infections lacked specificity. infected patients were most commonly admitted to intensive care units (54.9%) and frequently had chronic obstructive pulmonary disease (27.0%). the detection rates of abai and csue were both 59.8%, and those of ompa and bla - per1 were 100% and 0%, respectively. after genetic testing, antimicrobial resistance to amikacin, ampicillin / sulbactam, and 14 other types of antimicrobials was higher in abai- and csue - positive strains than in abai- and csue - negative strains (p<0.05).conclusionsthe findings of our study suggest that abai- and csue - positive acinetobacter baumannii strains are associated with a higher incidence of antibiotic resistance in 14 types of antimicrobials. |
neurological conditions like multiple sclerosis (ms) or stroke cause functional impairment and handicap which can have a major impact on patient quality of life. despite symptoms and disabilities varying based on underlying causes and individual manifestations, a major impairment in sensory function, orientation, and mobility commonly presents great challenges to the affected persons, their families, and healthcare providers. most patients wish to live independently despite condition - related restrictions. therefore, treatment focuses on symptom management and the prevention of acute episodes and disability. low - intensity rehabilitation improves quality of life, overall health, activity, and participation in social life [2, 3 ]. to enable individuals to reach their goal of living independently at home, specialised rehabilitation clinics provide care by multidisciplinary programs. the aim is to improve functionality, expand kinaesthetic competence in order to increase compensation of limitations, and improve quality of life [4, 5 ]. although specialised rehabilitation nursing care is an important aspect of rehabilitation programs, few studies have investigated the contribution on the effect of specialised rehabilitation nursing care within rehabilitation programs. through expertise, experience, and careful observations of clinical practice, nurses of a rehabilitation centre in switzerland developed and refined a standardised intervention (mobility - enhancing nursing intervention (mfp)) to specifically enhance patient safety, body perception, kinaesthetic competence, mobility, and functionality, as well as to reduce the burdens of care on relatives [6, 7 ]. mfp focuses on individual self - management and is an integrated part of the nursing care in the patients ' daily life at the centre. in this randomised controlled trial it was hypothesised that mfp would increase independence, quality of life, and fall - related self - efficacy in patients with ms, stroke, and brain injuries. the study was conducted in a specialised neurorehabilitation clinic in the german - speaking part of switzerland. all patients entering the clinic (from 2011 to 2013) were screened by registered nurses with special training for the following inclusion criteria : (1) diagnosed with ms, stroke, or brain injuries ; (2) german - speaking ; (3) aged 18 and older ; and (4) cognitively able to give written consent. mfp is a nursing intervention based on the assumption that learning takes place through movement. human development is seen to be based on the interaction between a person and the environment. tactile - kinaesthetic perception is important with regard to the way that the environment is perceived and fundamental to the development, organisation, and reorganisation of the brain. it was hypothesised that the way the patients perceived themselves, their environment, their body position changes, and hence cognitive - linguistic, social, emotional, and motor behaviour would be enhanced. for these purposes, the patients ' mattresses were placed on the floor, which enabled the patients to explore their environment safely without the risk of falling. additionally, the patients ' environment was arranged in accordance with a nursing assessment pertaining to the patients ' impairment and abilities, their goals in terms of improved mobility, and the mobility they would require in order to live at home as independently as possible. initially, most patients favoured a specific side to get up. the goal of the intervention was to teach the patients to get up step by step and to move independently over both sides. compared to standard mobilisation procedure from a bed which uses gravity, mfp care enables patients to overcome gravity which requires tailored support. constant tactile - kinaesthetic stimulation was applied by guiding the person from her current position, for example, standing, laying, or sitting in a wheelchair, to the floor. with successive gestures and position changes, the person was guided back into the original position using kinaesthetic. in order to implement the intervention, all nurses on the wards received training in two units of 3 and 5 days and ongoing clinical training (24 hours a month) on kinaesthetic principles. these principles deal with interaction, functional anatomy, human movement and function, effort, and environment. the main goal of the nurses ' training was to get familiar with the specific steps and the principles of kinaesthetic support of movement. in the intervention group, mfp was applied during 30 days in addition to the standard rehabilitation program in the control group, which was provided by physicians, physiotherapists, occupational therapists, and standard nurses. data were collected before randomisation (t0), after 15 days (t1) and at discharge (t2). further medical data were collected (diagnosis, length of stay, and discharge destination). therefore, the primary outcome was functionality. to measure functionality, the extended barthel index (ebi), a validated and common instrument in rehabilitation settings, the ebi includes 16 items that are rated on a 4- and 5-point likert scale (not possible, with support of a person, with low support, with facilities, and independent). secondary outcome variables were the need for nursing care after discharge, quality of life, and fall - related self - efficacy. the need for nursing care was measured with the self - care index (spi) which is based on nine functional items and one cognitive item [14, 15 ]. these items are part of the clinical assessment for acute care instrument (epa_ac), used in the swiss rehabilitation setting to plan necessary nursing care. the instrument includes 26 items that are rated on a 5-point likert scale (very poor to very good, very dissatisfied to very satisfied, not at all to an extreme amount, not at all to extremely, and never to always). the whoqol - bref yields a score for general quality of life in each of the four domains, physical, psychological, social, and environmental, with a score of 100 indicating maximum quality of life. internal consistency for the subscales ranges between an alpha of 0.70 and 0.86. to measure fall - related self - efficacy and fear of falls, the seven - item short version of the fall efficacy scale (fes - i) was used. the fes - i is a well established and validated (cronbach 's alpha was 0.85 in a sample of ms patients) instrument with a 4-point likert scale. a higher score is synonymous with more fear of falls and less self - efficacy [21, 22 ]. the sample size was calculated based on data obtained during a pilot study which showed a clinically relevant medium effect size of 0.54 for ebi. the power was set at 0.8, and alpha was set at 0.05 resulting in a required sample size of 126 in total or 63 per group for a 1 : 1 allocation. allowances were then made for an attrition rate of 30% ; hence the target was to recruit 162 participants or 81 per group. as the attrition rate was well below the estimated rate of 30% participants were then randomly allocated in blocks of ten to intervention or control group (1 : 1) using a computer - generated list for random numbers. a research assistant that was not involved in the delivery of the intervention collected the data for outcome measures. due to the nature of the intervention, data were analysed using spss version 19 (spss, inc., chicago, il). for all outcome measures (ebi, whoqol, and fes - i) changes between baseline (t0) and discharge (t2) were calculated. since length of stay showed a wide range among patients, changes for the ebi were calculated as mean changes per day (ebi - diff / day). to determine the demand for nursing care after discharge the percentage of participants with a self - care index (spi) below 32 points was calculated. in order to test the hypotheses, the changes were compared between the intervention groups (intervention and control) and between two diagnostic groups (ms and stroke). a two - way between - group covariance analysis was conducted to test the impact of the intervention on the rehabilitation outcomes. both the ebi score and the whoqol index at baseline were introduced as covariates. to test the effect on fall - related self - efficacy (fes - i) between april 2011 and march 2013, 782 patients were screened, 140 of whom were recruited and randomly assigned to the intervention group (n = 70) or the control group (n = 70). results are based on 126 participants included in final analysis (see figure 1). the characteristics of the two study groups were comparable at baseline (table 1). the mean age of the participants was 62 years (sd 13.6) and 49% were female and 95% (n = 133) lived at home prior to the clinic stay. patients were diagnosed with ms (59%), stroke (54%), and traumatic brain injury (n = 5). due to the small number of patients with brain injuries, this group was not included in the analysis. there was no significant difference between intervention and control group at baseline (mean = 40.6, sd 9.6 versus mean = 42.4, sd 11.79 ; p = 0.35). quality of life was high (mean 52.9 sd 24.9), approximately 8 points above the german norm rate for ms patients. there was no significant difference at baseline between the intervention and control group (mean = 49.6, sd 25.4 versus mean = 56.2, sd 24.1 ; p = 0.12). a significant main effect of the intervention was observed on functionality (ebi - diff / day : f(1,125) = 7.158, p = 0.008) (table 2). the mean increase in the ebi - diff / day score was 0.14 points higher (95% ci = 0.040.24, p = 0.006) in the intervention group (mean = 0.30, sd 0.31) than in the control group (mean = 0.16, sd 0.24). a significant main effect of the diagnostic groups was also observed (f(1,125) = 9.401, p = 0.003). the mean increase in the ebi - diff / day score was 0.24 points higher (ci = 0.140.33, p = 0.000) in the stroke group (mean 0.33, sd 0.30) compared with ms group (mean = 0.10, sd 0.20). the analysis showed no statistically significant interaction between the intervention and diagnostic group (f(1,125) = 0.222, p = 0.638). there was a significant effect of the ebi covariate at entry t0 (f(1,125) = 4.671, p = 0.034). the test for the second outcome, quality of life, showed a significant main effect of the intervention on whoqol - diff (f(1,116) = 4.06, p = 0.046). the mean increase in whoqol was 8.4 points higher (ci 0.1416.6, p = 0.045) in the intervention group (mean = 13.8, sd 19.6) than in the control group (mean = 5.4, sd 25). the analysis showed no statistically significant interaction between the intervention and diagnostic group (f(1,125) = 0.222, p = 0.638). no significant effect on fall - related self - efficacy (fes - i) between t0 and discharge was observed. there was also a significant difference in the self - care index (spi). the percentage of participants that remained below an index of 32 at discharge, indicating the need for nursing care after discharge, was significantly lower in the ig than in the cg (52.9% versus 80.6%, p = 0.001). the results of this first study investigating the effect of mfp carried out by specially trained nurses show that the intervention is effective to enhance mobility and quality of life of individuals with ms and stroke. the results strengthen the concept to integrate functional training into habitual daily routines, to create special, individualized context in order to speed up the rehabilitation process, and to produce sustainable effects on the patients ' functionality. to the best of our knowledge, little is known about the sensitivity to changes in either the extended barthel index or the barthel index. however, one can assume that a one - point change on a 4-point scale is clinically meaningful, because the graduations of the ebi are quite large. at discharge the detected change in the ig is almost double (13 points) the cg (6.8) and is presumably clinically significant for the patients. we propose that this change will have a clinically significant impact on the subjective patients ' perception of quality of life. even de souza. did not find a correlation between the level of disability and quality of life ; further research is needed to determine how enhanced mobility influences quality of life. we were surprised that the change in fear of falling and self - efficacy was not significant in this study. in interviews performed during data collection, patients explained how their new ways of moving around had made them aware of risks. during the long adaptation process, they experienced a wide range of emotions, ranging from frustration to success. these heterogeneous processes and the short observation period stretching slightly over a month could have led to this nonsignificant result. mentioned another difficulty with fes, as some patients may have difficulty estimating their self - confidence without performing specified activities. further research is needed to determine how patients estimate their self - efficacy during rehabilitation process. the clinical significance of the results was also shown by the difference of the spi, since the spi is used in swiss rehabilitation clinics to indicate the need of nursing care. the increase in self - care ability during the hospitalisation in the intervention group was twice that of the control group. among the control group participants, 80.6% of the individuals showed the need for additional nursing care after discharge. this percentage was 27.7% lower in the intervention group, where only 52.9% showed the same need for nursing care. therefore, it can be assumed that mfp has a positive effect on health care services, especially nursing home care. additional research is needed that looks at rehabilitation outcomes related to health care utilisation after discharge. to fully appreciate the clinical significance of the mfp intervention these results show a heterogeneous and individual experience and evaluation of the personal benefit and significance. further research is needed to explore these patient - centered changes. nurses are well aware that patients dealing with the aftermath of a stroke or in remission of ms need comprehensive treatments and care that are not limited to physiological retraining. the intervention is guided by principles of patient centeredness, negotiation of shared goals, and a perspective that includes family members. on one hand, this personalised, close contact may challenge both patient and nurse alike, especially during the intervention. on the other hand, it fosters a caring nursing relationship, which could explain why a considerable increase in quality of life was observed at discharge. the higher increase in the self - performance of activities of daily living combined with the higher increase in quality of life shows that mfp has the potential to boost the rehabilitation effect on a statistically and clinically significant level. the generalisation of the study findings is limited because of the fact that the design did not allow for blinding data collection. at the beginning of a rehabilitation process, people with brain injuries often were cognitively not able to give written consent. due to the decision of the ethics committee, further research is needed to investigate the effect of mfp and how nursing interventions combined with rehabilitative therapies contribute to the multiprofessional rehabilitation success. since mfp is only possible through specially trained nurses, it is necessary to further develop the role of rehabilitation nurses and to standardise patient - centred interventions such as mfp as a basis for further research. patient centeredness and empowerment to carry out everyday tasks are demanding challenges for both patients and nurses. the actual experiences of the patients during the intervention should therefore be investigated qualitatively, as the results from such studies could provide more insight into the basic nursing processes involved. | background. multiple sclerosis (ms) or stroke causes functional impairment which can have a major impact on patients ' life. objectives. this rct investigated the effect of a new nursing intervention (mobility enhancing nursing intervention mfp) designed to improve rehabilitation outcomes. method. the study took place in a rehabilitation clinic in switzerland. one hundred forty participants diagnosed with ms, stroke, and brain injuries were randomly assigned to control group (cg = standard care) or intervention group (ig). the ig combined standard care with 30 days of mfb. mfp placed patients on a mattress on the floor and used tactile - kinaesthetic stimulation to increase spatial orientation and independency. outcomes were functionality (extended barthel index, ebi), quality of life (whoqol), and fall - related self - efficacy (fes - i). results. there was a significant main effect of the intervention on functionality (ebi - diff / day mean = 0.30, versus mean = 0.16, p = 0.008). there was also a significant main effect on qol (whoqol - diff mean = 13.8, versus mean = 5.4, p = 0.046). no significant effect was observed on fall - related self - efficacy. conclusions. the positive effect of mfp on rehabilitation outcomes and quality of life suggests that this specialized nursing intervention could become an effective part of rehabilitation programs. the study was approved by the ethics committee of st. gallen (kek - sg nr. 09/021) and registered at clinicaltrial.gov nct02198599. |
early on, when the first two publications on genetically altered mice in the ras erk pathway became simultaneously available, what we call here the ras erk paradox appeared immediately clear : any manipulation of this pathway either causing an enhancement or a partial inhibition of cell signaling, would lead to learning and memory deficits (brambilla. in fact, not only the ablation from the mouse of ras - grf1, a cns specific neuronal activator of ras proteins, results in memory deficits but also the gene disruption of the neurofibromatosis type 1 (nf1) gene, the locus responsible for the expression of the ras - specific negative regulator neurofibromin, a gtpase activating protein, causes significant learning impairments. although these early studies did not provide any biochemical evidence supporting the expected contrasting cell signaling effect of the two mutations (ras - grf1 loss would attenuate ras activity while neurofibromin should enhance it), these genetic results initially suggested that any bidirectional alteration from the physiological neuronal erk activity would negatively impact on the brain s ability to correctly process information. shape, for instance the effect on memory mediated by corticosteroids, but the ras erk paradox is a unique case in which the modulation of an intracellular signal transduction pathway results in such a clear detrimental effect on cognitive functions. this observation was clearly underappreciated at that time, but in retrospect it was and it is still a formidable obstacle to the development of cognitive enhancers which, either directly or indirectly, would impact on erk signaling. one of the reasons why this question was not promptly investigated is that immediately after the ras - grf1 and nf1 ko publications, sl327 became available. this small chemical inhibitor of mek1/2, the kinases upstream of erk1/2, has been extremely useful in dissecting the effect of erk inhibition on behavior since it rapidly passes the brain later on, other similar drugs started to be used, e.g., pd184161, with similar effects. predictably, erk inhibition does block long - term experience- and drug - dependent behavioral plasticity (see the above articles for review). in addition, this pharmacological approach has also been instrumental in demonstrating a central role of the erk pathway in psychiatric conditions less dependent on memory functions such as chronic stress and depression (see for instance duman., 2007). however, this plethora of inhibition studies, generated in the last decade, has not helped us much in resolving the erk paradox but has simply confirmed that this pathway is a necessary (permissive) condition to cause long - term changes behavior. once again, the use of genetically altered animals has instead significantly contributed to define a much more complex role of ras erk in the brain. in 2002 the initial hypothesis that a general and non - cell - specific increase in erk signaling may lead to memory impairments had to be dismissed on the basis of two publications. firstly, the ideal system to test this hypothesis has been represented by the erk1 mutant mice which show a general enhancement of erk activity in the brain (mazzucchelli., 2002). this is due to a de - repression of erk2, the main map kinase. in normal conditions, erk1 acts as a built - in partial agonist, keeping erk2 activity tightly regulated (vantaggiato., erk1 mutant animals do not show any sign of cognitive deterioration as one would have predicted from the early studies on the nf1 ko mice. on the contrary, erk1 deficient mice manifest specific memory enhancing effects, including striatum - dependent operant conditioning long - term memory formation, increased fear memory consolidation and extinction, novel object recognition memory, and cocaine - dependent associative and non - associative learning but not spatial memory changes (mazzucchelli., 2002 ; interestingly, while two independently generated ras - grf1 ko lines seem to show behavioral responses which are the mirror image of those found in the erk1 mutants, a mutant showing a mild over - expression of ras - grf1 manifest a similar memory enhancing effect in most tasks but not, quite surprisingly, in spatial memory ones (fasano. thus, the results obtained with the analysis of the erk1 mutants indicate that a general enhancement of ras erk signaling can promote learning and memory functions. however, this observation is still in apparent contrast to the phenotype observed in the nf1 mice in which, supposedly, a general erk enhancement occurs as well. the explanation of this discrepancy was provided by two papers in 2002 and in 2008, both from the silva s lab. erk in the brain since they clearly demonstrated that the ras - dependent effect of neurofibromin ablation is specifically linked to an increase in gaba inhibition which is the likely cause of the learning and memory deficits. either using the global heterozygous nf1 mice or cell - specific promoters to drive cre expression in conditional nf1 mutants, silva and co - workers showed that the behavioral effect was compatible with an enhancement of ras activity in gabaergic interneurons that in turn may affect synaptic plasticity in key areas of the brain implicated in learning, such as the hippocampus. interestingly, the memory deficits could be partially rescued by either reducing (pharmacologically or genetically) ras activity (especially of the k - ras isoform) or by partially inhibiting gaba activity using receptor antagonists. these experimental observations are important since they provide a likely explanation why loss of neurofibromin in nf1 patients may lead to learning disabilities. importantly, they may also provide a rationale to understand other genetic diseases characterized by gain of function mutations in the ras erk pathway leading to mental retardation, the now called rasopathies or ras mapk syndromes (aoki. the clinical relevance of these disorders is such that a cogent question arising from the work on nf1 mutant mice requires an urgent answer : why do these changes in the ras erk activity only occur in gabaergic cells and not in all neurons ? indeed the data provided by the silva s 2008 paper indicate that a mutation restricted to glutamatergic pyramidal cells, using the camkii promoter - cre line has no impact on behavior. consistently, our own data on a conditional k - ras g12v knock - in mutant line also show that a pyramidal specific activation of this ras isoform has no effect on learning and memory (papale., 2010). on the contrary, the cre line driving expression under the synapsin i early promoter leads to significant learning impairments in both nf1 and k - ras12v mutants. firstly, expression analysis provided by the allen brain atlas (www.brain-map.org) of both nf1 and k - ras genes seems to indicate that their transcriptome levels are higher from late embryonic development (e18.5) to early post - natal stages (p4), a phase in which the synapsin i early promoter is already active while the camkii promoter is not. in addition, since during the post - natal maturation of gabaergic cells, the expression of nf1 and of k - ras is maximal while later during pyramidal cell development is down to lower levels the occurrence an increased inhibition would be favored. indeed, an indirect evidence that k - ras is acting early in development during a temporal window which coincides with gabaergic development comes from the transgenic mouse model in which the h - rasg12v mutation is expressed under the camkii promoter. in that case a forced over - expression in pyramidal neurons of this gain of function mutant leads to memory and plasticity improvements (kushner., 2005 ; kaneko., still needs to be seen but it is unlikely. indeed, to complicate the matter, a recently described targeted expression of the very same h - rasg12v via homologous recombination (gene knock - in) results in significant behavioral impairments which partially recapitulate the phenotypes observed in patients affected by the costello syndrome, one of the rasopathies (viosca., 2009) thus, all available data point to the importance of when and where : if ras erk signaling is predominantly active in the gabaergic compartment, then plasticity and memory impairments may occur while the opposite is true when erk activity is mainly activated in pyramidal cells. indeed the real scenario is probably even more complex than that if we take into consideration the global erk1 ko model in which memory improves despite gene ablation is not cell - type selective and the manipulation alters a protein hardly regulated in development. in this specific case, a possible explanation is that erk1 loss causes a differential effect between excitation and inhibition by favoring the former. if that is the real case still remain to be seen since appropriated experiments, either using conditional erk1 mutants or viral - mediated cell - specific gene knockdowns of erk1, have not yet been performed. however, one prediction which can already be made is that a specific enhancement of erk2 activity in gabaergic cells via erk1 ablation should recapitulate the nf1 ko phenotype by bypassing the requirement for an elevated ras activity. certainly, this prediction could be incorrect in the case that other ras - dependent signaling pathways, e.g., the pi3 kinase cascade, significantly contribute to the mental retardation phenotype observed in the nf1 mutants. one last comment is necessary on the exact mechanism by which neurofibromin and k - ras cause the enhancement of gabaergic activity. currently this is unclear but the possible explanations, not mutually exclusive are : (i) enhanced gaba release ; (ii) larger gaba synapses ; (iii) more gaba synapses ; (iv) enhanced gaba - mediated post synaptic signaling. in any case, it is clear that in cortical regions and in the hippocampus, future experiments targeting the ras erk signaling in a cell - specific manner and at different stages of development will provide substantial new evidence and will tell us whether the development of general cognitive enhancers based on erk manipulation will ever be a viable therapeutic option for memory impairments. most cortical and hippocampal regions function by a tight integration of excitatory and inhibitory signals. alterations of this balance, as we have seen above, may result in either a memory gain or a memory loss. however, not all brain areas work in this way. for instance, the striatum, the input nucleus of the basal ganglia system, is essentially constituted (> 95%) by gabaergic projection neurons, the so called medium spiny neurons (msn ; kreitzer and malenka, 2008). instead of generating an output activity toward the thalamus and the motor cortex by balancing excitation and inhibition within its structure, the dorsal portion of the striatum (the ventral one, the nucleus accumbens, may be slightly different) integrates two main neurotransmitter signals, the glutamatergic and the dopaminergic ones, and conveys their action on two distinct subclasses of msn : the direct pathway neurons, mainly expressing dopamine d1 receptors, and the indirect pathway neurons, mainly expressing d2 receptors. in normal conditions, a balanced activation of both pathways leads to an efficient activation of the thalamus and of the cortex. however, in certain neuropsychiatric diseases, one of the two pathways tends to dominate : an enhancement of the direct pathway leads to motor activation while that of the indirect pathway results in motor inhibition. not surprisingly, in recent years, the role of the ras erk pathway and downstream gene expression has extensively been investigated in the striatum, using both pharmacological (e.g., sl327) or genetic approaches. the scenario which has resulted from these studies is that in both the behavioral responses to drugs of abuse (drug addiction) and in a pathological condition resembling l - dopa induced dyskinesia (lid) in parkinson s disease, an aberrant hyperactivation of ras erk appears to be a key pathogenetic factor (murer and moratalla, 2011). in general terms, the most widely studied drugs of abuse, psychostimulants (e.g., cocaine and amphetamine), opiates (e.g., morphine and heroine), and cannabinoids (delta-9-tetrahydrocannabinol, thc) can cause both short - term changes of motor activity or long - term behavioral changes, which can include an enhanced locomotor activity or increased reward responses, as measured in conditioned place preference (cpp) or in self - administration procedures. the fact that pharmacological inhibition of erk blocks these responses is now well established but, for the same reasons of the memory studies discussed in the previous chapter, it provides little additional information beyond establishing a permissive role of this pathway in the process. on the contrary, mouse models of specific genes in the pathway have significantly contributed to outline an interesting scenario. for instance, the original report on erk1 ko mice that demonstrated an instructive role of erk signaling in memory consolidation, i.e., these mice showed better striatum - dependent memory, also showed that cpp responses to morphine can be significantly enhanced (mazzucchelli., 2002). this effect is not drug - specific since the same can be seen with cocaine (ferguson., 2006). similar drug - dependent enhancing effects have subsequently been seen in other mutant mice, most notably a striatal - specific dominant negative form of the transcription factor creb (killer creb) and an overexpressing line for ras - grf1 (fasano. the case of creb is particularly intriguing since the same dominant negative mutant, not only causes memory impairment when expressed in a hippocampus specific mouse line but also leads to instrumental learning deficits and ltp / ltd loss in a striatal - specific line (pittenger., 2002, 2006). thus, the manipulation of creb within a given brain area, the dorsal striatum, shows a stimulus - specific effect which is in contrast to what was observed for other mutations in the erk pathway, most notably ras - grf1, in which the gene disruption causes both memory loss and a reduced response to drugs (brambilla., 1997 ;, a likely possibility is that a repeated administration of a drug of abuse may lead to a compensatory upregulation of endogenous creb, as seen previously for hypomorphic mutations of the gene itself or after viral - mediated expression of either wt or other dominant negative mutants of creb in the nucleus accumbens (see carlezon., 2005). it is obvious that more sophisticated experiments will be required to fully understand the role of creb in striatum specific behavioral plasticity, including its expression specifically in either the direct or in the indirect pathway msn. in that respect, the use of bac transgenic mice expressing the green fluorescent protein (gfp) in the two msn compartments has enormously facilitated the analysis of the activation profile of the ras drugs like cocaine specifically activate erk1/2 in the direct pathway msn (girault., 2007 ; bertran - gonzalez., 2008). on the contrary, antipsychotics like haloperidol, uniquely induced erk1/2 activity in the indirect pathway. consistently with the molecular data, a specific activation of the direct pathway leads to motor activation whereas motor inhibition is seen when the indirect pathway is induced. l - dopa induced dyskinesia is a severe condition in which chronic administration (several years) of the gold standard treatment of parkinson s disease results in abnormal involuntary movements (aim). this pathological condition can be modeled in rodents by causing an unilateral loss of the substantia nigra pars compacta (snc) neurons with the neurotoxin 6-hydroxy dopamine (6-ohda) followed by repeated l - dopa injections. lid takes several years to appear in patients while in rodents the effect is almost immediate, after one or few injections of the therapeutic drug. in recent years, we have started to understand the molecular mechanisms underlying lid in rodents and the key event occurring in the dorsal portion of the striatum, the target region of the dopaminergic snc cells, is the supersensitization of d1 receptor signaling through the upregulation of golf (girault., 2007). in other words, lid is essentially characterized by an aberrant enhancement of the striatal direct pathway, a feature, as we have seen, shared with drug addiction (cenci, 2007 ; jenner, 2008 ; murer and moratalla, 2011). the intriguing similarities between lid and the responses to drugs of abuse are also evident for what erk signaling is concerned. indeed, this signaling pathway is massively upregulated in the striatum of dyskinetic animals. to our knowledge, the induction of phosphorylated erk1/2 in d1 receptor expressing msn is the strongest ever detected not only in brain but in the whole body, accounting to up 50-fold increase over basal levels (as a comparison, high dose of cocaine can lead to a 10-fold increase). this enormous activation of erk signaling is a combination of the engagement of the larger share of d1r expressing msn and also, to a lesser extent, of an increase of the signal in already activated cells (gerfen., 2002 ; the specificity of the cell - type implicated in erk activation was initially demonstrated pharmacologically but then it was confirmed using the bac transgenic mice mentioned above as well as by causing gene ablation of the d1 receptors (santini., 2008, 2009 ; 2009). as expected from the initial observations, pharmacological inhibition of erk using sl327 or a partial blockade of ras activity using statins results in a significant attenuation of lid onset (santini., 2007 ; more recently, also the ras - grf1 mutant mouse which was previously shown to be involved in memory and in drug addiction has been used to test the role of this molecule in lid. indeed, a strong reduction in the aim profile of ras - grf1 ko animals was observed together with a very significant attenuation of erk1/2 activity in the dorsal striatum of these animals (fasano. interestingly, that work also showed that a combined treatment with suboptimal doses of sl327 (10 mg / kg), a condition which per se is ineffective in wild - type animals, causes an almost complete reduction of the dyskinetic symptoms. all these data strongly support the notion that a specific targeting of neuronal components of the ras erk pathway, like ras - grf1, may lead to effective treatments of both drug addiction and lid. in that respect, the scientific community still lacks pharmacological tools which go beyond sl327 and can target other molecular components besides mek1/2. an interesting option is the development of cell permeable peptides that readily cross the brain blood barrier and cause reversible disruptions of protein protein interactions (patel. for instance, one can imagine that a ras - grf1 specific cell permeable peptide would be a valuable tool to study the role of this molecule in diverse behavioral processes, from memory formation, reconsolidation, and extinction, as well as for the development of treatments for the above mentioned diseases. at the same time, it is imperative to further advance in the development of cell - specific genetic tools to target crucial components of the ras erk pathway with a tight temporal and spatial control. one step in that direction is the availability of bac transgenics expressing cre recombinase in a cell and brain area specific manner. for instance, the recent report in which darpp-32, a signal integrator which in the striatum also controls erk signaling, was either specifically targeted in the msn of the direct or the indirect pathway lead to changes in the responses to cocaine / l - dopa or to haloperidol, respectively (bateup., 2010). also, a very significant leap forward would be represented by the combination of conditionally targeted mouse mutants with cell - specific cre delivery via viral vectors which would also allow us to achieve an excellent temporal control of gene expression. at present though, since some limitations still apply to validate viral vectors with bona fide cell - specific promoters, probably the best option would be to use bac transgenic lines expressing cre in combination with vectors conditionally expressing either dominant negative constructs, cell permeable peptides, or small interfering rnas (shrna), as recently suggested (papale., 2009). these multiple approaches should be able to address the major remaining question linking drug addiction, lid and the ras erk pathway in the striatum : why is only the direct pathway affected in these diseases ? the specificity certainly lies on the hyperactivation of the d1 receptors in response to l - dopa or to drugs like cocaine but this type of reasoning is rather circular and provides little explanation. thus, it is difficult to judge whether the activation of ras erk in d1r expressing cells is just a minor consequence of upstream events or plays a more instructive role. certainly, gene ablation of d1r completely block erk activation and downstream events in the striatum and affects not only lid but also learning and ltp in the hippocampus, as well as cocaine self - administration (caine. an interesting experiment in the direction to solve this problem would be to force over - expression of one of ras erk elements, e.g., ras - grf1 (which is normally in both msn types) in the indirect pathway, which is silent in lid and in response to cocaine, and see whether that is sufficient to readjust the system. in addition, it would be important to knockdown ras - grf1 individually in each compartment and to express constitutively active mutations in the pathway (e.g., ras g12v) and to verify the effect at the behavioral level. both loss and gain of function experiments will be crucial but certainly demanding from the technological point of view. however, in our opinion, they will represent a necessary new level of investigation to tackle the complexity of ras erk signaling in behavior which will keep us busy for at least additional 15 years. the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. | the role of ras erk signaling in behavioral plasticity is well established. inhibition studies using the blood brain barrier permeable drug sl327 have conclusively demonstrated that this neuronal cell signaling cascade is a crucial component of the synaptic machinery implicated in the formation of various forms of long - term memory, from spatial learning to fear and operant conditioning. however, abnormal ras erk signaling has also been linked to a number of neuropsychiatric conditions, including mental retardation syndromes (rasopathies), drug addiction, and l - dopa induced dyskinesia (lid). the work recently done on these brain disorders has pointed to previously underappreciated roles of ras erk in specific subsets of neurons, like gabaergic interneurons of the hippocampus or the cortex, as well as in the medium spiny neurons of the striatum. here we will highlight the open questions related to ras erk signaling in these behavioral manifestations and propose crucial experiments for the future. |
the superfamily database (1) provides predictions of the protein domains in amino acid sequences. the classification of these domains is built on the structural classification of proteins (scop) database (2) which groups domains of known structure hierarchically into classes, folds, superfamilies and families. the scop superfamily level groups together the most distantly related domains ; and this level is what superfamily is primarily based upon. the superfamily database contains domain assignments to 60% of the sequences of completely sequenced genomes, i.e. currently 64 eukaryotes, 327 bacteria (including 89 isolates) and 24 archae. our database also includes assignments for several sequence collections, i.e. uniprot (swissprot and trembl) (3) and the pdb (4) chains. is an expert curated library of profile hidden markov models (hmms) (5). hmms (6) are profiles based on multiple sequence alignments designed to represent a domain superfamily (or family). each of the models has a web page with a figure showing its amino acid composition, strongly conserved sites, hydrophobicity and regions in which insertions and deletions occur. since domains of a common superfamily are often diverged beyond easily detectable sequence similarity, the assignment of domain superfamilies is a non - trivial problem of remote homology detection. we use the sequence alignment and modeling (sam) hmm software package (7), as it is one of the best tools for remote homology detection (8,9), to build the model library, score the protein sequences and search the database for homologues. a program to produce domain assignments and the model library [in sam, hmmer (6) and psi - blast (10) formats ] are available for download. superfamily uses 10 894 models to represent the 1539 superfamilies in scop 1.69. the use of multiple models to represent a superfamily improves results (5). the superfamily web site, at, offers a variety of methods for navigating and analysing genome assignments. figure 1 gives an overview of the functionality and results that are available through the website. for example, the user can perform keyword searches for sequence identifiers, organism names, scop superfamily names, scop superfamily identifiers, scop unique identifiers, superfamily model numbers and pdb identifiers. further, up to 20 sequences can be submitted in the fasta format for domain assignment. these sequences are first passed through a blast filter to find any assignments already stored in the database. if no hits are found in our pre - computed database, the sequences are then scored against the model library. if this does not produce any significant hits the sequences are scored using prc (), which is an extremely sensitive profile summary of the functionality and results that are available as part of the superfamily analysis framework via the web interface. for each genome, the website displays a list of predicted superfamilies including links to the individual protein assignments. in addition, undirected domain occurrence networks for all superfamilies are available in two graphical representations, produced by graphviz (11) and vcn (12). nodes in the domain occurrence networks represent genomes and connections between genomes represent domain architectures which are common to both genomes. it is also possible to detect over- or under - represented superfamilies by comparing the domain composition of the given genome with a selectable list of other genomes. figure 2 shows an example of a superfamily assignment page which links to separate pages with the alignment between the sequence and the hmm which matched the domain. further, one can enter a query that returns all examples of a given domain 's combinations from all the genomes in superfamily. are the superfamily and family classification and associated e - values for two domains. links to further family details, alignments between the superfamily model and the protein, assignments for the human genome and domain combinations in which the superfamily domain occur in are included for each domain. all the domain assignments and information on domain combinations is available for download in the form of a mysql database dump. the domain assignments are also available through a protein distributed annotation server (das). this facility enables high traffic genomic servers, such as ensembl (13), to easily stay up to date with the live data in superfamily. a machine readable list of superfamily data sources (genomes) can be viewed at., we describe two major superfamily developments since the previous publication (1) : family level assignments and functional annotation of superfamilies. in the hierarchical organization of scop, each domain superfamily comprises one or more domain families. for example, the superfamily of nad(p)-binding rossmann - fold domains consist of 12 families, e.g. tyrosine - dependent oxidoreductases or coa - binding domains. sequence identity between members of a domain family is higher than between families of one superfamily. the newest release of superfamily now includes family level assignment in addition to the superfamily assignments, predicting 2845 families. the family level in the superfamily and pfam databases are similar, but not equivalent. the family level provides a wealth of functional information that is of use to structural, computational and bench biologists. we developed an algorithm (15) predicting family association for a domain, given its known association with a scop superfamily. from a statistical viewpoint, we test the hypothesis that assuming a query domain is in superfamily a, the query domain is in family a1. the general method to solve the superfamily to family level mapping can be considered to be a hybrid pairwise - profile method. the query domain and the member sequences of a family are each separately aligned to the superfamily model. we infer the pairwise alignment between the query domain and a family sequence by comparing the following two profile alignments : (i) query domain to superfamily model and (ii) family sequence to superfamily model. residues from each sequence are aligned to each other if they align to the same position in the model. a pairwise score can be calculated from the inferred alignment using the blosum 62 substitution matrix and affine gap penalties. the low gap open penalty, relative to blast which uses a value of 5, is due to the fact that the alignment comes from the superfamily model. the hmm is aware of other sequences in the superfamily and so is locally less refined on the query domain - family sequence pair. conserved sites in the hmm contribute more to the profile score than variable sites. for normal superfamily model scoring a relative weighting factor however, performance of the hybrid method was found to be best when the position - specific weights were not used. when the weights are included the hybrid method performs similarly to the original hmm score (15). to test the hypothesis, we calculate the pairwise score between the query domain and the best profile scoring sequence from family a1. from this result we subtract, the pairwise score between the query domain and the family sequence not in a1 which gives the best profile score. if a query domain has a strong score to one family and weak scores to the remaining families then discrimination between families is strong. conversely, if a query domain has comparable scores to more than one family, the distinction is weak. alignments and scores between every scop family member and each of the superfamily models are pre - calculated and stored in the superfamily database. only the alignment and score between the query domain and the superfamily model there is little additional computational cost involved in applying the family level sub - classification method. we consider family level domain classifications with e - values below 0.0001 to be strong classifications. the family level e - value function being used is e - value = k1+e(ln(n2es2)ln(n1es1)), where k, and are coefficients derived from a benchmark dataset (15), n is the length of the sequence in amino acids and s is the raw score. the 1 subscript refers to the hit with the highest score and subscript 2 denotes the second highest scoring hit. the second highest scoring hit must occur outside the family with the highest scoring hit. in the cases where there is no second highest scoring hit n2 is set to the average domain length (180) and because there is no alignment s2 is set to zero. one of the benefits of our hybrid method is the ability to infer the presence of new families from negative results. this ability has been used to make improvements to the scop hierarchy and should be of great utility to the structural genomics projects. in addition to the family level classification the method can for a given sequence suggest a closest homologue with a known 3d structure. this closest homologue is the pdb structure of the scop family member with the highest score. the family level data added to superfamily is being used for the functional annotation of genomes, individual sequence family studies, predictions of new family level targets for structural genomics projects, suggesting the most closely related structures for homology modeling, working with functional sets of domains, e.g. transcription factors (16) and further development of the gene ontology (go) (17) for scop. the hybrid method produces superior results to either profile or pairwise techniques (15). no updates to sub - level models, phylogenetic trees, neural networks etc, are required when the database is updated. the method scales up to the most complex genomes and has been applied to over 400 genomes and sequence collections. the aim of the hybrid method is to create a statistical technique for classifying domains into a pre - existing biological classification system. given an existing profile library with genomic assignments for one level of a classification scheme, sub - level assignments come at next to no computational cost compared to alternative methods. the hybrid method is general, so it could be applied directly to other databases such as gene3d (18), or indirectly to pfam. it is anticipated that the method will be of great use in future versions of scop which are expected to include a more fluid hierarchy. the definition of the function of a protein domain is still a matter of debate and can vary depending on the actual context of the biological problem being addressed. we developed a domain - centric scheme for functional annotation instead of the widely used whole - gene (17) or whole - protein (19) annotation. thus a particular protein with two domains can be assigned to two different functional categories, e.g. a protein could consist of a kinase and a small - molecule binding domain. our definition of domain function is a combination of the biological process and molecular function used in the go (17) and was modeled after the scheme used in the cluster of orthologous groups (cogs) database (19). our scheme is comprised of 50 detailed functional categories which map to seven general categories. these seven general functional categories are as follows : (i) information : storage, maintenance of the genetic code ; dna replication / repair ; general transcription / translation ; (ii) regulation : regulation of gene expression and protein activity ; information processing in response to environmental input ; signal transduction ; general regulatory or receptor activity ; (iii) metabolism : anabolic and catabolic processes ; cell maintenance / homeostasis ; secondary metabolism ; (iv) intra - cellular processes : cell motility / division ; cell death ; intra - cellular transport ; secretion ; (v) extra - cellular processes : inter-, extra - cellular processes, e.g. cell adhesion ; organismal processes, e.g. blood clotting, immune system ; (vi) general : general and multiple functions ; interactions with proteins, ions, lipids or small molecules ; and (vii) other / unknown : unknown function, viral proteins / toxins. supplementary data s1 describe the superfamily distribution in the 50 functional categories over all scop classes. the annotation describes the superfamily 's dominant molecular function, i.e. function as part of the protein, or dominant biological process, i.e. their role in the cell. each domain superfamily is associated with only one function, even though some superfamilies, especially large ones, may have a variety of functions. one example are immunoglobulin domains which are assigned to cell adhesion for common function in cell surface receptors, although in vertebrates this domain superfamily is also involved in the immune system. in cases where no one function was obviously dominant, the domain function was classified as general or several functions. we based our annotation on information from scop (2), interpro (20), pfam (14), swiss - prot (3) and literature. for validation, we used the existing annotations of go biological process and go molecular function for pfam domains in interpro, mapping them to scop superfamilies based on sequence similarity. this procedure largely confirmed our annotations for 647 and 667 domain superfamilies of known go process and go function, respectively. as further validation, we extracted the largest superfamilies (300), i.e. which have at least 25 members in one of the completely sequenced eukaryotes (21), and re - examined their annotations, consulting co - workers as an independent source of information. based on our experience, we estimate error rates of < 10% for abundant superfamilies and < 20% for other superfamilies. in total, we annotated all domain superfamilies of the scop classes 111, with an emphasis on the 1261 domain superfamilies of the primary classes 17. the scop classes are (1) all alpha proteins, (2) all beta proteins, (3) alpha and beta proteins (a / b), (4) alpha and beta proteins (a+b), (5) multi - domain proteins (alpha and beta), (6) membrane and cell surface proteins and peptides, (7) small proteins, (8) coiled coils proteins, (9) low - resolution structures, (10) peptides and (11) designed proteins. members of some superfamilies, particularly the large ones, may have a variety of functions. to date, we applied the functional annotation to several problems involving analyses of statistically over - represented domain combinations (21), domain recombination (22) and protein family expansions in relation to biological complexity (23). the annotation could also be used in superfamily - specific studies, improvements to the go annotation of scop and as part of the superfamily assignments. the general and detailed annotations for all superfamily domains in scop classes 17 are available for download from. a large part of the development of superfamily will be dictated by the changes to the scop hierarchy which is expected to become more fluid. the hybrid method outlined above will be invaluable for dealing with these changes to appear with release 1.73. we currently study a domain architecture approach to the phylogenetic distribution of superfamilies which we expect to produce large amounts of data useful for evolutionary studies. with respect to the model building process we currently investigate how to incorporate information on the most common domain combinations to improve assignment accuracy and coverage. | the superfamily database provides protein domain assignments, at the scop superfamily level, for the predicted protein sequences in over 400 completed genomes. a superfamily groups together domains of different families which have a common evolutionary ancestor based on structural, functional and sequence data. superfamily domain assignments are generated using an expert curated set of profile hidden markov models. all models and structural assignments are available for browsing and download from. the web interface includes services such as domain architectures and alignment details for all protein assignments, searchable domain combinations, domain occurrence network visualization, detection of over- or under - represented superfamilies for a given genome by comparison with other genomes, assignment of manually submitted sequences and keyword searches. in this update we describe the superfamily database and outline two major developments : (i) incorporation of family level assignments and (ii) a superfamily - level functional annotation. the superfamily database can be used for general protein evolution and superfamily - specific studies, genomic annotation, and structural genomics target suggestion and assessment. |
inflammatory disorder of the pituitary gland (hypophysitis) is a rare but increasingly recognized cause of pituitary dysfunction that should be considered in the differential diagnosis of sella turcica lesions. secondary hypophysitis is rare, while primary hypophysitis has traditionally been classified as lymphocytic (lh), granulomatous (gh), and xanthomatous hypophysitis (xh) [13 ]. xanthomatous hypophysitis (xh) has been recently described, and it is the rarest of the three types. xh is characterized by mixed inflammatory cell infiltrates consisting of foamy histiocytes, plasma cells, and small round mature lymphocytes infiltrating the anterior pituitary gland [36 ]. clinically the lesion may mimic a pituitary neoplasm as a result of mass effect and hypothalamic - hypophyseal endocrine dysfunction. we report here the case of a young woman who presented with a sellar mass on a head mri and endocrine dysfunction. a 31-year - old women was admitted to our department with a one - month history of headache and diabetes insipidus (di). at admission, furthermore, the patient had a positive history for ulcerative colitis under medical treatment. at the first physical examination, the patient appeared in good general conditions, had no fever, no hypertension, no signs of meningism, and no neurological deficits. the first pituitary function work up showed gonadotropic hormons in the lower normal range (nr) : cortisol levels : at 08 a.m. = 12.1 ug / dl (nr = 8.025.0.), at 02 p.m. = 3.6 ug / dl (nr = 1.017), at 10 p.m. = 1.6 ug / dl (nr = 1.05.0) ; prl = 13.8 ng / ml (nr = 3.027.0) ; fsh = 1.9 mui / ml (nr = 2.013.0 follicular phase) ; lh = 2.0 mui / ml (nr = 2.012.0 follicular phase) ; progesterone = 0.27 ng / ml (nr = 0.101.00 follicular phase) ; estradiol = 26.4 pg / ml (nr = 20.0200.0 follicular phase) ; tsh = 0.24 uiu / ml (nr = 0.255.00) ; ft3 = 2.49 pg / ml (nr = 24) ; ft4 = 1.25 ng / dl (nr = 0.61.7) ; gh = 1.1 ng / ml (nr = 04.7). a second preoperative endocrinological work up one month later showed reduced corticol levels, that is, cortisol (08:00 a.m.) = 0.9 ug / dl ; fsh = 7.9 mui / ml ; prl = 10.3 ng / ml ; lh = 10.1 mui / ml ; progesterone = 0.19 ng / ml ; estradiol = 7.9 pg / ml ; ft3 = 2.27 pg / ml ; ft4 = 1.41 ng / dl ; gh = 0.8 ng / ml. inflammatory indices such as wbc and haptoglobin were slightly increased (11.45e3/ul and 282 mg / dl, resp.). urine tests, including electrolytes, were within normal limits, except for low levels of phosphate, bun, creatinine, and magnesium. a first head gadolinium mri showed an increased volume of the pituitary gland with mild compression of the optic chiasm. a second head mri (multiplanar fse /+ gad, gre - t2) with thin slices on sellar region revealed a widening of the sella occupied by a solid lesion with necrotic core and a hemorrhagic spot. it extended into the suprasellar cistern determining a minimal displacement of the optic chiasm (figure 1). prior to surgery the patient underwent a complete pituitary function work - up (see above). an abdomen ct scan showed intestinal parietal thickening and pseudopolypoid nodular disease compatible with ulcerative images. endovenous intranasal desmopressin (ddavp - minirin) was started few days after the admission, and prophylactic hydrocortisone was given 48 hours prior to surgery. surgical exploration via the endoscopic transsphenoidal route revealed a cystic lesion containing pus - like fluid, which was drained after dural opening (figure 2(a)). the postoperative full histopathological work - up revealed an inflammatory infiltrate of numerous foamy histiocytes, multinucleated giant cells containing cholesterol clefts, and lymphocytes infiltrating between residual nests of normal pituicytes (figure 3). on immunohistochemical sections, the infiltrating foamy cells, were strongly positive for cd68, a macrophage marker (figure 4). there was no evidence of associated adenomas, granulomas, langherans ' cells or neutrophilic exudates. the rupture of a rathke 's cleft cyst was excluded by the pathologist as no cylindric ciliated epithelium could be seen. pituitary function did not improve ; 5 days after surgery she underwent a complete endocrinological work - up with the following findings : cortisol at 08 a.m. = 14.5 ug / dl, at 12 a.m. = 11.9, and at 12 p.m. = 12.4 ; prl = 1.6 ng / dl ; fsh = 6.1 mui / ml ; lh = 3.0 mui / ml ; tsh = 0.21uui / ml, ft3 = 1.69 pg / ml, ft4 = 1.5 ng / dl ; gh = 0.3 ng / ml, acth = 5.9 pg / ml (nr = 577) ; igf1 = 53 ng / ml (nr = 115500) ; c - peptide = 2.2 ug / ml (nr = 0.83.0). the patient continued to take ddavp, and she was started on cortone acetate 25 mg 1/2 cp 3/die. urine output required one daily administration of vasopressin (nasal spray). hormonal substitution therapy after discharge the patient underwent an endocrinological work - up at 1, 3, and 8 months after surgery ; serum prolactin gradually increased from 5.25 to 8.9 ng / ml. cortisol level increased from 0.9 ug / dl preoperativly to 18 ug / dl at 3 months and 31 ug / dl at 8 months postoperatively. fsh, lh, and tsh remained in the lower normal range while ft3 and ft4 slightly increased. a postoperative gadolinium - enhanced head mri at 8 months showed a normal pituitary gland with no pathological changes (figure 5). eight months postoperatively, the patient still required glucocorticoid and estrogen replacement therapy as amenhorrea still persisted. cortone acetate has been progressively reduced 12 months after surgery with cortisol levels remaining within normal range. recently, the patient decided to stop estroprogestinic drugs because of a pregnancy desire ; amenhorrea was still present. hypophystis is an infrequent inflammatory disease of the pituitary gland that may be misdiagnosed as a pituitary adenoma or other sellar masses as a ratcke 's cleft cyst. histologically hypophysitis includes three distinct histopathological subtypes : lymphocytic hypophysitis (lh), granulomatous hypophysitis (gh), and xanthomatous hypophysitis (xh) [13 ]. some authors classify the xanthogranulomatous (xgh) and necrotizing hypophysistis (nh) as autonomous entities [2, 3 ]. xgh has been recently defined as a new category by tashiro. for the presence of multinucleated giant cells, and it has also been described in association with adamantinomatous craniopharyngiomas. in terms of the site of inflammation, the latter is thought to be a different entity from lh because inflammation is confined to the hypothalamic neurohypophyseal system causing di [3, 7, 8 ]. for this reason, it is thought to be one of the causes of idiopathic di [7, 8 ]. on mr imaging, a thickening of the neurohypophysis or pituitary stalk is usually seen. since in idiopathic di a lack of normal early enhancement of the posterior pituitary is often described, the etiopathogenetic hypothesis is focused on the decreased posterior pituitary vascularity due to congenital lack, to poor development of the posterior pituitary arterial supply, or to secondary changes of vascular destruction. lh is the most common type and is characterized by infiltration of the anterior pituitary gland with lymphocytes and plasma cells and by fibrosis, most commonly observed in females during or after pregnancy [7, 9 ]. gh does not have a high predominance in females and is characterized by granulomas with epithelioid histiocytes and multinucleated giant cells ; it is different from systemic giant - cell disorders such as tuberculosis, sarcoidosis, or syphilis. few cases of gh with giant cell reaction caused by the rupture of a rathke 's cleft cyst have been described. nh consists of inflammation with necrosis of the hypothalamus, pituitary stalk, adenohypophysis, and infundibuloneurohypophysis, often accompanied by hypofunction and di. xanthomatous hypophysitis (xh) is a rare inflammatory disease that usually involves the anterior lobe of the pituitary gland. since the first report in 1998 by folkerth., a few cases of xh have been reported in literature [36, 12 ]. clinical and laboratory findings suggest an autoimmune basis for primary hypophysitis [2, 11 ]. the patient presented in this report had a history of ulcerative colitis, supporting this hypothesis, although autoimmune disorders are predominantly associated with lymphocytic hypophysitis. headache is a common complaint at presentation, while visual disturbances are uncommon [1, 2 ]. the clinical symptoms of primary hypophysitis are mainly due to inflammatory irritation of sellar and parasellar structures. di, probably caused by inflammatory infiltration rather than by compression of the posterior lobe and pituitary stalk, is rare in xh because inflammation is predominant in the anterior pituitary lobe while it occurs in 50% of patients suffering from lh and gh and corresponds to pituitary stalk thickening in mri studies [1, 7 ]. it can be assumed that in xh a circumscribed anterior pituitary lesion may lead to compression of the pituitary gland without alteration of pituitary stalk and optic chiasm. moreover, di is not often seen at the clinical onset of pituitary adenomas (pa). mr imaging is the procedure of choice in the evaluation of sellar masses, although diagnosis by mri alone can only be suspected : most xh cases appear as an enlargement of the pituitary gland with hypointense and round cystic lesions in t1-weighted images [1, 2, 4, 5 ]. at surgery, thick fluid material is found within the cystic lesion [6, 12 ]. (anjr, 2009) proposed a radiologic score to distinguish autoimmune hypophysitis from non secreting pa. in the differential diagnosis between hypophysitis and pa, the peculiar radiologic features of the first ones are a symmetric enlargement of pituitary gland, a homogeneous appearance both on pre- and post - gadolinium images, and an intense contrast enhancement. in contrast, pa are typically asymmetric showing heterogeneous enhancement and had a lower gadolinium uptake. moreover, a thickened pituitary stalk is typical for autoimmune hypophysitis [7, 13, 14 ]. others pathological entities to be considered in the differential diagnosis are pituitary abscesses (nowadays uncommon), secondary hypophysitis and noninfectious granulomatous processes. xh is characterized histologically by an inflammatory infiltrate of foamy histiocytes [3, 4, 6 ]. the basic structure of anterior pituitary is generally preserved [2, 6 ], without alteration of the pituitary stalk and optic chiasm. the overall prognosis for xh is good, but improvement of pituitary function after transsphenoidal surgery has been reported in less then 50% of the cases in the literature [1, 3 ]. chronic inflammation may result in destruction and fibrosis of the pituitary gland. in their initial description of xh, folkhert. reported two 30-year - old women presenting with menstrual dysfunction and a 12-year - old girl presenting with nausea, headache, and symptoms of di. subsequently, deodhare. reported about a 43-year - old female patient presenting with hyperprolactinemia, gh and cortisol deficiency, and a di. cheung. and tashiro. reported about three more cases of female patients affected by xh diagnosed after a transsphenoidal surgery., while reporting two cases of xh in two young men, have described the first documented case of xh with full recovery of pituitary function after surgery. gutenberg. in a study involving 21 lymphocytic, 6 granulomatous, and 4 xh cases have shown that endocrinological and neuroradiological improvement under pre- or post - operative high - dose corticosteroids is most often incomplete and transient. a prospective trial of glucocorticoid use in lh showed that methylprednisolone reduced mri mass in seven out of nine patients and improved anterior pituitary function in four patients. as far as xh is concerned, they showed no benefit after either pre- or post - surgical corticosteroids, while 50% of xh cases had an improvement of anterior pituitary dysfunction shortly after surgery and none suffered from endocrinological deterioration. our paper confirms the tendency of xh hypophysitis to cause a long lasting - though mild - hypopituitarism. while steroid therapy seems to be less effective in xh compared to other kinds of primary hypophysitis, surgical exploration is recommended in almost all pseudotumoral hypophysitis in order to relieve symptoms and secure a diagnosis [1, 14 ]. we suggest that in case of radiological or serological suspicion of inflammatory disease of the pituitary gland, a confirmatory biopsy through a transsphenoidal approach should be performed. in case of compression of sellar and suprasellar structures, surgical removal should be recommended. | background. hypophysitis is an inflammatory disease of the pituitary gland that may mimic pituitary tumors clinically and radiologically. case description. we report a case of a xanthomatous hypophysitis initially diagnosed as pituitary adenoma. a 31-year - old woman presented with headache, diabetes insipidus, and amenorrhea. a head ct scan showed no intrasellar changes, while an mri scan showed a sellar cystic mass. an endocrinological work up revealed mild hypocortisolism and diabetes insipidus (di). transsphenoidal surgery was performed. the intraoperative histological examination suggested a pituitary adenoma. the removed tissue showed central necrosis surrounded by accumulation of foamy cells and xanthomatous epithelioid cells. the patient made an uneventful postoperative recovery, nevertheless, di persisted and the adenohypophysis hypofunction did not recover. conclusion. we describe an unusual inflammatory lesion of the pituitary gland mimicking an adenoma. a high level of clinical suspicion of inflammatory disorders is necessary for correct diagnosis and optimal management. |
loss of teeth, which may be due to trauma, dental diseases, pathology, or otherwise not only alters the psychological thought of the patients but also disturbs the esthetics, phonetics, and functional occlusion. replacement of missing teeth is highly essential in order to restore the defect and regain function as best as possible. since ages, polymethyl methacrylate (pmma) has been used to fabricate the dentures and when facilities are available, metal cast / metal frame / metal base dentures are also fabricated to restore the defects. some problems with these prostheses are difficult to address, such as insertion in undercut areas, brittleness of methyl methacrylate which leads to fracture, and allergy to methyl methacrylate monomer. the innovation of the nylon - derived denture base material in the 1950s paved the way for a new type of dentures. flexible dentures are an excellent alternative to conventionally used methyl methacrylate dentures, which not only provide excellent aesthetics and comfort but also adapt to the constant movement and flexibility in partially edentulous patients. eighteen cases of complete / complete, complete / partial, collapsed vertical height, rehabilitated using two different types of denture base materials, are discussed. the present study was conducted at three dental centers, where patients selected were those who were not happy with their existing complete / complete [figure 1 ], complete / partial [figure 2 ], partial / partial dentures [figures 3 and 4 ], made of conventionally used poly methyl- methacrylate denture base material and were in search of some substitute. the problems with their existing dentures included irritating mucosa, foul smell, difficulty to wear and remove, frequent fractures / cracks, etc. (case 1 and 2) complete / complete flexible dentures (case 3) complete / partial flexible denture (case 4) partial / partial flexible denture (case-5) partial /partial flexible dentures velplast and flexite were the two nylon denture base materials used (as the materials were available) in the study. a total 18 cases were provided the requisite prostheses, which were observed for one and a half year for their function and acceptance by the patient as compared to their old prostheses. usual procedure was followed for the fabrication of complete / complete and partial dentures in making the impressions and recording the jaw relations. a questionnaire was prepared, which was narrated to the patients, and their consent was taken to participate with their experiences with the new dentures [appendix - i ]. once the maxillomandibular relations were recorded, the teeth were arranged as per directions of the manufacturer. small - size mechanical undercuts (diatorics) [figure 5 ] were made in each tooth so that flexible denture base material could easily be injected in these areas. wax carving was done and intraoral trial was taken for each patient to evaluate esthetics, phonetics, and functional occlusion. diatorics prepared in each tooth special flasks were used for flasking, dewaxing, and injecting molten velplast or flexite denture material as per directions of manufacturer. injection cast technique was used, which is highly sensitive to the position and size of sprue placement. once the casting was completed, the flask was allowed to bench cool.the sprues were removed and dentures polished. intraoral fit of the prostheses was checked for occlusal balancing, any overextension, etc., so that the functional relation of the prosthesis with intraoral structures could be established. all the cases were evaluated for the following parameters [tables 1 and 2 ] : mucosal irritation, halitosis, ease of insertion and removal and fractures / cracks frequency and comfort in function. all the patients were followed for check - ups at regular intervals of approximately 1 month. out of 18 cases, which ranged from 37 to 82 yrs, eight were female and ten were male in the study. only two cases (11%) reported halitosis, four cases (22.2%) reported debonding of teeth from the dentures after 10 and 11 months, respectively. as per questionnaire, the results were compiled and the data were analyzed by a nonparametric test, wilcoxon signed ranks test to study the change in various indices of functional observations and to draw the conclusion. when questioned about the preference among the two types of denture base material, 100% patients preferred the flexible dentures over the customary methyl methacrylate dentures. mucosal irritation (m) count wilcoxon signed ranks test (p - value) table 1a shows that with old dentures seven patients observed mucosal irritation at grade 4 which was highest and seven patients observed it at grade 3. same observation was 0 (zero) in almost all cases except one with new flexible dentures. table 1b shows that with old dentures six patients observed halitosis at grade 4 which was highest and five patients observed it at grade 3. this observation was reported at grades 4 and 2 by one patient each, respectively, with new flexible dentures. d and table 2 show that whereas the problem of insertion and removal and fracture had been taken care of, the comfort was higher with new flexible dentures and in all parameters, the p value was less than 0.001 and was of great importance statistically. insertion and removal (i) count it was evident that new flexible dentures were significantly better than the conventional methyl methacrylate dentures in all functional parameters observed in this study. the most commonly used material for the fabrication of complete / partial dentures so far has been pmma. this material is not ideal in every respect and it is the combination of virtues rather than one single desirable property that accounts for its popularity and usage. in spite of various advancements and research in dental materials, training, and techniques across the world, patients, who start wearing dentures at an early age due to various reasons, often get frustrated and start searching something better available for them. although, cast partial denture has been a viable substitute, the requirement of high skill in preparation, technique - sensitive casting procedure, heavy weight, and visibility of metal clasp made it more difficult and cumbersome alternative and net results have not been encouraging. the most recent preference in denture materials has been the use of nylon - like material for the fabrication of removable dental appliances. this material generally replaces the metal and the methyl methacrylate denture base material used conventionally to build the framework for standard removable partial dentures. it is nearly unbreakable, esthetically acceptable being colored like the gums, can be fabricated quite thin, and can form not only the denture base but the clasps as well. since, the clasps are built below the height of contours of teeth, they are practically indistinguishable from the gums that normally surround the teeth. it is much easier for the dentist to work with these materials while adjusting the prosthesis in patient 's mouth, using slow - speed grinding tools. also, the postinsertion maintenance is easy, which makes it a very all the 18 cases were prolonged denture wearers who had undergone frequent adjustments such as relining and rebasing of their old dentures, which were not functionally comfortable. all these patients were ready to participate in the study and all the questions were answered by them after thorough observations on a scale of 1 to 4 as compared to the same observations with their old pmma dentures. halitosis reported in two cases in the present study was attributable to the personal dietary and oral hygiene habits of these patients. once trained in good oral habits, halitosis started disappearing in these two cases also. according to tables 1 and 2 all the patients in the study experienced highest satisfaction with the new nylon - based dentures. however, de - bonding of the teeth was observed in four cases after 10 and 11 months of use and was attributed to faulty de - waxing procedure, where wax was not completely removed from the mechanical undercuts (diatorics) and the molten flexible denture base material could not flow into the areas created for mechanical locking. this new - generation nylon - based thermoplastic material has a predictable long - term performance. it also has a high creep resistance and fatigue endurance along with the excellent wear characteristics and solvent resistance. it has the flexibility to disengage forces on individual teeth and prevent transfer of forces to remaining natural teeth and the other side of the arch. the material is light in weight, heat resistant, and ductile and is injected at a temperature of 274 to 300c. all the patients in this study preferred flexible dentures over the customary methyl methacrylate dentures because they had bitter experience with their old dentures and this new material gave them hope for better quality of life. super polyamide denture base material was found to be more flexible (low flexural modulus) than pmma denture base, and hence seems to be promising. the rehabilitation of orofacial structures demands the restoration of esthetic and function irrespective of the individual 's dietary / parafunctional habits and structure left. the flexible dentures were found to fare significantly better as compared to the conventional methyl methacrylate dentures on the parameters taken in the present study. when questioned about the preference among the two types of denture base material, 100% patients preferred the flexible dentures over customary methyl methacrylate dentures. however, further long - term studies are recommended to assess the overall usefulness of the material. | although clinician 's skills and experience play a major role in designing and fabrication of the optimum prosthodontic restorations, the selection of denture resins is equally important, especially when the patient has been using the prostheses since long. eighteen cases who were not satisfied with their conventional acrylic dentures were selected. they were provided flexible dentures along with a questionnaire to precisely evaluate the advantages of new material. prosthodontic planning & observations regarding this material are discussed on various parameters. |
cardiovascular diseases (cvd) are generally believed as a major concern responsible for human mortality while being among the foremost causes of preventable death (1, 2, 3, 4). atherosclerosis is the major reason of cardiovascular diseases and is an important cause of arterial wall disorder which directly influences large and medium - sized arteries. elevated low density lipoprotein cholesterol (ldl) and reduced high density lipoprotein cholesterol (hdl) levels were previously known risk factors for developing atherosclerosis (2, 5, 6). obesity, as an important cause of cvds, is associated with diverse cardiovascular disease risk factors like hypertension, dyslipidemia, escalated insulin levels and finally an increased risk of cvd morbidity and mortality in adulthood (3, 7). modification of some socioeconomic behavioral habits like smoking, alcohol consumption, dietary patterns and physical activity especially from childhood and adolescence is important and effective measures for the prevention of cvds (8, 9). albeit the clinical emergencies of atherosclerosis are displayed in adulthood, early atherosclerotic changes are apparent in youth studied post - mortem. thus, effective prevention of atherosclerosis is advised to be started in childhood or adolescence (4, 10, 11, 12, 13). on the other hand the increasing prevalence of childhood obesity is a worldwide trend and is becoming globally a remarkable public health problem. a decline in physical activity and an increase in amount of calorie intake may be responsible for this increasing incidence. hence, lifestyle modification and weight control in childhood should be noticed enough to reduce the risk of cvd in adulthood (7, 14, 15, 16, 17). various chemical and herbal types of medications are used nowadays for the prevention and treatment of hyperlipidemia statins and fibrates are the most common chemicals used to treat plasma lipid disorders ; however concerns about their side effects like elevation of liver enzymes, gastrointestinal symptoms, predisposition to cholelithiasis, rhabdomyelosis, myopathy, renal dysfunction, etc and also lack of general agreement about their use in children and adolescents, have persuaded the scientists to find an appropriate substitution for them (11, 15, 18, 19). different studies have showed herbal drugs in treatment of dyslipidemia and prevention of ldl - oxidation thanks to their constituents like dietary fibers, vitamins, flavonoids, estrols, polyphenols, antioxidant compounds, etc as invaluable sources. although these effects are weaker than chemical drugs, their use in primary stages of dyslipidemia and atherosclerosis seems rational (11, 20, 21). hibiscus sabdariffa l. [roselle ] is a plant which belongs to the malvaceae family and is widely cultivated in the tropical areas like caribbean, australia, brazil, central america, india, africa, us and philippines. this plants is used from times as a traditional medicine due to its effects against kidney stones and urinary bladder stones, and also its antibacterial, antifungal, hypocholesterolemic, antispasmodic and antihypertensive effects. it is used as a folk medicinal plant in iran and has been recognized as sour tea (22, 23). h. sabdariffa l. is rich in polyphenols, anthocyanins, flavonoids that may justify its use in prevention of cardiovascular disorders. according to the antioxidant and antilipid peroxidation actions of hibiscus extract, it is considered that hibiscus anthocyanins and procatechuic acid may also have a role in amelioration or prevention of these clinical conditions (24, 25). the aim of this study a study was to evaluate the effect of hibiscus sabdariffa calices on dyslipidemia in obese adolescents. this randomized triple masked placebo controlled clinical trial was conducted from july 2010 to july 2011 in isfahan cardiovascular research institute, isfahan (iran) and registered in the iranian registry for clinical trials (irct201109122306n2). ninety (n=90) school - age adolescents (12 to 18 years old) who had at least one of these criteria : a) serum triglyceride more than 90 percentile b) serum total cholesterol more than 90 percentile c) ldl more than 90 percentile d) hdl less than 10 percentile, not using tobacco, no history of alcohol consumption or drug abuse, no history of metabolic diseases like diabetes, thyroid gland dysfunction, nephrotic syndrome, chronic pancreatitis, liver and gall bladder diseases and no drug consuming (which affects lipid profiles like statins and hormonal pills like estrogens, progestrones and oral contraceptives) were screened for inclusion in the study. exclusion criteria were lack of patients compliance with drug regimens at least for one week, pregnancy and lactation, drug sensitivity, presence of any disease which could interact with lipid profiles and need to take of any kind of drugs or other compounds which affects lipid profile like corticosteroids, androgens, estrogenes, progestines, thiazides, beta blockers and thyroid hormones. the study was approved by the institutional ethics committee of isfahan medical university of sciences and all participants and their parents or guardians read and signed a written informed consent form before participation (26, 27, 28). sample size was determined according the t - student formula. accordingly the most important variable in this study was considered as serum level of cholesterol which is a quantitative continuous variable and to detect at least 10 mg / dl decrease of this variable in case group (comparing to the controls), with 80% power and 5% type 1 error, suitable sample size was calculated 35 people in each group. to rectify anticipating losses of patients in terminating treatment period, sample size the participants divided into two groups of case and control using table of random numbers. a registered dietitian explained the same protocols for physical activity and food regimen for both groups. lipid parameters, fasting blood sugar and thyroid hormone measured before study to screen for eligible participants for the study. patients took 6 grams of powders per day (placebo or drug in identical packages) in divided doses for a complete 4 weeks period and the adherence to the therapy were checked after two weeks in the middle of study period for each patient separately. all lipid profile indicators and anthropometric factors were measured after the study period as well. venous blood samples obtained after eight hours of fasting and blood samples were frozen for a maximum of two weeks and stored at -70 c until the time of biochemical analysis. serum cholesterol levels were measured by enzymatic colorimetric methods using commercial kits obtained from parsazmoon (karaj, iran). h. sabdariffa l. was bought from isfahan (iran) medicinal plant market and air dried h. sabdariffa l. calices were blended into fine powder and then packaged into 2 gram sachets.. 1 gram of powder was added to 100 ml water and refluxed until water boiled. then the extract was filtered and 1ml of that was diluted with distilled water to 100 ml. thereafter 20 l of this solution poured into a test tube and 1.58 ml water and100 ml folin - ciocalteu reagent added and mixed for 5 minutes and then 300 ml of super - saturated sodium bicarbonate solution added. after two hours in room temperature, mixture absorbance measured in 765 nm using a spectrophotometer (jenway 6105, staffordshire, uk). a kolmogrov - smirnov test was performed to show normal distribution of all measured data. an independent t - test analysis was performed to verify whether significant differences existed between the anthropometric and biochemical parameters of the subjects in the cases and controls groups at the baseline. the paired samples t - test was used to compare differences between subjects of each group before and after the study. a multivariate general linear model test was performed to assess the changes over the time in the anthropometric and biochemical parameters between the two groups and also to offset the effect of confounding factors like age, gender and body mass index (bmi). this randomized triple masked placebo controlled clinical trial was conducted from july 2010 to july 2011 in isfahan cardiovascular research institute, isfahan (iran) and registered in the iranian registry for clinical trials (irct201109122306n2). ninety (n=90) school - age adolescents (12 to 18 years old) who had at least one of these criteria : a) serum triglyceride more than 90 percentile b) serum total cholesterol more than 90 percentile c) ldl more than 90 percentile d) hdl less than 10 percentile, not using tobacco, no history of alcohol consumption or drug abuse, no history of metabolic diseases like diabetes, thyroid gland dysfunction, nephrotic syndrome, chronic pancreatitis, liver and gall bladder diseases and no drug consuming (which affects lipid profiles like statins and hormonal pills like estrogens, progestrones and oral contraceptives) were screened for inclusion in the study. exclusion criteria were lack of patients compliance with drug regimens at least for one week, pregnancy and lactation, drug sensitivity, presence of any disease which could interact with lipid profiles and need to take of any kind of drugs or other compounds which affects lipid profile like corticosteroids, androgens, estrogenes, progestines, thiazides, beta blockers and thyroid hormones. the study was approved by the institutional ethics committee of isfahan medical university of sciences and all participants and their parents or guardians read and signed a written informed consent form before participation (26, 27, 28). sample size was determined according the t - student formula. accordingly the most important variable in this study was considered as serum level of cholesterol which is a quantitative continuous variable and to detect at least 10 mg / dl decrease of this variable in case group (comparing to the controls), with 80% power and 5% type 1 error, suitable sample size was calculated 35 people in each group. to rectify anticipating losses of patients in terminating treatment period, sample size the participants divided into two groups of case and control using table of random numbers. a registered dietitian explained the same protocols for physical activity and food regimen for both groups. lipid parameters, fasting blood sugar and thyroid hormone measured before study to screen for eligible participants for the study. patients took 6 grams of powders per day (placebo or drug in identical packages) in divided doses for a complete 4 weeks period and the adherence to the therapy were checked after two weeks in the middle of study period for each patient separately. all lipid profile indicators and anthropometric factors were measured after the study period as well. venous blood samples obtained after eight hours of fasting and blood samples were frozen for a maximum of two weeks and stored at -70 c until the time of biochemical analysis. serum cholesterol levels were measured by enzymatic colorimetric methods using commercial kits obtained from parsazmoon (karaj, iran). h. sabdariffa l. was bought from isfahan (iran) medicinal plant market and air dried h. sabdariffa l. calices were blended into fine powder and then packaged into 2 gram sachets.. 1 gram of powder was added to 100 ml water and refluxed until water boiled. then the extract was filtered and 1ml of that was diluted with distilled water to 100 ml. thereafter 20 l of this solution poured into a test tube and 1.58 ml water and100 ml folin - ciocalteu reagent added and mixed for 5 minutes and then 300 ml of super - saturated sodium bicarbonate solution added. after two hours in room temperature, mixture absorbance measured in 765 nm using a spectrophotometer (jenway 6105, staffordshire, uk). a kolmogrov - smirnov test was performed to show normal distribution of all measured data. an independent t - test analysis was performed to verify whether significant differences existed between the anthropometric and biochemical parameters of the subjects in the cases and controls groups at the baseline. the paired samples t - test was used to compare differences between subjects of each group before and after the study. a multivariate general linear model test was performed to assess the changes over the time in the anthropometric and biochemical parameters between the two groups and also to offset the effect of confounding factors like age, gender and body mass index (bmi). the total polyphenolic content of h. sabdariffa l. calyx after triple measurement was equivalent to 16.4 milligram gallic acid per gram. a total of 90 eligible volunteers were recruited for the study and 7 participants from the cases and 11 from the controls group dropped out of the study due to irregular consumption of drugs, changing usual diet or irregular attendance for measuring lipid profiles. the mean age of the subjects in the cases was 14.171.61 years and 14.251.59 years in the controls. there were no statistically difference in the mean age (p=0.826), gender (p=0.907), body mass index (p=0.648) and lipid profile between the cases and controls before the study. demographic data of all participants (age, gender and body mass index) are summarized in table 1. lipid profile outcomes before and after treatment in cases and controls are shown in table-2. the data showed a significant decrease in ldl, total cholesterol and triglyceride in cases (without any significant decrease in hdl). the p value results of the multivariate general linear model analysis of lipid profiles for triglyceride, total cholesterol, ldl and hdl were 0.01, 0.032, 0.005 and 0.782 respectively. this information reveals noticeable decrease in triglyceride, total cholesterol and ldl and no significant decrease in hdl (table 2). in this study no significant drug side effects was reported except some mild gastrointestinal symptoms (e.g. temporary constipation). considering the increasing need to find effective cholesterol lowering agents and the role of diet in reducing atherosclerosis risk, edible plants are markedly being considered as a resources of anti atherosclerosis agents (30). recently, many clinical studies proposed dyslipidemia as one of the major risk factors for coronary disease. preclinical observations demonstrate that hypercholesterolemia aggregates accumulation of oxidized low - density lipoprotein (ox - ldl) in the arterial wall, promoting endothelial cell dysfunction and therefore promotion of atherosclerosis (24, 31). h. sabdariffa l., an annual herbaceous shrub, is a traditional chinese rose tea, cultivated in tropical areas like africa, asia and central america used effectively in folk medicine. the chemical constituents reported in this plant are phenolic compounds, anthocyanins, flavonoids, protocatechuic acid, vitamin c and carotenoid (23, 24, 32, 33). different studies have showed variety of pharmacologic actions of this plant like antioxidative, antimutagenic, anticancer, hypolipidemic and liver protective (24, 32, 34 - 36). some studies on the cholesterol lowering effect of h. sabdariffa l. have previously been conducted on animals and humans (24, 37, 38). in a study conducted to establish the effect of anthocyanins content of h. sabdariffa l., it was observed that extracts of hibiscus flowers prevents ldl oxidation and macrophage death (39). another investigation about effects of h. sabdariffa l. extract powder and preventive diet in patients with metabolic syndrome showed antihypertensive and antihyperlipidemic impacts for this plant (40). another study revealed that the aqueous extract of h. sabdariffa l. calices modulates the production of monocyte chemoattractant protein-1 in humans and lower production of mcp1, which means the slower development of atherosclerosis (41, 42). also another study proved the effect of anthocyanin extracted from hibiscus in attenuating oxidized ldl - mediated foam cell formation involving regulation of cd36 gene (43). according to statistical data in cases and controls before and after consumption of h.s sabdariffa l. powder and placebo, the results show that using up the plant for one month decreases serum total cholesterol, triglyceride and ldl effectively. meanwhile, ldl downturn (p=0.005) by 7% fall and triglyceride downward trend (p=0.01) by 9% fall show more prominent decline in comparison with other parameters ; hence it shows the noticeable capacity of this herbal plant in modifying serum ldl and triglyceride. the short period of drug usage (a one month period) may justify the ineffectiveness of the plant on hdl, and longer exposures to the drug may be needed to have a more accurate conclusion. it is concluded that h. sabdariffa l. due to its polyphenolic and antioxidant compounds may have positive effects on serum lipids profile and presumably can be considered for future studies on prevention and treatment of dyslipidemia and atherosclerotic disorders in adolescents. roya kelishadi and shahin shirani supervised and helped for the clinical part of the work. all authors contributed in data analysis, manuscript preparation and read and approved the final manuscript. | conflict of interest : none declared.objectivewe aimed to evaluate the effects of hibiscus sabdariffa (hs) calices on controlling dyslipidemia in obese adolescents.methodologyin this triple blind randomized placebo - controlled clinical trial which was registered in the iranian registry for clinical trials (irct201109122306n2), 90 obese adolescents aged 12 - 18 years with documented dyslipidemia were randomly assigned in two groups of cases who received 2 grams of fine powdered calices of hibiscus sabdariffa per day for one month and controls who received placebo powder with the same dietary and physical activity recommendations and duration of exposure. full lipid profile and fasting blood sugar measured before and after the trial. data were analyzed using multivariate general linear model.findingsoverall, 72 participants (mean age of 14.211.6, 35 boys) completed the trial. the two arms of the study (cases and controls) were not statistically different in terms of age, gender, weight, body mass index (bmi) and lipid profile before the trial. serum total cholesterol, low density lipoprotein cholesterol and serum triglyceride showed a significant decrease in cases group but high density lipoprotein cholesterol level was not changed significantly.conclusionit is concluded that hibiscus sabdariffa calyces powder may have significant positive effects on lipid profile of adolescents which maybe attributed to its polyphenolic and antioxidant content. further studies are needed on dose - response and formulation optimization. |
in aneurysmal subarachnoid hemorrhage (sah), symptomatic vasospasm, and its associated delayed cerebral ischemia (dci), are not uncommon, and often result in significant morbidity and mortality5) ; however, a timely recognition of ischemic insults and the prompt use of interventions such as stat chemical vasodilators or balloon angioplasty are critical in order to prevent irreversible cerebral damage9). we present a case of successful and prompt detection of a symptomatic vasospasm using real - time continuous electroencephalograpy (ceeg). a 50-year - old woman without any significant medical history presented with a sudden - onset of severe bifrontal headache. initially, she was only mildly drowsy (hunt hess grade iii) and had a glasgow coma scale score of 13. initial brain computed tomography (ct) and ct angiography (cta) showed a diffuse subarachnoid hemorrhage (sah) in the basal cistern and bilateral sylvian fissures with thick hemorrhages without intraventricular hemorrhage (fisher grade 3), and a wide - neck aneurysm of 7.36 mm at the anterior communicating artery (acoa). management of blood pressure and intracranial pressure was in accordance with the latest stroke guidelines at the neurological intensive care unit (icu) (fig. 1). ceeg (grass technologies, west warwick, ri, usa), and daily transcranial doppler (tcd) were carried out from post - bleed days 5 to 9 (with the day of the sah as day 0) to detect vasospasm according to a predetermined ceeg protocol. her ceeg revealed diffuse theta slowing without asymmetry and tcd mean flow velocities increased to 100 cm / s and 106 cm / s in the right and left middle cerebral arteries (mca) respectively, resulting in respective lindegaard ratios of 3.03 and 3.31 on post - bleed day 5. on post - bleed day 6, the patient 's level of consciousness recovered completely, and was stable to be transferred out of the neurological icu. her neurological status was stable during the daily morning round (9 a.m.) and the hospital 's visiting hours (10 : 30 a.m.). her ceeg alpha / delta ratio (adr) was not remarkable at 10 a.m. from then on, there was a significant adr abnormalitiy, and the neurointensivist detected a progressive and rapid decrement of the adr on the left side (more in c3-p3 than in f3-c3) compared to the right at 11 : 45 a.m. the decrease of adr was more than 60% change from baseline (fig., the patient became acutely right hemiparetic (medical research council grade 3/5) with global aphasia. we attributed this worsening as symptomatic vasospasm in the mca territory based on quantitative ceeg and neurological examinations. during preparation for a corrective procedure, stat tcd was performed and the mean flow of the left mca was shown to be further increased (161 cm / s) with a lindegaard ratio of 5.03. stat conventional cerebral angiography without ct angiography or ct perfusion was performed, and it showed severe vasospasm in the anterior cerebral artery (aca) and mca of the left hemisphere and there was a residual sac on the clipped aneurysm (fig. successful chemical vasodialtion with intra - arterial (ia) infusion of 4 mg of nimodipine was carried out. the patient 's neurological deficits including her moderate motor aphasia and leg weakness on the right side remained in the period immediately after the procedure ; however, they were markedly improved compared to their severity before the procedure. on post - bleed day 7, her neurological status improved, but she still had minimal motor aphasia and leg weakness. on post - bleed day 8, the mean flow velocity of the left mca was markedly increased to 207 cm / s with a 6.27 lindegaard ratio in the morning. although the patient 's vasospasm was asymptomatic, we decided to perform conventional angiography because of the high probability of its change into symptomatic vasospasm. finally, she was discharged to home with only mild dysarthria after successful re - clipping of her acoa aneurysm. high - grade sah has a high risk of developing vasospasm and dci, from which secondary brain injury may develop, and result in death2). however, it is not possible to predict when or if vasospasm after sah will occur and this reality makes the condition difficult to treat. although daily tcd testing might help to detect vasospasm, the role of tcd as a real - time detection tool has been criticized for its poor temporal resolution10). the goal of neuromonitoring in neurological icu is to forestall secondary brain injury as soon as possible, and to prevent permanent injury by triggering timely interventions. a previous study reported that adr demonstrated the strongest association with dci compared with other indices and clinically useful cut - offs were defined 6 consecutive clip (one clip defined as 1 minute) with a > 10% decrease in adr from baseline and any single clip with a > 50% as reasonable values1). this association was based on eeg changes correlating with the degree of neuronal injury and cerebral blood flow impairment46). the onset of clinical symptoms due to vasospasm after aneurysmal sah may be preceded by decrement and asymmetry of adr on ceeg, which can be detected in real time1). since july 2013, our ceeg protocol for vasospasm has been applied in cases of high grade aneurysmal sah and its summary is as follows : 1) ceeg monitoring is performed using a 21-electrode longitudinal bipolar montage according to the international 1020 system. 2) the real - time quantitative ceeg (adr, alpha power, delta power, amplitude eeg, and density spectral array) is checked at least every 2 h. 3) the focus is on f3-c3-f4-c4 and c3-p3/c4-p4 in acoa aneurysm, on c3-p3/c4-p4 in mca - bifurcation aneurysm, and on c3-p3/c4-p4 and p3-o1/p4-o2 in aneurysm of the posterior communicating artery1). the interval from the last normal period to the detection time was about 75 minutes, which might be attributable to the ceeg monitoring protocol. conventional angiography with chemical angioplasty was always available in our hospital but, obtaining procedural consent from her family was somewhat delayed because they were not always present outside of the hospital 's visiting hours. as for vasospasm in aneurysmal sah, rapid treatment within a 2-hour window may be profitable for promoting not only angiographic but also clinical improvements79). we believe that this protocol - based treatment may lead to a rapid clinical response, which has facilitated the complete recovery of this particular patient. in this case, routine tcd before symptom onset did not predict vasospasm because of the impossibility of continuous monitoring, and there did not appear to be a relationship between clinical symptoms, mca flow, and lindegaard ratio after cerebral angioplasty (post - bleed day 8). the flow velocities are dependent on the vessels ' diameter and insensitive to vasospasm affecting distal vasculature8). moreover, hypervolemia can lead to high velocities, which increases the false - positive rate without having true dci due to cerebral vasospasm. in addition, tcd can be very limited in patients with poor acoustic windows. detecting a perfusion defect rather than real - time ceeg as well as daily routine tcd may help to detect vasospasm in aneurysmal sah. further, a protocol - based treatment algorithm may also reduce the permanent ischemic insult by reducing onset - to - detection time. multidisciplinary care including neurointerventionist, neurointensivist, neurosurgeon, neurologist and continuous monitoring could dramatically improve the clinical outcomes of patients with aneurysmal sah. | a continuous electroencephalography (ceeg) can be helpful in detecting vasospasm and delayed cerebral ischemia in aneurysmal subarachnoid hemorrhage (sah). we describe a patient with an aneurysmal sah whose symptomatic vasospasm was detected promptly by using a real - time ceeg. patient was immediately treated by intraarterial vasodilator therapy. a 50-year - old woman without any significant medical history presented with a severe bifrontal headache due to acute sah with a ruptured aneurysm on the anterior communicating artery (fisher grade 3). on bleed day 6, she developed a sudden onset of global aphasia and left hemiparesis preceded by ceeg changes consistent with vasospasm. a stat chemical dilator therapy was performed and she recovered without significant neurological deficits. a real - time and protocol - based ceeg can be utilized in order to avoid any delay in detection of vasospasm in aneurysmal sah and thereby improve clinical outcomes. |
xenopus extracts were supplemented with demembranated sperm nuclei and simultaneously released from their natural arrest in meiotic metaphase ii (figures 1a and 1b). over the next 2030 min, the sperm chromatin decondensed and was licensed for replication ; the dna was then assembled into interphase nuclei and the extracts entered s phase ; by 80 min, most of the dna had been replicated and the extracts entered g2 (figure 1b). chromatin samples, isolated by centrifugation through a sucrose cushion, were taken every 10 min. associated proteins were eluted from chromatin and analyzed by mass spectrometry. at each time point, the abundance of proteins was estimated from the extracted ion chromatograms of their corresponding peptides [3, 4 ]. the resulting temporal profiles were subjected to smoothing and normalized so that over the time series the maximum abundance of each protein was set to a value of 1 (figure s1a available online). we identified 606 nonredundant proteins on untreated chromatin, which were subject to further analysis. protein abundance is presented as a heat map, where red, black, and green indicate high, medium, and low abundance, respectively. figure 1c shows that there is good agreement between the relative levels of proteins measured by mass spectrometry and standard immunoblotting. our protocol can not measure absolute amounts of proteins, or compare levels between different treatments. an approximate comparison of protein levels between experiments can be derived from the number of different peptides detected. for example, geminin reduces the amount of mcm2 loaded onto dna as shown by immunoblotting, and although the heat map shows a relatively unchanged pattern, the numbers of mcm2 peptides detected is greatly reduced (figure 1c). some proteins showed only small dynamic changes on chromatin during interphase, most of which consisted of ribosomal proteins, chaperonins, and translation elongation factors, which were probably cytoplasmic contaminants. we therefore excluded from further analysis the 148 proteins with less than 15% variation on chromatin. to identify groups of proteins with similar temporal profiles of chromatin - association, we used fuzzy c mean (fcm) soft clustering. the mcm27 proteins (which peak on chromatin prior to entry into s phase) and the replication fork proteins (which peak on chromatin during s phase) could be separated when 12 clusters were used. figure 2 shows heat maps for all 458 chromatin proteins showing more than 15% variation in abundance sorted into the 12 fcm clusters. the 12 clusters were divided into three general types that have their peak abundance on chromatin early (e), intermediate (i), or late (l) in interphase. the early group, containing four clusters, were named e1, e2, e3, and e4 to reflect how rapidly their presence on chromatin decreased (e1 fastest, e4 slowest). the second group, where maximum abundance was at intermediate times, was represented by three clusters : i1 (containing the mcm27 licensing proteins), i2 (containing replication fork proteins), and i3 (where maximal abundance was more broadly in the middle of the time course). the third group reached its maximum abundance on replicating chromatin at later times, and its five clusters were named l1l5, reflecting the order in which they accumulated (l1 earliest, l5 latest). although the abundance data were highly reproducible between different runs (figure s1b), there was some variability in the assignment of proteins to the different fcm groups in three independent experiments. figure 2 and figure s1c show that the level of reproducibility of different fcm clusters was approximately proportional to how much the protein abundance changed during the time course. clusters e1, i1, i2, l4, and l5, where protein abundance dropped to virtually zero at certain times, were highly reproducible, whereas clusters e4 and l1, where protein abundance only ever fell to about 50% of peak values, were much less reproducible. we next used the david software to collect ontological terms associated with all the proteins. this revealed that all fcm clusters showed significant enrichment with particular sets of ontological terms, which in turn represented a small number of functional groups (figure 2). the statistical significance (p value) and the group enrichment score (a measure of the significance of all annotation terms in the group) of the terms figure 2 suggests that each fcm cluster contains several groups of functionally related proteins that associate with and/or dissociate from chromatin with similar dynamics during cell - cycle progression. many of the groups associate with chromatin as expected from their known function. the early e1 cluster contains m phase and dna condensation functional groups that play roles in late mitotic events. the e2 cluster, which falls slightly slower than e1, contains a series of proteins related to chromosome structure and metabolism (e2fg1e2fg3), as well as all essential members of the origin recognition complex (orc) that marks origins of replication [810 ]. clusters e3 and e4 contain ras small gtpase, rho type (e3fg2) and rab (e4fg4) functional groups consistent with the known role of rangtp in nuclear pore and nuclear envelope formation. the i1 cluster contains all of the mcm27 proteins (functional group mcm), which are loaded onto chromatin after orc and are displaced from dna as it replicates [1214 ]. the i2 cluster, which peaks during s phase, contains known replication fork proteins (dna replication, dna polymerase, and rf - c). the i3 cluster, which remained on chromatin slightly longer than the classical replication fork proteins in the i2 group, contains functional groups associated with nucleic acid metabolism that are likely to be involved in the processing of newly replicated dna. chromatin assembly factors and variants of histone core proteins are found in late cluster l2, as expected from the doubling of dna content during s phase. nuclear pore proteins, nuclear envelope proteins, and the nuclear lamins that show increasing association with chromatin throughout interphase were in the l4 and l5 clusters. functional groups involved in intracellular transport, including nucleocytoplasmic transport, are present in late fcm cluster l3. we also identified proteins that are not classically associated with chromatin but whose temporal profiles suggested specific roles in the building of chromatin and nuclei. these include proteins associated with intermediate filaments (e2, e3, and l1), actins (e3, l1, and l3), 14 - 3 - 3 proteins (l1), and protein catabolism (l1 and l2). geminin blocks the licensing of replication origins by preventing the cdt1-mediated loading of mcm27 onto chromatin [15, 16 ]. roscovitine is a cdk inhibitor that blocks replication initiation at licensed origins [17, 18 ]. figures 3a and 3b show how each protein identified in the replicating sample (y axis) was redistributed into temporal fcm clusters after inhibitor treatment (x axis). inhibition of replication with either drug had a system - wide effect on the chromatin proteome, and only 15%20% of proteins remained unaffected. although all clusters were affected by replication inhibition, the clusters that were most affected were ones that normally showed the greatest changes in abundance during s phase, particularly the intermediate clusters (i1i3), which are largely composed of replication proteins, but also the e4, l2, l3, and l5 clusters. approximately 30% of the members of each fcm cluster were not detected on chromatin at all after drug treatment (lost column in figures 3a and 3b). because we have not sampled the complete set of chromatin - associated proteins, some proteins may be lost because of a random sampling problem. however, in cases where a large proportion of proteins in a given cluster are lost (such as in the intermediate clusters), this is likely to be a genuine effect of the treatment. hierarchical clustering was then performed with a combined dataset from the inhibitor - free, geminin, and roscovitine experiments (figure 4 and figures s2 and s4). with the combined dataset, many proteins known to act together in protein complexes cluster next to each other on the same branch of the hierarchical tree (figure 4). examples of such clustering are the five tightly associated members of the orc, the six members of the mcm27 complex, a group of interacting nucleoporins (nups), and two groups of actin - related proteins. virtually all of the replication fork proteins in the i2 cluster were lost or were greatly perturbed when replication was inhibited (figure 4c, figure s4f). we chose two relatively uncharacterized proteins that showed interesting responses by mass spectrometry, duf87 (part of the fact chromatin - remodeling complex and previously implicated in xenopus dna replication) and ruvbl1 (pontin ; part of the ino80 chromatin - remodeling complex with reported roles in dna replication and repair [22, 23 ]) and raised antibodies to them. the antibodies were then used to immunoblot chromatin isolated at different times from extracts optionally treated with geminin or roscovitine. figure 4f shows that the immunoblotting profiles resembled those obtained by mass spectroscopy, further validating our approach. figure 3 shows that inhibition of replication affects many other proteins in addition to the replication proteins of clusters i1, 2, and 3. both geminin and roscovitine affected late cluster l5, which contains nuclear pore proteins. closer examination suggested that a relatively uncharacterized protein, elys / mel-28, was a new nuclear pore complex protein, and this was subsequently confirmed [2426 ]. inhibition of mcm27 loading delays nuclear pore complex assembly (figure 4f) and slows nuclear growth (figure 3c). this can be explained by mcm27 on chromatin stimulating the association of elys / mel-28 with dna. in contrast, roscovitine accelerated the association of nucleoporins with chromatin and therefore redistributed them into the l4 cluster (figure 4d). this may be a consequence of roscovitine preventing the release of mcm27 from dna, thereby facilitating increased loading of elys / mel-28. nuclear actins are involved in many basic nuclear processes, such as transcription and chromatin remodeling. we found the complete arp2/3 complex (involved in actin - filament nucleation and assembly) together with actin - capping proteins grouped in the early fcm cluster e3 (figure 2). proteins in this group are maximally loaded on replicating chromatin at the beginning of the time course and then gradually decreased. geminin and roscovitine changed this behavior so that no consistent decline in chromatin association occurred (figure 4e), suggesting that the release of these proteins from chromatin is dependent on dna replication. conventional actins were also found as a complex in the late cluster l1 and also were delayed in their chromatin association by geminin (figure 4e). a similar geminin - induced delay in chromatin loading of tubulins, keratins, and microfilament components (figures s2 and s4) suggests that building of the entire nuclear structure is affected by replication licensing. consistent with this idea, geminin (and to a lesser extent roscovitine) also affected the rate of growth of nuclei (figure 3e). some proteins were only detected on chromatin in the presence of roscovitine or geminin (figure s3). their absence from untreated chromatin may be a consequence of the random - sampling problem or may represent chromatin recruitment specific for the inhibition of replication. of potential relevance is the appearance of a dna repair functional group on chromatin treated with geminin and a phosphorylation the mcm27 complex plays a central role in dna replication and is differentially affected by the two inhibitors we use here. its loading onto dna is inhibited by geminin, whereas its activation as an essential component of the replication fork is inhibited by roscovitine. to investigate whether global effects of replication inhibition could be mediated by direct interaction with mcm27, we identified proteins coprecipitating with mcm2 or mcm3 on chromatin (table s2). as expected, the complete set of mcm27 proteins was the most abundant component of both the mcm2 and mcm3 precipitates, as well as the licensing proteins orc1 - 5 and cdc6, plus several replication fork proteins. in addition, other proteins whose abundance on chromatin was affected by replication inhibition were also coprecipitated with mcm2 and mcm3. these included ubf1, whose chromatin dynamics closely resemble orc, the complete arp2/3 complex together with capping proteins, plk1, and all members of the chromosome passenger complex (incenp, dasra a, aurora b, and survivin). a number of nucleoporins, including elys / mel28, were also identified in both mcm immunoprecipitates. the binding of these proteins to chromatin was delayed by geminin treatment (figure 4d), suggesting a functional connection to replication licensing. these observations led us perform experiments showing that the interaction between elys and mcm27 promotes nuclear pore complex assembly and helps coordinate dna replication with nuclear assembly. the large number of dynamic changes that occur in response to replication inhibition also support the idea that cell - cycle processes such as nuclear assembly and dna replication do not occur independently and in isolation but are part of a highly coordinated biological system. | summaryalthough the replication, expression, and maintenance of dna are well - studied processes, the way that they are coordinated is poorly understood. here, we report an analysis of the changing association of proteins with chromatin (the chromatin proteome) during progression through interphase of the cell cycle. sperm nuclei were incubated in xenopus egg extracts, and chromatin - associated proteins were analyzed by mass spectrometry at different times. approximately 75% of the proteins varied in abundance on chromatin by more than 15%, suggesting that the chromatin proteome is highly dynamic. proteins were then assigned to one of 12 different clusters on the basis of their pattern of chromatin association. each cluster contained functional groups of proteins involved in different nuclear processes related to progression through interphase. we also blocked dna replication by inhibiting either replication licensing or s phase cdk activity. this revealed an unexpectedly broad system - wide effect on the chromatin proteome, indicating that the response to replication inhibition extends to many other functional modules in addition to the replication machinery. several proteins that respond to replication inhibition (including nuclear pore proteins) coprecipitated with the mcm27 licensing complex on chromatin, suggesting that mcm27 play a central role in coordinating nuclear structure with dna replication. |
rab proteins constitute a subfamily of small gtpases that play important roles in the regulation of intracellular vesicle transport. rab gtpases represent a large family of small guanosine triphosphate (gtp)-binding proteins that comprise more than 60 known members. in mammalian cells, it is well established that different rab proteins localize on distinct membrane - bound compartments, where they regulate multiple steps in membrane traffic, including vesicle budding, movement, and fusion, through cycling between an active gtp - bound form and an inactive guanosine diphosphate (gdp)-bound form. gdp - gtp cycling is regulated by guanine nucleotide exchange factors (gefs) and gaps, which catalyze the activation and deactivation of gtpases, respectively. cytokinesis is the terminal stage of eukaryotic cell division in which the cytoplasm of a dividing cell is partitioned between 2 daughter cells. cytokinesis involves complex changes in cell shape, which require assembly and activation of a narrowing acto - myosin contractile - ring between the poles of the mitotic spindle. this contractile ring is a major driving - force for the physical partitioning of the cytoplasm. while the acto - myosin contractile - ring is vital for ingression of the cleavage furrow, it is not the sole mechanism that drives this process as incorporation of new membrane into the furrow is also required. thus, membrane trafficking is also crucial for abscission. a comprehensive picture of how membrane trafficking participates during cytokinesis has been presented. in fact, exocytic events have been documented with vesicles docking and fusing at the furrow area. it is believed that membrane input is critical during abscission for nascent daughter cell separation. in c. elegans, a role for rab gtpases in the later stages of cell division has been documented. sirna - mediated depletion of rab11, a rab protein involved in endocytic vesicle recycling, leads to cytokinesis alterations, including furrow regression and abnormal scission. indeed, endosomes are suitable candidates to provide membrane during cytokinesis, and both rab11 and its interacting protein rab11-fip3 localize to the cleavage furrow during cytokinesis. in mammalian cells, it has also been demonstrated that rab11- and fip3-containing recycling endosomes accumulate near the cleavage furrow and that rab11 is required for successful completion of cytokinesis. in addition, 2 other gtpases, arf1 and arf6 have also been implicated in cytokinesis. interestingly, it has been shown that fip3 is also required for arf6 recruitment to the midbody during late telophase. of note, when both rab11 and arf6 are knocked - down, the furrow rapidly regresses and the cells are unable to form a stable midbody, suggesting that rab11 and arf6 function synergistically in allowing the furrow to progress to the terminal stage of abscission. rab35 functions at an early endosome, prior to the relatively slow recycling endosome step regulated by rab11. rab11 and rab35 are localized to different subcellular compartments and control distinct endocytic recycling pathways. thus, it is likely that there are multiple endocytic routes that are individually essential for cytokinesis. in addition, rab35 is required during cytokinesis to concentrate pip2 at the cytokinetic bridge. furthermore, like rab11, rab35 is required during cytokinesis after furrow ingression to provide delivery of membrane vesicles derived from recycling endosomes to the cytokinetic bridge. in addition, it has been demonstrated that epi64b (a gap for rab35), acts as an effector of arf6 negatively regulating rab35 activation. this molecular mechanism controls the rab35 pathway, including the localization of rab35 at the intercellular bridge, and the completion of cytokinesis. in neurite outgrowth, another connection between rab35 and arf6 has been described, in response to nerve growth factor (ngf) stimulation. rab35 accumulates at arf6-positive endosomes and centaurin-2 (also known as acap2) is recruited to the same compartment in a rab35-dependent manner. however, in the case of rab4, it has been shown that this cycle (association and dissociation) is altered during mitosis and rab4 accumulates in the cytoplasm through phosphorylation by a mitotic kinase. mellman and collaborators have demonstrated that reversible phosphorylation - dephosphorylation of rab4 explains its localization during the cell cycle. indeed, a mutation of ser196 to glutamine or aspartic acid completely hampered rab4 phosphorylation in mitotic cells preventing its translocation to the cytoplasm. upon exit of cells from mitosis occurred dephosphorylation and reassociation of soluble rab4 with membranes. thus, phosphorylation of ser196 is directly responsible for the reversible translocation of rab4 into the cytosol in mitotic cells. in addition, when using a dominant positive mutant of rab4 that is permanently attached to the membranes, van der sluijs and collaborators, demonstrated that the membrane - bound pool of rab4 is targeted by a mitotic kinase. it has been shown that rab21 activity and integrins targeting to the cleavage furrow, a process regulated by rab21, are required for the completion of cell division. loss of rab21 gene expression in human cancer, leads to the accumulation of multinucleate cells. in addition, abnormal integrin traffic was linked with the generation of aneuploidy and cell transformation. in human prostate and ovarian cancer samples, loss - of - function experiments demonstrate that long - term depletion of rab21 is sufficient to induce chromosome number aberrations in normal human epithelial cells. it is also important to mention that rab6a is required for normal transit through mitosis since a blockage in metaphase has been observed in rab6a-depleted cells. leads to a similar phenotype to the one observed upon alteration of rab6a function. in our laboratory, we have evidence that rab24 not only colocalizes with gapcena at the centrosome but also co - immunoprecipitates with this protein. it has been proposed that gapcena may participate in a pathway involved in the metaphase / anaphase transition. interestingly, depletion of rab24 by a sirna causes a marked accumulation of cells in metaphase. it has been shown that rab6-interacting protein1 (r6ip1), a rab6-binding protein, also binds to rab11, suggesting that this protein links rab6 and rab11 function. notably, r6ip1 function is also involved in controlling metaphase and cytokinesis, 2 events of the mitosis in which rab6 and rab11 have been involved. preliminary results in our lab indicate that similar to rab6 and rab11, rab24 also interacts with r6ip1 (unpublished result). thus, this observation may explain, in part, the increased number of cells arrested in metaphase observed in rab24 depleted cells. it was demonstrated by the yeast 2-hybrid system that rab24 interacts with gabarap (a human homolog of the lc3 protein) and also with cyclophilin a. interestingly, in a more recent publication, it was shown that cyclophilin a is a centrosome protein that undergoes cell cycle - dependent relocation to the midzone and midbody during cytokinesis, implicating a role of this protein during mitosis. in addition, depletion of cyclophilin a leads to cytokinesis defects through an inability to resolve intercellular brigdes, culminating in delayed or failed cytokinesis. our results indicate that in cells transiently overexpressing rab24, the cells remain connected by a long cytoplasmic bridge and appear to be defective in abscission. chromatin bridges between daughter cells were also observed in rab24 silenced cells, but as described in the following section, we have evidence that defects in cytokinesis are likely a consequence of errors previously observed during segregation and congression of chromosomes (fig. 1). figure 1. a schematic diagram illustrating the pathways and rabs involved the exocytic / endocytic boundary in a cytokinesis cell : modes of transport from early or recycling endosomes to the intracellular bridge (midbody), may involve different pathways regulated by rab21, rab11 or rab35. moreover, arrive to midbody transport vesicles from the exocutic pathway (labeled with rab8). we also include in the scheme other rabs protein (such as rab4, rab5 and rab6) that regulate cell cycle as mentioned in the text. recent studies have shown the involvement of rab5 in an early stage of cell division by modulating the congression and segregation of chromosomes. silencing rab5 in u2os cells causes defects in chromosome congression and marked prometaphase delay, due to a reduction in the localization of the protein cenp - f to kinetochores. cenp - f, is a centromere - associated protein that contributes to the establishment of kinetochore - microtubule interactions. another group demonstrated, almost simultaneously, that rab5 is required for proper chromosome alignment before anaphase during drosophila mitosis. it was shown that rab5 associates in vivo with lamin altering lamin disassembly and causing relocation of mud (mushroom body defect) to the poles during mitosis. mud is the drosophila counterpart of nuclear mitotic apparatus protein (numa), which is known to be important for spindle formation and maintenance in vertebrate cells. thus, surprisingly, through 2 different mechanisms, there is strong evidence that rab5 is necessary for proper congression and alignment of the chromosomes in early stages of mitosis. in addition, it has been described that the protein dcdc5 (doublecortin domain - containing protein 5) plays an important role in mediating dynein - dependent transport of rab8-positive vesicles and in coordinating cell division. in a recent study that reveals genes associated with cell division through a complete phenotypic profile, it has been reported that silencing of the rab24 gene showed a binucleation phenotype and that the whole gene was essential for survival. we have recently demonstrated that the distribution of rab24 changes during the cell cycle (militello., 2013). in interphase, a fraction of rab24 presents a reticular pattern following the microtubules distribution whereas this protein localizes to the mitotic spindle at metaphase, and to the midbody and cleavage furrow during cytokinesis. in addition, we have demonstrated that rab24 can associate to microtubules assembled in vitro with cytosolic or purified tubulin. consistently, in a previous report, using the yeast two - hybrid system, it was shown that rab24 interacts with human 6 tubulin. interestingly, 6 tubulin is present in mitotic spindle - fiber but absent in cytoskeletal microtubules in naegleria gruberi. we have also observed that depletion of rab24 causes alterations in the symmetry and the aspect of the spindle during metaphase. furthermore, similar to rab5, depletion of rab24 leads to defects in proper chromosome alignment during metaphase. in addition, rab24 knock down significantly altered both mitotic and phase index with a concomitant increase in the number of binucleated cells, an indication of increased failure in cytokinesis. we have demonstrated that this protein is required for proper chromosome segregation and that the failures observed during cytokinesis, appear to be a result of errors in the segregation of the chromosomes. thus, it is tempting to speculate that these defects might be related to the interaction between rab24 and tubulin, at the mitotic microtubules. it is interesting to mention that both rab proteins (rab5 and rab24) have a high percentage (48%) of nucleotide sequence homology (database of pubmed gen). in addition, using a structural proteomic approach, lambright and collaborators have demonstrated the interaction between rabenosyn-5 (a rab5 effector) and rab24. thus, this connection between rab5 and rab24 or the structural analogies may account for some similar results described for both rab proteins. a possible interaction between rab24 and cenp proteins or a potential role of rab24 in degradation of nuclear lamin, are important topics for future investigations (fig. 2). figure 2. a schematic diagram depicting localization of different rab proteins required for mitosis (g2/prophase to metaphase). disassembly processes of the nuclear membrane and the congregation / segregation of chromosomes in the equatorial plane are regulated by rab5 and rab24 respectively. in this review we have presented enough evidence linking cellular transport proteins with cell division. as we mentioned above, through specific mechanisms, rab proteins regulate different cell cycle stages, including cytocinesis. | the superfamily of small gtpases serves as a signal transducer to regulate a diverse array of cellular functions. the members of this superfamily are structurally and functionally classified into at least 5 groups (ras, rho / rac, rab, arf, and ran) and they are involved in the control of cell proliferation and differentiation, regulation of the actin cytoskeleton, membrane trafficking, and nuclear transport. it is widely reported that members of the rab family participate in the control of intracellular membrane trafficking through the interaction with specific effector molecules. however, many rabs and other small gtpases have also been shown to function in cell division. in this review, we discuss current knowledge about rab proteins regulating different stages of the cell cycle, such as the congregation and segregation of chromosomes (during metaphase) and the final stage of cell division known as cytokinesis, in which a cell is cleaved originating 2 daughter cells. |
hand hygiene is recognized as the leading measure to prevent cross - transmission of microorganisms and to reduce the incidence of health care associated infections [1, 2 ]. despite the relative simplicity of this procedure, compliance with hand hygiene among health care providers is as low as 40% [35 ]. to address this problem, continuous efforts are being made to identify effective and sustainable strategies. one of such efforts is the introduction of an evidence - based concept of my five moments for hand hygiene by world health organization. these five moments that call for the use of hand hygiene include the moment before touching a patient, before performing aseptic and clean procedures, after being at risk of exposure to body fluids, after touching a patient, and after touching patient surroundings. this concept has been aptly used to improve understanding, training, monitoring, and reporting hand hygiene among healthcare workers. nurses constitute the largest percentage of the health care workers (hcw) and they are the nucleus of the health care system. because they spend more time with patients than any other hcws, their compliance with hand washing guidelines seems to be more vital in preventing the disease transmission among patients. in asia there is a paucity of studies [710 ] exploring this subject, although the prevalence of health care associated infections is high in this region ; especially medical and nursing student 's knowledge of standard precautions is rarely compared. the observance of hygiene by students is reported as being weak [12, 13 ]. therefore, it is absolutely essential to investigate and know nurse 's knowledge, attitudes, and practices about hand washing so that appropriate strategies can be developed to promote hand washing compliance. this cross - sectional study was conducted in navodaya medical college (nmc), one of the biggest teaching hospitals in raichur, india. nmc provides tertiary medical care for residents of raichur and patients referred from neighboring states. verbal consent was obtained from 98 medical and 46 nursing students who volunteered to participate. a self - administered questionnaire containing a set of questions regarding hand - hygiene knowledge, attitudes, and practices was distributed to all participants. this proforma of 25 questions includes multiple choice and yes or no questions. attitude and practice were assessed using another self - structured questionnaire which consists of 10 and 6 questions, respectively. respondents were given the option to select on a 1- to 7-point scale between strongly agree and strongly disagree. 1 point was given for each correct response to positive attitudes and good practices so that maximum score for attitude is 10 and for practice it is 6. a score of more than 75% was considered good, 5074% moderate, and less than 50% was taken as poor. z test was used to compare the percentage of correct responses between medical and nursing students. there were a total of 144 study participants (46 nursing students and 98 medical students). in this a majority (79%, 114 out of 144) had claimed to have received formal training in hand washing. a significant difference (p < 0.001) was observed between medical (73 out of 98, (74.2%)) and nursing (44 out of 46, (95.4%)) students who had received formal training in hand hygiene. when asked about the correct technique of hand washing, 89 out of 98 medical students (91.3%) and 45 of 46 nursing students (97.8%) said they knew the correct technique of hand washing. the knowledge on hand hygiene was moderate (107 out of 144, 74%) among the total study population. only 9% of participants (13 out of 144) had good knowledge regarding hand hygiene. (p = 0.023) the percentages of correct responses of the two groups of students to the individual questions on hand hygiene knowledge are given in table 1. nursing students had significantly (p < 0.05) better attitudes (52.1%) compared to medical students (12.9%). the percentages of correct responses of the two groups of students to the individual questions on hand hygiene attitudes are given in table 2. nursing students had significantly (p < 0.05) better practices (62.1%) compared to medical students (19.6%) and the difference was statistically significant (p < 0.05). the percentages of correct responses of the two groups of students to the individual questions on hand hygiene practices are given in table 3. in our study, both study groups had moderate knowledge on hand hygiene, which was a positive finding. studied the hand hygiene practices of 187 candidates during final mbbs osce (objective structured clinical examination) at the royal london hospital school of medicine and dentistry in uk and found that only 8.5% of candidates washed their hands after patient contact, although the figure rose to 18.3% when hand hygiene signs were displayed. the situation in healthcare centers of developing countries is even more unacceptable. in an earlier study from saudi arabia, adherence to hand hygiene was seen in 70% of medical students, 18.8% of nurses, and 9.1% of senior medical staff, but the technique was suboptimal in all. like most previous studies, our study showed that the overall compliance of hand hygiene by hcws was less than 50%. van de mortel. in 2010 compared the hand hygiene knowledge, beliefs, and practices between nursing and medical students. they found that the nursing students hand hygiene knowledge was significantly higher than that of medical students (p < 0.01) which is consistent with our study. our study shows the importance of improving the current training programs targeting hand hygiene practices among medical and nursing students. hand hygiene training sessions may need to be conducted more frequently for medical students with continuous monitoring and performance feedback to encourage them to follow correct hand hygiene practices. | background. hand hygiene is recognized as the leading measure to prevent cross - transmission of microorganisms. regarding hospital acquired infections, the compliance of nurses with hand washing guidelines seems to be vital in preventing the disease transmission among patients. there is a paucity of studies exploring this subject in asia. especially medical and nursing student 's knowledge of standard hand hygiene precautions is rarely compared. methods. a cross - sectional study was conducted among 98 medical and 46 nursing students in a tertiary medical college in india. knowledge was assessed using who hand hygiene questionnaire. attitude and practices were evaluated by using another self - structured questionnaire. z test was used to compare the percentage of correct responses between medical and nursing students. a p value less than 0.05 was considered significant. results. only 9% of participants (13 out of 144) had good knowledge regarding hand hygiene. nursing students knowledge (p = 0.023), attitude (p = 0.023), and practices (p < 0.05) were significantly better than medical students. |
this protozoan parasite has a global distribution, infecting humans and a wide range of mammalian hosts. the prevalence of giardiasis in humans in developed countries is 27%, while it may vary between 20 and 30% in developing countries. the spectrum of clinical manifestations in human giardiasis is relatively variable, ranging from the absence of symptoms to acute or chronic diarrhea, dehydration, abdominal pain, nausea, vomiting, and weight loss. children are especially affected, with more severe consequences than adults. however, the impact of giardia infection in children development is not clear. some studies showed detrimental effects on nutritional status and poorer cognitive function on children with giardiasis [58 ], while others showed that giardiasis did not affect childhood growth. host factors, such as immune status, nutritional status, and age, are recognized as important determinants for the severity of infection. however, studies on the possible association between g. duodenalis assemblages and the severity of the disease have proved thus far to be inconsistent. molecular tools have demonstrated that g. duodenalis is a species complex comprising at least eight assemblages (a h), among which only a and b were found infecting humans. previous studies focused on the prevalence of giardiasis in preschool children (3 - 4%) [13, 14 ], on g. duodenalis assemblages determination in humans [13, 15 ], animals, and water [15, 16 ], and, more recently, on prevalence and risk factors for g. duodenalis infection among children. the aim of this study was to determine whether there was any g. duodenalis in preschool children enrolled, the assemblages and their relation to clinical data from the city of lisbon. the population in study were all preschoolers (3306 children) attending that year the public preschools under the supervision of the lisbon city hall. the selection of the kindergartens where the study was conducted was decided by the lisbon city aiming at reflecting a wider socioeconomic of the families and geographic dispersion of the children. a total of 316 (46.1%, 316/685) preschool children, aged 36, attending the selected kindergartens of the network of public schools, were enrolled in this study. the enrolled children were those whose parents collected the stool samples and signed the informed consent. a meeting was held with the director of each school as well as with the parents to explain the study objectives, prior to sample collection. the parents / guardians were told to collect three stool samples in consecutive days without any pharmacologic induction and stored at 4c till monday morning, when the team went to the schools to collect all the samples. the fresh stool samples were screened for g. duodenalis and other intestinal parasites through microscopic analysis in saline and also in iodine. furthermore, the formolether concentration method was also performed to increase the sensitivity of the detection. positive samples for g. duodenalis were kept in filter paper (generation card kit, qiagen) and preserved at 20c for further analysis. dna was extracted from samples preserved in filter paper. a dna extraction protocol for dried blood spots (generation capture card kit, qiagen) changes to the original protocol included tripling the solution volumes used, except for the final elution step (100 l). for some samples, dna was reextracted using elution volumes of 25 l. all samples were amplified using primers targeting the small subunit ribosomal rna (ssu - rrna) [19, 20 ] and -giardin (bg) loci [21, 22 ]. amplification reactions were performed using 2 l of dna template in a final volume of 25 l, using illustra puretaq ready - to - go pcr beads (ge healthcare, uk). both positive (dna isolated from the portland-1 strain (atcc 30888dlgc promochem) and negative controls (no template added) were included in each series of pcr reactions. pcr products were visualized on 2% agarose gel stained with ethidium bromide. for sequence analysis, pcr products were purified using jetquick gel extraction spin kit/50 (genomed, germany) according to the manufacturer 's instructions. dna sequencing reactions were carried out in both directions using primers giarf / giarr for ssu - rrna gene fragment (175 bp) and those described previously for -giardin gene fragment (511 bp). sequences obtained in this study were aligned with previously published sequences of g. duodenalis isolates available in the genbank database, using clustalw. a questionnaire for clinical data relative to the children all children infected with g. duodenalis were assisted by a paediatric doctor from the tropical diseases clinic of the institute of hygiene and tropical medicine and treated for the infection with 15 mg / kg / day of metronidazole divided in three daily doses, for seven days. three weeks after the treatment new stool samples were obtained in order to confirm the treatment efficacy. if a child was infected with any intestinal parasite, the remaining members of the household were invited to collect their stools that were also screened for intestinal parasites. the present study was submitted and approved by the ethical committee of the institute of hygiene and tropical medicine, lisbon. written informed consent was obtained from all parents or the legal guardians of the children participating in the study. from the 316 children participating in this study, 166 (52.5%) were males and 150 (47.5%) were females. g. duodenalis cysts were found in 8 out of the 316 samples examined by microscopy (2.5%), corresponding to four of the schools included in this work. furthermore one family member (brother), from one of the infected children, was also infected with g. duodenalis. the nine positive samples for g. duodenalis were successfully amplified for ssu - rrna fragment gene, and for the bg gene only seven samples were amplified (77.8%, 7/9) (figures 1 and 2). ssu - rrna sequences obtained in this study were compared with homologous sequences found in genbank using blast. sequences obtained for bg gene were also compared with public sequences from genbank using blast. additionally, bg sequences were analysed for subassemblage discrimination according to the genetic polymorphisms described elsewhere. isolates 3, 4 and 5 belong to subassemblage a2, while the other isolate (6) belongs to subassemblage a3 (table 1). for the remaining two isolates, 7 and 8, belonging to assemblage b, it was not possible to determine the respective subassemblage due to the high nucleotide variability observed in the chromatogram. new stool samples were collected from all infected children after the complete treatment and analyzed. only four children reported symptoms (1, 3, 4, 5), including lack of appetite, abdominal pain, and flatulence (table 1). case number 9, the one with intermittent diarrhea, was the only case of moderate malnutrition (bmi 12 ; 2 < z score < 3). the medical examination was normal in four children (3, 6, 7, and 8). abdominal distension and pain on deep palpation were observed in the remaining five (table 1). from the three children infected with g. duodenalis assemblage b, two presented a normal medical examination with no symptoms (7 and 8), while the other one (1) presented abdominal distension and referred lack of appetite as a symptom. for the six children infected with assemblage a, two had a normal medical examination (3 and 6) and three referred no symptoms (2, 6, and 9). the number of children found infected with g. duodenalis in this study (8/316 ; 2.5%) was similar to other studies conducted in northern and centre of portugal where 3%, 3.7%, and 1.9%, respectively [13, 14, 17 ] were infected. these results are in agreement with the reported prevalence for this parasite in developed countries. the use of microscopy as the only diagnostic procedure for detecting g. duodenalis may be considered as a limitation of this study. however, the use of three stool samples allow the detection of over 90% of infection, which is very similar to the sensitivities recently reported for rapid diagnostic tests, while one stool sample will allow the detection of 60 to 80%, and the analysis of two stool samples will allow the detection of 80 to 90%. in this study at least two stool samples were obtained from all the children and three samples for more than 80% of the enrolled children. furthermore microscopy has the additional advantage of allowing the detection of other intestinal parasites. in our study assemblage a was more frequent, with six isolates, while assemblage b was detected in three children, which is the opposite pattern reported worldwide with assemblage b appearing more common [11, 12 ]. subassemblage determination was only possible for assemblage a positive samples, as b samples were impossible to subtype due to the presence of double peaks at specific position in the chromatogram. the importance of being able to subtype is especially relevant when a source of infection must be traced or when a distinction between reinfection / new infection is mandatory for therapeutic control. for instance, in this study, two brothers were infected with the same subassemblage (a2) of g. duodenalis, suggesting a common source of infection. another interesting data obtained from this work was the finding of a child (case 9) with malnutrition (moderate), which is a common situation in developing countries. this child also corresponds to the only one complaining of intermittent diarrhea. in this case while there has been a growing interest in the molecular characterization of g. duodenalis, there is still a lack of clear association between the assemblage and the clinical outcome, with contradictory results. a study conducted in the netherlands found a strong correlation between assemblage a and mild, intermittent diarrhea and assemblage b with severe and persistent diarrhea. other studies conducted in ethiopia and saudi arabia also suggest a correlation between the presence of symptoms and infection with the assemblage [28, 29 ]. on the other hand, a study performed in australia revealed that children infected with g. duodenalis isolates from assemblage a were 26 times more likely to have diarrhea than children with assemblage b. supporting these results other works in bangladesh and spain also showed a statistical association between assemblage a and symptomatic infections and between assemblage b and asymptomatic infections [30, 31 ]. in what concerns to portugal, the data obtained by sousa. supports that g. duodenalis belonging to assemblage a is strongly associated with symptomatic cases. in agreement with this, other work revealed a higher prevalence of assemblage b in asymptomatic children. none of the children studied presented diarrhea at the moment of the stool sample collection. however, of the four symptomatic children at enrolment, three were infected with a2 subassemblage with more evident symptoms when compared with the other symptomatic children carrying b assemblage. this study contributed with new and relevant data for the epidemiology of giardiasis in portugal and challenged the pattern of assemblage b being more frequent than assemblage a, eventually suggesting that a different epidemiological profile is detected in developed countries. the fact that a brother from an infected child was also infected reinforced the importance of when testing a child or a family member ; if positive, then the entire household should be screened. the study did not show an association between the clinical pattern observed and the assemblages. the low number of children infected with g. duodenalis does not justify the need for consistent request of stool parasitological analysis. however, special attention should be given to children reporting abdominal complains, considering that 4/9 of the infected children were symptomatic. | giardia duodenalis is the most prevalent intestinal protozoan infection especially in children. in portugal scarce data are available relative to this infection in preschoolers. the present study was conducted from april to july 2009 in public preschools in lisbon enrolling 316 children. stool examination was performed through microscopy. molecular analysis was conducted in all positive samples for g. duodenalis in order to determine the assemblage and subassemblage of this parasite. eight of the preschoolers studied children (2.5%, 8/316) were infected with g. duodenalis. additionally the brother of one of the infected children was also infected. genotyping analysis targeting ssu - rrna and -giardin loci revealed six infections with assemblage a and 3 with assemblage b. sub - assemblage determination was possible in four of the samples, with three a2 and one a3. the limited number of cases precluded an association of a determined symptom with an assemblage. the data presented here show the relevance of considering g. duodenalis analysis in children with intestinal complaints even in developed countries. |
in his letters to ladies, the american physician thomas ewell in 1812 advised women about, among other things, beautifying the skin. the state of the skin depends on the state of the body ; that to make it look well, you must make the body healthy. the connection between outer beauty and inner bodily health defined by skin has a long history. the conception of the skin as a receptacle for waste and as a layer of exchange with exhaling pores, and that it thus plays a key role in bodily health, was described already by plato. from the sixteenth century onwards, insensible and sensible perspiration appeared in writings on the body as purifying and beneficial for bodily health. by the early nineteenth century, however, the hygiene of body and skin had become a means to achieve both health and beauty. beauty and skin care for women were now defined by medical science and doctors advice. finally, for health reformer erasmus wilson the discovery of new anatomical parts inside the skin proved a central component in popular sanitary education from about 1840. although many authors of popular physiology books had discussed the role of skin cleanliness for health before this date, the defining role of skin anatomy for skin cleanliness was made only in 1835. that year french microscopists gilbert breschet and augustin roussel de vauzme published a treatise disclosing the anatomy of the sweat ducts, as was commemorated in an english publication on skin in 1837 : it was a great desideratum among english practitioners to have the true anatomy of the skin fully explained and demonstrated () but m. breschet and his confrre seem to have set the question at rest. erasmus wilson immediately adopted this new anatomy and physiology to explain the importance of healthy skin for sanitary purposes in popular handbooks on cleanliness. connecting skin as a regulator of animal heat, an organ for the removal of useless materials, and an organ of touch in one anatomical image, wilson turned skin into a tool for sanitary reform. his efforts to popularise skin anatomy presage the adoption of skin as a symbol for drainage of the body in a sanitary context. born the son of a naval surgeon and an artist, the london physician, skin specialist and sanitary reformer william james erasmus wilson became known to the general public for his philanthropy and popular books on the skin and cleanliness. wilson established himself as a surgeon, hygienist and productive author. in 1840 he started as a lecturer on anatomy and physiology at middlesex hospital where he set up his own practice. later, as a sub - editor of the lancet, wilson became interested in skin diseases and soon involved himself in medical establishment circles in london. a paper on the discovery of a new human skin parasite led to his acceptance as a fellow of the royal college of surgeons of england in 1843. a successful career as a surgeon and proponent of the study of skin diseased followed, with the foundation of a special journal on skin diseases, the journal of cutaneous medicine and diseases of the skin, and the establishment of a chair and museum of dermatology at the royal college of surgeons in 1869. in the medical arena, wilson s was one of a prominent london physician keen on developing the study and practice of dermatology. as one of london s elite practitioners and a wealthy philanthropist, wilson gained national fame around 1877 when he arranged for the obelisk known as cleopatra s needle to be transported to its rightful owner from egypt to british ground for around 10 000, using his profits from shrewd investments in gas and railways. this and his election as president of the royal society of the college of surgeons in 1881 were followed by publications in popular magazines such as vanity fair and the graphic. the obelisk can still be admired at the victoria embankment of the thames, as a prominent reminder of victorian nationalism and wilson s philanthropy. other projects more in keeping with his professional interests included his financial support for a new wing and chapel at the royal sea - bathing infirmary at margate. an essential part of wilson s public presence was his knowledge of healthy and diseased skin. in 1845 he published the best seller on the management of the skin as a means of promoting and preserving health. this pocket - size volume went through seven editions under different titles and remained in print for thirty - one years, mainly as healthy skin : a popular treatise on the skin and hair, their preservation and management. the book relates wilson s experiences with skin diseases and his involvement with popular physiology and health education. as the title suggests, wilson extensively discussed the anatomy of the skin and ways to preserve its health in order to maintain and control bodily health in general. in contrast, the abundant gory stories about all kinds of skin affections in the latter part of the book served as a harsh reminder of what could happen if one did not stick to wilson s rigid regimen of cleanliness. he introduced a view of the skin anchored in recently acquired anatomical knowledge, but also earlier concepts of cleanliness and health. his purpose was to supply a knowledge of that part of the economy of man which forms the exterior of his body and is more immediately under his own personal control namely, the skin, the nails, and the hair. against the background of the devastating asiatic cholera epidemics, wilson wanted to draw attention to the skin as the primary focus of cleanliness in every sense. public bathhouses and washhouses especially drew his attention. he commemorated the success of the first public bath in london at easton square in the parish of st pancras, which had drawn 674 866 people to the establishment since its opening in 1846 : [they ] have obtained the incalculable blessing of clean skins or clean linen. healthy skin, dedicated to hygienist edwin chadwick, thus fitted with the emergence of a quest for cleanliness and municipal reforms to improve conditions for the poor in britain s crowded cities. although wilson was not the first to discuss skin cleanliness, he promoted an understanding of the skin as an important cleansing agent in the context of recent insights into skin s complex cellular anatomy and at the service of sanitary reform of the labouring classes. he meticulously discussed the anatomical and physiological characteristics of the skin, such as the perspiratory system, which consisted of very minute cylindrical tubes travelling through the different layers of the skin, regulating the temperature of the body and removing noxious compounds. the anatomical knowledge of the layers, membranes, organelles and cellular structures of the skin became the foundation for the rules for the management of the skin. as wilson argued, by processes of formation and growth an action necessary, not merely to the health of the skin, but to that of the entire body, is established. wilson aimed to educate his readers on ways to keep the skin in a healthy state. anatomy, physiology, diseases, diet, clothing and above all bathing were discussed to this end. what was victorians understanding of the skin at the time wilson published healthy skin ? in general, people regarded the skin as a sensitive covering for the body. exhalant of waste matter from the system, as heat regulator, as a layer for absorption and as seat of sensation and touch. describing the (non - visual) anatomy and physiology of the skin, many medical authors relied on the works by the german christoph wilhelm hufeland and the french xavier bichat. popular magazines discussed a way of finding the pores in the skin with a glass as entertainment. and mothers worried about their babies health were taught how to care for their skin. in general, as a sensitive limit, the skin was understood to have an intimate relation to other excreting organs, such as the lungs, stomach, liver and bowels. relaxation, stimulation or irritation caused for example by bathing, rubbing or even clothing, influenced the functions of the skin and thus health in general. now wilson and other sanitarians moved skin hygiene into the context of the public health movement. wilson argued for skin cleanliness as the essence of both private hygiene and public health. he called for public health for the masses by creating public washhouses and bathhouses : they are the means of drawing the rich nearer to their poorer brethren ; and they enable us to fulfil () the divine injunction, to love one another. for wilson, however, public measures were founded in personal care of the skin. he directly addressed the reader s responsibility for keeping the skin healthy and vital : health lies in the equilibrium, and the duty of the individual to himself is to pursue that state as nearly as possible. he called cleanliness of the skin a value. without regular washing of the skin with water, a crust of impurities from the skin itself, from the atmosphere, and from the clothes would cover the skin. dust particles, soot, poisonous gases, miasmata and infectious vapours would adhere to the skin and cause problems of all kinds. a growing devotion for tubbing played on both the regime of cleanliness for the individual and the working classes. healthy skin set the agenda for the skin as the locus of cleanliness rituals, healthy bodies, and moral control of society. geographically, readership of healthy skin spread through britain and america while the book was also noticed on the continent. many subjects discussed in the book, such as proper diet and clothing, exercise and bathing, had appeared in popular health advice books and monthly family health magazines from the 1820s onwards. demographically, wilson likely had in mind a female audience for he often mentioned children and their health. his lady - readers with images of little animals inhabiting the oil glands of the skin. this elicited a completely different reaction from another reviewer, who even applauded wilson s discussion of the so - called entozoon folliculorum. wilson was praised for democratising the dangers of uncleanliness of the skin by showing how great unwashed : the delicate town - bred lady of fashion, in descending from her carriage, shrinks instinctively from the mass of rags, filth, and vermin, which is brought into continuity with her precious person by some pertinacious beggar ; ignorant all the while that her sebaceous follicles give board and lodging to a host of parasites, whose number may equal that of the various kinds of small deer that nestle in the matted hair and tattered garments of the fellow - being whom she regards with such loathing. the delicate town - bred lady of fashion, in descending from her carriage, shrinks instinctively from the mass of rags, filth, and vermin, which is brought into continuity with her precious person by some pertinacious beggar ; ignorant all the while that her sebaceous follicles give board and lodging to a host of parasites, whose number may equal that of the various kinds of small deer that nestle in the matted hair and tattered garments of the fellow - being whom she regards with such loathing. such moral use of knowledge of the skin s anatomy could thus serve a call to bridge class barriers. the medico - chirurgical review argued against wilson s discussing too minutely the anatomy of the skin and his popular descriptions of diseases and treatments, in particular his advocacy of hydropathy. a man of his professional status and standing should not busy himself with the popularisation of professional knowledge ; that would be useless and ridiculous. an extensive review in the british and foreign medical review argued that popular hygiene had been discussed much better by others before wilson, for example in the work of andrew combe. still, healthy skin was considered important to forward the bath and wash - house movement ; which, combined with those which are taking place of early shop - closing, of draining and ventilation, and of public places of recreation, we look upon as the most pleasing and favorable of the sign of the times. a review in the american medical examiner was wholly positive, praising the popular treatise as an excellent popular exposition and an agreeable work. in the religious and popular press, healthy skin was much praised and quoted. the american journal the biblical repository and classical review called healthy skin a medical work, yet of value to every person. other positive reviews appeared for example in the the living age and in the eclectic magazine, which reprinted articles from the spectator in 1846 and from the british jerrold s magazine in 1847. the review of 1846, praised wilson s work for its successful popularisation of the topic : it is a lucky subject ; for we all have a skin, and our health greatly depends upon its health. the book could not be too highly prized. an extensive editorial review in the methodist magazine the ladies repository, a monthly periodical on literature and religion, discussed wilson s findings on the skin at length. anatomical characteristics, interesting facts and advise about bathing and skin health appeared in the editorial. the importance of bathing was put in direct relation to the anatomical make - up of the skin : every moment of one s life a multitude of useless, corrupted, and worn - out particles evaporate through the numberless small vessels of the skin in an insensible manner, and if these are not promptly and properly removed you will have to pay for your indiscretion with compound interest (.) the way most people act would indicate that water was poisonous as a general wash, and that soap was a powerful irritant. away with such nonsense. there does not exist in the art of living a greater device for securing a vigorous and buoyant existence than bathing. every moment of one s life a multitude of useless, corrupted, and worn - out particles evaporate through the numberless small vessels of the skin in an insensible manner, and if these are not promptly and properly removed you will have to pay for your indiscretion with compound interest (.) the way most people act would indicate that water was poisonous as a general wash, and that soap was a powerful irritant. away with such nonsense. there does not exist in the art of living a greater device for securing a vigorous and buoyant existence than bathing. besides his promotion of bath and washhouses, wilson s vivid and appealing description of the perspiratory system was quoted over and over again throughout the second half of the nineteenth century. in a time of growing concern about drainage, sewage, and ventilation in the middle - class house and the city his discussion of the tasks performed by the skin, perspiration in particular, hit a nerve. from the 1840s, calls for skin cleanliness became a tool for hygienists in the sanitary reform of the home and the city. in a passage from healthy skin, wilson drew an analogy of the skin to the city s sewage system that was immensely popular for the remainder of the nineteenth century. in popular physiology and health books on human anatomy, authors connected wilson s drainage analogy to pictures of the microscopical make - up of the skin, as well as to experimental proof of the importance of skin for health. images of the skin with its own plumbing finally played a crucial role in the dissemination of the idea of a healthy functioning skin within sanitary reform. for victorians around 1850 architecture, science, engineering, politics and economics all influenced the body in one way or another. public health authorities and sanitary reformers acted through engineering, control, regulation and sewage systems to ensure proper living conditions in cities and lowering of mortality rates. in particular, the sewerage doctrine by the famous sanitarian edwin chadwick included measures for drainage of waste waters from houses through pipes. this was the scene for wilson s writings : a society preoccupied with water and wastes ; a society moving towards modern sanitation, less stench, less noxious gases, less death. one of the most frequently quoted passages from wilson s book on healthy skin was his calculation of the length of the marvellous and extended perspiratory system. he used this passage to stress the importance of the skin and to endorse washing as a way to manage and control health : to arrive at something like an estimate of the value of the perspiratory system in relation to the rest of the organism i counted the perspiratory pores in the palm of the hand, and found 3528 in a square inch. now, each of these pores being the aperture of a little tube of about a quarter of an inch long, it follows, that in a square inch of skin on the palm of the hand there exists a length of tube equal to 882 inches, or 73 feet. surely such an amount of drainage as seventy - three feet in every square of skin, assuming this to be average for the whole body, is something wonderful, and the thought naturally intrudes itself : could we need a stronger argument for enforcing the necessity of attention to the skin ? () to obtain an estimate of the length of tube of the perspiratory system of the whole surface of the body, i think that 2800 might be taken as a fair average of the number of pores in the square inch, and 700 consequently, of the number of inches in length. now, the number of square inches of surface in a man of ordinary hight and bulk, is 2500 ; the number of pores, therefore, 7 000 000, and the number of inches of perspiratory tube 1 750 000, that is, 145 833 feet, or 48 600 yards, or nearly twenty - eight miles. to arrive at something like an estimate of the value of the perspiratory system in relation to the rest of the organism i counted the perspiratory pores in the palm of the hand, and found 3528 in a square inch. now, each of these pores being the aperture of a little tube of about a quarter of an inch long, it follows, that in a square inch of skin on the palm of the hand there exists a length of tube equal to 882 inches, or 73 feet. surely such an amount of drainage as seventy - three feet in every square of skin, assuming this to be average for the whole body, is something wonderful, and the thought naturally intrudes itself : could we need a stronger argument for enforcing the necessity of attention to the skin ? () to obtain an estimate of the length of tube of the perspiratory system of the whole surface of the body, i think that 2800 might be taken as a fair average of the number of pores in the square inch, and 700 consequently, of the number of inches in length. now, the number of square inches of surface in a man of ordinary hight and bulk, is 2500 ; the number of pores, therefore, 7 000 000, and the number of inches of perspiratory tube 1 750 000, that is, 145 833 feet, or 48 600 yards, or nearly twenty - eight miles. this passage touched the imagination of many audiences throughout the second half of the nineteenth century. it was used over and over again in anglo - american medical, popular and educational literature well into the second half of the nineteenth century. the strength of the calculation relied upon the idea of such an immense length of drainage tubes within such a thin layer of the body. in industrialised societies, the advent of the steam train and steamship was changing people s experience of distance and time. as the essex surgeon john coventry noted in reference to wilson s calculations of man s twenty - eight miles of perspiration tubes : () an hour s railway ride, forsooth ! () we shall arrive at something like an appreciation of the importance of keeping this pipeage pervious. wilson s popular call for the importance of the skin found fertile soil in health education literature for women, workers and children. the american physician calvin cutter, for example, copied large parts of wilson s writings on skin cleanliness in his textbooks on anatomy and physiology designed for schools and colleges. keeping wilson s calculations on perspiration in mind, management of skin cleanliness was of interest not only to the female audience, but also for men s work practices. in the farmer s magazine, the passage on the perspiratory system was transposed to the health of dairy cattle, as to demonstrate the importance of cleaning the cows. in the context of sanitary reform, wilson s popularisation of experimental and microscopical researches on the skin provided a confirmation of the ancient belief that clogged skin pores caused diseases and disaster. as the reverend charles hole stated in his practical moral lesson book, think of the twenty - eight miles of drainage in the human body being blocked up by uncleanliness ! is it not a wonder that unclean persons have any health at all?. imagine what would happen if such an extended system of drainage for the body was clogged up with dirt. wilson secured his arguments for skin cleanliness first by his popular translation of recent french experimental research on the physiology of the skin. physiologists developed a special interest in the role of the skin for balancing and the exchange of gases and fluids from around the 1830s. in the following decade french and german physiologists performed animal experiments in an attempt to explain the respiratory and perspiratory functions of the skin. in healthy skin, wilson discussed the experiments on animal temperature by fourcault, as well as experiments done by alfred becquerel and gilbert breschet. these experiments showed that rabbits, horses and birds whose skin was covered with a thick layer of impermeable varnish apparently asphyxiated and died the impact of these experiments was reinforced by part of fourcault s experiments recounting the striking story of a boy who s skin was covered in gold on the occasion of the election of pope leo x and died soon after. figure 1:microscopical depiction of skin in healthy skin : a popular treatise on the skin and hair, their preservation and management (1853). reproduced with kind permission of the wellcome library, london. microscopical depiction of skin in healthy skin : a popular treatise on the skin and hair, their preservation and management (1853). wilson s entertaining calculations had serious consequences and the functions of skin should not be underestimated. in an article in the scientific american from 1863 on the wonders of the skin both fourcault s experiments and wilson s calculations were discussed, without mentioning their names however, concluding : the skin, although so simple in appearance, affords a beautiful illustration of the infinite skill and wisdom of the great creator, not only in its wonderful structure, but with respect to all its varied functions. in addition to the link with experimental research on the skin, wilson also managed to connect the newly introduced microscopical imaging of the skin and its sweat ducts to his calculations. following the diagrammatic illustrations of breschet and de vauzme, wilson incorporated a recent standard microscopical depiction of a cross section of skin in his popular work (figure 1). from now on, in works on human physiology wilson s calculations of the perspiratory ducts were connected to the discoveries and visual diagrammatic representations of breschet and de vauzme. this combination of visual imagery with recent experimental research and wilson s calculations found its way to popular literature on the skin. the authors of the family cyclopaedia of 1859, for example, presented a diagram of the structure of the skin with wilson s numbers on drainage as useful knowledge. figure 2:cross - section of london holborn viaduct, showing the city sewerage. a section through the roadway of holborn viaduct, london : looking east, showing the middle level sewer. wood engraving after w. haywood, 1854. cross - section of london holborn viaduct, showing the city sewerage. a section through the roadway of holborn viaduct, london : looking east, showing the middle level sewer. reproduced with kind permission of the wellcome library, london. soon after the appearance of healthy skin, hygienists positioned wilson s attention to the skin as a tool in the moral and political call for cleanliness of the masses. in an article in the lancet in 1846, essex surgeon john coventry addressed the the mischiefs of uncleanliness and the public importance of ablution. contrary to what this title might merely suggest, coventry mainly called for attention to hygiene, and ablution in particular, while seeking to elucidate the various evils of dirtiness as deduced from the structure and functions of the skin. drawing on many of wilson s ideas, coventry reaffirmed that the skin, so consentaneous in its operations, influenced health in many ways and, therefore, must be kept in the utmost proper condition. most importantly, coventry drew a direct connection between the broader issue at hand, namely the state of hygiene and sanitation in britain, to the situation of skin cleanliness and health. the skin, being the grand drainage - pipe of body reflected the state of the sanitation and sewage in the city : having traced so large an amount of disease and misery to uncleanliness, how deeply must we regret that london is still the great unwashed, as tightly packed as the hides in a tanyard. from year s end to year s end, the skins of the metropolis, glazed and varnished, reek in their accumulated sordes, to the sad detriment of comfort, comeliness, health, and longevity. having traced so large an amount of disease and misery to uncleanliness, how deeply must we regret that london is still the great unwashed, as tightly packed as the hides in a tanyard. from year s end to year s end, the skins of the metropolis, glazed and varnished, reek in their accumulated sordes, to the sad detriment of comfort, comeliness, health, and longevity. the analogy between the skin as indispensable bodily drainage system and the necessity of proper city sewage and bathing facilities was crucial. while wilson himself in early editions of healthy skin alluded to a similar metaphor, his discussion of the skin in his book on the turkish bath was even more striking. in the eastern, or turkish bath : its history, revival in britain and application to the purpose of health (1861), wilson referred to the skin as a sanitary commissioner, draining the system of its impurities. in a slightly different way, coventry s article in the made a connection between the skin (pores) and cleanliness of body and society at large : [w]e hope that every facility may be afforded for public bathing ; that cleanliness may no longer be viewed as a luxury accessible only to the wealthy, but that, before the ensuing parliamentary session, the pores, as well as the ports, of our mother country may be rid of their imposts, and unreservedly thrown open. [w]e hope that every facility may be afforded for public bathing ; that cleanliness may no longer be viewed as a luxury accessible only to the wealthy, but that, before the ensuing parliamentary session, the pores, as well as the ports, of our mother country may be rid of their imposts, and unreservedly thrown open. the work by edwin chadwick also focused on the parallels between skin and subterranean drainage. in the well - known report on the sanitary conditions of the labouring classes, the term drainage obviously occurs many times. yet in a later article dedicated specifically to skin cleanliness, chadwick described the skin as the last stage of the sewage of the body. he argued for the education of children in schools as the first step towards hygienic reform. his crusade against the sanitary evil of personal filth required a regular cleaning of the skin as a disciplinary ritual for children. thus, the skin becomes for the body what drainage and sewage should be for the city. this is why wilson s quote about the skin s twenty - eight miles of drainage was such a powerful and almost tangible explanation. these tubes directly connected cleanliness of the body to the house, and the house to the city. from the 1870s, as medical historian annmarie adams has shown, physicians of the domestic sanitation movement applied principles of physiology to architecture and in so doing treated body, house, and city as part of a single system. obsessed with drainage, sanitarians urged the removal of all waste from the house and the city via well - designed intestines, while, as pamela gilbert has argued, analogies of body and home cleanliness were also directed towards women in an effort to prevent the spread of epidemic disease. skin had been a particularity gratifying subject for analogy ever since erasmus wilson articulated its physiology as a tool for sanitary reform by stressing drainage, plumbing and exhalation. with its sweat ducts taken as drains and sewers of the body, the anatomy of the skin was visually transposed onto the sewerage system of the house and the london city in cross - section imagery (compare figures 1 and 2). when chadwick in 1875 wrote on the importance of clean streets for the prevention of disease and mortality, he alternately stressed skin cleanliness for children and the dangers of excrement - sodden street surfaces. moving between the skins of playing children and the urban filth in the street, chadwick put skin cleanliness on the same level as city cleanliness. the surfaces of the body and the street, both had to be rid of the evils of waste. skin cleanliness as proposed by wilson and other sanitarians stressed evacuations of waste and symbolised the promise of control over health and disease. to act on skin cleanliness i will next discuss how soap advertisements from the pears soap company became a visual analogy for the removal of unwanted moral wastes and the consequent promise of health and beauty. lithograph after h. stacy marks ; after 1881. reproduced with kind permission of the wellcome library, london. lithograph after h. stacy marks ; after 1881. reproduced with kind permission of the wellcome library, london dirt, as lord palmerston defined it, is matter in the wrong place. to remove this to the right place, so far as the human being is concerned, and to remove it without detriment to the health of the skin, is the function of soap. dirt, as lord palmerston defined it, is matter in the wrong place. to remove this to the right place, so far as the human being is concerned, and to remove it without detriment to the health of the skin, is the function of soap. if skin provided the necessary drainage of waste materials through its plumbing, soap was indispensable to remove unwanted matter. soap manufacturers such as pears presented the soap bar as a medium that would rid the skin of all unwanted dirt, thereby turning skin into locus of control over body, race and beauty. supported by the scientific knowledge of the workings of the skin, erasmus wilson and others became prominent public figures in pears soap advertising. the ideal of a white, morally uplifted, unspoiled, civilised, beautiful skin could be achieved by removing skin cleanliness became part of mass - media advertising in the pears campaign after 1865. around the middle of the nineteenth century, the london - based firm a. & f. pears developed from a small enterprise into a large, well - known commercial business, partly due to the remove of the excise on soap in 1853. the industrial production of everyday products such as soap created a culture of mass consumption from the 1850s onwards. a resourceful man with insights into publicity, pears business associate thomas barratt completely changed the marketing and distribution system for pears. one of barratt s famous stunts involved the distribution and circulation of coins imprinted with the name pears throughout britain. in the 1880s, pears spent around 30 00040 000 pounds per year on advertising their soap. barratt also used the authority of prominent skin specialists, chemists and doctors in the pears campaign. specialist on the skin, wilson was the perfect candidate to endorse pears transparent soap and his name appeared repeatedly in pears advertisements from the 1860s on (see figure 4). wilson s endorsements for pears soap originated partly from his own medical works. in healthy skin indispensable aid and as a tool to preserve the skin in health. in an article on toilet soaps in the journal of cutaneous medicine, he also advocated the use of soap for beauty purposes : we once knew a beautiful woman, with a nice complexion, who had never washed her face with soap all her life through ; her means of polishing were, a smear of grease, eg. cold cream, then a wipe, and then a lick with rose - water. of course we did not care to look too closely nor to approach too closely such an avowal ; and we have met in the world with persons so unfortunate as to be unable to bear soap to their skin at all. soap is to the skin what wine is to the stomach, a generous stimulant ; and a solvent to boot of the surface which holds the dirt. we once knew a beautiful woman, with a nice complexion, who had never washed her face with soap all her life through ; her means of polishing were, a smear of grease, eg. cold cream, then a wipe, and then a lick with rose - water. of course we did not care to look too closely nor to approach too closely such an avowal ; and we have met in the world with persons so unfortunate as to be unable to bear soap to their skin at all. soap is to the skin what wine is to the stomach, a generous stimulant ; and a solvent to boot of the surface which holds the dirt. an article of the nicest and most careful manufacture, and scentless or scented, one of the most refreshing and agreeable of balms to the skin. within a few years, this statement would be quoted repeatedly in pears advertisements in newspapers, magazines and on posters in the city streets and objects such as bookmarks. the close connections between wilson and pears did not go unnoticed. on several occasions, wilson was accused of commercial exploitation of his name and reputation. in a letter to the editor in the lancet in 1864, wilson defended himself against some critical questions posed by an anonymous reader. in response to the accusation that he sold his name to all kinds of hairwashes and pomades wilson responded that he could not escape the commercialisation of his name : it has happened to others as well as myself, and we see daily the names of eminent men associated with popular remedies of various kinds prepared from their prescriptions. i can only say that i have not encouraged this practice in my own instance. with regard to pears in particular, a letter on the issue of an implied partnership between wilson and pears explains the sensitivities. apparently, mr pears had met with a member of the medical profession and stated that he had a partnership with mr erasmus wilson. the aim of the author of the letter was to firmly deny all communication between pears and wilson : i know that mr wilson has never had any communication with messrs. i had some months ago to compel the firm under threat of proceedings by injunction to alter at a great expense the whole of their advertisements and this they have done and with the exception of this i know that mr wilson has never had anything to do with them indeed i doubt if there is such a person as pears in existence and i only regret i was unable to remove from the advertisement all mention of mr wilson s name, but i could not do so as a statement to the effect of that made in the advertisement was incautiously inserted in a work edited by mr wilson. i had some months ago to compel the firm under threat of proceedings by injunction to alter at a great expense the whole of their advertisements and this they have done and with the exception of this i know that mr wilson has never had anything to do with them indeed i doubt if there is such a person as pears in existence and i only regret i was unable to remove from the advertisement all mention of mr wilson s name, but i could not do so as a statement to the effect of that made in the advertisement was incautiously inserted in a work edited by mr wilson. despite this statement we may assume that wilson did not take any action to remove his name from the advertising campaign, since later advertisements still featured his name. wilson s appearance in the pears advertisements, wanted or unwanted, created a visual connection between medical expertise, skin cleanliness and soap. in soap advertising, control over dirt removal was visually encouraged in three ways. first, advertisements reinforced the discourse of the removal of the undesired behaviour of the lower and working classes. this is represented, for example, in the so - called you dirty boy advertisement (see figure 5). illustrated london news, 14 december 1889, 778. reproduced with kind permission of the courtauld institute of art. reproduced with kind permission of the courtauld institute of art. the humorous image of a young, ragged boy having his ears washed by an old woman was based on a famous statue by london - based artist giovanni focardi (18421903) and commissioned by the a. & f. pears company for the paris world fair as pears owned the right to make copies and use them as advertisements for pears soap, small - scale terracotta versions were spread around great britain and united states. for audiences of the time, the power of the representation lay in its irresistibly droll performance that everybody recognised : for most of us have had grandmothers, and more of us have violently objected to having our faces washed. in paris alone, hundreds of thousands of visitors admired the statue. it figured prominently in the stand of pears at the international health exhibition in london 1884 (see figure 6). pears thus often played on the contrast between the innocence of children versus disobedient youth. to rid the body of its bad behaviour through the skin was of a type with the religious cleansing of sin (see figure 3). figure 6:you dirty boy statue by giovanni focardi as part of the pears soap stand at the international health exhibition. illustrated london news, 2 august 1884, 101. reproduced with kind permission of the courtauld institute of art. you dirty boy statue by giovanni focardi as part of the pears soap stand at the international health exhibition. illustrated london news, 2 august 1884, 101. reproduced with kind permission of the courtauld institute of art. a second analogy to the removal of unwanted matter in soap advertising concerns the representation of ethnic stereotypes and the whitening of the skin of uncivilised black people. in historical analyses of soap ads, the colonial context and racial connotations as ann mclintock argued with the image of a black toddler washing himself : in the victorian mirror, the black child witnesses his predetermined destiny of imperial metamorphosis but remains a passive racial hybrid, part black, part white, brought to the brink of civilization (see figure 7). figure 7:advertisement for pears soap. pears transparent soap for improving the complexion. pure, fragrant, durable., 1884 (folding sheet, 22 cm). reproduced with kind permission of the british library board (evan. pure, fragrant, durable., 1884 (folding sheet, 22 cm). reproduced with kind permission of the british library board (evan. highly humorous dual picture of the black boy washing his black body, but not his face, white with pears soap. the effectiveness of soap on the skin, as testified to by authorities like erasmus wilson, was so refreshing and balmy that it cleansed only those parts in contact with the water. dirty boy ad, where the face was the only thing left to be rid of dirt. the implicit message of the ads was that pears soap could relieve the skin of unwanted dirt and skin colour. the connection between a healthy skin system and (white) beauty was indicated with the slogan matchless for the complexion. in beauty guides the connection between healthy skin and beauty was promoted with physiological facts about the workings of the skin system. in what our girls ought to know, for example, natural history teacher mary studley in a chapter on how to become beautiful referred to the lesson that all beauty must be organic when discussing skin. to stimulate the excretory power of the skin using water was good physiological management for a girl to obtain a radiant halo. pears advertising alluded to this connection between the physiological management of the skin system and beauty resulting from that. one ad typically quoted both erasmus wilson about the beneficial workings of soap for the skin and adelina patti on her experience with the soap for the complexion. opera singer patti, like actress lillie langtry, figured prominently in many pears soap advertisements. since patti was one of the most photographed women of the victorian era, the public connection between her beauty and pears soap was easily reinforced. with its radiant, enamel - like surface gleaming amid the heavy dark - line engravings, the face was the focal point in advertisements that held the promise of a beautiful complexion (see figure 8). advertisements for pears soap can be taken as constitutive of late victorian visual body culture. soap advertisements generated an ideal of control over waste removal analogous to the subterranean sewage systems in homes and city streets. paradoxically, while the abundance of soap advertisements stressed a visual healthy skin, cleanliness lost its visibility. yet the multi - layered artistic exposition of skin referred to control over skin colour, beauty and behaviour. the reality of modern city life for the middle class (women especially) meant that there was a real possibility of coming into contact with dirt, visible or invisible. the modern - life painting by george william joy the bayswater omnibus (1895) shows a scene in which pears soap advertisements are visible in the background (see figure 9). in the cramped space of the omnibus, the fair skin of the fashionable young woman on the viewer s left of the painting and the milliner to her left, run the chance of getting dirty on the trip. joy, 1895. reproduced with kind permission of the museum of london (www.museumoflondonimages.com). due to copyright restrictions the high - resolution version is only available in the printed version of the journal. joy, 1895. reproduced with kind permission of the museum of london (www.museumoflondonimages.com). due to copyright restrictions the high - resolution version is only available in the printed version of the journal. in another narrative painting by george earl, going north, king s cross station (1893), a pears soap ad offers the promise of washing away the dirt that comes from contact with people of various social classes and the filth of the city for fashionable middle - class women. during the nineteenth century, people s conception of skin was greatly influenced by developments in sanitation for living spaces. for physicians like erasmus wilson, the analogy between the sewerage of the skin and that of the house or city proved an effective way of promoting his popular message to the rising middle class. draining body and society of unwanted matters, reminded to city life and the constant flux of wastes that demanded control. in addition to deepening our understanding of skin cleanliness and victorian body culture, this article demonstrates the ways in which visual representations of healthy skin functioned in household handbooks, popular literature and city life. the popularisation of microscopical imagery and its analogy to municipal sewage systems defined skin cleanliness as way to rid body, city and society of unwanted dirt, disease and contact. a focus on skin reveals the connections between the personal space of the body, the home and the city. promoting the practice of cleaning the skin strengthened the links that reformers sought to make between the health of the individual, the city and society at large. | the concept of a healthy skin penetrated the lives of many people in late - nineteenth - century britain. popular writings on skin and soap advertisements are significant for pointing to the notions of the skin as a symbolic surface : a visual moral ideal. popular health publications reveal how much contemporary understanding of skin defined and connected ideas of cleanliness and the visual ideals of the healthy body in victorian britain. characterised as a sanitary commissioner of the body, skin represented the organ of drainage for body and society. the importance of keeping the skin clean and purging it of waste materials such as sweat and dirt resonated in a britain that embraced city sanitation developments, female beauty practices, racial identities and moral reform. by focusing on the popular work by british surgeon and dermatologist erasmus wilson (180984), this article offers a history of skin through the lens of the sanitary movement and developments in the struggle for control over healthy skin still in place today. |
1) is a newly approved orphan antiepileptic drug (aed) [1, 2 ]. a characteristic feature of the drug is the presence of a chiral center at c-3. stp is marketed as a racemic mixture of two enantiomers : r(+)-stp and s()-stp of which r(+)-stp has about 2.4-fold greater anticonvulsant potency and it is also associated with ~3-fold faster elimination rate than s()-stp. the compound has been under investigations for nearly 4 decades [6, 7 ] ; its clinical development was delayed due to the inhibitory effect of stp on hepatic cytochrome - p450 (cyp-450). stiripentol, (diacomit ; biocodex inc.) has been approved by the european and canadian marketing authorization for the treatment of dravet syndrome or severe myoclonic epilepsy in infancy (smei) in conjunction with sodium valproate and clobazam when seizures are not adequately controlled with the association of these two medications [9, 10 ]. stp inhibits gaba metabolism through the blockade of gaba - transaminase activity and reduces synaptosomal uptake of gaba, leading to an increase in gaba brain content. stp, at relevant clinical concentrations (30300 m), markedly increases the mean open duration of gabaa receptor dependent chloride channels by a barbiturate - like mechanism. stability testing of pharmaceutical dosage form of stp or any other drug, (i.e., purity and stability of the active ingredient and/or final product) is imperative for the optimum and efficient delivery of its therapeutic activity to the patients. the purpose of stability testing is to investigate changes in the quality of a drug substance or a drug product with time under the influence of environmental factors, such as temperature, humidity, and light. moreover, stability testing establishes the recommended storage conditions and shelf life for the drug product [14, 15 ]. this is actually due to the fact that presence of impurities and/or potential degradation products may alter the pharmacological and/or toxicological characteristics of the active drug [16, 17 ]. in particular, pharmaceuticals are significantly sensitive to environmental conditions, which are usually varied during the different stages (i.e., manufacturing, transportation and storage) of the finished products. based on the aforementioned importance of stability testing, it is vital to investigate degradation pathways and the intrinsic stability of the drug, compatibility of the drug with the excipients in the dosage form, and the long - term effects of the environment. recently, the need of establishing a stability - indicating assay method for the stability testing has become more clearly mandated in the official guidelines at the international conference on harmonization (ich) and united states pharmacopeia (usp). a thorough literature survey revealed that only few high performance liquid chromatography (hplc) methods were reported for the determination of stp to study its pharmacokinetics [7, 18 ], enantioseparation, and its estimation in plasma. consequently, a stability - indicating method for determination of stp in its bulk drug and pharmaceutical capsules is important. accordingly, the present study describes for the first time a comprehensive stability - indicating study for stp. this study aims to develop and fully validate a rapid, simple, and accurate hplc - dad method to quantify stp in the presence of its degradation product in bulk and pharmaceutical dosage form. additionally, structure elucidation techniques including the h - nmr, c - nmr, and mass spectrometry were used to identify stp degradation product. stiripentol reference standard (purity ~99.6%) was purchased from sigma - aldrich co. (st. louis, mo, usa). diacomit capsules (biocodex, montrouge, france), labeled to contain 250 mg (as the anhydrous base) per capsule, were procured from the local market. hplc - grade solvents and reagent - grade sodium hydroxide, hydrochloric acid, hydrogen peroxide, potassium dihydrogen phosphate, and orthophosphoric acid were purchased from merck (darmstadt, germany). ultrapure water of 18 was obtained from a millipore milli - q uf and purification system (millipore, bedford, ma, usa) was used throughout the study. hplc apparatus consists of a waters breeze system consisting of 1525 binary pump with online degasser, 717 + autosampler, 5ch thermostated column compartment, and a 2996 photodiode array detector (dad) (waters corporation, milford, ma, usa). the chromatographic separations were performed on a symmetry c18 column (3.5 m, 75 mm 4.6 mm i.d) manufactured by waters corporation, milford, ma, usa. the column temperature was kept constant at 25 2c. separations were performed in isocratic mode using a mobile phase consisting of acetonitrile and 50 mm potassium dihydrogen phosphate buffer (60 : 40, v / v), and ph was adjusted to 4.1 0.1 with 10% phosphoric acid solution. the mobile phase was filtered through a millipore vacuum filtration system equipped with a 0.45 m filter (millipore, bedford, ma, usa), degassed by ultrasonic water bath prior to its use. the flow rate of the mobile phase was 1 ml / min, and the sample injection volume was 20 l. the dad detector was set at scan range of 210400 nm to allow investigation of any impurities and/or degradation products with shorter wavelength. additionally, quantitation of stp and its acidic degradation product was performed at 262.5 nm. prior to each run, the hplc - dad system was allowed to warm up for nearly 30 min and the baseline was monitored until it was stable before the samples were injected. peak identity was confirmed by retention time comparison and comparison of the spectra obtained from the dad detector. stp stock solution (1 mg / ml) was prepared by dissolving 25 mg of stp reference standard material into 25 ml methanol in a 25 ml volumetric flask and completing the volume properly. this stock solution was later diluted with methanol to produce a working standard solution of 100 g / ml. an eight - point calibration curve (1, 2, 4, 6, 10, 15, 20, and 25 g / ml) was constructed by plotting the peak area of stp (y) versus stp nominal concentration (x). slope, intercept, and r values were calculated as regression parameters by linear regression. the linear regression equation was used to calculate the concentrations of stp in aqueous solutions and dosage form based on their peak area. sample solutions were prepared by diluting the working solution with mobile phase to obtain final concentrations of stp. twenty capsules (diacomit 250 mg capsules ; batch number 2611) were weighed and average weight was determined. capsules were emptied and two portions (250 mg each) were separated where one portion was transferred to a 500 ml volumetric flask. a volume of 250 ml of methanol was added, the contents were mechanically shaken for 10 min and ultrasonicated for 5 min, and the volume was diluted to 500 ml with methanol. this solution (0.5 mg / ml) was diluted as required for analysis. the remaining 250 mg powder was used as it is for thermal and photodegradation studies. thermal degradation of the drug substance in its bulk form and capsules was carried out in both solid and solution state. powder samples of standard stp (10 mg) and diacomit capsules (10 mg of capsule content) were kept in a controlled - temperature oven at 80c for 48 hours. solutions were then diluted with mobile phase to obtain a concentration of 10 g / ml, and a volume of 20 l of each solution was injected into the hplc system. meanwhile, a volume of 1 ml of stp working solution (100 g / ml) and capsule extract solution (100 g / ml ; prepared as previously mentioned in section 2.5.) was transferred to small round bottom flasks and subjected to reflux at 80c for 48 hours. the solutions were cooled to room temperature (25 5c) and diluted with mobile phase to yield final concentrations of 10 g / ml ; and a volume of 20 l of each solution was then injected into the hplc system. aliquots of 1 ml of stp working solution (100 g / ml) and capsule extract solution (100 g / ml) were transferred to small round bottom flasks. in the preliminary forced degradation testing, it was observed that acid degradation takes place rapidly and the drug was almost fully degraded when 0.5 n hydrochloric acid was used. therefore, in later experiment, acid degradation was performed with a maximum of 0.1 n hydrochloric acid. ultimately, solutions were individually mixed with 4 ml of (0.005 n, 0.01 n, 0.05 n, and 0.1 n hydrochloric acid) and/or (0.05 n, 0.1 n, 0.5 n, and 1 n sodium hydroxide). the prepared solutions were heated for 1, 2, and 3 hours at 60c in a water bath. the samples were cooled to room temperature (25 5c), neutralized with an amount of acid or base equivalent to that of the previously added. the resulting neutral solutions were then diluted with mobile phase to 10 g / ml and a volume of 20 l was injected into the hplc system. four aliquots of 1 ml stp working solution (100 g / ml) and capsule extract solution (100 g / ml) were transferred into small round bottom flasks. the contents were then mixed separately with 4 ml of (1, 6, 10, and 30%) hydrogen peroxide solutions, and the reaction mixtures were kept at ambient temperature for 2 hours with intermittent shaking. the resulting solutions were diluted with mobile phase to 10 g / ml and a volume of 20 l of each solution was injected into the hplc system. powder samples of standard stp (10 mg) and diacomit capsules (10 mg of capsule content) were uv irradiated (peak intensity of 254 nm) for 72 hours. solutions were then diluted with mobile phase to obtain a concentration of 10 g / ml, and a volume of 20 l of each solution was injected into the hplc system. similarly, an aqueous solution of stp (1 mg / ml) and capsule extract solution (1 mg / ml) were exposed to the uv light for 72 hours and diluted with mobile phase to 10 g / ml and a volume of 20 l of each was then injected into the hplc system. acid degradation was carried out by refluxing stp (40 mg) in methanol (15 ml) and 8 n hcl (5 ml) for 3 h at 60c using the same procedure discussed in section 2.6.2. the acid degradation product (dstp) was extracted from the residue using diethyl ether which was subsequently evaporated under reduced pressure. crude dstp was then purified via column chromatography (silica gel 60 ; 0.0630.200 mm) using chloroform as a solvent. the collected eluate was evaporated under vacuum to give pure dstp, which was identified through h - nmr, c - nmr, and mass spectrometry. purified dstp was dissolved in deuterated chloroform and the nmr measurements were performed at 25c on a bruker ac-500 nmr spectrometer operating at 500 mhz for h and 125.76 mhz for c. tetramethylsilane (tms) was used as an internal standard and chemical shift values were recorded in ppm on the scale. the h - nmr data were represented in terms of chemical shifts, multiplicity, and number of protons. the c - nmr data were represented as chemical shifts and type of carbon. for structure elucidation of dstp, an agilent 6410 triple quadrupole mass spectrometer (agilent technologies, usa) equipped with an electrospray ionization interface (esi) coupled to an agilent 1200 hplc (agilent technologies, usa) was used. agilent 1200 series system consists of g1311a binary pump, g1322a degasser, and g1367b hip - als autosampler, and g1316a thermostated column compartment was used. purified dstp was dissolved in acetonitrile (5 g / ml), and a connector was used instead of the column to allow direct injection of the sample. mobile phase composed of two solvents : a is hplc grade water and b is acetonitrile mixed in the ratio 50 : 50 v / v%. ms parameters were optimized to scan in ultrascan mode. for screening of mass signals of dstp the source temperature was set to 350c nebulizer gas pressure of 55 psi and dry gas flow rate of 12 l / min. four sets of stp working solution (100 g / ml) and capsule extract solution (100 g / ml), 1 ml of each, were subjected to acid degradation using 4 ml of 0.005 n, 0.01 n, 0.05 n, and 0.1 n hydrochloric acid (procedure described in section 2.6.2). (0, 15, 30, 60, 120, 180, and 240 min). the samples were cooled to room temperature (25 5c) and neutralized with an amount of base equivalent to that of the previously added acid. the resulting neutral solutions were then diluted with mobile phase to 10 g / ml and a volume of 20 l was injected into the hplc system and the peak area of stp was compared with a freshly prepared standard solution. the initial method development was conducted on pure drug using working standards solution protected from light. optimized chromatographic conditions were established after a number of preliminary experiments for selecting the most efficient mobile system, separation column, and detection wavelength range. selection of the proper system was based on its ability to give good separation between the pure stp and its possible impurities and/or degradation products. different mobile phase compositions and ph ranges were tested to achieve a more symmetric peak and shorter retention time for stp. mobile phase was modified between 20 : 80% and 70 : 30% acetonitrile : 1 mm phosphate buffer and ph was changed in the range of 37. ultimately, optimum chromatographic separation was achieved as described in section 2.2. under these chromatographic conditions, the run time of each sample was 6 min, and the retention time of stp was 1.80 0.07 min (n = 3) (figure 2). an advantage of the current method is its simple sample preparation as the procedure did not include significant error - prone experimental manipulations (e.g., chemical derivatization, etc.) which would negatively affect the results. this supported the avoidance of internal standard use, which is usually added to increase the method efficiency when there are multisteps in sample preparation. the method was fully validated in accordance with the recommendations of ich and is rugged and adequately sensitive for routine stp analysis. using the above - mentioned optimum chromatographic conditions, three independent calibration curves were constructed correlating the detector signals with the corresponding stp concentrations. each curve was generated by 8 concentration points ; each concentration was injected in triplicates. the standard deviation values of each concentration point (triplicates) did not exceed 2%. the method revealed a good linear calibration fit in the range of 125 g / ml. system suitability tests for the principle peak and its degradation product were evaluated using stp solution of 12 g / ml. the capacity factor k = 0.8884 indicates that the stp peak is well resolved with respect to the void volume. the asymmetry factor at 5% of peak height (b / a) was 1.3079 for stp peak which reflects good peak symmetry. theoretical plate number (n) of 5008.09 for the column used in the study (3.5 m, 75 mm 4.6 mm i.d) thus demonstrates acceptable column efficiency. all previous results assured the adequacy of the proposed hplc method for routine analysis of stp. the lower limit of quantitation (loq) is the lowest concentration of the standard curve which can be measured with acceptable accuracy and precision for the analyte. the limit of detection (lod) and the lower limit of quantitation (loq) were calculated based on the following equations : (1)lod=3.3s, loq=10s, where is the standard deviation of the intercept (115.82 3300) of regression line and s is the slope (30875.64 248.86) of regression line of the calibration curve. the lod and loq values were 0.081 and 0.242 g / ml, respectively. accuracy and precision were determined by the recovery study of known amounts in accordance with ich recommendation. five consecutive injections of stp sample solutions (2, 6, 10, 15, and 20 g / ml) showed excellent intraday accuracy (98.74101.64%) and precision (0.642.32%) (table 2). interday accuracy and precision data of back - calculated concentration of calibration samples for stp were evaluated in triplicates by recovery studies using the standard addition method (table 3). in order to measure the extent of the method robustness, the most critical parameters were interchanged while keeping the other parameters unchanged, and in parallel the chromatographic profile was observed and recorded. robustness of the proposed method was determined using stp concentration of 10 g / ml and changing the flow rate (0.91.1 ml / min) and column temperature (2c) and change in the ph of mobile phase (0.2). no significant changes in assay value were observed (table 3) by changing the chromatographic conditions which confirms the robustness of the method. ruggedness of the method was determined by using mobile phase components from two different manufactures and changing analysts. there was no significant change in the retention time of stp which was observed ; % rsd was 0.310.63% indicating the ruggedness of the method. the stability of the drug in solution during analysis was determined by repeated analysis of samples during the course of experimentation on the same day and also after storage of the drug solutions (calibration samples) for 24, 48, and 72 hours under laboratory bench condition (25 1c) and under refrigeration (4.0 0.5c). the mean % rsd between peak areas for the samples stored under refrigeration (4.0 0.5c) and at laboratory temperature (25 1c) was found to be 0.58% and 0.93%, respectively, suggesting that the drug solution can be stored without any degradation over the time interval studied. the ich guideline entitled stability testing of drug substances and products requires the stress testing to be carried out to elucidate the inherent stability characteristics of the active substance and provide a rapid identification of differences that might result from changes in the manufacturing processes or source sample. an ideal stability - indicating method is one that quantifies the standard drug alone and also resolves its degradation products. as described in the experimental section, different stress conditions were applied : boiling, acid / base forced degradation, oxidation, and irradiation with uv light. from this investigation, it was clear that, in case of boiling, base - forced degradation (1 n naoh), oxidation (h2o2), and uv irradiation (solid state and solution), stp was stable under the employed stress conditions as no degradation products were observed in their chromatograms (figures 3(a) and 3(b)) which confirms the data from european medicines agency (emea). however, in case of acidic conditions, one degradation product was observed, dstp, at a retention time of 4.25 min (peak resolution from stp peak was 5.89) (figures 3(c)3(e)). the intact stp concentration was calculated and found to be degraded by 76.03, 83.51, 84.87, and 95% in case of acid degradation using 0.1 n hydrochloric acid after 1, 2, 3, and 5 hours, respectively. acidic degradation of stp was studied using h - nmr, c - nmr (figures 4 and 5), and mass spectral data. h - nmr spectrum of dstp showed a signal integrating to one proton at 4.09 ppm which was assigned to be cl ch with concomitant disappearance of the signal of oh functionality at 1.56 ppm and the signal of the methine proton (oh ch) at 3.80 ppm. additionally, c - nmr of dstp showed signal at 74.8 ppm which was assigned to be cl ch with concomitant disappearance of the signal of oh ch at 83.9 ppm. moreover, mass spectrum (esi) of dstp showed peak at m / z 217 [m-36 + h ]. uv spectra of stp (figure 1) and dstp are identical indicating that there is no change in the conjugation system upon this acidic degradation of stp. structure elucidation of dstp explained its delaying in hplc elution due to its lower polarity compared to stp. accordingly, the aforementioned analytical data of dstp suggested that upon acid degradation of stp the hydroxyl moiety was protonated with hydrochloric acid and subsequently substituted with chloride ion to give the halogenated derivative dstp (scheme 1). h - nmr (cdcl3) of stp : (ppm) = 0.88 (s, 9h, t - butyl), 1.56 (s, 1h, oh), 3.80 (d, j = 7.0 hz, 1h, choh), 5.86 (s, 2h, och2o), 6.05 (dd, j = 15.7, 7.85 hz, 1h, ch = ch c6h3), 6.39 (d, j = 15.5 hz, 1h, ch = ch c6h3), 6.67 (d, j = 8.0 hz, 1h, ar h), 6.72 (d, j = 7.95 hz, 1h, ar c - nmr (cdcl3) of stp : (ppm) = 26.5 (c(ch3)3), 32.8 (c(ch3)3), 81.9 (ch oh), 101.1 (och2o), 107.1, 108.3, 121.1 (ar ch), 126.9, 131.5 (ch = ch c6h3, ch = ch c6h3, ar ms m / z (esi) : 217 [m-18 + h ]. h - nmr (cdcl3) of dstp : (ppm) = 0.83 (s, 9h, t - butyl), 4.09 (d, j = 9.5 hz, 1h, ch cl), 5.74 (s, 2h, och2o), 5.92 (dd, j = 15.4, 7.95 hz, 1h, ch = ch c6h3), 6.23 (d, j = 15.5 hz, 1h, ch = ch c6h3), 6.54 (d, j = 8.0 hz, 1h, ar h), 6.60 (d, j = 7.96 hz, 1h, ar c - nmr (cdcl3) of dstp : (ppm) = 26.8 (c(ch3)3), 36.4 (c(ch3)3), 74.8 (ch cl), 101.2 (och2o), 105.9, 108.3, 121.6 (ar ch), 125.7, 130.6, 132.5 (ch = ch c6h3, ch = ch c6h3, ar c), 147.8, 148.1 (ar c). ms m / z (esi) : 217 [m-36 + h ]. the degradation of stp was investigated in acidic condition using 0.005, 0.01, 0.05, 0.1 n hcl. the drug was found to be extremely labile for higher concentrations of hcl (0.05 and 0.1 n). a gradual decrease in stp concentration was observed with increasing time at lower hcl concentrations (0.005 and 0.01 n) (figure 6(a)). a linear relationship was observed by plotting the 1og of residual molar concentration of stp versus time (figure 6(b)) which indicates first - order kinetics according to the following equation : (2)lnct=kt+lnc0. here, ct is the remaining stp molar concentration, c0 is the initial molar concentration of stp, k is the apparent first order rate constant with a negative sign, and t is the time. the half - life (t1/2) of the first - order reaction was found to be t1/2 = 1.42 h according to the following equation : (3)t1/2=ln(2)k. it is evident from the results obtained previously that the proposed method gave satisfactory results with the analysis of stp in bulk. thus, stp - containing capsules were subjected to the analysis by the proposed method. the same procedure for determination of drug dosage form the peak area of five injections was compared by a standard solution of stp with the same concentration level and the mean recovery percentage was calculated to be 99.34 0.59%. this acceptable value indicated the applicability of the method for the routine quality control of stp capsules without interference from the excipients. this was evidenced from the good label claim percentage as well as the absence of any peaks in the chromatogram of the capsule extract solution (figure 2). the present study represents the first report that deals with the development and validation of a stability - indicating hplc - dad method for determination of stp. this study is a typical example of development of a stability - indicating assay, established following the recommendations of ich guidelines. the proposed method showed acceptable accuracy, precision, and selectivity. from the economical point of view, the method involved the native uv - absorbing property of stp, rather than expensive derivatizing analytical reagents. stp showed great stability at the various stress conditions (thermal, alkaline, oxidative, and photodegradation). a single degradation product, dstp, was only formed with the exposure of stp to acidic degradation using hcl. structure elucidation of the acidic degradation product using h - nmr, c - nmr, and ion trap mass spectrometry revealed substitution of oh functionality of stp with chloride following treatment of stp with hydrochloric acid. additionally, kinetic study of the acidic degradation reaction demonstrated that stp undergoes first - order kinetics. the method is suitable for the determination of stp in bulk and capsule form without any interference from the degradation products, and it is recommended for routine use in quality control industry laboratories. | a rapid, simple, sensitive, and accurate isocratic reversed - phase stability - indicating high performance liquid chromatography method has been developed and validated for the determination of stiripentol and its degradation product in its bulk form and pharmaceutical dosage form. chromatographic separation was achieved on a symmetry c18 column and quantification was achieved using photodiode array detector (dad). the method was validated in accordance with the ich requirements showing specificity, linearity (r2 = 0.9996, range of 125 g / ml), precision (relative standard deviation lower than 2%), accuracy (mean recovery 100.08 1.73), limits of detection and quantitation (lod = 0.024 and loq = 0.081 g / ml), and robustness. stiripentol was subjected to various stress conditions and it has shown marked stability under alkaline hydrolytic stress conditions, thermal, oxidative, and photolytic conditions. stiripentol degraded only under acidic conditions, forming a single degradation product which was well resolved from the pure drug with significantly different retention time values. this degradation product was characterized by 1h - nmr and 13c - nmr spectroscopy as well as ion trap mass spectrometry. the results demonstrated that the method would have a great value when applied in quality control and stability studies for stiripentol. |
raw materials used to form the glass - ceramics were over 99.9% purity in sio2, caco3, cahpo42h2o, h3po4, mgo, and caf2, and 200 g of the composition of cao 44.9 weight percent (wt%), sio2 34.2 wt%, p2o5 16.3 wt%, mgo 4.6 wt%, and caf2 0.5 wt% were batched. the batched sample underwent ball milling for 24 hours with absolute methanol as a solvent and was dried. after drying in the mixer, each batch was placed in a platinum crucible and melted at 1,500-1,600 for two hours. the glass - ceramic was manufactured through rapid cooling by pouring the material into a water bath and grounding the contents using spex milling for 15 hours to make around 1 - 2 m sized glass powders. as a result in addition, high purity caco3 (99.99%, kojundo chemical co., ltd, sakado - shi, japan), sio2 (99.9%, kojundo chemical co., ltd, sakado - shi, japan) and b2o3 (99.9%, kojundo chemical co., ltd, sakado - shi, japan) were used, and the composition of cao 45.7%, sio2 45.7% and b2o3 8.6% in the mole ratio was named as cs10b. the ratios of a - w gc and cs10b were 1:4, 1:9 and 1:19 in p20b80, p10b90, and p5b95, respectively and the composition was cao 45.54 wt%, sio2 43.40 wt%, p2o5 3.26 wt%, b2o3 8.00 wt%, mgo 0.92 wt% and caf2 0.10 wt% in p20b80 and cao 43.57 wt%, sio2 45.30 wt%, p2o5 1.62 wt%, b2o3 9.00 wt%, mgo 0.46 wt% and caf2 0.05 wt% in p20b90 and cao 43.51 wt%, sio2 45.92 wt%, p2o5 0.81 wt%, b2o3 9.50 wt%, mgo 0.23 wt% and caf2 0.025 wt% in p5b95. p20b80, p10b90, and p5b95 were maintained at 1,450 for two hours, and glass powders were produced by melting and cooling them rapidly. sponge infiltration slurry was used to manufacture a sponge - type porous implant by mixing polyvinyl alcohol and the glass powders. the slurry was infiltrated three times by using 10 g of four appropriate - sized sponges (4 5 cm) once each time. after making both the inner and outer parts infiltrated evenly, blocked pores were pierced with an air gun before the infiltration solution dried. the glass - ceramics manufactured through this method were dried by open air, and sponges were burnt at 600 to make sponge - type porous implants. fifty - one new zealand white male rabbits with an average weight of 3.3 0.3 kg were kept individually in standard cages under 12 hours on / off light cycles. room temperature and humidity all animals were divided into four groups by implant materials, i.e., p20b80 (18 rabbits), p10b90 (16 rabbits), p5b95 (9 rabbits), with added autografts (8 rabbits). the study was approved by the institutional animal care and use committee at the clinical research institute seoul national university hospital. care was taken to avoid unnecessary stress and discomfort to the animals throughout the experimental period. surgical procedures were performed to the lumbar vertebra (l5-l6) on both sides of intertransverse processes under general anesthesia as described by boden.13) three milliliters of glass - ceramics were implanted on each side of the intertransverse processes. this procedure was described in our previous reports.11,12) after the placement of the glass - ceramics, fascial tissue layers were repositioned and sutured after irrigation. manual palpation to determine the fusion status was performed by stressing across the fusion site. formation of hca on the surface of the specimens was observed by scanning electron microscopy (jsm5600, jeol, akishima, japan). immediately after euthanasia, fusion success was evaluated by holding the upper and lower transverse processes of the 5th and 6th vertebrae. twisting, bending, and folding motions were performed. only specimens with immobility against the applied forces were judged to be successful fusions.11,12) before tensile testing, all remaining muscle and ligaments including the intervertebral discs, posterior longitudinal ligaments, and the thecal sac were removed. holes were made in the l5 and l6 vertebral bodies with a 3.2-mm - diameter drill bit, and then a 3.0-mm - diameter steel rod was inserted.11,12) an instron electromechanical materials testing system (instron 5582, instron co., norwood, ma, usa) was used to test the l5 and l6 uniaxial tensile forces at a displacement rate of 0.5 cm per minute11,12) after fitting with a calibrated load - cell of 10 n. the ultimate tensile load, i.e., the break point, was read from the peak load to failure in the load - displacement curve. a posteroanterior radiograph was completed for each animal immediately postsurgery at 45 kv for 12 milliseconds.11,12) additional plain radiographs were obtained for all surviving animals at 2, 4, 6, 8, 10, 12 weeks postsurgery. we examined whether clear opacity in the border of blocks of each implant immediate postsurgery became vague compared to adjacent areas, whether clear radioopacity among blocks presurgery became similar with that of neighboring blocks, and whether fusion mass connecting adjacent transverse processes was formed. we calculated the degradation rate of the glass - ceramics radiomorphometrically by comparing changes in the area occupied by the glass - ceramics between the initial and final radiographs which was described in previous reports.11,12) the percentage of the area of glass - ceramics was evaluated in the final radiographs (fig. histological sections were carried out in the same way as reported in previous studies.11,12) lumbar spines from each group were fixed for 24 hours in 10% formalin. a sagittal section from the right and left sides undecalcified tissues were embedded in methacrylate and sectioned 4 m in width parallel to the fusion mass with a diamond cutter (exakt300cp, exakt co., norderstedt, germany).13) slides were stained using hematoxylin and eosin. satisfactory osteoconductivity was defined as new bone was detected in pores or adjacent to the ceramics. the degree and the pattern of bony fusion between bone and implant and the surface change of the fusion mass were observed with a light microscope. moreover, the degree of bone growth was evaluated by classifying parts of the fusion mass into an implant, new bone, and soft tissues. null hypotheses of no difference were rejected if p - values were less than 0.05. raw materials used to form the glass - ceramics were over 99.9% purity in sio2, caco3, cahpo42h2o, h3po4, mgo, and caf2, and 200 g of the composition of cao 44.9 weight percent (wt%), sio2 34.2 wt%, p2o5 16.3 wt%, mgo 4.6 wt%, and caf2 0.5 wt% were batched. the batched sample underwent ball milling for 24 hours with absolute methanol as a solvent and was dried. after drying in the mixer, each batch was placed in a platinum crucible and melted at 1,500-1,600 for two hours. the glass - ceramic was manufactured through rapid cooling by pouring the material into a water bath and grounding the contents using spex milling for 15 hours to make around 1 - 2 m sized glass powders. as a result in addition, high purity caco3 (99.99%, kojundo chemical co., ltd, sakado - shi, japan), sio2 (99.9%, kojundo chemical co., ltd, sakado - shi, japan) and b2o3 (99.9%, kojundo chemical co., ltd, sakado - shi, japan) were used, and the composition of cao 45.7%, sio2 45.7% and b2o3 8.6% in the mole ratio was named as cs10b. the ratios of a - w gc and cs10b were 1:4, 1:9 and 1:19 in p20b80, p10b90, and p5b95, respectively and the composition was cao 45.54 wt%, sio2 43.40 wt%, p2o5 3.26 wt%, b2o3 8.00 wt%, mgo 0.92 wt% and caf2 0.10 wt% in p20b80 and cao 43.57 wt%, sio2 45.30 wt%, p2o5 1.62 wt%, b2o3 9.00 wt%, mgo 0.46 wt% and caf2 0.05 wt% in p20b90 and cao 43.51 wt%, sio2 45.92 wt%, p2o5 0.81 wt%, b2o3 9.50 wt%, mgo 0.23 wt% and caf2 0.025 wt% in p5b95. p20b80, p10b90, and p5b95 were maintained at 1,450 for two hours, and glass powders were produced by melting and cooling them rapidly. sponge infiltration slurry was used to manufacture a sponge - type porous implant by mixing polyvinyl alcohol and the glass powders. the slurry was infiltrated three times by using 10 g of four appropriate - sized sponges (4 5 cm) once each time. after making both the inner and outer parts infiltrated evenly, blocked pores were pierced with an air gun before the infiltration solution dried. the glass - ceramics manufactured through this method were dried by open air, and sponges were burnt at 600 to make sponge - type porous implants. fifty - one new zealand white male rabbits with an average weight of 3.3 0.3 kg were kept individually in standard cages under 12 hours on / off light cycles. room temperature and humidity all animals were divided into four groups by implant materials, i.e., p20b80 (18 rabbits), p10b90 (16 rabbits), p5b95 (9 rabbits), with added autografts (8 rabbits). the study was approved by the institutional animal care and use committee at the clinical research institute seoul national university hospital. care was taken to avoid unnecessary stress and discomfort to the animals throughout the experimental period. surgical procedures were performed to the lumbar vertebra (l5-l6) on both sides of intertransverse processes under general anesthesia as described by boden.13) three milliliters of glass - ceramics were implanted on each side of the intertransverse processes. this procedure was described in our previous reports.11,12) after the placement of the glass - ceramics, fascial tissue layers were repositioned and sutured after irrigation. manual palpation to determine the fusion status was performed by stressing across the fusion site. formation of hca on the surface of the specimens was observed by scanning electron microscopy (jsm5600, jeol, akishima, japan). immediately after euthanasia, fusion success was evaluated by holding the upper and lower transverse processes of the 5th and 6th vertebrae. twisting, bending, and folding motions were performed. only specimens with immobility against the applied forces before tensile testing, all remaining muscle and ligaments including the intervertebral discs, posterior longitudinal ligaments, and the thecal sac were removed. holes were made in the l5 and l6 vertebral bodies with a 3.2-mm - diameter drill bit, and then a 3.0-mm - diameter steel rod was inserted.11,12) an instron electromechanical materials testing system (instron 5582, instron co., norwood, ma, usa) was used to test the l5 and l6 uniaxial tensile forces at a displacement rate of 0.5 cm per minute11,12) after fitting with a calibrated load - cell of 10 n. the ultimate tensile load, i.e., the break point, was read from the peak load to failure in the load - displacement curve. a posteroanterior radiograph was completed for each animal immediately postsurgery at 45 kv for 12 milliseconds.11,12) additional plain radiographs were obtained for all surviving animals at 2, 4, 6, 8, 10, 12 weeks postsurgery. we examined whether clear opacity in the border of blocks of each implant immediate postsurgery became vague compared to adjacent areas, whether clear radioopacity among blocks presurgery became similar with that of neighboring blocks, and whether fusion mass connecting adjacent transverse processes was formed. we calculated the degradation rate of the glass - ceramics radiomorphometrically by comparing changes in the area occupied by the glass - ceramics between the initial and final radiographs which was described in previous reports.11,12) the percentage of the area of glass - ceramics was evaluated in the final radiographs (fig. histological sections were carried out in the same way as reported in previous studies.11,12) lumbar spines from each group were fixed for 24 hours in 10% formalin. a sagittal section from the right and left sides was taken for processing and evaluated separately. undecalcified tissues were embedded in methacrylate and sectioned 4 m in width parallel to the fusion mass with a diamond cutter (exakt300cp, exakt co., norderstedt, germany).13) slides were stained using hematoxylin and eosin. satisfactory osteoconductivity was defined as new bone was detected in pores or adjacent to the ceramics. the degree and the pattern of bony fusion between bone and implant and the surface change of the fusion mass were observed with a light microscope. moreover, the degree of bone growth was evaluated by classifying parts of the fusion mass into an implant, new bone, and soft tissues. null hypotheses of no difference were rejected if p - values were less than 0.05. of the fifty - one rabbit specimens initially used in this study, eight experimental animals were lost immediately after surgery and during the healing phase. four groups were designated according to the implanted material or autograft and the number of specimens were evaluated per group. group names and their numbers are as follows : p20b80 group, 17 specimens ; p10b90 group, 11 specimens ; p5b95 group, 7 specimens ; and the autograft group, 8 specimens. after soaking in sbf for 1 day, an hca layer covered the whole surface of 25% of the specimens in the p20b80 group. however, an hca layer appeared 3 days after soaking in sbf in the p10b90 group and 5 days after soaking in sbf for the p5b95 group. moreover, the aggregate of the hca layer for groups p10b90 and p5b95 was smaller in comparison with that of the p20b80 group (fig. solid bone fusion was detected in 5 of 17 specimens (29.4%) in the p20b80 group, 0 of 11 (0.0%) in the p10b90 group, 1 of 7 (14.3%) in the p5b95 group, and 8 of 8 (100.0%) in the autograft group. the autograft group recorded significantly higher fusion rates than all the implanted materials groups of p20b80, p10b90, and p5b95 (p < 0.0001, 0.0016 and 0.0014, respectively). the fusion rate for the p20b80 group was higher than those of the p10b90 and p5b95 groups. the uniaxial tensile test showed 214.9 46.8 n of the ultimate tensile load for the p20b80 group, 187.1 44.9 n in the p10b90 group, and 230.3 13.9 n in the p5b95 group. the ultimate tensile load of the control group, i.e., the autograft, was slightly higher than those of the other groups although there was no significant difference. the degree of resorption increased at 12 weeks compared to that immediately postsurgery in the implanted material p20b80, p10b90, and p5b95 groups, and the implants formed one mass (fig. 3). in addition, the radio - opaque area was inside the fusion mass, and it was, therefore, considered that new bone grew into it and bony fusion was in the process. the proportions of the area occupied by the glass - ceramics at the final radiographs to the area occupied by the glass - ceramics at the initial radiographs were 76.6% 7.0% in the p20b80 group, 74.2% 9.0% in the p10b90 group, and 75.8% 5.8% in the p5b95 group. the radiomorphometry of the three kinds of cao - sio2-p2o5-b2o3 glass - ceramic areas revealed no statistical difference with time. according to the histological study, satisfactory osteoconductivity was found in all of the three types of cao - sio2-p2o5-b2o3 glass - ceramics. in the p20b80, p10b90, and p5b95 groups, new bone was formed around the ceramics inserted between the upper and lower transverse processes continuously. undecalcified examination revealed that the glass - ceramic implants had very low opacity, and the brown ceramic implant was absorbed losing nearly all its original pattern. new bone had osteoblasts and osteocytes in the adjacent and central areas, respectively, and the formation of lamellar bone was observed with both osteocytes arranged to be in concentric circles and mature bone matrices (fig. the border of the composite plane between implants and bone became vague when the implants were absorbed. it is assumed that the glass - ceramics were absorbed during the formation of bone tissues on the ceramic implants, and the absorption occurred mainly in areas in contact with bone. after soaking in sbf for 1 day, an hca layer covered the whole surface of 25% of the specimens in the p20b80 group. however, an hca layer appeared 3 days after soaking in sbf in the p10b90 group and 5 days after soaking in sbf for the p5b95 group. moreover, the aggregate of the hca layer for groups p10b90 and p5b95 was smaller in comparison with that of the p20b80 group (fig. based on manual palpation, solid bone fusion was detected in 5 of 17 specimens (29.4%) in the p20b80 group, 0 of 11 (0.0%) in the p10b90 group, 1 of 7 (14.3%) in the p5b95 group, and 8 of 8 (100.0%) in the autograft group. the autograft group recorded significantly higher fusion rates than all the implanted materials groups of p20b80, p10b90, and p5b95 (p < 0.0001, 0.0016 and 0.0014, respectively). the fusion rate for the p20b80 group was higher than those of the p10b90 and p5b95 groups. however, the difference was statistically insignificant. the uniaxial tensile test showed 214.9 46.8 n of the ultimate tensile load for the p20b80 group, 187.1 44.9 n in the p10b90 group, and 230.3 13.9 n in the p5b95 group. the ultimate tensile load of the control group, i.e., the autograft, was slightly higher than those of the other groups although there was no significant difference. the degree of resorption increased at 12 weeks compared to that immediately postsurgery in the implanted material p20b80, p10b90, and p5b95 groups, and the implants formed one mass (fig. 3). in addition, the radio - opaque area was inside the fusion mass, and it was, therefore, considered that new bone grew into it and bony fusion was in the process. the proportions of the area occupied by the glass - ceramics at the final radiographs to the area occupied by the glass - ceramics at the initial radiographs were 76.6% 7.0% in the p20b80 group, 74.2% 9.0% in the p10b90 group, and 75.8% 5.8% in the p5b95 group. the radiomorphometry of the three kinds of cao - sio2-p2o5-b2o3 glass - ceramic areas revealed no statistical difference with time. according to the histological study, satisfactory osteoconductivity was found in all of the three types of cao - sio2-p2o5-b2o3 glass - ceramics. in the p20b80, p10b90, and p5b95 groups, new bone was formed around the ceramics inserted between the upper and lower transverse processes continuously. undecalcified examination revealed that the glass - ceramic implants had very low opacity, and the brown ceramic implant was absorbed losing nearly all its original pattern. new bone had osteoblasts and osteocytes in the adjacent and central areas, respectively, and the formation of lamellar bone was observed with both osteocytes arranged to be in concentric circles and mature bone matrices (fig. the border of the composite plane between implants and bone became vague when the implants were absorbed. it is assumed that the glass - ceramics were absorbed during the formation of bone tissues on the ceramic implants, and the absorption occurred mainly in areas in contact with bone. hydroxyapatite is clinically applied in cases of bone defects and spinal fusion.13 - 15) however, its resorbing properties and mechanical strength are considered relatively low. although calcium phosphate ceramics with tricalcium phosphate are known to have high resorbing properties,16) their low mechanical strengths have been commented as a distinct disadvantage. because bioactive glass - ceramics can control resorption and mechanical strength according to their composition, this study also tried to regulate their resorbing properties with b2o3 content. the correlation that a higher b2o3 content leads to higher resorption was displayed in our previous study. in the in vitro study using sbf, p20b80 (p2o5 3.26 wt%) formed an hca layer after soaking for 1 day, while p10b90 (p2o5 1.62 wt%) and p5b95 (p2o5 0.81 wt%), with their relatively low p2o5 contents, began to form a layer in proportion to the p2o5 content and its amount was also proportional to the p2o5 content. however, the difference in the amount of the hca layer produced after five days was not significantly different between the two compounds. the difference in the time in forming the hca layer was notably considered to be attributed to the p2o5 content of each glass - ceramic. although p2o5 is inessential for the nucleation of hca, it can earlier increase the amount of the hca layer as it accelerates hca formation. with regards to this point, decreased p2o5 content could influence osteoconductivity. for these reasons, the increased b2o3 and decreased p2o5 content that were used to improve the resorption of the glass - ceramics was considered to decline eventually in fusion rates as displayed in the in vivo study. this study measured the two - dimensional area of the implanted glass - ceramics in radiographic images to assess their resorption. because the connection of trabecular bone found in complete fusions was unclear in groups p20b80, p10b90, and p5b95, the autograft group was found to have bony bridging in the upper and lower transverse processes at 12 weeks ; and, thus, solid bony fusion formed. the rate of the absorbed area was only around 25% and, thus, it was considered an inadequate resorption rate. when observing the measured border of the radioopaque lesions, the implanted glass - ceramic material remained as debris after the area of the absorption of the porous structure became radio - opaque. gross inspection of the glass - ceramics implants revealed that the porous structure was nearly lost, and the absorption of the glass - ceramics used in this study was much higher than that in previous studies. in our previous study, the fusion rate of compositions with relatively higher p2o5 rates and a - w gc was 80%-90%.11) for this study, the fusion rate of p20b80 with the highest level of p2o5 was recorded as only 29.4%, and most of the p10b90 and p5b95 groups showed pseudarthrosis. conversely, the histological examination showed excellent osteoconductivity found in all three glass - ceramics. in other words, newly formed bony tissues were bonded directly to the implanted glass - ceramics and remodeling also proceeded. thus, it is believed that the decreased p2o5 content affected osteoconductivity to some degree. therefore, a higher b2o3 content, rather than osteoconductivity itself, led to poorer early porous structures for increased resorption and was considered to be a major cause of pseudarthrosis. in general, for bone defect filling, stem cells are supplied in neighboring parts of the implanted glass - ceramics, and the vascular structure grows into it easily. thus, this resorption characteristic is considered to be highly advantageous. in the case of intertransverse process fusions, because the transverse processes mainly consist of cortical bone with very small amounts of stem cells and the distance between transverse processes is relatively long, some parts close to the bone can form new bone by receiving stem cells. however, the middle area between the transverse processes is difficult to attain fusion as this area needs the connection of new bones produced from both transverse processes. therefore, to obtain intertransverse process fusion, porous structures and osteoconductivity of the osteoconductive scaffolds should be maintained sufficiently during the period needed for newly formed bone to grow and be connected in neighboring areas. for intertransverse process fusion with regards to this study, the three types of glass - ceramics used had excellent osteoconductivity but could not maintain the porous structure fully to prevent pseudarthrosis. properties of the ideal biodegradable material include : (1) the ability to maintain strength and stability of porous structures during degradation until achieving fusion in the early stage, (2) the ability to be replaced completely into the new bone, (3) and having adequate reconciling resorption and new bone formation rates. however, materials meeting all of these conditions have yet to be invented. for one reason, the absorption of porous glass - ceramics is performed through a slow degradation of the debris made by the destruction of the porous structure rather than by the maintenance of the porous structure, which is critical for osteoconductivity. unfortunately, the destruction of a porous structure actually negatively influences various aspects of osteoconductivity. moreover, the environment for bone formation in the implanted area varies according to anatomical location. however, even when implants are made in the same anatomical location, large differences in time and success rates of bone formation can depend on age and the health condition of the recipients. therefore, in a favorable environment for bone formation, glass - ceramics with high absorption rates can produce an ideal situation, obtaining not only bony fusion but also absorption. conversely, the same glass - ceramics in an unfavorable environment for bone formation can experience pseudarthrosis due to absorption taking place before new bone formation can occur. from all of these results, osteoconductivity and sufficient porous structure maintaining properties rather than resorbing properties are considered to be more advantageous for bony fusion in an environment unfavorable for new bone formation. furthermore, resorption rates faster than new bone formation growth rates need careful attention as an imbalance may lead to failure. however, in cases using bone morphogenetic protein with high osteoinductivity and stem cells with properties of both osteoinductivity and osteogenesis, osteoconductive bio glass - ceramics with high resorbing properties are expected to be used as carriers. in conclusion, the high resorbing properties of osteoconductive porous glass - ceramics have a material advantage of completely dissolving within the human body. however, their ability to be rapidly resorbed before bony fusion in their role as osteoconductive scaffolds is a disadvantage that must be taken into consideration. | backgroundbioactive glass - ceramics have the ability to directly bind to bones and have been widely used as bone graft substitutes due to their high osteoconductivity and biocompatibility. cao - sio2-p2o5-b2o3 glass - ceramics are known to have good osteoconductivity and are used as bone graft extenders.methodsthis study aimed to evaluate the effects of the resorbing properties of glass - ceramics in bone fusion after producing and analyzing three types of cao - sio2-p2o5-b2o3 glass - ceramics with high osteoconductivity that had enhanced resorption by having an increased b2o3 content. the three types of cao - sio2-p2o5-b2o3 glass - ceramics with b2o3 contents of 8.0, 9.0, and 9.5 weight % were designated and grouped as p20b80, p10b90, and p5b95, respectively. glass - ceramic types were tested for fusion rates and bone formation by employing the lumbar 5 - 6 intertransverse process fusion model in 51 new zealand male rabbits. bioactivity was assessed by soaking in simulated body fluid (sbf).resultsin vitro study results showed sufficient hydroxycarbonate apatite layer formation occurred for p20b80 in1 day, for p10b90 in 3 days, and for p5b95 in 5 days after soaking in sbf. for the rabbit lumbar spine posterolateral fusion model, the autograft group recorded a 100% fusion rate with levels significantly higher than those of p20b80 (29.4%), p10b90 (0%), and p5b95 (14.3%), with high resorbing properties. resorbing property differences among the three glass - ceramic groups were not significant. histological results showed new bone formation confirming osteoconductivity in all three types of glass - ceramics. radiomorphometric results also confirmed the resorbing properties of the three glass - ceramic types.conclusionsthe high resorbing properties and osteoconductivity of porous glass - ceramics can be advantageous as no glass - ceramics remain in the body. however, their relatively fast rate of resorption in the body negatively affects their role as an osteoconductive scaffold as glass - ceramics are resorbed before bony fusion. |
degenerative cervical spondylosis, herniated cervical discs, and ossification of the posterior longitudinal ligament (opll) can result in chronic compression of the spinal cord. surgical treatment for this condition posterior approaches to decompression, such as laminectomy and laminoplasty, are typically indicated for cervical myelopathy3,6,7,10,16,19). cervical expansive laminoplasty was originally carried out using spinous processes as spacers. since development of classic open - door laminoplasty with the use of sutures, the procedure has been modified to reduce complications such as restenosis, axial symptoms, and segmental motor paralysis1,12,13,14,20). various modifications to expansive laminoplasty have been developed and clinical outcomes are typically satisfactory in patients with cervical spondylotic myelopathy (csm). in fact, one of the most popular modifications is a bilateral hinge - type laminoplasty procedure called the double - door laminoplasty. development of surgical implants for expansive laminoplasty has resulted in surgeons using various kinds of lamina spacers. previously, we introduced a new type of modified expansive double - door laminoplasty that utilizes a titanium miniplate system for an box - shape cervical expansive laminoplasty14). this method showed much higher canal expansion rate than that of other cervical spacers such as hydroxyapatite (ha) (apaceram) and centerpiece8,9,14)(fig. so this new method was expected to show good clinical outcome but it was not studied up to date. in this study between june 2008 and july 2013, a total of 87 patients with opll and csm underwent laminoplasty at our institute with box - shape cervical expansive laminoplasty (60 pts), apaceram (1 pt), centerpiece (20 pts). all patients presented myelopathy in a physical exam and cord compression was observed by mri. a total of 48 patients (opll(36 ts), csm(12 pts)) that underwent box - shape cervical expansive laminoplasty with miniplate or maxpacer were enrolled to this study. male patients were more common than females (m : f=35:13) and the average age was 62.83 (max 81, min 30) (table 1). a french - door (double - door) laminoplasty horizontal amputation of the spinous processes was performed and bilateral lamina exposure was carried out. midline laminotomy was subsequently achieved with a drill and lateral outer cortical bone drilling was performed to facilitate elevation. when performing lateral outer cortical bone drilling, the surgeon must find the lamina - facet junction, which is a landmark for drilling. it is important to drill medially to the facet joint to have adequate spinal canal area. because a narrow drilling space can induce lamina fracture during elevation, adequate space is important to avoid fracture. after drilling, the ligament flavum was split centrally and each lamina and ligamentum flavum was opened bilaterally until the lamina stood straight. after proper positioning of the lamina, miniplates were applied to the space between both lamina. clinical outcomes of patients who received box - shape cervical expansive laminoplasty were assessed at postoperative day (pod)-6 months using the joa scoring system, performed by a single observer in all 48 patients. clinical outcomes were compared to patients with opll (36 pts), csm(12 pts) before and after surgery. postoperative ct scans were performed in 39 patients at 177 levels for a total of 354 screws to investigate the position of intralaminar screws. between june 2008 and july 2013, a total of 87 patients with opll and csm underwent laminoplasty at our institute with box - shape cervical expansive laminoplasty (60 pts), apaceram (1 pt), centerpiece (20 pts). all patients presented myelopathy in a physical exam and cord compression was observed by mri. a total of 48 patients (opll(36 ts), csm(12 pts)) that underwent box - shape cervical expansive laminoplasty with miniplate or maxpacer were enrolled to this study. male patients were more common than females (m : f=35:13) and the average age was 62.83 (max 81, min 30) (table 1). a french - door (double - door) laminoplasty was used in all cases discussed. horizontal amputation of the spinous processes was performed and bilateral lamina exposure was carried out. midline laminotomy was subsequently achieved with a drill and lateral outer cortical bone drilling was performed to facilitate elevation. when performing lateral outer cortical bone drilling, the surgeon must find the lamina - facet junction, which is a landmark for drilling. it is important to drill medially to the facet joint to have adequate spinal canal area. because a narrow drilling space can induce lamina fracture during elevation, adequate space is important to avoid fracture. after drilling, the ligament flavum was split centrally and each lamina and ligamentum flavum was opened bilaterally until the lamina stood straight. after proper positioning of the lamina, miniplates were applied to the space between both lamina. clinical outcomes of patients who received box - shape cervical expansive laminoplasty were assessed at postoperative day (pod)-6 months using the joa scoring system, performed by a single observer in all 48 patients. clinical outcomes were compared to patients with opll (36 pts), csm(12 pts) before and after surgery. postoperative ct scans were performed in 39 patients at 177 levels for a total of 354 screws to investigate the position of intralaminar screws. for patients given the box - shape cervical expansive laminoplasty, the total average joa score improved from 11.49 to 14.22 by pod-6 months. in the 36 patients with opll, scores improved from 11.32 to 14.3. in the 12 patients with csm, scores improved from 12 to 14 (table 2). most patients ' joa scores improved and there were none with joa scores that decreased (fig. of the 48 patients that received the box - shape cervical expansive laminoplasty, postoperative ct scans were performed in 39 patients at 177 levels for a total of 354 screws. the total malposition rate of intralaminar screws was only 3.4% (12 out of 354). 4), center to medial invasion, center to lateral invasion, and pulling out were 4%, 2%, 0%, 4%, and 1%, respectively (table 3). but, hardware - related neurological complications did not occur in any of the cases. for patients given the box - shape cervical expansive laminoplasty, the total average joa score improved from 11.49 to 14.22 by pod-6 months. in the 36 patients with opll, scores improved from 11.32 to 14.3. in the 12 patients with csm, scores improved from 12 to 14 (table 2). most patients ' joa scores improved and there were none with joa scores that decreased (fig. of the 48 patients that received the box - shape cervical expansive laminoplasty, postoperative ct scans were performed in 39 patients at 177 levels for a total of 354 screws. the total malposition rate of intralaminar screws was only 3.4% (12 out of 354). the rates of medial invasion (fig. 4), center to medial invasion, center to lateral invasion, and pulling out were 4%, 2%, 0%, 4%, and 1%, respectively (table 3). but, hardware - related neurological complications did not occur in any of the cases. this method achieves expansion of the spinal canal and preservation of the posterior structures for stability11,13,14,18,19,20). the relationship between degree of spinal canal expansion and clinical outcomes was not known and current techniques for canal expansion are not sufficient5,9). however, maintaining spinal canal expansion is critical. so we developed the box - shape cervical expansive laminoplasty for use with the miniplate or maxpacer. the box - shape cervical expansive laminoplasty allowed for maximal expansion of the spinal canal, and neurologic deterioration caused by restenosis or hinge reclosure did not occur14). edmund frank. reported a technique using titanium miniplates that permitted adequate decompression of the cervical spinal cord and bilateral cervical nerve roots. deutsch and harel. showed successful laminoplasty using a ti - mesh lp miniplate system without complications in five patients. reported performing laminoplasty using a novel titanium system that provided secure laminar fixation, thus minimizing the risk of canal restenosis, better preserving motion and decreasing axial neck pain2,4,17). in this study, we investigated clinical outcomes associated with our procedure. we show that those who received boxshape cervical expansive laminoplasty had an average joa score increase from 11.49 to 14.22 at pod-6 months. one patient, however, suffered from c5 palsy at pods 5 to 7, but they completely recovered by pod-6 months. although excessive opening of the spinal canal results in risk of nerve root kinking and epidural scar tissue21,22), the major finding of this study is that maximal expansion of the spinal canal leads to improvements in joa scores of patients with severe opll or csm with narrowed canals. the defining characteristic of this operation is the use of intralaminar screws to create maximal standing at each lamina. however, the double - door laminoplasty to create maximal lamina standing is a difficult technique and insertion of intralaminar screws at the proper position can be tedious. proper positioning of the intralaminar screw is important for holding the remodeled lamina - miniplate. to determine whether screws were inserted in the proper intralaminar position, we reviewed postoperative ct scans of the surgical methods at pod-6 months. of the 39 patients, scanned at 177 levels in 354 screws, the malpositioning rate of intralaminar screws was only 3.4%. no hardware - related complications occurred in patients after pod-6 months and there were only 4 screws that invaded the medial wall. however, this does not present a problem because the ligament flavum is a good protector of medially invaded screws. to avoid improper screw insertion, it is essential to have an anatomical understanding of the spinous process and medial lamina wall. additionally, even though the miniplate screw is selftapping, it does not necessarily mean that the screw will penetrate to the proper insertion point. creating a screw pathway with a small power drill can make correct insertion of the intralaminar screw easier. one limitation to this study is that the follow - up period was only 6 months, and posterior neck pain (vas score) and range of cervical motion were not analyzed. and comparative study should be performed to assess clinical benefits by comparing other cervical laminoplasty methods. the box - shape cervical expansive laminoplasty provided maximal canal expansion and demonstrated excellent clinical benefits. this method is easy to perform, provides a rigid construct, and intralaminal screws are placed in the proper position without inducing neurologic deficits. | objectivebox - shape cervical expansive laminoplasty is a procedure that utilizes a miniplate or maxpacer to achieve maximal canal expansion. this method is expected to show much larger canal expansion and good clinical outcome. so we investigated the clinical and radiological outcome of box - shape cervical expansive laminoplasty.methodsbetween june 2008 and july 2013, we performed cervical expansive laminoplasty in 87 and 48 patients using the box - shape cervical expansive laminoplasty, respectively. we analyzed the clinical results of these operations using the japanese orthopedic association (joa) scoring system and by assessing the position of intralaminar screws with postoperative computed tomography (ct) at pod-6 months.resultsa total of 48 patients with ossification of the posterior longitudinal ligament (opll) (36 pts), cervical spondylotic myelopathy (csm) (12 pts) were enrolled. overall joa scores improved from 11.49 to 14.22 at pod-6 months (opll : 11.32 -->14.3 ; csm : 12 - ->14). postoperative ct scans were performed in 39 patients at 177 levels for a total of 354 screws. the malpositioning rate of intralaminar screws was 3.4% and hardware - related neurologic complications did not occur.conclusionbox-shape cervical expansive laminoplasty creates maximal spinal canal expansion and leads to improved cervical myelopathy. the use of intralaminar screws to fix the remodeled lamina - facet does not represent a significant difficulty. |
the sounds heard by tinnitus sufferers can be a ringing, buzzing, chirping, roaring, and/or a large variety of other types of sounds. the sound may be intermittent or constant and may localize to the right ear, left ear, either ear, or neither ear but instead may be perceived in the head. epidemiology studies report that 15%20% of the adult populations experience some form of tinnitus, either temporarily or permanently. many people can cope with chronic tinnitus, but for 1 - 2% of the population, it is a severe handicap significantly impairing their quality of life. according to the american tinnitus association, fifty million americans suffer with tinnitus and tinnitus is the most prevalent service connected disability among veterans (exceeding posttraumatic stress disorder and traumatic brain injury). the department of veterans affairs (va) has reported that tinnitus disability claims have exceeded 840,000 and the cost to compensate veterans for tinnitus is over $ 1.28 billion annually. a wide variety of therapies exist for tinnitus, but none have been consistent in providing relief. this is due, in part, to the fact that tinnitus patients have not been clustered in such a way that determines the best treatment approach for individual patients. for instance, loud noise exposure is the most prevalent and direct cause of tinnitus and loud noise exposure can induce primary neuropathy of the viiith craniofacial nerve (primary auditory neurons) whether the hearing loss recovers or not. furthermore, patients with normal hearing and tinnitus reveal deafferentation of high threshold viiith nerve fibers with normal low and mid threshold fibers, indicating that only a subset of neurons may drive tinnitus. these and other evidences demonstrate that tinnitus is a signal for the presence of dysfunctional auditory neurons. however, not all auditory neuronal dysfunctions lead to tinnitus and therefore tinnitus may represent a specific type of dysfunction among a specific population of neurons. tinnitus may start within the viiith nerve but the generating loci may shift with time to more central (brainstem and cortex) regions. this suggests that the particular auditory neuronal dysfunction that drives tinnitus will also affect the central nervous system. this is plausible, given that certain lesions to the viiith nerve result in the reorganization of tonotopic maps in the cortex and more important for tinnitus, results in an increase in neuronal excitability from the brainstem to the cortex. recent literature has shown that up to 77% of the tinnitus population may present with psychiatric comorbidities [2, 8, 9 ]. among those psychiatric disorders, anxious and depressive symptoms seem to be the most common complications with tinnitus, with a lifetime prevalence of depression and anxiety significantly higher in tinnitus patients than in the general population. when considering psychological comorbidities such as anxiety / depression with tinnitus, the disease may be further categorized into compensated versus decompensated tinnitus. in compensated tinnitus, the patient copes well with the tinnitus and there is little or no psychological strain. on the other hand, in decompensated tinnitus, the tinnitus perception is considered uncontrollable and interferes with the patient 's quality of life, causing associated emotional and psychological distress. in compensated tinnitus patients, the perception of auditory sounds is normally extinguished in a short time through the habituation mechanism : the superior brain (involving the frontal gyri, cingulate gyrus, and parietal cortices) activates thalamic filters to switch off the signal, often independently of the resolution of the dysfunction that generated the tinnitus (peripheral auditory nerve dysfunction and neural changes of the central auditory system). this mechanism was initially identified by dehaene and changeux as components a global neuronal workspace that is activated by normal hearing subjects when required to consciously process task stimuli and make behavioral responses. it has recently been proposed that engagement of the global workspace is essential for the conscious experience of a tinnitus sound. in other words, the perception of tinnitus requires active awareness of the sensation, and shifting attention extinguishes the perception of the sounds. this understanding plays a significant role in medical therapy for tinnitus, as is evidenced in the use of masking techniques. in decompensated tinnitus, there is usually a negative emotional reaction such as fear, anxiety, or tension associated with the perception of the sounds. psychology literature illustrates that negative appraisals of events and situations produce distorted misinterpretations of the events, leading to memory and attention strategies that recall negative elements in the environment during the event [13, 14 ]. based on this model, fagelson proposed that the individual suffering from tinnitus could develop emotional disorders such as anxiety and depression that were triggered or heightened by an inappropriate interpretation of a sensory event. while fagelson studied a cohort who also suffered from ptsd (posttraumatic stress disorder) and this disorder was not included in our patient population, the finding of these investigators can only be compared with ours with qualification. in this context, negative emotional reinforcement may interfere with the habituation mechanism by drawing more attention to the perception and prolonging the event. although current literature is still inconclusive on whether tinnitus leads to anxiety and depression or vice versa, substantial physiological evidence through pet and fmri studies suggests that negative emotions and continued perception of tinnitus are amplified in a vicious negative feedback loop. and hazell and jastreboff describes continued perception of tinnitus as being supported by the amygdala, which is also activated by negative emotions. when tinnitus and negative emotions are present together, there is amplification (via increased neuronal excitability) and chronification (via neural plasticity mechanism) of signals, resulting in persistence of both emotional and tinnitus symptoms. because of the strong association of tinnitus with psychiatric disorders and indications that veterans are particularly vulnerable to experiencing tinnitus, anxiety, and depression, this study aimed to further evaluate comorbid anxiety and depression associated with tinnitus in a veteran population. ninety - one male veterans who reported subjective tinnitus were enrolled in a vamc tinnitus clinic from 2010 to 2013. a retrospective chart review of case history, audiometric thresholds, self - assessment of tinnitus handicap, and icd-9 codes was conducted on all patients. data from the medical records included demographic information, tinnitus case history, audiologic case history, pure - tone thresholds, and information contained in self - assessment inventories. the diagnoses of anxiety and depression were established through screening and intake examinations conducted by the behavioral medicine service at the vamc. icd-9 codes for anxiety and depression were used to identify patients with these diagnoses and were recorded as categorical data for analysis (e.g., 1 = anxiety, 0 = no anxiety). pure - tone averages (ptas) at 500, 1000, and 2000 hz were used to assess hearing status. for the purpose of analyzing the effect of anxiety and depression on tinnitus, the patients were divided into five groups : tinnitus with anxiety, tinnitus without anxiety, tinnitus with depression, tinnitus without depression, and tinnitus with both anxiety and depression (see table 1). spss (version 21 ; ibm corporation, armonk, ny) was used for statistical analysis. among the 91 male veterans with subjective tinnitus, 72/91 (79.1%) were diagnosed with anxiety ; 19/91 (20.9%) were without anxiety ; 54/91 (59.3%) were diagnosed with depression ; 37/91 (40.6%) were without depression ; and 53/91 (58.2%) were diagnosed with both anxiety and depression. table 1 lists the median age, standard deviations, and age ranges among five groups. there is no significant age difference among the five groups, with the median age being 64. the mean ptas showed no differences among the five groups or between ears (p > 0.05). the average hearing loss was around 38 db hl among all the groups (figure 1). a bivariate plot of the ptas and the thi scores was performed and indicated that both the ptas and thi scores displayed substantial variability, and the comparison of means demonstrated that self - assessed handicap between the groups was independent of auditory thresholds (data no shown). the thi is a 25-item self - report questionnaire that has functional, emotional, and catastrophic subscales. figure 2(a) shows that patients with anxiety had elevated total thi scores as compared to patients without anxiety (p 0.05). figure 2(b) shows that patients with either anxiety or depression had significantly increased functional thi scores (p 0.05) (figure 2(d)). interestingly, patients with both anxiety and depression do not have worsening subscale scores as compared to patients with either condition (figure 2). there is no significant difference between patients with neither anxiety nor depression versus patients with no anxiety or no depression only in terms of thi and subscale scores (figure 2). functional thi scores had the strongest correlation with both anxiety and depression, followed by emotional scores. the data also demonstrated that anxiety has higher correlation to more severe tinnitus in terms of total thi score than depression. tinnitus characteristics such as occurrence (e.g., intermittent and persistent), lateralization (unilateral and bilateral), and years of suffering were evaluated among patients with and without anxiety / depression (see table 3). the prevalence of anxiety was higher among patients with intermittent tinnitus relative to those who suffer with persistent tinnitus. however, depression was more prevalent among those who suffered with persistent tinnitus relative to intermittent tinnitus. the prevalence of anxiety was higher among patients who suffered with unilateral tinnitus relative to those with bilateral tinnitus. in contrast, the prevalence of depression was higher among patients with bilateral tinnitus compared with patients with unilateral tinnitus. interestingly, both anxiety and depression tend to be more prevalent after 10 years of suffering with tinnitus. these results suggest that the characteristics of the tinnitus perception may be associated with comorbid conditions. subjective tinnitus, as opposed to objective tinnitus with a physical stimulation generator within the body, is by far the more common type of tinnitus experienced by patients. various studies have shown a positive correlation between the severity of tinnitus and quantitative measures of anxiety and depression. unterrainer. showed that comorbid depression was one of the best predictors for perceived severity of tinnitus, greater than duration, pitch, locus of control, or the perception of tinnitus as an illness. according to zger., increased severity of anxiety and depression is associated with more severe tinnitus. this finding was replicated by zeman. using the thi questionnaire, underscoring the usefulness of the thi as a screening instrument for comorbid depression and anxiety. the same study also found that 15 out of the 25 items of the thi are significantly related to the beck depression inventory (bdi) and explain more variance of bdi than the total thi score, although the authors suggest the thi should still be considered a one - factor instrument in explaining quality of life. similar to the above studies, we demonstrated positive correlations between the severity of tinnitus and anxiety or depression in a va population. furthermore, tinnitus patients presented with anxiety (79.1%) and depression (59.3%) were considerably higher than the civilian population [2, 8, 9, 22 ]. this finding might suggest that veterans with tinnitus present with a higher prevalence of anxiety and depression than the general population ; however more extensive analysis of larger veterans cohorts will likely be needed to clarify such a predilection. when comparing anxiety against depression, granjeiro. showed that patients with depression had milder tinnitus symptoms while patients with anxiety had more moderate tinnitus symptoms, and patient with both anxiety and depression together had the most severe tinnitus symptoms. the same study also showed that higher anxiety and depression scores correlated with higher tinnitus severity scores. in addition, our study showed that anxiety has higher correlation to more severe tinnitus than depression, consisting with granjeiro. however, our study showed that patients with both anxiety and depression do not exhibit worsening tinnitus symptoms as compared to patients with anxiety or depression alone, which was different from the above study. interestingly, folmer. found no correlation between depression and loudness of perceived tinnitus, but there was a positive correlation with severity of tinnitus. this finding is in line with the model that the severity of tinnitus as elucidated by the questionnaires as well as associated emotional distress is less related to the causes of the condition and more to the cognitive factors of perception and reaction to tinnitus [20, 24 ]. when looking at the reverse effects of tinnitus on anxiety and depression, gomaa. found that the duration of tinnitus had positive correlation on both severity of depression and severity of anxiety, but the severity of tinnitus did not affect severity of anxiety and depression. in contrast to most literature, ooms. found no correlation between depression and tinnitus and proposed that high correlations between the thi and bdi scores were due to significant content overlap. the thi was validated against the bdi in its original development, and langguth. further countered ooms 's argument with a study using quality of life measurements that indicated high thi scores reflecting a significant impairment in quality of life, independent of potential overlap in single items between the thi and bdi. our results showed that there were no differences in the mean ptas among the five groups or in between ears in all the groups. the lack of association between the pta results and anxiety or depression suggests that these variables are independent in our veteran population. subjective tinnitus has been assumed to be caused by or associated with damage to the auditory system, both peripherally and centrally. early theories of tinnitus neural mechanisms suggested a peripheral generation model, where the origin of the phantom sound resides in the inner ear. the idea was based on the fact that cochlear damage from traumatizing sounds and ototoxic agents induced both hearing loss and tinnitus. however, this idea was countered when bilateral auditory nerve sectioning did not always eliminate tinnitus. current measurements with magnetoencephalography (meg) show increased spontaneous firing rate (sfr) of neurons in several auditory structures including the dorsal and ventral cochlear nucleus, the inferior colliculus, and auditory cortices, but no signals were seen in peripheral nerve fibers. this evidence points to a central generation model, where all forms of tinnitus, even those triggered by cochlear damage, have origin in the central auditory system (cas). the connection between peripheral damage and central auditory changes is explained as an alteration in the normal balance between excitatory and inhibitory nerve transmission brought about by loss of inhibition, which leads to increased firing rate [28, 31 ]. auditory regions of the cerebral cortex have also been studied in relation to the tonotopic reorganization model of tinnitus., a lack of afferent signals from a specific region of the cochlea due to hearing loss leads to a rapid reduction in activity within the corresponding section of the auditory cortex, and neural plasticity allows new connections to adjacent cortical fields. as a consequence of this reorganization, a disproportionately large number of neurons become sensitive to lower or high frequencies bordering on the area of hearing reduction. functional mri have shown increased signals in the middle and superior frontal gyri, the cingulate gyrus, the precuneus, and the parietal cortices in tinnitus patients, verified by eeg and meg studies. the nonauditory structures are evidenced to play the greatest role in understanding the reaction to tinnitus, the subjective reporting of tinnitus severity, persistence of tinnitus perception, and physiological relationship to comorbid psychological factors such as anxiety and depression. functional imaging studies show that the subgenual anterior cingulate cortex (sgacc) plays an important role in both coping styles and depression. the most recent study by vanneste. showed that tinnitus patients using a maladaptive coping style show increased scores on the bdi and thi and experienced louder sounds and more distress in comparison to tinnitus patients using adaptive coping styles. these evidences further suggest why tinnitus can be associated with major depression, anxiety, and other psychosomatic and/or psychological disturbances. standard care of subjective tinnitus involves education / counseling, sound therapy (either hearing aids or sound generators), and intervention to reduce the distress (relaxation therapy or cognitive behavioral therapy (cbt) or both). the approach to using cbt for tinnitus patients follows the theory that relaxation and cognitive restructuring of thoughts may promote improved and habituated responses to the phantom noise, thus decreasing the distress level and perceived severity of tinnitus. the cochrane collaboration in 2007 reviewed 6 trials of cbt for tinnitus with 285 participants. although the data analysis did not demonstrate any significant effect in the subjective loudness of tinnitus or comorbid depression, there was a significant improvement in quality of life, as assessed by the thi. this outcome further supports the idea that anxiety and depression are comorbidities that affect the perception and response to tinnitus. at present no specific therapy for tinnitus is acknowledged to be satisfactory for all patients. due to the high comorbidity of tinnitus and psychiatric illnesses, a wide range of pharmacological agents, including anticonvulsants, benzodiazepines, tricyclic antidepressants (including amitriptyline, imipramine, and nortriptyline), and selective serotonin reuptake inhibitors (ssris), have been used. however, there is debate about whether psychoactive drugs act on the central auditory system and reduce tinnitus directly, whether they act by treating concomitant psychological illnesses, or whether they have a simultaneous effect of both the psychological disturbance and tinnitus. the cochrane study in 2012 evaluated six trials involving 610 patients with the object of assessing the effectiveness of antidepressants in the treatment of tinnitus and whether any benefit is due to a direct tinnitus effect or a secondary effect due to treatment of concomitant depressive states. they concluded that all trials assessing tricyclic antidepressants showed slight improvement in tinnitus, but the effects may have been attributed to methodological bias. the ssri drug trial showed possible benefit for the subgroup that received higher doses of ssris, and the observation merits further investigation. the trial investigating trazodone showed an improvement in tinnitus intensity and quality of life, but it did not reach statistical significance. overall, the cochrane collaboration concluded that further research is required because only one of the studies they reviewed met the high quality standard. in conclusion, this study verified that strong correlations exist between the degree of tinnitus and anxiety and depression in a veteran population. although tinnitus and anxiety / depression involve distinct perceptual events, they may share many mechanisms of the central nerve system, particularly those comprising the auditory cortical pathways and limbic system. a multidisciplinary team comprising psychiatrists and audiologists is needed to evaluate and manage the tinnitus patients with psychiatric comorbidity. we will further determine mechanisms of tinnitus, including whether particular neuronal dysfunction that drives tinnitus may also lower the threshold of susceptibility for anxiety / depression or vice versa. | objective. the mechanisms of tinnitus are known to alter neuronal circuits in the brainstem and cortex, which are common to several comorbid conditions. this study examines the relationship between tinnitus and anxiety / depression. subjects and methods. ninety - one male veterans with subjective tinnitus were enrolled in a veterans affairs tinnitus clinic. the tinnitus handicap inventory (thi) was used to assess tinnitus severity. icd-9 codes for anxiety / depression were used to determine their prevalence. pure tone averages (pta) were used to assess hearing status. results. descriptive analyses revealed that 79.1% of the 91 tinnitus sufferers had a diagnosis of anxiety, 59.3% had depression, and 58.2% suffered from both anxiety / depression. patients with anxiety had elevated total thi scores as compared to patients without anxiety (p < 0.05). patients with anxiety or depression had significantly increased functional and emotional thi scores, but not catastrophic thi score. significant positive correlations were illustrated between the degree of tinnitus and anxiety / depression (p < 0.05). there were no differences in pta among groups. conclusions. a majority of patients with tinnitus exhibited anxiety and depression. these patients suffered more severe tinnitus than did patients without anxiety and depression. the data support the need for multidisciplinary intervention of veterans with tinnitus. |
acute kidney injury is a common clinical syndrome that is characterized by a rapid decline in kidney function, often triggered by glomerular disease and/or tubulointerstitial disease and associated with high morbidity and mortality [14 ]. in addition to serum creatinine, 2-microglobulin is a biomarker that can be used to determine underlying causes of acute kidney injury. serum 2-microglobulin derives from cellular membrane turnover, since 2-microglobulin forms the invariant light chain portion of major histocompatibility complex (mhc) class i in membranes of all cells [710 ]. as a single - chain small polypeptide (mw = 11.8 kda), 2-microglobulin is filtered almost completely through the glomeruli of the healthy kidney [11, 12 ] and then reabsorbed by the renal proximal tubules. only a small amount of 2-microglobulin can be detected in the urine under normal physiological conditions. therefore, levels of serum and urinary 2-microglobulin reflect the functions of glomeruli and proximal tubules. in patients with acute kidney injury, an increase in serum creatinine, together with an increase in urinary 2-microglobulin, strongly suggests proximal tubule injury. on the other hand, an increase in serum creatinine with elevated serum 2-microglobulin is seen in patients with decrease in glomerular filtration rate. elevated urinary 2-microglobulin was associated with tubular injury caused by viral infection, ischemia, and toxicity from medications or heavy metals [15, 16 ]. measurement of urinary 2-microglobulin is convenient and has been applied in routine clinical practice (renalvysion bostwick laboratories, uniondale, ny) to evaluate the tubular function. using immunohistochemical staining for cd133 in kidney biopsies, we demonstrated that urinary 2-microglobulin is a sensitive indicator for tubular injury. however, a direct correlation between urinary 2-microglobulin and proximal tubule injury has not yet been fully established due to lack of a specific proximal tubule injury marker. recently, kidney injury molecule-1 (kim-1) was shown to be a biomarker for proximal tubule injury [1820 ]. kim-1 is a type 1 cell membrane glycoprotein comprised of extracellular, transmembrane and intracellular domains [21, 22 ]. kim-1 is undetectable in the healthy kidney. with different forms of kidney injury, mrna and protein levels of kim-1 are upregulated and expressed [20, 23, 24 ] in injured tubules [23, 2527 ]. kim-1 binds to surface - specific epitopes on the apoptotic bodies and cellular debris derived from injured tubular cells, facilitating macrophages to engulf dying cells. in addition, the extracellular domain of kim-1 can be cleaved by metalloproteinases into a soluble shedded part and short membrane bound fragments that are released into urine. moreover, the expression of kim-1 in injured tubule cells is limited to the proximal tubular cells. using immunohistochemistry, kim-1 staining in kidney tissue has been shown to accurately detect proximal tubular injury associated with ischemic and toxic type of tubular injury, allograft rejection in renal transplantation, and acute pyelonephritis [20, 26, 28 ]. the present study was designed to examine the correlation between increase in urinary 2-microglobulin and proximal tubule injury assessed by kim-1 staining in kidney biopsies. between january 2009 and december 2012, a total of 5494 patients had urinary 2-microglobulin measurements by renalvysion analysis (bostwick laboratories). among these, nine renal biopsies either contained less than 10 glomeruli per histological section or had insufficient tissue to perform immunohistochemical staining and were excluded from the present study. the remaining forty - six biopsies met selection criteria and were included in the present study. briefly, 10 ml fresh urine was collected from each patient and urine ph was adjusted to ph 68 with 1 m naoh. the specimen was delivered to lab by overnight shipping and the level of 2-microglobulin was measured next day. the majority of immunohistochemical staining was performed in archived unstained slides prepared at the time of renal biopsy. in those biopsies in which unstained slides were not available, additional new slides were prepared from archival tissue from formalin - fixed paraffin - embedded blocks. immunohistochemical staining for kim-1 was conducted on paraffin slides using dako autostainer (dakocytomation, carpinteria, ca). to remove paraffin, sections were heated at 60c for 60 min, placed into xylene baths for 3 min, 3 times, followed by 100% alcohol, for 3 min, 3 times, and then washed by running water for 30 s. antigen retrieval was carried out using a tris - edta buffer at ph 8.0 for 20 min at 99c and cooled down for 20 min at room temperature, raised by tap water. slides were placed into h2o2 for 15 min and then placed in tris buffer (ph 7.6). finally, slides were placed into dako autostainer programmed by thermo scientific ultravision lp detection system (kalamazoo, mi). staining procedures included 5 min ultra v block, 30 min incubation with primary kim-1 monoclonal antibody (akg7 clone at 1 : 10 dilution, kindly provided by dr. joseph v. bonventre from renal division brigham and women 's hospital, boston), 8 min primary antibody enhancer, 10 min hrp polymer (equivalent to secondary antibody), and 5 min of chromogen dab to achieve brown staining. granular staining in the cytoplasm and membrane of proximal tubular epithelium was considered positive. staining intensity of kim-1 was scored from 0 to 3 + as follows : 0, absence of staining ; 0.5, focal weak fine granular staining ; 1 +, weak fine granular staining cytoplasm and/or membrane in entire tubular luminal surface ; 2 +, moderate granular staining ; and 3 +, strong coarse granular staining. parameters related to tubular injury including serum creatinine, granular and cellular casts, and urine cytological findings were also recorded. staining with kim-1 was evaluated by two pathologists who were blinded to the clinical data, final pathological diagnosis and patient outcome. linear regression analysis was used to correlate values of urinary 2-microglobulin and kim-1 staining scores. odds ratio analysis was applied to express the association of urinary 2-microglobulin level and intensity of kim-1 staining. data were also stratified according to urinary 2-microglobulin level as normal (< 300 g / l) versus increased to kim-1 staining (positive or negative) and evaluated by 2 2 contingency table (table 2). the sensitivity, specificity, predictive value for positive result, and predictive value for negative result were calculated as follows : sensitivity = true positive/(true positive + false negative) ; specificity = true negative/(true negative + false positive) ; predictive value for positive result = true positive/(true positive + false positive) ; predictive value for negative result = true negative/(true negative + false negative);the age of the patients was expressed as mean sd. sensitivity = true positive/(true positive + false negative) ; specificity = true negative/(true negative + false positive) ; predictive value for positive result = true positive/(true positive + false positive) ; predictive value for negative result = true negative/(true negative + false negative) ; among 5494 patients with urinary 2-microglobulin measurement, renal biopsy was obtained out in 56 patients (56/5494, 1.0%). among these, thirty patients had increases in urinary 2-microglobulin ; it was in normal range in the remaining 16 patients. the demographic details for 35 patients (m : f = 22 : 13) with positive kim-1 staining in proximal tubules are shown in table 1. the age of patients at the time of biopsy was 54.9 10.7 years ranging from 16.0 to 87.0 years. glomerular diseases included membranous nephropathy, antineutrophil cytoplasmic antibody- (anca-) associated glomerulonephritis, diabetic nephropathy, focal and segmental glomerulosclerosis, iga nephropathy, and lupus nephritis. in addition to glomerular disease, five patients had tubular injury (figures 1(c) and 1(d)), two of which were toxic type of injury with cytoplasmic vacuoles (figure 1(d)). of the 26 patients with elevated urinary 2-microglobulin and positive kim-1 staining in proximal tubules, 18 patients had increased serum creatinine, ranging from 1.50 mg / dl to 6.00 mg / dl (table 1). since the elevated urinary 2-microglobulin is associated with proximal tubular injury, in these 18 patients it was considered that the proximal tubular injury is the primary cause of increase in serum creatinine. nine patients with normal urinary 2-microglobulin had positive kim-1 staining in proximal tubules, 6 of which had increase in serum creatinine, ranging from 1.10 mg / dl to 3.20 mg / dl. granular casts were presented in 2 patients, who also had increase in both urinary 2-microglobulin and serum creatinine. thirty - five of 46 biopsies showed positive kim-1 staining with a granular pattern in the cytoplasm and/or apical membrane of tubular epithelium, ranging from finely granular to coarsely granular (figure 1(b)). the corresponding h&e slides of kidney biopsy showed flattening and simplification of tubular epithelium, ranging from focal to diffuse, overall focal tubular injury with varying degree, and mild tubular injury in the majority of the kidney biopsies. staining intensity ranged from 0.5 + to 3 + with 1 + staining in 18 patients (18/35, 51.4%). the relation between value of urinary 2-microglobulin and kim-1 staining score is shown in figure 2. there was a significant correlation between levels of urinary 2-microglobulin and kim-1 staining (r value = 0.293, p < 0.05) (figure 2). odds ratio was 7.229, implying that levels of urinary 2-microglobulin are strongly associated with intensity of kim-1 staining. confidence interval at the 95% range was 2.868 to 12.01. among 35 biopsies with kim-1 positive staining, using positive kim-1 staining as the end point, sensitivity was 86.6%, specificity was 43.7%, positive predictive value was 74.2%, and negative predictive value was 63.6%. we found that increased urinary 2-microglobulin was significantly correlated with kim-1 staining in proximal tubules, indicating that urinary 2-microglobulin is a sensitive (sensitivity 86.6%) assay for detecting tubular injury. further, because kim-1 is a selective proximal tubule injury marker, the present study also demonstrated that measurement of urinary 2-microglobulin was a reliable assay to detect proximal tubule injury. therefore, in patients with acute kidney injury with increase in serum creatinine, measurement of urinary 2-microglobulin is also helpful to identify the underlying tubular dysfunction, that is, proximal tubule injury. acute tubular injury is a common clinical diagnosis. in a patient with classic clinical presentations such as hypotension together with a rapid rising of serum creatinine, the diagnosis of severe tubular injury (acute tubular necrosis) can be made and does not require kidney biopsy to initiate treatment. however, the diagnosis of glomerular disease accompanied by tubular injury is not easy to establish, as both may contribute to elevated serum creatinine. tubular injury was the secondary diagnosis in 5/35 patients but other evidence of tubular injury such as tubular casts and an increase in tubular cells by urine analysis was found in 22/35 patients. moreover, 2 of 4 patients with secondary diagnosis of acute interstitial nephritis showed tubulitis, a feature associated with tubular injury. these findings indicate that glomerular diseases associated with varying degree of tubular injury could be missed in renal biopsies due to sampling. in a prior study, using cd133 staining in injured tubular epithelium, it was demonstrated that urinary 2-microglobulin was a sensitive indicator for tubular injury. as a progenitor marker in addition, cd133 positive cells are present not only in proximal tubular cells but also in distal tubular cells and/or atrophic tubules. therefore, a careful morphological correlation is needed to exclude false positive staining. by comparison, kim-1 is a selective proximal tubule injury marker that only presents in patients with proximal tubule injury [1820 ]. therefore, kim-1 is a more specific marker than cd133 for detecting proximal tubular injury. in the present study, kim-1 was able to detect patients with mild, focal tubular injury evidenced by urine analysis as an increase in number of tubular cells ; some patients presented with normal levels of serum creatinine (table 1). this was also supported with the finding in the present study that more than half of kim-1 staining score was weakly positive (1 +). a good correlation of urinary 2-microglobulin and kim-1 staining (r value of 0.293) indicates that measurement of urinary 2-microglobulin is a reliable assay for proximal tubule injury. similar to cd133 staining, kim-1 was also able to identify two major types of tubular injury : ischemic and toxic type (figures 1(c) and 1(d)). as shown in table 2, tubular injury was an additional diagnosis included in 5 of 26 biopsies (25%) (table 1), and all showed positive kim-1 staining in proximal tubules. using kim-1 staining as a proximal tubule injury marker in the present study, nine patients, rather than six patients with normal urinary 2-microglobulin, presented with positive kim-1 staining, which were considered false positive. with low specificity, clinical interpretation of elevated urinary 2-microglobulin should be closely correlated with clinical history, (e.g., history of hypotension, recent history of medications use, etc.) and clinical presentation, including value of serum creatinine, as well as urine cytological findings. the diagnosis of tubular injury with renalvysion was made not only based on the elevation of urinary 2-microglobulin and serum creatinine but also combined with morphological findings of tubular casts and the number of sloughed tubular epithelial cells in urine analysis. renalvysion is a urine - based analysis that combines urine chemistry and cytology to detect glomerular disease and tubular injury. elevation of urinary 2-microglobulin over 300 g / l was considered to be abnormal by renalvysion. g / l and positive kim-1 staining in biopsies were considered to be false positive. however, the actual values of urinary 2-microglobulin in 2 patients were marginally high in 280 g / l and 290 g / l. the remaining 7 patients were found to have levels less than 210 g / l, which is the measurement threshold of the assay. because the confidence interval at the 95% range in lower end was 2.868 in present study, the normal range of urinary 2-microglobulin applied in renalvysion should be reconsidered to be 2.868 g / l. g / l should be considered as abnormal and belongs to true positive for kim-1 staining. therefore, corrected sensitivity is 87.1%, specificity is 46.6%, positive predictive value is 77.1%, and negative predictive value is 63.6%. our study has multiple limitations. only a small number of patients had follow - up renal biopsies after renalvysion (56/5494, 1.0%), raising concern for selection and ascertainment biases. as shown in table 1, these patients had glomerular disease and/or tubular - interstitial disease. the reason for this might be that renal biopsy is usually avoided in patients with a clear clinical diagnosis of severe tubular injury. a larger study cohort involving patients with severe tubular injury is needed to better correlate renal histopathology and immunohistochemistry. owing to limited sampling by biopsies, the retrospective nature of the study represents an additional limitation. in summary, measurement of urinary 2-microglobulin is a sensitive assay to screen patients with proximal tubule injury. our data indicate a correlation of urinary 2-microglobulin with results of immunohistochemical staining of kim-1 in renal biopsy and consequently with proximal tubular injury. | objective. after filtration through glomeruli, 2-microglobulin is reabsorbed in proximal tubules. increased urinary 2-microglobulin indicates proximal tubule injury and measurement of 2-microglobulin in urine is useful to determine the source of renal injury. kidney injury molecule-1 (kim-1) has been characterized as a selective proximal tubule injury marker. this study was designed to evaluate the correlation of urinary 2-microglobulin concentration and kim-1 expression as evidence of proximal tubule injury. methods. between 2009 and 2012, 46 patients with urine 2-microglobulin (renalvysion) had follow - up kidney biopsy. diagnoses included glomerular and tubule - interstitial disease. immunohistochemical staining for kim-1 was performed and the intensity was graded from 0 to 3 +. linear regression analysis was applied to correlate the values of urinary 2-microglobulin and kim-1 staining scores. p < 0.05 was considered statistically significant. results. thirty patients had elevated urinary 2-microglobulin. kim-1 staining was positive in 35 kidney biopsies. there was a significant correlation between urinary 2-microglobulin and kim-1 staining (p < 0.05). sensitivity was 86.6%, specificity was 43.7%, positive predictive value was 74.2%, and negative predictive value was 63.6%. conclusion. increased urinary 2-microglobulin is significantly correlated with kim-1 staining in injured proximal tubules. measurement of urine 2-microglobulin is a sensitive assay for proximal tubule injury. |
diabetes can cause acute and long - term complications, and is as well, a global risk factor for cardiovascular disease.1 lifestyle and behavior, as well as individual genetic predisposition, influence the risk of diabetes and prevalence in a community.2 public knowledge is the basis for lifestyle and behavior in a given community.3 therefore, the knowledge particular to diabetes is a key determinant of the lifestyle of a community, and can discriminate those who are at risk from those who are free of risk. diabetes is increasingly prevalent in ethnic minority groups globally.2,4,5 recently, lorga reported that 16.7% of the karen rural community had an abnormally high blood glucose level (> 100 mg / dl), and that there was a considerable proportion of prediabetic persons (13.04%) in a cross - sectional survey among the karen minority. the karen ethnic rural community along the thai myanmar border is a hard - to - reach population for health promotion services. karen people use their native language and most of the senior adult population in the community can not read thai script. moreover, in a district like thasongyang, the population pattern is very dynamic and unstable because of a large migration across the border.7 karen people are indigenous to, and tend to stay along the thai - myanmar border such as area around tak province. there is limited literature to guide the assessment of health literacy and knowledge of diabetes in such a community.8 the objective of the study was to assess the knowledge of diabetes of the rural karen residents, and compare the diabetes knowledge between male and female respondents. this study was approved by the ethical board of the boromrajonani college of nursing nakhon lampang, lampang, thailand, and conducted with the verbal informed consent of participants. it was conducted in thasongyang, which is a district in the far northwest of thailand, by the narrow moei river on the border of myanmar. all participants were thai citizens but most of the participants could not read thai script. none had any prior known diagnosis of diabetes. diabetes knowledge was assessed by a questionnaire that was developed by consensus between native researchers and researchers from the boromrajonani college of nursing nakhon lampang. the questionnaires were delivered in the karen language by researchers speaking both thai and the karen language, and were completed by the interviewers. fasting plasma glucose (fpg) measurement was preceded by overnight fasting for 8 hours before the test. body weight and height were measured by the public health officers using a standard measuring scale. body mass index was calculated by the ratio of body weight in kilograms divided by the square of the height in meters. continuous variables were summarized as median, maximum, and minimum values, as the distribution was not normal. the nonparametric test for trend analysis was run by nptrend in stata version 11 (stata corp, college station, tx). yes, no, and not sure answers in the questionnaires were recoded as categories in order to compare by rank sum test. a correct answer was coded to be 1, and an incorrect answer, or the answer of not sure, was coded as zero. statistical significance was defined as p value less than 0.05, with a 95% confidence interval. diabetes knowledge was assessed by a questionnaire that was developed by consensus between native researchers and researchers from the boromrajonani college of nursing nakhon lampang. the questionnaires were delivered in the karen language by researchers speaking both thai and the karen language, and were completed by the interviewers. fasting plasma glucose (fpg) measurement was preceded by overnight fasting for 8 hours before the test. body weight and height were measured by the public health officers using a standard measuring scale. body mass index was calculated by the ratio of body weight in kilograms divided by the square of the height in meters. continuous variables were summarized as median, maximum, and minimum values, as the distribution was not normal. the nonparametric test for trend analysis was run by nptrend in stata version 11 (stata corp, college station, tx). yes, no, and not sure answers in the questionnaires were recoded as categories in order to compare by rank sum test. a correct answer was coded to be 1, and an incorrect answer, or the answer of not sure, was coded as zero. statistical significance was defined as p value less than 0.05, with a 95% confidence interval. we assessed the diabetes knowledge briefly in three parts (1) general knowledge, (2) knowledge about the risk factors, and (3) knowledge about the symptoms, diagnostic signs, and complications of diabetes mellitus. one - third of the population answered correctly (range 27.09%41.81%), and another one - third gave no answer to these questions (range 33.44%39.80%), whereas another one third gave a wrong answer. among the karen community, 36.45% knew that old age is a risk factor for diabetes, 37.12% knew that they had diabetic relatives, 39.80% knew that obesity can lead to diabetes, 37.12% answered that exercise can prevent diabetes, 25.09% reported that pregnant women can have diabetes, and 41.81% said they should not eat excess fatty food for fear of diabetes. regarding the symptoms, diagnosis and complications of diabetes, less than 30% knew that fasting blood glucose level should be less than 100 mg%. a few of the rural karen ethnic residents could correctly answer questions in the third part of the questionnaire. among the respondents, 33.78% answered that diabetic patients could have thirst and polyuria, 34.78% knew that numbness of the limbs might link to diabetes, 35.12% recognized the importance of foot care in people living with diabetes, 32.78% answered that untreated diabetes could lead to blindness, and 34.78% knew that slow wound healing might be due to diabetes. overall, one - third of the sample population gave the correct answers (range 32.78%34.78%) while the other two - thirds were either unsure or gave wrong answers. overall, public knowledge of diabetes among karen ethnic rural community is at the lower extreme (figure 1). diabetes knowledge was compared between male and female by median sum of the knowledge scores. overall knowledge level in female participants was also lower than in male participants (figure 2). the median sum of the knowledge scores was 6 (interquartile range : 211) among males, and 3 (interquartile range : 18) among the females (p for trend = 0.009). there was a significant difference in level of knowledge among females and males. we assessed the diabetes knowledge briefly in three parts (1) general knowledge, (2) knowledge about the risk factors, and (3) knowledge about the symptoms, diagnostic signs, and complications of diabetes mellitus. one - third of the population answered correctly (range 27.09%41.81%), and another one - third gave no answer to these questions (range 33.44%39.80%), whereas another one third gave a wrong answer. among the karen community, 36.45% knew that old age is a risk factor for diabetes, 37.12% knew that they had diabetic relatives, 39.80% knew that obesity can lead to diabetes, 37.12% answered that exercise can prevent diabetes, 25.09% reported that pregnant women can have diabetes, and 41.81% said they should not eat excess fatty food for fear of diabetes. regarding the symptoms, diagnosis and complications of diabetes, less than 30% knew that fasting blood glucose level should be less than 100 mg%. a few of the rural karen ethnic residents could correctly answer questions in the third part of the questionnaire. among the respondents, 33.78% answered that diabetic patients could have thirst and polyuria, 34.78% knew that numbness of the limbs might link to diabetes, 35.12% recognized the importance of foot care in people living with diabetes, 32.78% answered that untreated diabetes could lead to blindness, and 34.78% knew that slow wound healing might be due to diabetes. overall, one - third of the sample population gave the correct answers (range 32.78%34.78%) while the other two - thirds were either unsure or gave wrong answers. overall, public knowledge of diabetes among karen ethnic rural community is at the lower extreme (figure 1). diabetes knowledge was compared between male and female by median sum of the knowledge scores. overall knowledge level in female participants was also lower than in male participants (figure 2). the median sum of the knowledge scores was 6 (interquartile range : 211) among males, and 3 (interquartile range : 18) among the females (p for trend = 0.009). there was a significant difference in level of knowledge among females and males. the number of diabetes cases is rising worldwide, and the burden of this is more severe in low- and middle - income countries.9,10 belonging to an ethnic minority has been reported as a risk factor for the development of type 2 diabetes.2 disadvantaged people in these groups are more prone to the consequences of a silent epidemic.11 it is important to assess the public knowledge of diabetes, especially in minority ethnic populations. today, rising public health education and reduced disparity between groups are compulsory for the prevention and care of diabetes in all settings.12 risk perception of a community is the basis for healthy behavioral changes within a community.13 perception of risk among people at risk can prevent the disease thus, we assessed the perception of diabetes risk factors among the karen ethnic rural community. we found only one - third of the study sample had knowledge about diabetes risk factors (table 2). most in the karen community did not know that elderly age, being overweight, and physical inactivity are risk factors for diabetes. initiation of measures to educate this minority population, including use of mass media, and printed materials in the local language, might be a useful strategy for risk communication. knowledge of diabetes symptoms is important for early diagnosis and accessing care. recognizing the symptoms of diabetes and its complications may bring the diabetic individuals to seek health care service. symptoms of diabetes complications are common presentations, even though these might be indicators of late diagnosis, or poor glycemic control. individuals lacking knowledge of these may not seek medical care in time, and consequently will have complications of diabetes.14 we assessed such knowledge in karen ethnic community (table 2). the histogram of knowledge scoring was skewed to the left and peaked at lower end of the scoring (figure 1). in contrast, the similar histogram for public knowledge of diabetes in singapore, was reported as totally skewed to the right side.15 the knowledge distribution among the karen ethnic sample was at the lower end, and pointed out a need for intervention. in fact, diabetes and its consequent cardiovascular disease risk are higher in women.7 there is also increasing concern about gestational diabetes;16 however, our observations pointed out that diabetes knowledge among female karen participants was lower than among males. this type of health inequality has also been reported in many other diseases.17 type 2 diabetes is more common in disadvantaged groups of women, such as those with least income and who are least educated.18 risk behaviors for type 2 diabetes have been highly prevalent even in the developed setting;19 moreover, poor women in the developing world tend to delay seeking care until symptoms are too severe.17 in the particular setting of this study, it is likely that women usually work in the home at housekeeping, and become housebound and sedentary. a television education program in the local language might reach these women.19 lack of diabetes knowledge can cause missed diagnosis, late diagnosis, and higher incidence of diabetes complications. creation of a peer - group gathering place for women, such as a health education club for mothers, and exercise club for housewives, may promote women s knowledge of diabetes and sharing of information. in contrast to the current study findings, in a previous study, the public knowledge among thai people was reported to be fairly high and there was no gender gap between thai men and women.20 it is obvious that diabetes health education is poor among the karen ethnic community. this might be due to a language barrier, which is a major obstacle to educating minority groups, as in other parts of asia.21 existing evidence suggests that such a situation calls for a culturally tailored and well - designed health education strategy.22 as this was a pilot study swiftly conducted to provide important information, there were weaknesses in the current study. the questionnaire used in this study was made easily comprehensible for rural karen residents, and study findings may not be generalizable to other settings. albeit, the findings are the first of their kind to our knowledge, and are meant to assist in guiding further studies. despite these limitations, this investigation contributed to our understanding of the health disparity in an ethnic minority population. it provided insights into the need for health education interventions for the prevention of diabetes in a rural ethnic community. the result of this study showed that karen ethnic men and women were less knowledgeable about all aspects of diabetes. public education about diabetes in this setting should be started urgently and carefully, to overcome language and cultural barriers and prevent diabetes epidemic among these rural people. our study finding is expected to be a step forward, in minimizing the disparity in diabetes health education. | background and purposethe public knowledge of diabetes is important for prevention of disease. this study aimed to evaluate knowledge of diabetes, risk factors, and the common warning signs of diabetes and complications among community participants in a rural karen ethnic community.methodsparticipants were asked to answer a questionnaire regarding their knowledge of diabetes. fasting blood glucose testing, blood pressure measurement, and body mass index (bmi) assessment were provided to the participants. the study was conducted at thasongyang district, tak province, thailand.resultsa total of 299 karen rural residents were included in the study. the median age was 45 years and median fasting blood glucose was 88 mg / dl. the response rate to the questionnaires was 91.97%. half of the participants knew diabetes is a noncommunicable disease needing lifelong treatment. overall, one - third of the community participants could correctly answer the knowledge assessment questions regarding risk factors and common features of diabetes. whereas the other two - thirds either gave a wrong answer or were not sure. female participants had poorer diabetes knowledge than the males.conclusionthe public knowledge of diabetes, as represented by this sample of the karen ethic community, is alarmingly low. there is significant gender difference in knowledge level. culturally tailored and gender - sensitive diabetes health education interventions are urgently needed in this minority ethnic community. |
it is caused by the protozoan parasite of genus plasmodium and transmitted by the female anopheles mosquito. so far, only 5 of the 170 species of this parasite have been found which are the cause of disease in humans. they consist of plasmodium falciparum, plasmodium knowlesi, plasmodium ovale, plasmodium vivax, and plasmodium malariae. the latest released statistics by december 2014 showed 198 million cases of malaria in 2013 comprised of estimated 584,000 deaths. malaria mortality rates have fallen by 47% globally since 2000 and by 54% in the who african region. since the 17th century, the bark of the cinchona tree which was the source of quinine had been the first effective western treatment for malaria. however, chloroquine replaced it from the 1940s, although quinine is still used under certain circumstances. the resistance against antimalaria drugs is a drawback of standard drugs like chloroquine, sulphadoxine - pyrimethamine, and even artemisinin. cinnamon consists of cinnamaldehyde compounds, volatile oils, tannins, mucilage, limonene, and safrole that possesses antibacterial, antiseptic, antiviral, and antifungal properties. senhaji. in 2005 tested different extracts of cinnamon like the aqueous, hexane, methanol, and ethanol on gram positive and negative bacteria as well as yeast, leishmania, and toxoplasma with positive results. more recently, nkanwen and colleagues in 2013 tested the bark of cinnamon for antiplasmodial activity and found that it had an inhibitory effect on plasmodium falciparum enoyl - acp reductase enzyme. metabonomics is the recent omics that studies simultaneously all the metabolites and small molecules in biological fluids, cells, and tissues. it uses high throughput technology like h nuclear magnetic resonance (hnmr) and liquid chromatography mass spectrometry (lc - ms). it plays the most important role in direct observation of the physiological status of an organism or the cell and is a faster and more affordable way of testing drugs and their mechanism of action. earlier studies have reported antiplasmodial effect of cinnamon extract and its result on one of its enzymes. it was decided to study the metabolome of plasmodium falciparum after exposure to cinnamon extract by hnmr spectroscopy. cinnamomum cassia obtained from mumbai, india, were ground into a fine powder. 50 grams of cinnamon powder was dissolved in 500 ml of distilled water and boiled for 3 hours and then filtered through a gauze. the obtained extract was concentrated into an oily extract using a rotary machine and then lyophilized to 12.48 grams of cinnamon powder. briefly, the parasites were cultured in 7 ml rpmi 1640 medium with 5% serum, 10% hematocrit, hypoxanthine, and gentamicin (complete medium) in 75 ml flasks. the medium was changed every 48 h and flasks were incubated at 37c with 5% co2, 5% o2, and 90% n2. large - scale cultivation of plasmodium falciparum for metabonomics was carried out by the method modified by radfar. with 24 h changes of 75 ml of complete medium enriched with 5% albumax and 0.5% hematocrit. daily monitoring of the culture assisted in increasing its parasitemia to 60% and the ic50 dose of cinnamon extract. after 48 hours, they were harvested by centrifugation at 800 g for 5 min. parasites reaching 5% were then diluted to reduce the parasitemia to 0.5%, and the haematocrit was adjusted to 1.5%. this suspension was then added (100 l per well) to microplates predosed with 90 l of different concentration of cinnamon or artemisinin and incubated for 48 hours at 37c in mixed gas of 5% co2, 5% o2, and 90% n2 ; after that thin smears were prepared from each well, stained with giemsa stain for determination of percentage of parasitemia and ic50 detected by microscopy. parasites were isolated by adding 40 times the volume of 0.02% saponin in phosphate buffered saline (pbs) on ice for 30 min and then centrifuged at 4,000 g for 20 min at 4c. the cells were washed three times with 1xpbs and pellet collected by centrifugation in the above conditions and final centrifugation was carried out at 14,000 g for 5 min at 4c ; the cells were counted in a hemocytometer and stored at 20c. the samples containing parasites sonicated in a sonifier (soniprep 150) at 9 khz for 5 min in pulse were then centrifuged at 10000 g for 10 min, and the pellet dissolved in 200 l of 1.8 mm cold perchloric acid and ph adjusted by addition of 5.4 m koh to 6.8 and kept on ice for 60 min to precipitate the acid. the parasite extract was then centrifuged for 10 min at 10,000 g and the ph once again adjusted to 6.8 and lyophilized. 1 ml of d2o and 0.01% tsp was added to the lyophilized powder and spectroscopy was performed using 2-dimensional noesy (nuclear overhauser spectroscopy) conditions. the spectra from hnmr were fourier transformed by mestrec software. to obtain regression values, the variables of the signal intensities and chemical shifts were integrated and were inserted into the excel file. pls is a supervised method to obtain a model using regression in multivariate techniques via linear combination of original variables in which x is the normal intensities from the hnmr spectra and the y matrix comprises 0 for cinnamon treated and 1 for controls. orthogonal signal correction (osc) filters removed noise from the spectrum ; only one factor was removed and pls was applied after osc. metabolites corresponding to these resonances were then identified using chemical shift assignments of spectra of differentiating metabolites of sera based on comparison with chemical shifts of metabolites in human metabolite database data bank (hmdb) (http://www.hmdb.ca/metabolites) and in other published data. the lyophilized cinnamon was redissolved in rpmi medium and tested on 5% plasmodium falciparum and ic50 of 1.25 mg / ml was obtained with significance p < 0.001 (figure 1). parasite extract obtained from large - scale cultivation of plasmodium falciparum was analyzed by hnmr. the spectra of the cinnamon treated pasmodium falciparum and controls were superimposed in figure 2. the chemical shifts (ppm) of the spectra were converted into figures and then analyzed using osc - pls in matlab. the metabolites were entered into the metaboanalyst database and differentiating metabolic cycles were recognized (figure 6). cinnamon has ic50 of 1.25 mg / ml on plasmodium falciparum in vitro with ic50 of 1.25 mg / ml obtained with significance p < 0.001. the altered metabolites comprise succinic acid, glutathione, l - aspartic acid, beta - alanine, and 2-methylbutyryl glycine (table 1). the most significant biochemical pathways which have changed are discussed below (figure 6). the alanine, aspartame, and glutamate pathway which is one of the amino acid cycles is the first one to be affected. there are very early reports about the ability of the parasite to fix carbon dioxide and then synthesize alanine, aspartame, and glutamate. but amino acid uptake by the parasite from the infected erythrocytes is confirmed. when culturing plasmodium falciparum in vitro seven amino acids have to be supplied exogenously ; they are isoleucine, methionine, cysteine, glutamate, glutamine, proline, and tyrosine. proteases act on amino acids especially aspartic proteases, as probable drug targets till a few years ago. it is seen that cinnamon affects the parasite 's amino acid biosynthesis essential for its survival. the second most important pathway affected is pantothenate and coenzyme a biosynthesis associated directly with the tricarboxylic acid (tca) cycle. reports have shown that infected human erythrocytes exhibit higher coenzyme a biosynthesis than uninfected ones. human erythrocytes are capable of complete synthesis of pantothenate and coa unlike rat and avian erythrocytes. cinnamon affects this cycle which is imperative in tca and requires the carriage of carbons within the cell and entry of pyruvate to the tricarboxylic acid (tca) cycle. it was thought that plasmodium falciparum utilized glucose in an unconventional manner, but further work by metabolic approaches has proved that both asexual and sexual blood stages utilize a conventional tca cycle to catabolize glucose and glutamine [23, 24 ]. the next important cycle to be affected is lysine biosynthesis which has been reported in the parasite, plasmodium falciparum. lysine is generally implicated in posttranslational modifications and is also involved in diverse cellular functions. high throughput technology has identified 421 acetylation sites in 230 proteins in the human malaria parasite plasmodium falciparum during its active proliferation in erythrocytes. lysine - acetylated proteins are distributed in the nucleus, cytoplasm, mitochondrion, and apicoplast. there are also reports of histone lysine methylation, regulated by methyltransferases and demethylases, which play an important role in chromatin structure and gene expression in a wide range of eukaryotic organisms. the set - domain protein methyltransferase superfamily includes all but one of the proteins known to methylate histones on lysine. bioinformatic analysis has shown that nine set - domain - containing genes were found in the malaria parasite plasmodium falciparum and its sibling species. most set - domain genes and histone methyl - lysine marks displayed dynamic changes during the parasite asexual erythrocytic cycle, suggesting that they constitute an important epigenetic mechanism of gene regulation in malaria parasites. they are considered as recent targets for designing of antimalarial drugs and cinnamon extract affects them. glutathione metabolism is the last and most important cycle which has shown a change in the metabolome of plasmodium falciparum to cinnamon. it is reported that plasmodium falciparum employs a complex thioredoxin and glutathione system based on the thioredoxin reductase / thioredoxin and glutathione reductase / glutathione couples. plasmodium falciparum thioredoxin reductase reduces thioredoxin and a range of low molecular weight compounds, while glutathione reductase is highly specific for its substrate glutathione disulfide. since plasmodium spp. lack catalase and a classical glutathione peroxidase, their redox balance depends on a complex set of five peroxiredoxins differentially located in the cytosol, apicoplast, mitochondria, and nucleus with partially overlapping substrate preferences. moreover, p. falciparum employs a set of members belonging to the thioredoxin superfamily such as three thioredoxins, two thioredoxin - like proteins, a dithiol and three monocysteine glutaredoxins, and a redox - active plasmoredoxin with largely redundant functions. it is seen that glutathione metabolism is disturbed by the cinnamon extract. it can be concluded that cinnamon has an inhibitory effect on plasmodium falciparum in vitro with ic50 of 1.25 mg / ml with significance of p < 0.001. the altered metabolites are succinic acid, glutathione, l - aspartic acid, beta - alanine, and 2-methylbutyryl glycine and the main metabolic cycles affected were alanine, aspartate, and glutamate pathway, pantothenate and coenzyme a biosynthesis, lysine biosynthesis, and glutathione metabolism, all of which are important as drug targets. | malaria is responsible for estimated 584,000 deaths in 2013. researchers are working on new drugs and medicinal herbs due to drug resistance that is a major problem facing them ; the search is on for new medicinal herbs. cinnamon is the bark of a tree with reported antiparasitic effects. metabonomics is the simultaneous study of all the metabolites in biological fluids, cells, and tissues detected by high throughput technology. it was decided to determine the mechanism of the effect of aqueous extract of cinnamon on the metabolome of plasmodium falciparum in vitro using 1hnmr spectroscopy. prepared aqueous extract of cinnamon was added to a culture of plasmodium falciparum 3d7 and its 50% inhibitory concentration determined, and, after collection, their metabolites were extracted and 1hnmr spectroscopy by noesy method was done. the spectra were analyzed by chemometric methods. the differentiating metabolites were identified using human metabolome database and the metabolic cycles identified by metaboanalyst. 50% inhibitory concentration of cinnamon on plasmodium falciparum was 1.25 mg / ml with p < 0.001. the metabolites were identified as succinic acid, glutathione, l - aspartic acid, beta - alanine, and 2-methylbutyryl glycine. the main metabolic cycles detected were alanine and aspartame and glutamate pathway and pantothenate and coenzyme a biosynthesis and lysine biosynthesis and glutathione metabolism, which are all important as drug targets. |
malignant gastric outlet obstruction by an unresectable or recurrent carcinoma causes nausea and vomiting, prevents oral ingestion, and seriously compromises the quality of life of the patient. bypass surgery, i.e. gastrojejunostomy, has been the treatment of choice for malignant gastric obstruction for a long time. malignant gastric obstruction also affects gastric cancer patients who have undergone a distal gastrectomy owing to locoregional tumor relapse. in such cases, bypass surgery is often impossible, because the gastric remnant is too small to create an anastomosis and the adhesion formed by the previous surgery makes it technically difficult. postgastrectomy patients with malignant gastric outlet obstructions have been managed with parenteral nutrition and decompression with the placement of a nasogastric tube. this treatment requires the patients to be hospitalized and is unmet to their clinical needs, because current advances in chemotherapy have markedly prolonged the survival of gastric cancer patients suffering recurrence. recently, self - expandable metallic stent (sems) placement has emerged as a minimally invasive alternative treatment and has shown promising results in cancer palliation [3, 4 ]. the wallflex duodenal stent (boston scientific co., natick, mass., usa) is a new type of enteral stent, approved in japan in april 2010. this stent is made of nitinol instead of stainless steel, which markedly improves its flexibility while maintaining the patency of the lumen. the stent also has looped distal ends to reduce the risk of mucosal injury and a flared proximal end to minimize the risk of stent migration. these modifications allow safer endoscopic stenting at the duodenum and extend the indications for enteral stenting to at - risk sites, such as the jejunum and enteral anastomoses after surgery. however, few data are available on whether the wallflex duodenal stent is applicable to the management of malignant gastric outlet obstructions in postgastrectomy patients. we here report a patient with malignant stenosis of the jejunal limb of a roux - en - y gastrojejunostomy. the patient underwent endoscopic stenting with the wallflex duodenal stent and swiftly resumed her diet and a good quality of life. the patient was diagnosed with unresectable gastric cancer and induction chemotherapy was initiated with s-1 (ts-1 ; taiho pharmaceutical co., ltd., tokyo, japan) plus cisplatin because computed tomography scans revealed that the tumor had invaded the pancreas. after two cycles of chemotherapy, the tumor had shrunk markedly and was resected with a distal gastrectomy with a d2 lymphadenectomy and a partial resection of the pancreas. gastrojejunostomy using a roux - en - y procedure was selected as the anastomotic procedure to avoid anastomotic obstruction owing to local recurrence at the pancreatic head. a histological examination of the surgical specimens showed that the tumor had invaded the pancreas, but the surgical margin was negative. the patient 's disease was classified as stage iv (t4n2h0p0cy0m0) according to the japanese classification of gastric carcinoma, the second english edition. the patient underwent postoperative s-1 chemotherapy for 1 year, but a recurrent tumor appeared in the vicinity of the gastrojejunostomy 42 months after the initial surgery (fig. the disease was controlled for 10 months by outpatient - based chemotherapy : six cycles of weekly paclitaxel, four cycles of docetaxel, and three cycles of irinotecan. however, 52 months after the initial surgery, the patient developed severe dysphagia and was hospitalized. on admission, the patient was only able to take a small amount of liquid, and her performance status was evaluated as 1 according to the eastern cooperative oncology group grading and 70% on the karnofsky index. a fluoroscopic examination revealed that the jejunal limb of the gastrojejunostomy had narrowed on a length of 5.2 cm (fig. (xq240 ; olympus, tokyo, japan) with a diameter of 0.9 cm was passed through the narrowed segment, and tumor exposure was not found in the involved jejunum (fig. abdominal computed tomography showed that the recurrent tumor had regrown to a size of 5.0 cm and involved the jejunal limb of the gastrojejunostomy. we made a diagnosis of stenosis of the roux - en - y jejunal limb following distal gastrectomy and decided to relieve the stenosis by endoscopic stenting after we had obtained the patient 's informed consent (given the risk of the procedure). a wallflex duodenal stent with a diameter of 2.2 cm and a length of 9.0 cm was selected. a guidewire was inserted through the working channel of the endoscope and advanced through the stenosis. a delivery catheter was inserted along the guidewire until it passed the distal end of the stenosis. the time required for stent insertion and total endoscopic manipulation was 12 and 35 min, respectively. no treatment - related complications, including pain, hemorrhage, or perforation, were noted. the patient could then resume a regular diet without obstructive symptoms such as nausea or vomiting. follow - up fluoroscopy on the third day after treatment revealed that the stent was fully expanded and that the contrast medium had passed smoothly (fig. she rejected further treatment, including chemotherapy or radiation therapy, and eventually died of cancer recurrence 201 days after the stent placement. oral ingestion was sustained for 194 days, which corresponded to 96.5% of the patient 's survival time. she remained at home for 165 days, with an infusion - free time of 148 days. malignant gastric outlet obstruction caused by an unresectable carcinoma was traditionally considered to be an indication for gastric bypass surgery as a palliative treatment. although gastrojejunostomy relieves obstructive presentations, it often takes time to achieve stable oral ingestion, resulting in a prolonged hospital stay. a systematic review of gastrojejunostomy for the palliation of gastric outlet obstruction showed that the clinical success rate was 72%, the mean hospital stay was 13 days, the early and late major complication rates were 4 and 17%, respectively, and the minor complication rate was 33%. the endoscopic placement of semss has emerged as an alternative minimally invasive technique for the palliative treatment of patients with unresectable malignant gastric outlet obstructions [3, 4 ]. since keymling. initially reported the utility of semss for gastric outlet obstruction in 1993, reports have accumulated supporting this as a safe and effective procedure, with high technical and clinical success rates. however, previous types of semss, which had sharp ends and low flexibility, often caused severe stent - related complications, including hemorrhage and perforation, especially at the curvature of the duodenum or the colon [9, 10 ]. the wallflex duodenal stent is a new sems that was developed for pyloroduodenal stenoses, which was approved in japan in april 2010. [11, 12 ] showed that the technical success rate for the placement of this stent to relieve gastric outlet obstruction was 93.598%, and the clinical success rate was 8485%. the rates for short - term complications, including pain, perforation, and hemorrhage, were only 4, 2, and 2%, respectively. these results suggest that stenting with a wallflex duodenal stent to relieve gastric outlet obstruction is both safe and clinically effective and could replace bypass surgery as a procedure for the palliation of patients with this condition. in the present case, stent placement was the only way to resolve the patient 's gastric outlet obstruction, which was caused by cancer recurrence, because the patient had undergone a distal gastrectomy. we were concerned about the safe and successful placement of the stent, because limited data are available on the safety of sems placement in postgastrectomy patients [13, 14 ]. song. placed semss in 39 patients with malignant anastomotic obstructions after gastrojejunostomy and reported their results. in their series, stent placement was technically achieved in all patients, and 35 patients (90%) obtained relief from their obstructive symptoms. however, aspiration pneumonia, stent migration, and reobstruction occurred in 2, 4, and 2 patients, respectively, with a total complication rate of 23.1%. kim. also reported the results for 39 postgastrectomy patients who underwent sems placement., 2 patients suffered from stent - associated perforation. on the basis of previous studies, we infer that sems placement is more risky in postgastrectomy patients than in patients with unresectable primary gastric cancers, although the treatment is technically feasible. because it prevents severe reflux esophagitis and may reduce anastomotic leakage, roux - en - y gastrojejunostomy is currently becoming a preferred procedure in japan for reconstruction after distal gastrectomy. the jejunal limb of the roux - en - y procedure is free of the retroperitoneum and may form a sharply angulated adhesion. this differs considerably from cases of antropyloric stenosis caused by unresectable gastric cancer or billroth i anastomosis, in which the duodenum is physiologically fixed to the retroperitoneum and the curvature is natural. in our patient, we ensured that the jejunal limb was straight by passing a gastrofiberscope through the stenotic segment. in this special postsurgical setting, for the safe placement of a wallflex duodenal stent, it is critical that the position of the distal end is confirmed using fluoroscopy and/or endoscopy. several studies have shown that patients with unresectable gastric cancers associated with gastric outlet obstruction have a poor prognosis, with a median survival of 3399 days after stent placement [1, 3, 4 ]. these short survival times clearly suggest that the palliation for these patients should not be time - consuming. van hooft. prospectively analyzed 51 consecutive patients with malignant gastric outlet obstruction and showed that, after stent insertion, obstructive presentations disappeared immediately and oral ingestion was resumed within 8 days in all patients. in the present case, oral ingestion was resumed 1 day after the intervention and the patient 's intake improved from liquid only to a full diet. the patient survived for 201 days after stenting, despite her refusal of maintenance chemotherapy. she maintained oral ingestion for 194 days (96.5% of her survival time), stayed at home for 165 days (82.1% of her survival time), and remained infusion free for 148 days (73.6% of her survival time). these data demonstrate convincingly that stent placement satisfactorily contributed to the improvement of the quality of the rest of her life. in conclusion, endoscopic stenting appears to be an effective and safe treatment for the palliation of patients with malignant gastric outlet obstruction. although the safety of stenting in intestinal segments interposed by an anastomosis must be confirmed, the present case suggests that the new knitted nitinol stenting device will extend the indications for endoscopic stenting to include roux - en - y gastrojejunostomy following distal gastrectomy. | a 69-year - old japanese woman with a history of distal gastrectomy with a roux - en - y reconstruction for advanced gastric cancer was admitted to our hospital complaining of severe dysphagia. on admission, the patient was only able to take liquids, and a firm, fist - sized tumor was palpable in her left upper abdomen. an endoscopic examination disclosed stenosis of the jejunal limb of the gastrojejunostomy. abdominal computed tomography revealed that a recurrent tumor, 5.0 cm in diameter, was compressing the jejunal limb of the gastrojejunostomy. a knitted nitinol self - expandable metallic stent (wallflex duodenal stent) was placed endoscopically at the stenotic jejunum from the gastrojejunostomy. the time required for stenting and total endoscopic manipulation was 12 and 35 minutes, respectively. no stent - related complications were observed. the patient could resume oral ingestion 1 day after endoscopic stenting and was discharged on the fifth day after treatment. she survived for 201 days after stenting. she continued oral ingestion for 194 days and stayed at home for 165 days. the wallflex duodenal stent allows safe endoscopic stenting, even in cases of malignant stenosis of a gastrojejunostomy following distal gastrectomy. this stenting device will extend the indications for endoscopic palliation of gastric cancer patients with gastric outlet stenosis. |
prosthetic articulators are mechanical devices used in dentistry to assist in the fabrication of prosthodontic restorations and appliances. casts of the maxillary and mandibular teeth are fixed to two arms that reproduce the motion of the mandible respect to the maxilla (figure 1). typically, the upper arm is constrained by conjugate profiles that simulate the mandibular condyles, and by a pin that slides over an inclined table, whose effect is to reproduce the contact between incisal teeth during the protrusion movement. conventional articulators usually allow the adjustment of the inclination of the incisal table but, since the surface in contact with the incisal pin is a flat plane, the end of the pin must follow a simple straight trajectory. this fact advocates the adoption of an incisal table that exploits also an adjustable curvature, so that the original shape of the incisors could be replicated with higher precision. such outlook is also supported by experimental data : when sliding upper and lower casts of a healthy man, the envelope of motion of the incisal pin engraves a concave hole in a mold block placed on the incisal table [2, 3 ]. this work presents a kinematical analysis of the protrusive movement by means of multibody simulation software [47 ] and demonstrates the advantage of a new incisal table that features a curved contact surface. numerical simulations show that the reconstructed profile of the incisors is an inexact approximation of the original shape if conventional flat incisal tables are used. indeed, the adjustment of the inclination of the plane is enough to obtain a limited set of profiles featuring an almost flat concavity, not adjustable, whereas profiles with more varied curvatures might be desirable, even for the same table pitch. if curvatures are ignored, as in traditional flat tables, the concavity of the incisal guidance can be underestimated, therefore causing the maxillary incisors to be thicker. this inaccuracy, if modeled prosthesis are not corrected, may affect the occlusion mechanism [813 ]. finally, we propose a compliant device that can be used to implement the concept of the curved incisal table using a small amount of mechanical parts : a thin deformable section is bent up to the desired curvature by turning a knob which bends the surface, hence obtaining the approximation of the needed curvature. a three - dimensional model of the articulator has been implemented in our in - house multibody software chrono::engine, customized with special features for the analysis of such problems. the model is made of two moving parts, mandible and maxilla, and of two holonomic constraints for anterior and posterior guidance (figure 1). to obtain the highest precision, dynamic and kinematic analyses are computed using a special method which uses a precise nurbs reproduction of the contact surfaces. the design of the simulated mechanical parts is inspired to an adjustable arcon - type articulator featuring values for the condylar guide eminence angles, shape of condylar guides, pitch and vertical tilting of the incisal table, and length of the incisal pin. the shape of the teeth can be modified in order to study patients with different anterior guidance. the protrusion can be adjusted via an automated simulation cycle, which runs through 68 mm of protrusion imposed by a virtual actuator. the reciprocal motion of the two parts (maxillary and mandibular arches) is studied using two different modes for the posterior guidance, a sliding constraint (curvilinear glyph) represents the condylar guides, while anterior guidance is represented by a sliding constraint between surfaces of maxillary and mandibular incisors. this mode simulates the physiological kinematics of the occlusion ; figure 2(a) shows the superimposition of multiple snapshots of teeth and the relative motion of maxilla and mandible. for the posterior guidance, a sliding constraint (curvilinear glyph) is used for the condylar guides, while anterior guidance is obtained by means of a sliding constraint between the incisal pin and the incisal table. this mode simulates the mechanical behavior of the articulator as constrained by the incisal table, ignoring the effect of the contact between incisors. b - flat, a conventional flat incisal table is used, thus giving approximate results. in mode b - curved, ideal, an exact incisal table is used, obtained as the envelope of the hemisphere at the apex of the incisal pin during a simulation performed with mode a. the conjugate profile of the incisal pin is concave : the surface has a downward curvature, denoted with the s symbol in figure 3. simulations for mode a show that the incisal pin, driven by contact between true incisors and motion of condyles in the glenoidal fossa, describes a trajectory that, in general, exploits some kind of variable curvature. the error caused by the adoption of a conventional flat incisal table can be measured by comparing results coming from the ideal mode a to simulations performed in mode b - flat. in the latter case, the top end of the incisal pin is constrained on the flat surface of the incisal table : despite all the admissible settings of alignment for a standard incisal table, it will be difficult to obtain a close approximation of the profile of the original incisal guide, represented by true contact between healthy teeth. in fact, if a flat table is rotated in order to obtain a good approximation of the incisal guide for the initial part, the thickness of the maxillary incisors will be underestimated. on the other hand, if the table is rotated so that the last part is approximated more accurately (figure 2(b)), the thickness of the maxillary incisors will be overestimated in the middle part. moreover, dynamical simulations show that, even if the amount of the thickening may be small, at the point of contact the direction of surface normals may be noticeably different from the one of the ideal case, and the effect would be a different direction of reaction forces. such error in force orientation may range in the 20 interval : this could cause negative side effects from the gnatological point of view. various simulations have been automatically performed to investigate the main parameters which may affect the curvature of the incisal table. hence, a range of tests has been simulated for different values of eminence angles, different shapes of condylar guides, and different teeth profiles. for all these cases, simulations have been performed in mode a, hence by enforcing contact between real incisors and contact in the curvilinear condylar guides. in this way, the ideal profile of the incisal table is obtained as the envelope of the incisal pin (figures 3 and 4). the profile of the ideal incisal tables has been analyzed in terms of curvature, so that the effects of the parameters have been recognized and studied. from the abovementioned analyses, the most relevant results can be summed up as follows. in general, given a spherical incisal pin with radius ra, and denoting the radius of the curvature of the trajectory of the center of the pin with rc, the radius of the table is rp = ra + rc. kinematic analyses show that rc can be as low as 2 mm. in all cases, the curvature is more noticeable in the first part of the protrusion and progressively decreases during the protrusion up to a flex (null curvature) about 7 - 8 mm away from the position of centric occlusion. changes in the shape of the condylar guides have small effects either on the average inclination of the ideal incisal table or on its curvature ; on the other hand, changes in the shape of the incisal guide have noticeable consequences on the shape of the ideal incisal table. there is almost a linear relation between the average inclination of the ideal incisal table and the average inclination of the incisal guidance of real teeth. also, for growing values of incisal guidance curvature there are growing values of incisal table curvature. given the requirement of a simple and intuitive method to adjust the articulator, a single parameter, namely, the curvature, could be used to approximate the ideal anterior guidance using a bendable table (figure 5). this practical implementation of curved incisal table exploits two zones : a flexible a section with uniform curvature (whose radius rp may be adjusted up to rp = depending on user needs) and a straight b section, without curvature. moreover, the entire profile can be rotated forward / backward by an angle. the case rp = would correspond to null curvature, as in the case of flat tables for conventional articulators. in order to test the precision of this kind of profile, simplified because it can be easily implemented by means of a mechanical device featuring adjustable curvature, the multibody software performed simulations (figure 6) which compared the results of this mode with the results corresponding to the ideal case. the approximation that has been introduced by switching from an ideal incisal guide, featuring the exact nonuniform curvature, to the simplified profile, is so low that it can be neglected : from our simulations, the approximation is usually lower than 0.1 mm. figures 7 and 8 show a preliminary design for an incisal plate that can be adjusted in terms of curvature. the variable curvature, on the first 7 mm of contact, is obtained by bending a metallic compliant mechanism, where the surface that must be curved can be made of a thin deformable layer exploiting selective flexibility. the user could turn a knob until the desired curvature is obtained ; yet we remark that this device can still work in a null - curvature configuration : in such a case, the effect would be identical to a conventional articulator. the compliant surface is a critical part of the design because it must withstand strong curvatures without breaking or plasticizing. since polymeric materials can feature early plasticization, steel foils have been preferred. however, curvature of a steel foil is limited by the yield strength y : to avoid plasticizing, the maximum bending stress, in the case of maximum curvature, must be max eb(2y)1. in order to keep the rp limit as low as possible, we adopt high - strength laminated steel with young modulus e = 200 gpa and y = 900 mpa. since rp = ra + rc, where the curvature rc of the trajectory of the center of pin must be as low as 4 mm to approximate the discussed kinematic requirements, we used a foil thickness b = 0.1 mm and a pin radius ra = 10 mm. larger ra values would allow even larger b thickness still satisfying (1), but at a cost of making a large assembly whose usage might look uncomfortable and atypical to the user (most conventional articulators have small pin radii, about 2 - 3 mm). on the other hand, lower values of b would allow even smaller rp values (thus lower ra and a more compact pin), but the surface would be too delicate. moreover, we used a pack of 20 layered foils for a total thickness of the incisal table of btot = 20 b = 2 mm ; this avoids that the pressure of the pin over the table could cause damages such as wear or unwanted deformations. multibody simulations made possible to obtain the ideal shape of the incisal table by computing the conjugate profile of the incisal pin during the simulation of an ideal, physiological protrusion. software analysis showed the superior precision of a prosthetic articulator featuring an adjustable curvature of the incisal table, when compared to a conventional articulator with a simple flat incisal table. a prototype of table with adjustable curvature, where the user can modify the profile of the table by acting on a knob, is currently under testing and clinical results will follow. | this study illustrates the effectiveness of an advanced incisal table surface, featuring adjustable curvature, in the sake of more accurate articulator kinematics in anterior teeth reconstruction. prosthetic articulators, used by dental technicians in reconstructive dentistry, are adjustable instruments that simulate the motion of mastication between dental casts : usually, the forward motion (protrusion) of the mandible is guided by sliding a pin over a flat table in order to recreate those movements when incisal teeth are missing. however, such protrusion is an approximation of the exact motion, since flat incisal tables have a limited set of adjustments. customized software has been developed in order to simulate the kinematics of articulators in three - dimensional space : animations and measures of the envelope of teeth profiles show the unfeasibility of reconstructing with good approximation the profile of incisive teeth, when a simple flat ' incisal table is used. a new incisal table with an adjustable curvature has been proposed, simulated, and built, and computer simulations demonstrated the superior precision of the new design when compared to a conventional articulator which uses a flat incisal table. |
compared with the more common inguinal and femoral hernias, obturator hernias are rare with an incidence of 0.051.4% of all abdominal wall hernias. probably due to a wider pelvis and a more triangular obturator canal with a greater transverse diameter as well as previous history of pregnancy, obturator hernias are more common in elderly, emancipated women. since obturator hernias often become only clinically apparent when incarcerated, signs of acute small bowel obstruction can be found in 90% of patients with newly diagnosed obturator hernia. owing to unspecific clinical signs and therefore often delayed diagnosis, advanced age of the patient, comorbidities and associated chronic disease mortality has been described as high as 47% [24 ]. a 97-year - old woman presented to the emergency department (ed) with a sudden onset of pain in the right lower abdomen with radiation to the right thigh. in her past medical history, the patient had bilateral open inguinal hernia repair and an open appendectomy. no inguinal or femoral hernia was palpable, but coughing and internal rotation of her right hip caused increasing pain (howship romberg sign). computed tomography (ct) scan revealed an obturator hernia on the right side with a herniated small bowel loop with early signs of incarceration (fig. 1). we performed an emergency laparoscopic transabdominal preperitoneal plasty (tapp) to repair the hernia. during laparoscopy no herniated small bowel was present, but an erythema of a small bowel loop was seen not justifying a resection. figure 2:intra - operative view after opening the peritoneum, showing the corona mortis (a) just above the obturator hernia orifice (b), the obturator vein with underneath the obturator artery (c) and the obturator nerve (d). intra - operative view after opening the peritoneum, showing the corona mortis (a) just above the obturator hernia orifice (b), the obturator vein with underneath the obturator artery (c) and the obturator nerve (d). apart from the obturator nerve and vessels, we detected a vessel crossing the pubic bone : the corona mortis (fig. this vessel was spared from extra manipulation and the hernia closed with a monocryl - prolene mesh that was fixed with titanium tacks. obturator hernia is a rare form of abdominal wall hernia published the first time by de ronsil in 1724. a compression of the obturator nerve by the hernia can cause pain or cramps from the groin to the knee. the symptoms can be even enforced by adduction and internal rotation of the thigh, which is called the howship in addition, compression of the obturator nerve can lead to an absent adductor reflex in the presence of a positive patellar reflex (hannington kiff sign). pain from the groin to the knee might get mistaken for neuromuscular pain, as joint pain is common in elderly patients. quite often diagnosis is missed and patient undergoes surgery for bowl obstruction and obturator hernia is diagnosed during the operation. if suspected preoperatively, the diagnostic modality of choice is nowadays the ct scan of the abdomen. for the treatment of an obturator hernia, the advantages of laparoscopic versus open abdominal surgery, namely less post - operative pain, earlier oral intake, shorter hospitalization, fewer risk of an incisional hernia and smaller wounds are well known. furthermore, with the technical progress in laparoscopic surgery, transabdominal and extraperitoneal approaches are possible and have been described to be successful in obturator hernia repair [7, 8 ]. in our case, the transabdominal (tapp) approach was chosen because of clinical and radiographic signs of incarceration. by the transabdominal approach a good visualization and assessment of the concerning bowel loop is possible. as demonstrated by the positive clinical course, our patient with 97 years of age has been benefiting from the minimal invasive procedure. this so - called crown of death has an incidence of up to 40%. as an increasing number of abdominal hernias are repaired by a laparoscopic approach, surgeons should be aware of its existence and spare the vessel as severe bleedings can occur. in summary, obturator hernias are rare and often present to the ed when incarcerated. especially in elderly, thin women presenting with bowel obstruction and hip pain this exclusive type of hernia | obturator hernias are a rare form of abdominal wall hernias. we present a case of a patient with an obturator hernia diagnosed by the classical signs of lower abdominal pain, a positive howship romberg sign (painful internal rotation of the hip) and a computed tomography scan showing a herniated loop of small bowel. during the emergency laparoscopic hernia repair (transabdominal preperitoneal approach) a variant vessel, the corona mortis, was detected. |
international travel has increased in the last six decades worldwide with the highest increase noted in tropical and subtropical areas. international tourist arrivals grew by 4% in 2012 and for the first time in history exceeded one billion. in 2012, a 6% increase was recorded for africa, equivalent to 3 million more travelers, reaching 50 million for the first time ever. travelers to tropical and subtropical countries can be exposed to various infectious diseases and may facilitate their importation into countries where these diseases are not endemic [2, 3 ]. approximately 8% of travelers to developing countries require medical care during or after travel ; the main diagnoses include acute diarrhoea, malaria, and dengue fever. sub - saharan africa was one of the most common regions (26.7%) where illnesses were acquired, as recorded by the geosentinel surveillance network. the importation of vaccine - preventable diseases has been recognized as an important travel - related problem. vaccine preventable diseases (vpds) accounted for 1.5% of ill returned travelers, with enteric fever, acute viral hepatitis, and influenza as the most common diagnoses. malaria is one of the most important travel associated diseases due to its widespread geographic distribution and its potential fatal outcome if untreated, especially in nonimmune individuals. malaria diagnosis accounted for 29% of those with fever and disproportionally in travelers returning from africa ; according to the global data base, the majority of the infections were acquired in sub - saharan africa and 60% of them were due to plasmodium falciparum. as a result of growing international travel, health consultants are increasingly likely to be consulted for advice before travel or by ill returned travelers. previous studies have revealed that a large proportion of travelers do not seek pretravel advice [712 ]. the current study aimed to assess pretravel consultation seeking practices and provision of vaccinations and malaria prophylaxis among international travelers departing from athens international airport and visiting african countries. a prospective questionnaire - based study was conducted from november 1, 2011 to april 30, 2013. travelers leaving from the departure gates of athens international airport were invited to participate in the survey. selection criteria were being a greek resident, 19 years old, and traveling to africa. permission was given by the international airport authority, the airlines flying to the above destinations, and the hellenic center for disease control and prevention (hcdcp). data included information about demographic and travel characteristics, and pretravel consultation (vaccination, malaria prophylaxis, general preventive measures, and source of pretravel consultation). up to 5 travelers were interviewed per day, depending on the selection criteria. high risk areas and those with moderate - high prevalence of malaria were defined according to published sources [6, 13 ]. the definition of high risk travelers with regard to exposure to malaria in endemic areas was based also on travel (area and place of stay, duration and purpose of travel, and activities) and travelers ' characteristics (age, behaviour, and medical history). adequate vaccine and malaria prophylaxis is considered according to the national hellenic centre for disease control and prevention guidelines, which are in accordance with the world health organization and us centers for disease control and prevention guidelines [1416 ]. urban accommodation was defined as cities with population of 5,000 people or more, whereas rural accommodation was defined as villages with up to population of 5,000 or staying in the countryside (hellenic statistics authority : personal communication). short - term travel was defined as a trip of < 1 month duration, while long - term travel was defined as a trip of 1 month. an organized trip was defined as a guided, package trip, mainly to popular tourist destinations. travelers vfrs were classified as those immigrants to greece who return to their homeland, a lower income country, to visit friends or relatives according to a definition outlined by the us centers for disease control and prevention (cdc). chi - squared test and fisher 's exact test were used for comparison between categorical variables. the standard multiple logistic regression was conducted (if p value < 0.15 in univariate analysis) to examine the relation between pretravel preparation and independent factors. multivariate analysis was used in order to identify factors influencing the attitude to pretravel consultation. odds ratios (ors) and their 95% confidence intervals (cis) were calculated. a total of 360 travelers participated in the survey (98% participation rate) ; 60%, 35%, and 5% were traveling to sub - saharan africa, north africa, and southern africa region (based on united nations ' classification), respectively ; more than one quarter of them visited for the first time a tropical or subtropical country. the five most common african countries were egypt (35%), ethiopia (13.3%), kenya (8.3%), ghana, and nigeria (7.2% each). sixty - two percent of travelers visited malaria endemic areas. the majority of international travelers were male, of greek nationality and with tertiary education. one - third of the travelers were foreign born ; 56.1% of them were born in north africa (mainly in egypt) and ethiopia. outdoor activities and contact with animals were reported by 67 (18.6%) and 15 (4.2%), respectively. those traveling for leisure and vfrs were more likely to be involved in outdoor activities than those traveling for work (31.9% and 29.9%, resp. half of all travelers reported having sought pretravel consultation and most of them sought consultation 1528 days prior to departure (table 1). the majority of those who pursued health information (61.5%) had a history of previous travel to a tropical country (p value < 0.001), traveled to sub - saharan africa (89.4%) (p value < 0.001), and stayed in local houses (56.1%) (p value < 0.001). of those traveling for business, leisure, and vfrs, 60%, 24.4%, and 12.2% sought pretravel advice, respectively (p value < 0.001). vaccination (previous vaccination or vaccination prior to the current trip) according to destination is described in table 2. a total of 572 vaccines were administered to 136 (37.8%) of travelers (mean number of vaccines : 4.2, range : 114) ; 122 (89.7%) and 14 (10.3%) received travel and routine vaccines, respectively. the vaccines most commonly administered were hepatitis a, yellow fever, and tetanus / diphtheria vaccines. more than 90% of travelers visited areas of high endemicity for hepatitis a, and nearly 80% of them areas endemic for typhoid fever. hepatitis a and typhoid vaccination rates were lower than expected (60% and 55%, resp.). all travelers who received meningococcal vaccine traveled to sub - saharan africa (16.1%) ; retrospective analysis of data based on travelers ' and travel characteristics, revealed that this rate was lower as expected by 25%. similarly, all of those to whom yellow fever vaccine was administered traveled to sub - saharan africa ; this rate was considered slightly lower than expected (10%). rabies vaccine was administered to only 1.9% of all travelers and only to travelers visiting sub - saharan africa. vaccination in relation to purpose of travel revealed that more of those who traveled for recreation received hepatitis a vaccine than business travelers, vfrs, and tourists (54.2% versus 47.5% and 5.8%, resp.) (p value < 0.001), whereas more business travelers were administered typhoid fever vaccine than vfrs and tourists (40.6% versus 2.2%, 0%, resp.) similarly, more business travelers received meningococcal vaccine than tourists and vfrs (32.2% versus 11.1% and 1.5%, resp.) yellow fever was delivered to 44.8% of business travelers, 37.5% of tourists, and 6.6% of vfrs (p value < 0.001). the association between tetanus / diphtheria, poliomyelitis, hepatitis b, hepatitis a, typhoid, yellow fever and meningococcal vaccination, and destination and more specifically with sub - saharan africa was statistically significant (p value < 0.001). nearly 22% (77) of travelers who visited malaria endemic areas did not receive any antimalarials. there was a statistically significant association between malaria chemoprophylaxis, general advice, and destination. most of those who traveled to sub - saharan africa (66.8%) were prescribed chemoprophylaxis than to other destinations (p value < 0.001) ; 55.2% and 44% of them received mefloquine and atovaquone / proguanil, respectively. in terms of purpose of travel, more business travelers (66.9%) received antimalarials than tourists (51.4%) and vfrs (6.6%) (p value < 0.001). the association of purpose of travel (recreation, work, and vfrs) with age, gender, education, employment, destination, place and area of stay, and duration of travel was statistically significant (p value < 0.001). multivariate analysis was used in order to investigate the association of several factors with failing to pursue pretravel health consultation among travelers to african destinations. having a history of previous travel to a tropical country, having an elementary or secondary level of education, traveling for visiting friends and relatives, k traveling for short duration and traveling with someone were significant determinants for not pursuing pretravel consultation (table 5). during the period 2009 - 2010 nearly 34,000 travelers from greece visited countries of africa. most of these destinations are endemic to a variety of infectious diseases which may cause considerable morbidity and mortality. the aim of the current survey is to study the profile of greek travelers visiting africa, and their pretravel health seeking practices including vaccine and malaria prevention. the profile of the participants in the present study revealed that more than two - thirds of international travelers were travelers with employment and a history of previous travel to tropical and subtropical countries and with tertiary level of education. travelers with this profile are expected to be more receptive to awareness about health prevention. however, the overall level of practice regarding vaccinations and malaria chemoprophylaxis was low. in regard to the demographics of the study population the results were comparable to those of other similar studies ; most of the travelers were men and young or middle aged [712 ]. the percentage of those traveling alone was higher compared to that of other studies [8, 10 ]. similar rate was noted in a spanish study. in regard to purpose of travel, the results of the present survey showed that the number of those traveling for the purpose of work or vfrs was higher, whereas the rate of those traveling for leisure was lower compared to the results of other studies [7, 8, 10, 11 ]. vfrs traveled predominantly to north africa, which may reflect the composition of the african vfr population in greece, and those who traveled for recreation and work visited mainly sub - saharan africa. the majority of business travelers stayed for long period of time, which explains the fact that most of them stayed in local houses. compared to a previous greek study the current survey shows a changing profile of greek travelers to developing countries ; this may be due to the influence of economic crisis in greece which is reflected on immigration and international travel. in the current study, less than half of the travelers reported having sought pretravel consultation ; similar rates were found in other studies [713 ]. inadequate pretravel preparation, visiting rural areas, traveling for longer periods of time, and staying with local people in developing countries predispose vfrs, compared with other travelers, to many largely preventable infectious diseases [36, 20 ]. vaccination rate in the current survey was low ; however it was comparable with the results of other studies [8, 1012 ]. similarly, this was the most common vaccine administered in similar studies [8, 1012 ]. although more than 90% of travelers visited areas endemic for hepatitis a, vaccination rate for hepatitis a was lower than expected as shown by retrospective analysis of travel and travelers ' characteristics. a possible reason may be previous vaccination as part of the national immunization program of greece or previous travel to tropical or subtropical countries. however, lack of knowledge about risk destinations may be another reason [21, 22 ]. typhoid vaccine was delivered to 17.5% of travelers ; this rate was lower compared to the results of other studies [8, 12 ]. although vfrs accounted for more than one - third of the travelers and more than 80% of them stayed for 1 month and nearly all of them at local houses, only a few of them (< 2%) received typhoid fever vaccine ; this may be the result of inadequate pretravel health seeking practices of vfrs as shown by a previous greek study. retrospective analysis based on travel and travelers ' characteristics showed that the vaccination rate against typhoid fever was lower than expected (55%). meningococcal vaccine was administered to those traveling to sub - saharan africa. nearly two - thirds of the travelers to sub - saharan africa intended to stay for long period of time and for work ; as shown by retrospective analysis of data this rate was lower by 25%. meningococcal vaccination should be considered in all travelers to countries in the sub - saharan meningitis belt in particular for those traveling during the dry season or those staying in the area for longer periods and living with or being in close contact with the local population for whom the risk of infection may be greater [23, 24 ]. yellow fever vaccine was administered only to travelers visiting sub - saharan africa (47.9%) ; lower vaccination rate was justified due to previous immunity as shown by retrospective analysis. rabies vaccine was administered to only 1.9% of all travelers and only to travelers visiting sub - saharan africa (3.3%). only 0.1% of travelers vfrs and 0.6% of long - term travelers were vaccinated against rabies despite traveling to a rabies - endemic country. travelers ' and travel health providers ' lack of knowledge about the risks of rabies exposure may be related to the low vaccination rates against rabies, as shown in recent studies [25, 26 ] ; results from a previous greek survey showed that travel health consultants ' knowledge regarding preexposure rabies prophylaxis was deficient. in the current study, vaccination with routine vaccines such as tetanus / diphtheria was comparable to the rates of another study. previous vaccination coverage through the national vaccination program of greece which is provided by health professionals other than travel health consultants may be responsible for low vaccination rates with routine vaccines. since influenza is considered as the most common travel - associated, vaccine - preventable infectious disease it is important to increase travelers ' awareness of influenza risk distribution and prevention measures. furthermore specific recommendations are needed which address the indications for influenza vaccination in travelers. malaria risk was assessed according to destination countries, area and place of stay, purpose, and duration of travel. the rate of malaria chemoprophylaxis in the current study was comparable to the rate in other studies [810, 12, 30 ]. retrospective analysis of data based on travel and travelers ' showed that approximately 22% of all travelers who visited malaria endemic areas did not receive any antimalarials. only 6.6% of vfrs and 67% of business travelers who traveled to malaria endemic areas were prescribed chemoprophylaxis, whereas approximately 8% of business travelers who traveled for more than six months to malaria endemic areas had no information about sbet ; for travelers visiting high risk areas and staying in remote locations for prolonged period of time sbet is indicated. these results suggest that malaria prophylaxis was inadequate, in particular with regard to special groups, such as vfrs and business travelers which may be related to their lack awareness about the risk of the disease [22, 30 ]. travel medicine providers were underutilized with only one half of all travelers to african destinations reporting having sought pretravel consultation. inadequate vaccination coverage and malaria prevention of travelers to high risk destinations suggest that public health strategies should be developed in order to increase travelers ' awareness about the risk of exposure to travel - related infectious diseases and the importance of pretravel consultation. in particular, there is a special need for education initiatives to target high risk groups of travelers such as travelers vfrs and business travelers. travel health providers should increase their efforts through training and continuing professional development in order to propagate safe and healthy travel. | background. international travel to africa has grown dramatically over the last decade along with an increasing need to understand the health issues for travelers. the current survey aimed to assess vaccination and malaria prevention of travelers visiting africa. methods. a questionnaire - based survey was conducted from of november 1, 2011 to of april 30, 2013 at athens international airport. results. a total of 360 travelers were studied ; 68% were men. their mean age was 39.9 years. previous travel to tropical countries was reported by 71.9% of them. most frequent destination was sub - saharan africa (60%). most of them traveled for 1 month (62%). the main reason for travel was work (39.7%). only 47% sought pretravel consultation. hepatitis a, typhoid, and meningococcal vaccines were administered to 49.8%, 28%, and 26.6%, respectively, and malaria chemoprophylaxis to 66.8% of those who visited sub - saharan africa. a history of previous travel to a tropical country, elementary level of education, and traveling for visiting friends and relatives, and for short duration were significant determinants for not pursuing pretravel consultation. conclusions. the current survey revealed important inadequacies in vaccine and malaria prophylaxis of travelers departing to africa. educational tools should be developed in order to improve awareness of travelers to risk destinations. |
during april 2002april 2012, brucellosis was discovered in 13 beef cattle herds and 4 ranched bison herds in the gya (figure 1). additionally, from comingling of cattle herds at the time of transmission and transfer of ownership of some animals between infection and detection, 3 more infected cattle herds were identified. in each of the 17 herds, infection was detected by serologic testing and confirmed by culture of tissues collected at slaughter of > 1 animals. the source of infection of each cattle or bison herd was determined through extensive epidemiologic investigations by state and federal animal health authorities. these investigations included serologic testing of all cattle herds adjacent to or in contact with the infected herd, testing of all herds from which the infected herds had received animals in the preceding years, interviews with owners and managers to determine the history of comingling with wildlife, and comparison of dna test results of the isolates with those from wildlife and domestic animals. the transmission event in 2002 positive domestic cattle and ranched bison herds (combined) detected each year, greater yellowstone area, usa, 19892012. we subjected 248 b. abortus isolates from affected cattle and bison herds and surrounding wild elk and bison to a 10-loci variable number tandem repeat assay. we analyzed results using a minimum spanning tree model (8). cattle and bison herd sizes varied from 300 animals, and seroprevalence ranged from 0.2% to 20% (table). for 8 herds, infection initially was detected through required testing of cattle going through markets or to slaughter. four infected herds were detected by routine testing because of the location of herds in the brucellosis - endemic area, 1 herd was detected because of testing of area herds in proximity to a previously infected herd, and 4 herds were detected by testing required for interstate transport or change of ownership. this was considered a single transmission event, and statistics are given for the combined herds. herd located 0.8 km from site where elk feeding ground had been until 2003. this herd was discovered infected in carbon county, mt, in 2007, but animals had been transported from park county in 2005. index cow aborted in 2005 and did not calve in 2006. examination of the data from these herds reveals several facts. with few exceptions, herds had low seroprevalence at time of detection (14 of 17 herds : < 10% ; 11 of 17 herds : < 3%). these findings probably are due to rigorous surveillance and the widespread use of vaccination in gya herds. the attenuated live vaccine, strain rb51, is efficacious in decreasing abortions but does not prevent infection (9). the herd with highest seroprevalence (herd no. management actions may have increased risk for exposure (i.e., allowing elk to feed with cattle and placing cattle in pasture with elk during late winter or spring). in wyoming and idaho, 7), owners or investigators reported that elk were sharing the premises with cattle.. 7 s owner and his son were employed at a nearby elk feeding ground, where their duties included removal of elk fetuses, which created the potential for transmission through fomites. information obtained from state wildlife agencies indicated that in every transmission case, serologic surveillance of elk in the area showed some level of infection in that species. on the basis of the 10-loci variable number tandem repeat assay, the b. abortus isolates recovered from cattle and farmed bison are very closely related to and sometimes indistinguishable from isolates from wild elk (figure 2). minimum spanning tree generated from variable number tandem repeat (vntr) data for 348 brucella abortus isolates in the national veterinary services laboratory database. nodes are color coded according to the source of the isolate, and segments of nodes represent isolates from different animals with the same vntr profile. the numbers represent the herd designations as indicated in the table (note that herd no. seventeen instances of brucellosis transmission from elk to livestock were reported during the last decade. this crescendo of interspecies transmission in all 3 gya states and involving ranches in proximity to and remote from elk feeding grounds suggests a change or combination of changes in risk factors in the gya ecosystem. until the discovery of increasing prevalence in non feeding ground elk (20062008) (10), b. abortus infection was not believed to have been self - sustaining in these populations (6). this belief was supported by high seroprevalence in populations in proximity to feeding grounds, with a marked decrease in prevalence proportional to distance from feeding grounds. in the last decade, however, seroprevalence in some non feeding ground elk herds has increased to levels similar to those of feeding ground herds, suggesting that brucellosis is now self - sustaining in these populations (11). several factors are likely to have contributed to changes in elk distributions and the resulting increases in brucellosis in some populations and its transmission to livestock. these include population and density increases (11,12), changes in land management that created safe havens for elk (13), and reintroduction of wolves to the gya (14). other factors that might have had local or general effects include climatic and snowfall changes (15), reduction of habitat by urbanization, and increased use of motorized backcountry vehicles. changes in elk and cattle brucellosis surveillance during the last decade do not account for the disease spread in elk or increased transmission to livestock. if brucellosis continues to increase among free - ranging elk populations remote from feeding grounds, the area to which brucellosis is endemic is likely to expand and the risk for transmission to livestock and the public will increase, in part reversing the hard - fought gains of the past 75 years in eliminating the disease in the united states. gaining a better understanding of ecologic and sociologic changes in the gya and their impact on the epidemiology of this wildlife livestock | bovine brucellosis has been nearly eliminated from livestock in the united states. bison and elk in the greater yellowstone area remain reservoirs for the disease. during 19902002, no known cases occurred in greater yellowstone area livestock. since then, 17 transmission events from wildlife to livestock have been investigated. |
radioelectric asymmetric brain stimulation technology with its treatment protocols has shown efficacy in various psychiatric disorders.110 the aim of this work was to highlight the mechanisms by which these positive effects are achieved. functional magnetic resonance imaging (fmri) is used to measure the hemodynamic response (changes in blood flow and blood oxygenation) that results from neural activity in the brain.1113 fmri images have the advantage of good spatial resolution, about 23 mm.14 however, as the hemodynamic response typically peaks after 45 seconds,15,16 this method suffers from a low temporal resolution.17 images are usually separated by 14 seconds, and the stimuli or the cognitive and motor tasks are carried out during the fmri session. it has previously been observed that the changes in psychological1,310 and motor behavior activity1820 resulting from radioelectric asymmetric conveyor (reac) brain stimulation pulse (bsp) stimulation are relatively long lasting, the persistence of effects has been observed for a few years. although the duration of the reac stimulus is brief (500 milliseconds), it was hypothesized that changes in brain activity following stimulation would be observable using fmri, despite the low temporal resolution of this imaging modality. this preliminary study used unpaid right - handed healthy volunteers, in accordance with the helsinki declaration. ten subjects were examined (six females, four males, aged 3048 years, mean age 39 years), who underwent brain fmri examination before and after a 500-millisecond reac - bsp. prior to fmri examination, all subjects were informed about how to properly perform the finger - tapping motor task (figure 1a). an nt intera 1.5 t mri scanner (philips, amsterdam, netherlands) was used to perform fmri to evaluate the cerebral areas involved during a simple motor task (finger tapping)21 (figure 1a) before and immediately after a 500-millisecond reac - bsp. the area of interest was visualized using a survey protocol with images obtained along the axial, sagittal, and coronal planes, on which a volumetric t1-weighted gradient echo sequence (t1 three - dimensional turbo field echo, repetition time = 13, echo time = 3, flip angle = 30) and an fmri sequence (t2 echo planar imaging - fast field echo, repetition time = 3000, echo time = 50, flip angle = 90) were placed. before administering reac - bsp, the subjects were placed in a supine position and asked to perform a 30-second task that consisted of a self - paced right fingers to right thumb opposition movement task, alternating with equal periods of rest. subjects performed 96 dynamic sequences during fmri acquisition, which were divided into blocks of ten (30 seconds each) with the elimination of the first six sequences during rest. as a control, this sequence was repeated after an hour for each subject. following the administration of the reac - bsp, subjects were brought back to the magnet room to repeat the fmri examination (figure 1). fmri data were analyzed using statistical parametric mapping 2 (spm2 ; the wellcome trust center for neuroimaging, london, uk) software package,22 using the familywise error rate correction routine23,24 that automatically sets the correct value of p = 0.05 at the voxel (a volumetric picture element representing a value on a regular grid in three dimensional space) level. the reac25,26 is an innovative technology for biostimulation and/or bioenhancement techniques that uses weak radio frequencies to modulate brain activity. the model used in this study (convogliatore di radianza modulante crm ; asmed, florence, italy) is specific for noninvasive brain stimulation techniques. reac treatment has proved its efficacy in ameliorating several stress - related disorders,1,69 depression,5,9,10 anxiety,5,9 bipolar disorders,4 and also in some forms of dementias3 and impaired motor control.18,20 the reac procedure is painless, and no adverse effects from its use have been reported. the reac pulse used in the current study consisted of a single 500-millisecond radiofrequency burst at 10.5 ghz. at a distance of 150 cm from the emitter, a specific absorption rate of 7 w / kg was obtained (and the density of radioelectric current flowing to the subject during the single radiofrequency burst was j = 7 a / cm. the electromagnetic field surrounding the device was approximately 20 w / m. the reac pulse was applied by touching the tip of the metallic reac probe to a specific area of the ear located at the top of the lower third of the scapha of the ear pavilion, according to the neuro postural optimization protocol.7,9 with these parameters, the radioelectric pulses emitted by the reac were much weaker than those from a mobile phone. in the united states, the federal communications commission has set a special absorption rate limit of 1.6 w / kg, averaged over a volume of 1 g of tissue, for the head. in europe, the limit is 2 w / kg, averaged over a volume of 10 g of tissue. the world health organization has classified mobile phone radiation on the international agency for research on cancer scale into group 2b possibly carcinogenic. an nt intera 1.5 t mri scanner (philips, amsterdam, netherlands) was used to perform fmri to evaluate the cerebral areas involved during a simple motor task (finger tapping)21 (figure 1a) before and immediately after a 500-millisecond reac - bsp. the area of interest was visualized using a survey protocol with images obtained along the axial, sagittal, and coronal planes, on which a volumetric t1-weighted gradient echo sequence (t1 three - dimensional turbo field echo, repetition time = 13, echo time = 3, flip angle = 30) and an fmri sequence (t2 echo planar imaging - fast field echo, repetition time = 3000, echo time = 50, flip angle = 90) were placed. before administering reac - bsp, the subjects were placed in a supine position and asked to perform a 30-second task that consisted of a self - paced right fingers to right thumb opposition movement task, alternating with equal periods of rest. subjects performed 96 dynamic sequences during fmri acquisition, which were divided into blocks of ten (30 seconds each) with the elimination of the first six sequences during rest. as a control, this sequence was repeated after an hour for each subject. following the administration of the reac - bsp, subjects were brought back to the magnet room to repeat the fmri examination (figure 1). fmri data were analyzed using statistical parametric mapping 2 (spm2 ; the wellcome trust center for neuroimaging, london, uk) software package,22 using the familywise error rate correction routine23,24 that automatically sets the correct value of p = 0.05 at the voxel (a volumetric picture element representing a value on a regular grid in three dimensional space) level. the reac25,26 is an innovative technology for biostimulation and/or bioenhancement techniques that uses weak radio frequencies to modulate brain activity. the model used in this study (convogliatore di radianza modulante crm ; asmed, florence, italy) is specific for noninvasive brain stimulation techniques. reac treatment has proved its efficacy in ameliorating several stress - related disorders,1,69 depression,5,9,10 anxiety,5,9 bipolar disorders,4 and also in some forms of dementias3 and impaired motor control.18,20 the reac procedure is painless, and no adverse effects from its use have been reported. the reac pulse used in the current study consisted of a single 500-millisecond radiofrequency burst at 10.5 ghz. at a distance of 150 cm from the emitter, a specific absorption rate of 7 w / kg was obtained (and the density of radioelectric current flowing to the subject during the single radiofrequency burst was j = 7 a / cm. the reac pulse was applied by touching the tip of the metallic reac probe to a specific area of the ear located at the top of the lower third of the scapha of the ear pavilion, according to the neuro postural optimization protocol.7,9 with these parameters, the radioelectric pulses emitted by the reac were much weaker than those from a mobile phone. in the united states, the federal communications commission has set a special absorption rate limit of 1.6 w / kg, averaged over a volume of 1 g of tissue, for the head. in europe, the limit is 2 w / kg, averaged over a volume of 10 g of tissue. the world health organization has classified mobile phone radiation on the international agency for research on cancer scale into group 2b possibly carcinogenic. following the reac - bsp, a significant reduction in the amplitude of motor task - induced cortical area activation, and a reduced statistical significance (z value) of the areas that remain activated, was observed in all subjects. as shown in figure 2, images taken prior to the reac - bsp in a representative female subject show large clusters of activation that include the premotor cortex contralateral to the limb used in the motor task (figure 1a), the adjacent primary motor area, and the somatosensory cortex. clear activation may also be observed in the ipsilateral premotor cortex, the bilateral visual cortex, the prefrontal cortex, and the superior cerebellar cortex ipsilateral to the limb used in the task. in control, following the reac - bsp, the same task resulted in statistically significant activation only in the contralateral primary motor area and the dorsolateral prefrontal and superior cerebellar regions ipsilateral to the involved limb. a similar phenomenon may be observed in a representative male subject (figure 3). the effects of the reac - bsp were observed as a significantly reduced signal amplitude following the motor task (figure 1a) in the ipsilateral superior cerebellar, premotor, and primary motor cortex, compared to that observed prior to the reac stimulation. in the fmri session following the reac - bsp, the disappearance of the larger clusters of activation that were present during the motor task (figure 1a) prior to the reac - bsp was observed, with activity persisting a small area of the medial premotor and motor cortex. based on available data, the efficacy of transcranial magnetic stimulation (tms), another technique of brain stimulation, is not comparable with other therapeutic strategies such as reac. reac has shown great stability of results, as demonstrated in a study on bipolar disorder with a follow - up period of about 4 years,4 whereas tms has been shown to be efficient in the same psychiatric disorder only in the short - term. also, in over 10 years of observation by the authors, many patients affected with recurrent depression achieved complete stabilization. however, some patients with obsessive - compulsive disorder, which was sometimes treated with reac, significantly worsened with tms after an initial improvement. more than 16 cases of tms - related seizure associated with single - pulse and repetitive tms have been reported in the literature;31 other risks include fainting, syncope, pains, headache, minor cognitive changes, and psychiatric symptoms. up until now, there have not been any noted negative side effects of reac. occasionally, a slight sense of vertigo has been reported when getting up from the examination table after reac, but this feeling can not be attributed to the treatment with any certainty. in the current study, an event - related approach was used and it was demonstrated that the modulation of brain activity induced by a single, brief reac pulse can clearly be measured using fmri, allowing for further indepth study of brain activity modulation in human subjects. in all the subjects examined, a significant reduction in the amplitude and area of the cortical activation induced by a simple motor task (figure 1a) was observed. this modulation was evident as a reduction in the z significance value between the area of activation before and during the motor task. these findings might be explained by the occurrence of remodeling, which would involve not only a decrease of activation, but also a positive adjustment in the mapping of the areas involved in the execution of motor tasks. these findings suggest that the reac pulse modulates activity only in those regions that are acutely necessary to the completion of the requested task, with the exclusion or inhibition of secondary circuits involved in somatosensory processing, motion planning, or executive feedback. the significant reduction in the amplitude of motor task - induced cortical area activation after reac - bsp substantially means that to perform the same task the subject uses less brain areas and, in this view, it is nt important if this phenomenon is related to primary areas, associative areas, or both. given the magnitude of the changes observed in all brain activity, it is thought that, as with other brain stimulation techniques,2831 the reac - bsp reshapes the ionic fluxes of the main neurotransmitters such as glutamate and gamma - aminobutyric acid. the testing of this hypothesis requires further study using a larger sample size and a variety of motor tasks. the goal of this preliminary study was to explore the possibility of using fmri to investigate the effects of reac - bsp on brain activity. the findings are encouraging, as they open new perspectives into the ability to study and understand the effects and potential benefits of reac - bsp upon the electrical activity of the brain. | purposeradioelectric asymmetric brain stimulation technology with its treatment protocols has shown efficacy in various psychiatric disorders. the aim of this work was to highlight the mechanisms by which these positive effects are achieved. the current study was conducted to determine whether a single 500-millisecond radioelectric asymmetric conveyor (reac) brain stimulation pulse (bsp), applied to the ear, can effect a modification of brain activity that is detectable using functional magnetic resonance imaging (fmri).methodsten healthy volunteers, six females and four males, underwent fmri during a simple finger - tapping motor task before and after receiving a single 500-millisecond reac-bsp.resultsthe fmri results indicate that the average variation in task - induced encephalic activation patterns is lower in subjects following the single reac pulse.conclusionthe current report demonstrates that a single reac - bsp is sufficient to modulate brain activity in awake subjects, able to be measured using fmri. these initial results open new perspectives into the understanding of the effects of weak and brief radio pulses upon brain activity, and provide the basis for further indepth studies using reac - bsp and fmri. |
fecal specimens from 6 clinical cohorts in salvador, northeastern brazil, were analyzed (table 1). adult cohorts comprised hiv - infected patients with gastroenteritis (105 persons) and without (49 persons) gastroenteritis. child cohorts included 359 children with gastroenteritis and 204 healthy children from child - care centers. nasal swab specimens were obtained from controls attending child - care centers and from children with gastroenteritis and concomitant respiratory symptoms. all specimens were stored at 30 to 80c until further processing. rt - pcr, reverse transcription pcr ; gastroenteritis was defined as acute diarrhea with > 3 watery stools in the previous 24 h and within 13 d before hospital admission. all hospital units were located within the hospital professor edgard santos, federal university of bahia. samples only considered if positive in nested rt - pcr as in (3) and in strain - specific real - time rt - pcr. # reports of diarrhea in the preceding 2 wk served as an exclusion factor. viral rna was purified from 200 mg fecal specimen and 140 l nasal swab specimen suspended in phosphate - buffered saline by using the viral rna mini kit (qiagen, so paulo, brazil) as described (7). a nested reverse transcription pcr (3) detected cosavirus. after nucleotide sequencing of all pcr - positive specimens, we developed a specific real - time rt - pcr for quantifying viruses from brazil (table 2). assay optimization and quantification relied on photometrically quantified crna in vitro transcripts, as described (8). rt - pcr, reverse transcription pcr ; utr, untranslated region ; fam, fluorescein ; r, g / a ; ddq1, deep dark quencher 1 ; y, c / t. rt - pcr reactions were carried out using the qiagen one - step rt - pcr kit as described by the manufacturer (qiagen, so paulo, brazil), 300 nmol / l of each primer, 200 nmol / l of the probe (real time rt - pcr assay), 1 g bovine serum albumin, and 5 l rna extract. second - round reactions used the platinum taq dna polymerase kit as described by the manufacturer (invitrogen, so paulo, brazil) with 2.5 mol / l mgcl and 1 l of first - round pcr product. real time rt - pcr amplification involved 55c for 15 min, 95c for 15 min, and 45 cycles of 95c for 15 s and 58c for 30 s (fluorescence measured).nested rt - pcr involved 30 min at 50c ; 15 min at 95c ; 10 cycles of 20 s at 94c, 30 s starting at 60c with a decrease of 1c per cycle, and 50 s at 72c ; and 40 cycles of 20 s at 95c, 30 s at 54c, and 50 s at 72c ; and a final elongation step of 5 min at 72c. second - round reactions used 3 min at 94c and thermal cycling as for the first round. in the adult cohorts, 1 of 105 hiv - infected persons with gastroenteritis had positive results for cosavirus. in the control group of 49 hiv - infected adults without gastroenteritis, of children with gastroenteritis, 13 (3.6%) of 359 patients were cosavirus positive. the proportion of cosavirus - positive controls without gastroenteritis, sampled in 2008, was significantly higher : 65 (49.2%) of 132 controls were cosavirus positive (149.1 ; p 1 of these pathogens could be detected. a) co - infections with established viral causes of diarrhea in children with gastroenteritis who were positive for cosv. viral rna and dna were detected by real - time pcr (methods available upon request) in the same eluates used for cosv detection. b) boxplot generated with spss v19 (spss, munich, germany) of log10 cosv rna concentrations per gram of feces in children with gastroenteritis and healthy control children from a child - care center in 2008 and 2011. the whiskers represent an extension of the 25th or 75th percentiles by 1.5 the interquartile range. cosavirus concentrations were low in outpatient and control children (10 copies / g) on 2 sampling occasions (table 1) and did not differ significantly (analysis of variance f 2.0 ; p = 0.14 ; figure 2, panel b). low cosavirus concentrations in the enteric tract could result from swallowing viruses originating in the respiratory tract. therefore, we tested for cosavirus in 96 nasal swab specimens from gastroenteritis patients, including 3 nasal swab specimens from persons with cosavirus - positive fecal specimens and 65 nasal swab specimens from cosavirus - positive controls sampled in 2008. none of the nasal specimens from controls with positive fecal specimens and only 1 nasal specimen from a patient unrelated to the 3 aforementioned persons was weakly positive (10 rna copies / ml), which refutes this hypothesis. to analyze whether a preceding point - source infection caused high cosavirus prevalence in the controls without gastroenteritis sampled in 2008, we determined the genomic sequence of the 5 untranslated region pcr amplicons and phylogenetically analyzed the sequence (genbank accession no. maximum nucleotide distance within these cosaviruses was up to 22.5% in the analyzed 398-nt fragment, making a recent point - source infection unlikely. human cosavirus infections were reported previously from a limited number of persons and geographic areas (36). in brazil, the 3.6% detection rate in children with gastroenteritis was comparable to the 1.8% rate in a cohort study of gastroenteritis patients in china (6). although the 6.5% detection rate in 1 control cohort in brazil was compatible with the 1.7% rate in 60 healthy controls in china, the combined 33.8% prevalence detected in controls from 3 different samplings in brazil was much higher. nonetheless, the prevalence was comparable to the 43.9% detected in 41 healthy southeast asian children in the only other cohort study (3). detecting cosavirus in 1 of 154 adults in brazil was compatible with finding a single cosavirus - positive patient among 1,000 adults with gastroenteritis in scotland, confirming that cosaviruses are rare and probably neither pathogenic nor commensal in adults (3). the higher prevalence of cosavirus found in controls than in patients, the frequent co - infections with established pathogens, and the unusually low rna virus concentrations give evidence against cosavirus involvement in human gastroenteritis. viruses that replicate in the human gut generally reach concentrations 1,000- to 100,000-fold higher than those of cosavirus. this finding is exemplified by genetically related picornaviruses (aichi viruses, parechoviruses, and cardioviruses) and established enteric pathogens (e.g., noroviruses and rotaviruses) (812). notably, the aforementioned study on cardioviruses included the same specimens from brazil, which indicates that poor sample quality was not a factor. these low concentrations would be compatible with absence of replication in the enteric tract and passive virus ingestion, e.g., from nutritional sources, drinking water, or the respiratory tract. however, nutritional patterns of the tropical countries in which cosavirus have been detected certainly differ. furthermore, in brazil, adults are unlikely to have a completely different diet from infants and children. moreover, the unprecedented detection of cosavirus in a respiratory tract specimen makes ingestion of viruses from nutritional sources alone unlikely, although a link to fluid droplets from drinking water in the respiratory tract is hypothetically possible. another explanation for low cosavirus rna levels in fecal samples is that a cosavirus infection occurred early in the person s life and produced partial mucosal immunity and limited subsequent cosavirus replication in the gut. this is exemplified for viruses transmitted by the fecal oral route by up to 100-fold higher fecal shedding of vaccine rotavirus and poliovirus among seronegative persons than among seropositive or previously vaccinated persons (13,14). however, this explanation would be incompatible with the high prevalence of cosavirus in many control children, who were generally older than patients. prolonged low concentrations of picornavirus shedding has been demonstrated, e.g., by detectable hepatitis a virus rna up to 3 months after acute infection (15). nonetheless, this circumstance is unlikely to explain the low cosavirus concentrations, given the overall high number of persons with positive results. although our study extends the known geographic occurrence of cosavirus, whether it is a human pathogen remains to be determined. future studies would be enhanced by serologic analyses and investigations focusing on nutrition and drinking water in tropical countries. | to determine possible cosavirus association with clinical disease, we used real - time reverse transcription pcr to test children and hiv - positive adults in brazil with and without gastroenteritis. thirteen (3.6%) of 359 children with gastroenteritis tested positive, as did 69 (33.8%) of 204 controls. low prevalence, frequent viral co - infections, and low fecal cosavirus rna concentrations argue against human pathogenicity. |
seborrheic dermatitis (sd), a common skin disorder that typically presents as erythematous plaques or patches and can vary from mild dandruff to dense, diffuse, adherent scale affecting the areas where sebaceous glands are prominent, including the scalp, face and chest. its precise etiology remains unknown, though it is associated with genetic, environmental, and general health factors. its role could be supported by the positive correlation between yeast density on the skin and the severity of sd and a high efficacy of antifungal agents in the treatment of this disorder. while sd rarely causes serious complications, it almost always leads to marked aesthetic deterioration and psychological distress due to its chronic and relapsing nature. despite high frequency of sd, surprisingly, only few studies have examined the effect of sd on quality of life (qol) [46 ]. although the topical treatment of sd with corticosteroids or antifungals is effective, there is a high risk of relapse and poor patient compliance, and therefore in some cases it is necessary to resort to systemic antifungals, especially in severe disease. various oral antifungals have been used to control sd with variable success rates. among them, itraconazole was the most frequently reported oral treatment particularly for severe sd, but therapeutic efficacy was evaluated only by clinical signs and symptoms in previous studies [7,911 ]. assessing qol can help in providing patients better service, by acknowledging their real needs and interfering with treatment decisions. because qol is a very important aspect in health and no study exists in the literature which assess the effect of treatment on qol in sd patients, this randomized controlled trial was set up to determine the impact of oral itraconazole on qol in patients with moderate to severe sd. this was a randomized placebo controlled, double - blind trial carried out with patients attending the outpatient ward of the dermatology department at the razi hospital tehran, between june 2012 and march 2014. patients were informed clearly and understandably about the possible risks or benefits of the trial and informed consent was provided by each patient included in the study. the approval of the study was obtained by the local and university ethics committees and performed in accordance with the helsinki declaration of 1964, as revised in 2013. inclusion criteria were clinical diagnosis of sd made by a dermatologist, moderate to severe sd (seborrheic dermatitis area severity index [sdasi ] = 4 plus : 3 anatomical sites involved and/or recurrent sd and/or disease unresponsive to conventional topical therapy) and age 18 years. exclusion criteria included the following : any concomitant skin disease (e.g. acne, rosacea, contact dermatitis) or hiv+/aids, recent use of antiseborrheic treatment (2 weeks for topical and 4 weeks for systemic therapy), history of allergy to azoles, renal, liver or cardiac disease, patients using drugs interacting with itraconazole (e.g., cyclosporine, isoniazide, omeprazole, warfarin, statins), and pregnant / lactating women. both itraconazole and placebo were in capsule form and identical in appearance and were delivered to patients by a third person recruited for this purpose. the study investigator and patients were blind to intervention, and that was maintained until patients completed their last follow up visit. the qol was assessed at the start and end of the study by a standard dermatological life quality index (dlqi) questionnaire (persian version) filled by patients, which consisted of 10 questions each referring to the previous week. each question was scored from 3 (very much) to 0 (not relevant or not at all). a questionnaire including the patient s basic profile was filled in the first visit for assessing other secondary outcomes like effect of age, sex, education level, disease duration, number of relapses and body mass index (bmi) on qol. this scoring system was modified from psoriasis area severity index (pasi) and the comert study that had already been used in a previous study by ghodsi. the treatment was administered in two different phases. in the first phase (initial treatment), topical 1% hydrocortisone ointment once daily and 2% ketoconazole cream twice daily plus either oral itraconazole 200 mg / day or oral placebo was prescribed for one week. topical treatment was included in the study for ethical issues. in the second phase (one month after initial treatment), which was maintenance period, oral itraconazole 200 mg / day or oral placebo was given on the first two days of every month (400 mg / month) for three months. no other topical treatment, including therapeutic shampoos (except moisturizers) or oral medications, was allowed during this study period. during the second phase, in cases of relapse or flare of disease, patients of either group were allowed to use topical treatment (1% hydrocortisone ointment once daily and 2% ketoconazole cream twice daily) for a maximum of three days as a rescue regimen after informing the study investigator. the difference in sdasi, symptom severity and dlqi scores before and after therapy was compared by using paired t - test. comparisons of mean age, disease duration, number of relapses and bmi between itraconazole and placebo groups were performed using unpaired t - test. the pearson correlation was used to assess the link between dlqi and other secondary parameters. sixty - eight patients who met study criteria were randomly assigned to itraconazole and placebo groups. consequently, 57 patients completed the study and were included in the analysis (figure 1). the main characteristics of patients at baseline and at the end of study are summarized in table 1. although dlqi scores decreased significantly in both groups (table 1), patients taking itraconazole had greater reduction compared to placebo (p = 0.001) (figure 2). the most compromised elements of qol were clinical symptoms, feelings (question 1, 2 of dlqi) and daily activities (question 3, 4) (figure 3). qol was not affected by age, sex, educational level, bmi, disease duration and number of relapses (p > 0.05) but was impaired significantly with high disease severity (sdasi) (p = 0.002) and presence of symptoms (itching and burning sensation) (p = 0.001 each). furthermore, dlqi was not significantly different between the two groups of itraconazole and placebo (p = 0.089). the most common site involved in our study was the scalp, but the location with significant effect on quality of life was the face (including forehead, eyebrows, nasolabial folds and cheeks collectively compared with scalp and trunk) (p = 0.017). one patient in the itraconazole group as opposed to four patients in the placebo group required rescue medication in the second phase of the study. side effects like nausea, mild abdominal pain and diarrhea were reported by only three patients in itraconazole group. in our study, we showed that quality of life is significantly improved by systemic itraconazole in moderate to severe sd patients. one of the important findings of our study was that patients in the itraconazole group had experienced a 73.36% reduction in dlqi after four months treatment, compared with 29.16% in the placebo group, and this effect of itraconazole on severe sd (mean dlqi before 6.72 and after 1.79) is comparable to systemic isotretinoin in acne patients (mean dlqi before=6.7, after=2.8). the mean dlqi score in our study was similar to harlow (mean dlqi : 5.9, n=20) but lower than szepietowski (mean 6.925.34). the difference could be explained by the different regional and social backgrounds and disease severity level in these studies. there is marked impairment of qol in patients with more severe disease, with a greater effect observed in women and in young patients (age subgroup : 2635 years) with higher educational level (master and phd level), and this is almost parallel to previous studies [45 ]. interestingly, in our study, disease duration and number of relapses had no effect on qol. here, adaptations of the patients to the nature of their disease and coping more efficiently with the disease and therapies over a certain period of time may have had effect. the most common site involved in our study was the scalp, but qol impairment was higher significantly in facial disease. as the appearance plays important role in the society, a patient with a red and flaky face may feel more embarrassed. the assessment of the impact on quality of life in patients with skin diseases is important for clinical management. it is essential to detect patients at a higher risk of experiencing a worse quality of life in order to treat him / her in a more integrated way. to our knowledge, this trial was the first using a randomized, placebo - controlled design that investigated the effect of itraconazole on the quality of life in moderate to severe sd patients. therapeutic efficacy of itraconazole has already been studied in many previous clinical trials [7, 911, 13 ]. although study designs and measurement scales were different, the results regarding effect of itraconazole were almost similar in these studies, and this fact shows the marked efficacy of itraconazole in the treatment of severe sd. first, no fungal culture was performed and therefore clinical outcome could not be correlated directly to malassezia colonization. second, the power of the study is limited by the monocentric design and small sample size of our study, therefore, a larger, multi - centric study is warranted to evaluate the therapeutic effect of itraconazole or other newer therapies for sd patients. third, the short period of follow up ; a longer study period would have been beneficial. fourth, it is well known that sd may worsen or improve due to many factors such as seasonal variation, host immunity, and host mood status ; these factors might have changed the clinical condition in some patients. fifth, although the effect of topical therapy declined mainly after two weeks, it might have affected partly over the final result of clinical picture and dlqi. pulse therapy with itraconazole can dramatically improve quality of life and disease severity. due to the complex interactions among diseases, the patients qol and therapeutics, physicians must couple subjective assessment of qol with objective measurements for the proper management of severe sd. | background : few studies have examined the effect of seborrheic dermatitis (sd) and/or its consequent therapy on a patient s quality of life. itraconazole has been suggested as an effective therapy for severe sd but its impact on quality of life (qol) in these patients has never been studied before.objective:the study aimed to verify the efficacy of the itraconazole on the quality of life in patients with moderate to severe sd.methods:a randomized, double - blind, placebo controlled trial was planned to describe the effect of sd per se on qol and to determine the impact of oral itraconazole or placebo on qol of sd patients. sixty - eight patients with moderate to severe sd participated in the study to receive either itraconazole or placebo. dermatology life quality index was used to evaluate their quality of life before and after treatment. itraconazole 200 mg / daily or placebo was prescribed for one week and then the first two days of every month for the following three months. fifty - seven patients completed the study.results:significant improvement was observed in qol of both itraconazole and placebo groups, but itraconazole group showed significantly higher improvement as compared to placebo (p=0.001). qol was impaired significantly with high disease severity (p=0.002) and facial involvement (p=0.017).conclusions : itraconazole significantly improves the qol in patients with moderate to severe sd. |
adequate debridement of the root canal plays an important role in the success of root canal treatment. mechanical instrumentation of root canals produces a smear layer comprising organic and inorganic substances, such as dentin particles, necrotic debris, microorganisms, and odontoblastic processes. despite the controversies regarding the smear layer [3, 4 ], the general consensus is that the smear layer adversely affects the root canal seal [5, 6 ]. the effectiveness of citric acid for removal of the smear layer was demonstrated in the 1970s [7, 8 ].. reported an irrigation solution comprising a mixture of 3% doxycycline, 4.25% citric acid, and 0.5% polysorbate 80 detergent, named mtad. the quantity of smear layer removed by an irrigation solution is related to its ph and the exposure time. traditionally, irrigants are delivered by a large syringe and needle to facilitate their insertion. therefore, several mechanical devices have been developed to improve the penetration and effectiveness of irrigants. some of these irrigation techniques include manual irrigation with needles and cannulas and the use of machine - assisted agitation systems with sonic and ultrasonic energy sources. self - adjusting file (saf) was introduced in 2010 and claimed to be successful in difficult - to - clean parts of the root canal with continuous flow of the irrigant. the aim of this study was to evaluate efficacy of mtad and citric acid solutions used with saf system on smear layer. twenty - three freshly extracted single - rooted human teeth with a straight canal, stored in saline solution, were used. radiographs of the teeth were taken in the buccolingual and mesiodistal projections to analyze the shape of the root canals and to detect possible anatomic variations. the coronal parts of the teeth were cut with a high - speed diamond bur to standardize the root lengths and to provide direct access to the root canals. number 15 k files (dentsply - maillefer, ballaigues, switzerland) were introduced further into the root canals until their tips were visible at the apical foramen. 5 ml, 5.25% sodium hypochlorite (naocl) was used for irrigation between the instruments. a saf file (redent - nova, israel) irrigation was performed continuously during the instrumentation using a special irrigation apparatus (vatea irrigation device, redent - nova, israel). the saf file instrumentation with irrigation was performed for a total of 4 minutes in each root canal : group 1 : 10 roots were used. 5.25% naocl was used for 3 minutes (at a flow rate of 5 ml / min, 15 ml in total) and then 5 ml mtad was used for 1 minute ; group 2 : 10 roots were used. 5.25% naocl was used for 3 minutes (at a flow rate of 5 ml / min, 15 ml in total) and then 5 ml 20% citric acid was used for 1 minute ; group 3 (control group) : 3 roots were used. 5.25% naocl solution were used for 4 minutes (at a flow rate of 5 ml / min, 20 ml in total). 5.25% naocl was used for 3 minutes (at a flow rate of 5 ml / min, 15 ml in total) and then 5 ml mtad was used for 1 minute ; group 2 : 10 roots were used. 5.25% naocl was used for 3 minutes (at a flow rate of 5 ml / min, 15 ml in total) and then 5 ml 20% citric acid was used for 1 minute ; group 3 (control group) : 3 roots were used. 5.25% naocl solution were used for 4 minutes (at a flow rate of 5 ml / min, 20 ml in total). finally, all roots were irrigated with 5 ml distilled water, then dried with sterile paper points, and left to dry at a room temperature for 24 hours. all roots were grooved longitudinally on the external surface with a diamond disc in the buccolingual plane, avoiding penetration of the root canals. the specimens were fixed on metal holders and coated with gold and viewed with fei quanta 400f field - emission scanning electron microscope (fei company, hillsboro, or, usa). the smear layer was evaluated from images at 2000x magnification based on the scale of hlsmann. : score 1, no smear layer and all dentinal tubules were open ; score 2, a small amount of smear layer and some dentinal tubules were open ; score 3, homogeneous smear layer covering the root canal wall and only a few dentinal tubules were open ; score 4, complete root canal wall covered by a homogeneous smear layer and no open dentinal tubules ; and score 5, heavy homogeneous smear layer covering the complete root canal. scores 1 and 2 represent clean canal wall. the kruskal - wallis test was used for statistical evaluation and mann - whitney u test was used for multiple comparisons. the saf, operated with mtad - naocl and citric acid - naocl, resulted in clean canals and most of the specimens revealed scores 1 and 2 (figures 1 and 2). the cleaning rates of groups 1 and 2 are shown in tables 1 and 2. control group exhibited heavy smear layer covering the root canal walls (figure 3). no significant difference was found statistically in the smear layer on the dentine wall among the coronal, middle, and apical thirds in groups 1, 2, and 3 based on comparisons within each group (group 1, p = 0.378 ; group 2, p = 0.065 ; group 3, p = 1.00) (kruskal - wallis test). mean scores and statistical equivalence related to the thirds of teeth in the three groups are shown in table 3. in the coronal third, there was a statistically significant difference among the three groups (groups 1 - 2, p = 0.005 ; groups 13, p = 0.007 ; groups 2 - 3, p = 0.005). group 3 (control) was statistically different from the other groups also in the middle (groups 13, p = 0.009 ; groups 2 - 3, p = 0.007) and apical thirds (groups 13, p = 0.009 ; groups 2 - 3, p = 0.009) (mann - whitney u test). similar to hand and rotary instrumentation, the saf system produces a smear layer when using naocl alone, but when alternating the application of mtad and 5.25% naocl and 20% citric acid and 5.25% naocl, the canals were rendered virtually free of debris and smear layer, with the most pronounced benefit realized in the apical third of the root canal, as confirmed by the present study. in recent years, numerous researchers [1519 ] studied saf system using different evaluation methods and reported successful results as our study in shaping and irrigating root canals. however, as a result of a microbiological and scanning electron microscopy study, paranjpe. found out insufficient apical preparation and irrigation when using the saf system. this result could be explained by differences in the sample and the testing methods. in the past, different concentrations of citric acid were used to remove the smear layer [21, 22 ]. di lenarda. reported no or negligible difference in smear layer removal with citric acid and edta, the most common chelating agent. in a study of mancini., the efficacy of 42% citric acid, mtad, and 17% edta was tested on removing the smear layer. the irrigation solutions were delivered via a nickel - titanium needle (stropko niti flexi - tip ; sybronendo, orange, ca), which penetrated within 1 to 2 mm of the working length. in direct contradiction with our study, none of these three solutions removed the smear layer in the apical third of the root canal. numerous investigations [2528 ] revealed that extended exposure to acids results in excessive demineralization. therefore, 4 min of 20% citric acid application instead of 40% was used with a final flush of saline in the current study. although we used a lower concentration of citric acid (20%), the smear layer was successfully removed in the majority of our specimens. this success can be attributed to the continuous irrigation and vibration action of the saf system. in the study of metzger., the use of the saf system and irrigation with edta and naocl resulted in smear layer - free canal walls in the apical third of 65% of the specimens. the vatea peristaltic pump used in the saf system delivers a continuous flow of irrigant, which enters the canal through the hollow file. according to the manufacturer, the motion of the file agitates the irrigant to such an extent that it effectively reaches the apical part of the canal with sonic activation. we thought that continuous replacement of irrigant could also explain the excellent cleaning efficiency observed in this study., mtad irrigation was achieved using a 28-gauge needle placed into the canal space 1 to 1.5 mm short of the working length and a cotton - wrapped barbed broach was placed to the end of the working length and left there for a few minutes. these researchers reported smear - free dentine walls in the apical third of the canals. this success might be due to the direct contact of the fresh solution and replacement of the older solution by the cotton wrapped barbed broach. similarly, direct contact of fresh irrigant with dentine walls might facilitate the saf system irrigation with mtad in our study. as zehnder emphasized, optimal cleaning requires direct contact of the irrigant with the root canal surface. in a study by tay., biopure mtad and edta were applied as a final irrigant, and to increase contact and penetration, a gutta - percha point was used to agitate the solution. the authors reported that the smear layer was successfully removed using this technique, regardless of the irrigant. in the present study, metzger. claimed that the saf file has a scrubbing action on the canals, which clearly results in a very clean surface even in the unreachable parts of the canal by activation of the irrigant in the apical third of the canal. in a recent study of melo ribeiro., oval saf was used with continuous naocl irrigation on oval - shaped root canals. these researchers reported that the percentage of remaining debris and uninstrumented canal perimeter was significantly lower in the saf group than in the rotary group. previously, de - deus. explained this result by the ability of saf instrument to adapt itself to the cross - section of the canal and the mechanical debridement efficacy of its continuous irrigation system. although there is general agreement regarding the necessity of removing the smear layer, the optimal irrigation solution and removal technique remain under debate.the present findings revealed the effectiveness of the saf system with two different irrigation solutions, suggesting that this methodology may be a useful alternative to conventional methods. in the limitations of this study, it can be concluded that using the saf system and continuous irrigation action with edta and mtad solutions could overcome the difficulty of removing smear layer even in hard - to - reach regions of the root canal. | mechanical instrumentation of root canals produces a smear layer that adversely affects the root canal seal. the aim of this study was to evaluate efficacy of mtad and citric acid solutions used with self - adjusting file (saf) system on smear layer. twenty - three single - rooted human teeth were used for the study. canals were instrumented manually up to a number 20 k file size. saf was used to prepare the root canals. the following groups were studied : group 1 : mtad + 5.25% naocl, group 2 : 20% citric acid + 5.25% naocl, and group 3 : control (5.25% naocl). all roots were split longitudinally and subjected to scanning electron microscopy. the presence of smear layer in the coronal, middle, and apical thirds was evaluated using a five - score evaluation system. kruskal - wallis and mann - whitney u tests were used for statistical analysis. in the coronal third, group 2 exhibited the best results and was statistically different froms the other groups (p 0.05). the solutions used in group 1 and 2 could effectively remove smear layer in most of the specimens. however, citric acid was more effective than mtad in the three thirds of the canal. |
delayed cerebral ischemia (dci) is the major cause of morbidity and mortality in patients suffering from spontaneous subarachnoid hemorrhage (sah). besides increasing importance of newer aspects like brain injury, inflammation, and microthrombosis and their influence on dci, cerebral vasospasm (cvs) remains an important therapeutically target. therefore, monitoring of cerebral blood flow (cbf) and oxygenation is a substantial and to improving issue in multi - modal intensive care [111 ]. diverse therapeutically approaches have focussed bedside brain monitoring for early detection of hypoperfusion of the brain. primarily, it is the measurement of intracerebral pressure (icp), partial tissue oxygenation (tipo2), regional cerebral blood flow using thermal diffusion flowmetry and micro - dialysis being invasive measurement method. as a noninvasive measurement tool one or two channel eeg adhesive electrodes are used to monitor the sedation depth [2, 1113 ]. as another noninvasive but discontinuous bedside measurement method transcranial doppler (tcd) is also available, with well - known deficits like investigator dependence and weak correlation of elevated blood flow velocity and symptomatic cvs [14, 15 ]. near - infrared spectroscopy (nirs) enables continuous and noninvasive measurement of regional cerebral oxygen saturation (rso2) via absorption of near - infrared light by oxyhemoglobin (hbo), deoxyhemoglobin (hb), and cytochrome oxidase [1, 1618 ]. nirs is frequently used in cardiac and vascular surgeries and during neuroendovascular procedures as well, achieving good and reliable results, though, its application is confined to the periprocedural and short time of the postprocedural stage [16, 1921 ]. long - term measurement of rso2 for early detection of hypoperfusion of the brain after sah is not yet widespread, so that the aim of our prospective study was to examine the applicability and the diagnostic value of nirs used continuously in sah patients over the estimated cvs period. in this study we used the invos 5100c oximeter (somanetics), which provides a continuous, noninvasive and real time measurement of cerebral oxygenation (figure 1(a)). the near - infrared wavelengths are generated by a light source of the sensor and penetrate the skin and the bone. within the brain tissue in 3 cm depth the light is either absorbed or reflected to the two detectors of the sensor (figure 1(b)). since hemoglobin within the detection field consists of venous blood in about 75%, arterial blood in about 20%, and capillary blood in about 5%, clinical interpretation of the values can be considered as a venous measurement. the portion of reflected data gives the relative concentration of deoxyhemoglobin and the overall level of hemoglobin, from which the regional oxygen saturation (% rso2) is calculated. the continuous measured data are displayed on the monitor with an update every five seconds. this study was approved by the ethics committee of the university of schleswig - holstein, campus kiel. we included patients with spontaneous sah, diagnosed by ct scanning or analysis of cerebrospinal fluid via lumbar puncture within 24 h after occurrence of symptoms in our study. after diagnosis, digital subtraction angiography (dsa) or ct angiography was performed for detecting the bleeding source. after treatment of the bleeding source (clip occlusion or coil embolization), all patients were treated in the neurosurgical icu with continuous measurement of the arterial blood pressure, pulse rate, oxygen saturation, and continuous recording of electrocardiogram. the neurological condition in the continuing course was assessed by the physician at the icu closely, using the hunt and hess scale (h&h). cvs was determined as acceleration of blood flow velocity > 120 cm / second. sedated and intubated patients with mechanical ventilation received intracranial probes for continuous measurement of icp and tipo2 additionally. external ventricular drainage was done in cases of occlusive hydrocephalus or hydrocephalus like ventricular system. in cases of elevated icp due to insufficient sedation, one - channel eeg electrodes (bis) after admission of the patients in the icu, the forehead of the patients was cleaned up with alcohol pads, and self - adhesive oximetry strips were applied bilaterally to measure the baseline value of rso2 in 3 cm depth (figure 1(c)). after setting the baseline value, we started the continuous measurement of rso2 over the estimated cvs period up to the 12th day after onset. measured data were saved in the memory device of invos 5100c and additionally in a memory stick. in cases of decrease of rso2 with an obvious cause for example, removing the strips for personal hygiene, dysfunction of the strips, or removing for neuroimaging an event mark button was pressed to illustrate this event and to allow correct correlation during data analysis. the oximetry strips could be removed during nursing activities and were reapplied at the same position. once the self - adhesive behaviour of the strips diminished, fixation was done by a regular wound plaster to obtain further measurement (figure 1(d)). desaturation below 50% or decrease of rso2 by 20% from the baseline value was estimated to be critical ; rso2 below 40% or decrease by 25% was assumed to be associated with dci and neurological deficits. for correct analysis of the measured values, intrinsic factors were determined to be mean arterial blood pressure (map), hemoglobin (hb), peripheral oxygen saturation (sao2), partial carbon dioxide pressure (pco2), and core temperature (t). extrinsic factors were head positioning, correct position and adhesion of the strips, and correct connection of the strips to the invos device. in case of decrease of rso2 intrinsic and extrinsic factors were controlled and equalized, before terming the rso2 value pathologic and performing further diagnostic evaluations. for analysis, the recorded data were transferred and represented graphically in the microsoft office excel program. furthermore, changes of rso2 values were matched with the values of icp, tipo2, and tcd and the results of additional neuroimaging. continuous measurement of rso2 was performed in nine patients (7 females, 2 males). dsa revealed an aneurysm as the source of hemorrhage in 7 patients (three aneurysms of the anterior communicating artery (acoa), one middle cerebral artery (mca) aneurysm, one aneurysm of the posterior cerebral artery (pca), and one aneurysm of the pericallosal artery). the bleeding source was unclear in one case after dsa (sah of unknown origin). in the last case dsa was not performed due to poor clinical condition with cessation of cbf diagnosed by perfusion - weighted ct scanning (pw - ct) and brain death two days after admission. five aneurysms were treated with clip occlusion, and two aneurysms were embolized with coils. the distribution to the h&h and fischer (f) scale was as follows : h&h1:n = 1, h&h2 : n = 3, h&h3:n = 0, h&h4:n = 3, h&h5:n = 2 ; f1:n = 1, f2:n = 0, f3:n = 3, f4:n = 5. the clinical course was uneventful in 7 patients without occurrence of cvs and ischemic strokes. in these patients, nirs measured constant and stable rso2 values without any significant alterations. case 1a 70-year - old female patient presented with sah h&h grade 5, fisher grade 4 due to a ruptured left - sided pca aneurysm with intracerebral and intraventricular hemorrhage. the aneurysm was treated surgically, and the patient remained sedated and intubated, receiving mechanical ventilation postoperatively. in the continuing course, tcd showed elevated blood flow velocities of both mca and aca arteries of up to 200 cm / second. despite triple h therapy and nifedipine application, nirs showed left - sided decrease of rso2 below 40% on day 5 after onset (figure 2). intrinsic and extrinsic factors were normal at that time (map 127 mmhg, sao2 99%, fio2 60%, t 38.3c, pco2 35%, and hb 11.3 g / dl). left frontal applied icp probe showed no significant changes at the same time (icp 11 mmhg, cpp 118 mmhg). subsequently performed native ct and pw - ct scans showed neither perfusion deficits nor ischemic stroke (figures 3(a) and 3(b)). a 70-year - old female patient presented with sah h&h grade 5, fisher grade 4 due to a ruptured left - sided pca aneurysm with intracerebral and intraventricular hemorrhage. the aneurysm was treated surgically, and the patient remained sedated and intubated, receiving mechanical ventilation postoperatively. in the continuing course, tcd showed elevated blood flow velocities of both mca and aca arteries of up to 200 cm / second. despite triple h therapy and nifedipine application, nirs showed left - sided decrease of rso2 below 40% on day 5 after onset (figure 2). intrinsic and extrinsic factors were normal at that time (map 127 mmhg, sao2 99%, fio2 60%, t 38.3c, pco2 35%, and hb 11.3 g / dl). left frontal applied icp probe showed no significant changes at the same time (icp 11 mmhg, cpp 118 mmhg). subsequently performed native ct and pw - ct scans showed neither perfusion deficits nor ischemic stroke (figures 3(a) and 3(b)). two days later, icp increased slowly and reached the maximum of 39 mmhg on day twelve after onset. in parallel to this right - sided rso2, values decreased as well. newly performed ct scan showed a marked left hemispheric ischemic stroke with shift of the midline strictures and signs of brain herniation (figure 3(c)). in consideration of the poor clinical condition, the age, and occurrence of distinct ischemic stroke, we decided to limit the therapy. case 2a 42-year - old male patient presented with sah h&h grade 2 and fisher grade 3 due to a ruptured aneurysm of the acoa (figure 4(a))., the patient suffered from headaches, but he was alert without neurological deficits at all times. in the continuing course, tcd showed elevated blood flow velocities of the left aca and mca up to 220 cm / second. although performed magnetic resonance angiography (mra) showed radiological spasm of the left ica and aca (figure 4(b)), the clinical condition of the patient remained stable without deterioration. further on, nirs showed stable rso2 values without significant desaturation. in line with performed mild triple - h therapy and oral nifedipine application, tcd values normalized in the continuing course, and the patient could be discharged without any neurological complaints. at the six months follow - up examination, the patient was still in a good condition, and he was working again. mra showed normalized vascular patterns (figure 4(c)). a 42-year - old male patient presented with sah h&h grade 2 and fisher grade 3 due to a ruptured aneurysm of the acoa (figure 4(a))., the patient suffered from headaches, but he was alert without neurological deficits at all times. nirs showed normal and stable rso2 values (figure 5). in the continuing course, tcd showed elevated blood flow velocities of the left aca and mca up to 220 cm / second. although performed magnetic resonance angiography (mra) showed radiological spasm of the left ica and aca (figure 4(b)), the clinical condition of the patient remained stable without deterioration. further on, nirs showed stable rso2 values without significant desaturation. in line with performed mild triple - h therapy and oral nifedipine application, tcd values normalized in the continuing course, and the patient could be discharged without any neurological complaints. at the six months follow - up examination, the patient was still in a good condition, and he was working again. case 3 in this case, it was a 14-year - old girl, who was found comatose at home with dilated and nonresponsive pupils. pw - ct showed cessation of cbf, and tcd showed abnormal reverberating flow, indicating distinct increased icp. nirs strips were applied for two days and measured interestingly rso2 values of 63%. over the measurement period of about 48 hours rso2 values were very stable within the range of 6070%, (figure 6) without common fluctuations as seen in other patients. two days after onset the patient was pronounced brain dead. in this case, it was a 14-year - old girl, who was found comatose at home with dilated and nonresponsive pupils. pw - ct showed cessation of cbf, and tcd showed abnormal reverberating flow, indicating distinct increased icp. nirs strips were applied for two days and measured interestingly rso2 values of 63%. over the measurement period of about 48 hours rso2 values were very stable within the range of 6070%, (figure 6) without common fluctuations as seen in other patients. the results of several studies have shown that monitoring of rso2 decreases the risk of procedure - related cerebral desaturation and improves the outcome in those patient groups [2327 ]. however, continuous measurement of cerebral oxygenation with nirs after spontaneous sah is not sufficiently investigated. just a few studies focused on the application of nirs after sah [1, 17, 18 ]. while mutoh, yokose, and zweifel used the niro 200 monitor (hamamatsu) in their studies, comparable studies with the invos system (sommanetics) are to the best of our knowledge not available. the mentioned studies draw the conclusions that application of nirs can provide continuous and long - term measurement of rso2 and can give relevant information about the cerebral autoregulation and the effectiveness of performed therapy on cvs. concluding the results of our study, we can postulate that measurement of cerebral oxygenation with nirs is a save and easy to use noninvasive additional measurement tool, which is applicable for long - term measurements in sah patients. it seems to be useful especially in alert patients, in whom invasive probes are not usually used. in general, performance of rso2 measurement was done without major problems in our study, whereby the nursing personnel needed repeatedly well instruction to obtain a smooth functioning process. although our examined patient group is relatively small, the displayed cases show in an illustrative manner the diagnostic relevance of measured values. in case 1, nirs detected early relevant hypoperfusion of the aca territory way ahead of increased icp. the interpretation of measured elevated blood flow velocities by tcd is controversial concerning radiological and symptomatic cvs, as already discussed in other studies [14, 15 ]. this case illustrated pointedly the diagnostic value of nirs for early detection of dci in a sedated and intubated patient. vice versa, rso2 values remained consistently stable in case 2 with proven cvs by tcd and mra. the patient developed no neurological impairments, so that cvs could be termed radiological. in this case, nirs was a very important additional measurement tool to control the gathered findings in view of dci in an alert patient. even though the neurological examination by a physician probably remains the easiest and most reliable procedure to detect cvs related deteriorations in alert patients, early illustration of changes in cerebral oxygenation before clinical manifestation is desirable and the investigators examined six brain dead patients with nirs and measured relatively normal rso2 values and brain desaturation dependent on the level of mechanical ventilation. hence, the authors suggested an additional extracranial contribution of measured rso2 values and restricted the diagnostic relevance of nirs in the diagnostic protocol of brain death. although new generations of nirs devices claimed to exclude the extracranial component effectively, and the tissue oxygenation index was shown to be independent of hemoglobin concentration, skull thickness, and the area of the cerebrospinal fluid layer underlying the sensors [18, 29 ], our findings remain poorly explained, raising further questions in this area of research. a possible explanation could be that nirs measured the remaining desoxgenated haemoglobin in the brain, which does not underly the blood circulation. thus, this attempted explanation could state the relative constant rso2 values without alterations. according to this particular case, it is arguable that application of nirs is unsuitable in brain dead patients. different aspects should be considered, which could lead to limitation of its applicability and interpretation. first of all, it is to note that the measurement of cerebral oxygenation mainly involves the aca territory. the important mca territory is only partly captured, if the strips are applied to the forehead. it remains to prove if application of the strips to the temples after head shaving can provide reliable rso2 values of the mca territory. another problem is the restricted applicability due to limited space on the forehead if the sedation depth has to be measured with eeg electrodes in patients with increased icp. a solution for this problem could be the combination of nirs strips and eeg electrodes, which is an object of development. this could be a problem for continuous and long - term measurement, since infections and fever occur relatively frequently in sedated and intubated patients at the icu. hence, measured data could be unsuitable for decision making towards further imaging and therapy. furthermore, it is to note that application of nirs strips requires a compliant patient. measurement and data interpretation of an agitated patient is nearly impossible, if the patient dislocates and tears the strips off repeatedly. finally, there is a lack of common consensus about the threshold values in view of necessity of intervention [2, 22, 30, 31 ]. however, it has already been postulated and is also our opinion that nirs is suitable for recording trends. our selected threshold values were based on the information of the manufacturer, which is the result of a large number of other studies. it is desirable to have a noninvasive and continuous measurement method of cerebral oxygenation with reliable values. despite the promising course of our study, further large prospective studies are required to answer the open questions and to test our observations. measurement of rso2 with nirs is a safe, easy to use, noninvasive additional measurement tool for cerebral oxygenation, which is used routinely during vascular and cardiac surgical procedures. nirs is applicable over a long - time period after sah, especially in alert patients without invasive probes. extrinsic and intrinsic factors have to be considered exactly to enable correct data interpretation. under consideration of our relative small patient group, however, further large studies are needed to test our observations and to obtain recommendations for its application in sah patients. | objective. the aim of our prospective study was to investigate the applicability and the diagnostic value of near - infrared spectroscopy (nirs) in sah patients using the cerebral oximeter invos 5100c. methods. measurement of cerebral oximetry was done continuously after spontaneous sah. decrease of regional oxygen saturation (rso2) was analyzed and interpreted in view of the determined intrinsic and extrinsic factors. changes of rso2 values were matched with the values of icp, tipo2, and tcd and the results of additional neuroimaging. results. continuous measurement of rso2 was performed in nine patients with sah (7 females and 2 males). mean measurement time was 8.6 days (range 212 days). the clinical course was uneventful in 7 patients without occurrence of cvs. in these patients, nirs measured constant and stable rso2 values without relevant alterations. special findings are demonstrated in 3 cases. conclusion. measurement of rso2 with nirs is a safe, easy to use, noninvasive additional measurement tool for cerebral oxygenation, which is used routinely during vascular and cardiac surgical procedures. nirs is applicable over a long time period after sah, especially in alert patients without invasive probes. our observations were promising, whereby larger studies are needed to answer the open questions. |
dental caries is the most common chronic disorder in children, and toothache is a usual complication. odontogenic pain occurs as a result of physical stimulation or inflammatory mediators provoking nociceptive efferent neural fibers. nociceptive fibers are richly distributed in trigeminal nerve fibers which are responsible for periapical and pulp tissues innervations. it has been shown in studies that irritation of the affected tooth initiates inflammatory reactions in gingival mucosal tissues. pulp damages lead to cell apoptosis and inflammation. depending on duration and intensity of stimuli and body reactions, these damages may lead to a variety of changes from reversible pulpitis or progression through to irreversible pulpitis and necrosis. stimulation of the pulp results in different biologic reactions due to a nonspecific inflammatory response in body, which are presented with polymorphonuclear leukocytes degranulation, protease inhibitor activation, and histamine, bradykinin, and arachidonic acid metabolites release. it has been shown that neuropeptides can enter from pulp to periodontal ligament (pdl) space through communication pathways such as apical foramen and accessory canals. thus, pulp - related pain leads to expression of pro - inflamatory neuropeptides in adjacent gingival crevicular fluid (gcf). these neuropeptides are found in dental pulp and the periodontium, and their raised levels have been reported in decayed painful teeth. different studies have used various methods including radioimmunoassay, enzyme immunoassay (eia or elisa), and immunohistochemistry to measure these mediators, but among these methods, elisa is known as a significantly safe and effective method. the present study aimed to compare the level of sp and nka as pain mediators available in gcf, between intact and decayed permanent teeth, using elisa method. evaluating the mediators and clarifying their role in the neurophysiology of pulpitis may lead the research into more effective ways to alleviate the symptoms of pulpitis. this survey was a cross - sectional study conducted on 20 children (13 girls and 7 boys) aged between 7 and 12 years referred to pediatric dentistry department of dentistry faculty of tehran university of medical sciences, tehran, iran, between 2012 and 2013. inclusion criteria were the presence of a painful first molar tooth diagnosed with irreversible pulpitis by clinical or radiographic examination. the control tooth was an intact molar in the opposite quadrant in the same jaw. exclusion criteria were considered to be antibiotic use during the last month, taking analgesic or anti - inflammatory drugs during the last month, positive history of any systemic disease which may affect oral tissues, any evidence of gingivitis, abscess, bleeding during periodontal probing, any radiographic evidence for periapical or periodontal defect, carious, painful, or previously restored the first molar in the opposite side of the patient 's jaw, uncooperative patient, and parents disconsent with taking part in the study. then, eligible patients were asked about their child 's demographic information (name and family name, date of birth, and gender), medical and dental histories, and the presence of dental pain. the location of the carious and intact molar teeth (upper or lower jaw) was also documented. after giving brief information about the aims and method of the study, patients without recent radiographs underwent radiographic examination to rule out any proximal decay in the test and control teeth. the intensity of the pain was evaluated by visual analog scale (vas) which is one of the most reliable tools to assess and report pain in children age group. sampling was done in each patient in the eight following steps : isolation of the tooth with the use of cotton roll and suctiondrying the area by air tooth dryer for 10 sremoving the biofilm with using sterile cottonputting the absorbent paper points in gingival crevice.for this purpose, gamma - sterilized paper number 30 was used. the paper (aria dent co. tehran, iran) was placed in gingival crevice of buccal surface of the tooth along the linear axis till a resistance was felt and it remained there for 30 s. in case, the paper was contaminated with saliva or blood during or before its placement ; it was changed with a new one.all samples were taken between 9 and 11 am from all patients.placing every paper in a propylene microtube containing 300 l of phosphate - buffered saline (pbs) right after sampling.pbs is a water - based saline solution which is used in biologic studies for its isotonic and nontoxic nature. utilizing pbs tablets is one of the ways for preparing this solution (43).placing samples in a freezer immediately after sampling and transferring frozen samples to immunology laboratory of tehran university of medical scienceskeeping samples in 70c until laboratory analysis was doneanalyzing the gcf samples in laboratory. isolation of the tooth with the use of cotton roll and suction drying the area by air tooth dryer for 10 s removing the biofilm with using sterile cotton putting the absorbent paper points in gingival crevice. for this purpose, the paper (aria dent co. tehran, iran) was placed in gingival crevice of buccal surface of the tooth along the linear axis till a resistance was felt and it remained there for 30 s. in case, the paper was contaminated with saliva or blood during or before its placement ; it was changed with a new one. all samples were taken between 9 and 11 am from all patients. placing every paper in a propylene microtube containing 300 l of phosphate - buffered saline (pbs) right after sampling. pbs is a water - based saline solution which is used in biologic studies for its isotonic and nontoxic nature. utilizing pbs tablets is one of the ways for preparing this solution (43). placing samples in a freezer immediately after sampling and transferring frozen samples to immunology laboratory of tehran university of medical sciences keeping samples in 70c until laboratory analysis was done analyzing the gcf samples in laboratory. in this study, we used elisa method and elisa reader machine (dana, da3200) for measuring sp (cyman kit, usa) and nka (phoenix kit, usa) concentrations. samples were placed in room temperature for an hour and were then centrifuged for 20 min for complete solvation of absorbed particles in buffer solution. to measure sp levels, we first prepared eia buffer, washed buffer, and standards were created according to the elisa kit instructions. unlinked proteins were washed the next day, and a junctional substance was added to the wells which would bind to the intended antibody. unbound molecules of this substance were then washed, out and a color - producer substance was added to allow the use of optical densitometry and software analysis to determine the level of antigen. for measuring nka levels, all the same steps were performed, and standard density solutions were prepared by following the manufacturer 's instructions as well. five test tubes were prepared, and 900 l of standard attenuator solution was poured in the first laboratory tube, and each of other laboratory tubes contained 500 l of it. five hundred microliters of the high density standard solution was then added to the first test tube and mixed by shaker. five hundred microliters of the first laboratory tube solution was poured to the second one, and it continued to the 5 one. final achieved densities were 10, 5, 2.5, 1.2, and 0.6 pg / ml, respectively. wells of the microplate were previously covered by anti - nka antibody ; then, 50 l of the prepared standard solution from the five test tubes was poured in the first five wells of the plate, and the same procedure was done for the rest wells with 50 l of each sample. twenty - five microliters of primary antibody and biotinylated peptide were added to each well afterward, and samples were incubated in room temperature for 2 h. unlinked proteins were washed, and 100 mcl of sa - hrp solution (junctional substance) was added to the wells. samples were incubated in room temperature for an hour, washed again, and the color - producer substance was added in the last step. the plate was transferred to elisa reader machine (dana, 3200 model) to measure nka level according to color densitometry. final measures were reported as level of sp and nka (in picogram) in each milliliter of gcf. sampling was done in each patient in the eight following steps : isolation of the tooth with the use of cotton roll and suctiondrying the area by air tooth dryer for 10 sremoving the biofilm with using sterile cottonputting the absorbent paper points in gingival crevice.for this purpose, gamma - sterilized paper number 30 was used. the paper (aria dent co. tehran, iran) was placed in gingival crevice of buccal surface of the tooth along the linear axis till a resistance was felt and it remained there for 30 s. in case, the paper was contaminated with saliva or blood during or before its placement ; it was changed with a new one.all samples were taken between 9 and 11 am from all patients.placing every paper in a propylene microtube containing 300 l of phosphate - buffered saline (pbs) right after sampling.pbs is a water - based saline solution which is used in biologic studies for its isotonic and nontoxic nature. utilizing pbs tablets is one of the ways for preparing this solution (43).placing samples in a freezer immediately after sampling and transferring frozen samples to immunology laboratory of tehran university of medical scienceskeeping samples in 70c until laboratory analysis was doneanalyzing the gcf samples in laboratory. isolation of the tooth with the use of cotton roll and suction drying the area by air tooth dryer for 10 s removing the biofilm with using sterile cotton putting the absorbent paper points in gingival crevice. for this purpose the paper (aria dent co. tehran, iran) was placed in gingival crevice of buccal surface of the tooth along the linear axis till a resistance was felt and it remained there for 30 s. in case, the paper was contaminated with saliva or blood during or before its placement ; it was changed with a new one. all samples were taken between 9 and 11 am from all patients. placing every paper in a propylene microtube containing 300 l of phosphate - buffered saline (pbs) right after sampling. pbs is a water - based saline solution which is used in biologic studies for its isotonic and nontoxic nature. utilizing pbs tablets is one of the ways for preparing this solution (43). placing samples in a freezer immediately after sampling and transferring frozen samples to immunology laboratory of tehran university of medical sciences keeping samples in 70c until laboratory analysis was done analyzing the gcf samples in laboratory. in this study, we used elisa method and elisa reader machine (dana, da3200) for measuring sp (cyman kit, usa) and nka (phoenix kit, usa) concentrations. samples were placed in room temperature for an hour and were then centrifuged for 20 min for complete solvation of absorbed particles in buffer solution. to measure sp levels, we first prepared eia buffer, washed buffer, and standards were created according to the elisa kit instructions unlinked proteins were washed the next day, and a junctional substance was added to the wells which would bind to the intended antibody. unbound molecules of this substance were then washed, out and a color - producer substance was added to allow the use of optical densitometry and software analysis to determine the level of antigen. for measuring nka levels, all the same steps were performed, and standard density solutions were prepared by following the manufacturer 's instructions as well. five test tubes were prepared, and 900 l of standard attenuator solution was poured in the first laboratory tube, and each of other laboratory tubes contained 500 l of it. five hundred microliters of the high density standard solution was then added to the first test tube and mixed by shaker. five hundred microliters of the first laboratory tube solution was poured to the second one, and it continued to the 5 one. final achieved densities were 10, 5, 2.5, 1.2, and 0.6 pg / ml, respectively. wells of the microplate were previously covered by anti - nka antibody ; then, 50 l of the prepared standard solution from the five test tubes was poured in the first five wells of the plate, and the same procedure was done for the rest wells with 50 l of each sample. twenty - five microliters of primary antibody and biotinylated peptide were added to each well afterward, and samples were incubated in room temperature for 2 h. unlinked proteins were washed, and 100 mcl of sa - hrp solution (junctional substance) was added to the wells. samples were incubated in room temperature for an hour, washed again, and the color - producer substance was added in the last step. the plate was transferred to elisa reader machine (dana, 3200 model) to measure nka level according to color densitometry. final measures were reported as level of sp and nka (in picogram) in each milliliter of gcf. twenty patients enrolled in the present study, 7 (35%) patients were male and 13 (65%) were female. the patients average age was 9.4 1.4 (with a range of 7 - 12 years). demographic data of participants are demonstrated in table 1. among 40 evaluated teeth, 26 belonged to the lower jaw, and the remaining teeth (14 teeth) were located in the upper jaw. demographic data of participants the pain intensity described by vas was reported severe (5) by all patients. mean concentration of sp mediator were 2.65 0.56 and 1.83 0.65 in gcf of painful decayed and healthy intact teeth, respectively. these measures were reported for nk level 2.29 0.29 and 1.61 0.35, respectively. as demonstrated in table 2, kolmogorov smirnov test showed a normal distribution for sp and nka level variables among decayed and intact teeth (p > 0.05). paired t - test was then used for comparison both of these variables between decayed and healthy teeth. the mean levels of sp and nka difference in gcf were significantly different between healthy and painful decayed teeth (p < 0.05). comparison of substance p and neurokinin a concentrations between gingival crevicular fluid of decayed and intact teeth correlation test between substance p and neurokinin a levels to test the relevance between sp and nka, correlation test was performed which showed no significant relation between two variables in gcf. sp and nka are two neuropeptides among tachykinins with a same origin which are released from trigeminal nerve endings due to physical or chemical stimuli and are the main pain - related neurotransmitters. existence of these mediators in gcf of intact teeth may indicate their role in physiologic processes. pulpitis causes sp and nka release from sensory endings of pulp which are vasodilators and increase inner pressure of pulp. communicating canals between pulp cavity and pdl including apical foramen, accessory canals, canals of the furcation area, and dentinal tubules direct these neuropeptides into pdl space and into gcf, in turn. on the other hand, since sensory nerves of pulp and periodontal tissues have the same origin, sensory stimulation of pulp may have a provoking effect on periodontal tissue innervations too. since it has been previously shown in studies that gingivitis and periapical or periodontal defects can alter the level of sp and nka in gcf, the study did not include patients with evidence of the above - mentioned disorders. samples also were taken in the same time of the day from all participants to avoid probable changes of gcf amount secretion affected by circadian rhythm. sp and nka levels were shown to have significant higher concentrations in gcf of decayed teeth which were affected by pulpitis. considering above clarification of the mechanism and cause of this additional concentration around decayed teeth, obtaining such a result seems rational and is also supported by some previous surveys. in their two distinct studies reported higher level of sp found in gcf of decayed teeth in comparison to the control group. the nature of pain was different in these studies as it was induced by pushing elastic separators and using simulator machine, which varies in origin with the pain caused by pulpitis and pulp disorders. another group of studies has also compared the level of pain mediators such as sp between painful and intact teeth and supported its role in toothache by finding its higher level in gcf of decayed teeth. while it has been shown that local anesthetic ingredients can affect the result of such studies by slowing the release of neuropeptides from dental pulp as well as lowering sp concentration in pulp tissue and diluting it. some of these studies were performed on samples of coronal pulp tissue taken after local anesthesia administration. 's study was the nearest to ours in regard to methodology but was carried out in adult setting. reported results of the study were in line with our findings both for sp and nka. they had found higher levels of these mediators in gcf of decayed teeth than what we did, and this quantitative difference is attributable to the sampling method as they used periopapers which have larger absorbing surface and thus higher rate of absorption. the duration of sampling was also different in both studies as it was 30 s in ours and 1 min in awawdehal 's. variability in the laboratory methods was another cause. besides presenting sp and nka as effective mediators contributing in dental pain induction, this study highlighted the fact that each of these mediators participates in pain induction independently. these include the small sample size as many of the eligible patients had used medications stated in our exclusion criteria. it was not possible to collect longitudinal data on the changes in these mediators following treatment due to limited resources in our busy public clinic. further studies are suggested to support the pivotal role of sp and nka in dental pain process with larger populations and applying a variety of changes in sampling - related factors such as number of times and duration of sampling as well as evaluating after - treatment conditions. making a clearer understanding of the exact role of these important mediators in dental pain process can lighten the path for future studies on drugs and methods used for dental pain alleviation. for better understanding of responsible substances and mechanisms of dental pain and better finding of dental pain - reducing ways, this study aimed to evaluate levels of sp and nka in gcf of carious and healthy permanent teeth, comparatively. significant higher levels of both sp and nka in gcf of painful carious teeth were observed, which is in line with previous studies ' findings. the authors of this manuscript declare that they have no conflicts of interest, real or perceived, financial or non - financial in this article. the authors of this manuscript declare that they have no conflicts of interest, real or perceived, financial or non - financial in this article. | background : it is shown that neuropeptides can be transported from pulp chamber to periodontal ligament through apical foramen and accessory canals. therefore, clinical pulpal pain leads to expression of preinflammatory neuropeptides such as substance p (sp) and neurokinin a (nka) in gingival crevicular fluid (gcf). this study aimed to evaluate levels of sp and nka in gcf of carious and healthy permanent teeth, comparatively.materials and methods : this cross - sectional study was performed on twenty children referred to department of pediatric dentistry, tehran university of medical sciences, who had a painful permanent first molar. sampling was done by sterile paper cone from gcf of the mentioned teeth and the intact tooth of the other side of the jaw in the same patient. values of sp and nka were measured by elisa test.results:the mean concentration of sp in gcf of painful carious and healthy teeth was 2.65 0.56 and 1.83 0.65 pcgr / ml, respectively. this value was 2.29 0.29 and 1.61 0.35 pcgr / ml for nka concentration in carious and healthy teeth as well.conclusion:significant higher levels of both sp and nka in gcf of painful carious teeth were observed, which is in line with previous studies findings. |
acute coronary syndrome (acs), which is commonly known as atherosclerotic heart disease, is the most common type of heart disease and the cause of heart attacks. acs is a term used to describe any condition that results in a sudden reduction in blood flow to the heart. if the plaque ruptures as shown in figure 1, it can lead to a blood clot that blocks the blood supply to the heart and triggers a heart attack. it may also block blood supply to the brain, which could trigger a stroke. intravascular ultrasound (ivus) imaging is a unique imaging clinic tool that provides a real time cross - sectional inside view of a coronary artery in living individual and thus allows a complete study of its morphology, such as arterial wall, lumen, and plaque. the ivus method helps in the diagnosis and treatment of acs, as far as a tissue characterization and plaque volume calculation are available. as the first step in a diagnosis of acs, the inner and outer coronary plaque boundaries in the ivus image have to be detected for evaluating the quantitative assessment of the coronary plaque compositions. currently, these coronary plaque boundaries are manually drawn and the area of that plaque is also evaluated manually by a medical doctor. after that, the volume of the plaque is estimated by integrating the calculated areas. a manual processing of the ivus images by a medical doctor is a time consuming task and a difficult task and might suffer from intra- and interobserver variability. this is not only because a large number of the ivus images must be processed, but also because the ivus images tend to a heavy speckle noise. this fact motivates the development of automatic image processing technique addressing detection of the coronary plaque boundary with high accuracy. a probabilistic segmentation for identification of luminal boundary (lb) has been proposed by ruiz.. those methods however do not automatically work because the method needs an initial area created by a user, and the method needs a set of training data manually segmented by an expert. this method also needs a set of training data which is manually segmented by an expert. in our previous works [5, 6 ], fuzzy inference model employed is takagi - sugeno (t - s) fuzzy model. in, membership functions (msfs) in the antecedent parts of the fuzzy rules were allocated adaptively. the fuzzy inference was used because it has several advantages over the conventional methods in the boundary calculation of image, for example, sobel 's method, prewitt 's method, and robert 's method. the fuzzy inference can handle problems with imprecise, noisy, inconsistent, and incomplete data set. ivus images often have noise and the plaque boundaries are usually missing in several areas. we have employed the t - s fuzzy inference to restore the missing boundaries by inference. because the ivus image has heavy speckle noise, a speckle noise reduction and an edge enhancement of coronary plaque boundary are very important tasks in the case of preprocessing of ivus image. as the representative conventional noise reduction methods, the median filters, morphology analysis, and bilateral filters are well known, but at the same time the coronary plaque boundary also becomes dull unexpectedly by applying those methods. above all those methods, perona - malik diffusion (pmd) filter is known as an effective edge - preserved smoothing method and is broadly used in [3, 5, 6, 1318 ]. in [5, 6 ], when the normal pmd filter is applied to ivus image, the coronary plaque boundary can not be preserved on several areas. the diffusion direction and its strength are very important factors to enhance the image edges and to reduce the speckle noise. this is because the diffusion direction and its strength in the normal pmd filter used in [5, 6 ] have not been set properly and the plaque boundary direction in ivus image was not considered. in this paper, we propose a modified pmd filter to reduce the speckle noise and enhance a coronary plaque boundary by considering the plaque direction in ivus image. after applying the modified pmd filter to ivus image, the coronary plaque boundaries are detected by the takagi sugeno (t - s) fuzzy model. the accuracy of the proposed method has been verified through the experiments using the real ivus images. the intravascular ultrasound (ivus) method is one of the applications of ultrasound technology which has many applications in medical diagnosis. the ivus method is a catheter based medical imaging technique that produces cross - sectional images of blood vessel and is particularly useful for the diagnosis of atherosclerosis. ivus method is further used in the coronary arteries to observe within the blood vessel all the way through to the surrounding blood column, visualizing the coronary plaque, determining the amount of plaque built up at any particular point in the coronary artery in living individual. the progressive accumulation of plaque within the artery wall over decades is the setup for vulnerable plaque which, in turn, leads to heart attack and stenosis of the artery. the ivus method uses a specially designed thin catheter with the ultimately miniaturized ultrasound probe attached to its distal end (see figure 2). the probe rotates in the arterial lumen in order to receive an ultrasound radio frequency (rf) signal reflected from the plaque and the vascular wall. the ivus images are acquired by means of high - frequency, single - use probes based on various mechanical and electronic phased array systems. the ivus image is generated by using the amplitude information from the received ultrasound rf signals. in the first step, the sampled rf signal is first transformed into an 8-bit luminal intensity signal in all radial directions by taking the absolute value of the signal. as the second step, because the rf signal propagating in the tissue is affected by attenuation due to depth, it is necessary to compensate it by time gain compensation function. as the third step, in order to reduce the noise effect and spurious harmonic components outside the band of interest, the rf signal can be filtered by a band - pass filter. after taking the envelope of this signal, data is normalized. as the last step, in order to expand the effective dynamic range of our digital images in terms of saturation, we can optionally apply the gamma correction using the gamma value, the gradient of the linear region on the gamma value curve. as the final result, we get a tomographic cross - sectional image of a coronary artery as shown in figure 3. a b - mode image displays a real time ultrasound cross - sectional image of a thin section of a blood vessel where currently a catheter probe is rotating. in quantitative assessment of coronary plaque, the following two boundaries are calculated in the ivus b - mode image. one is a luminal boundary (lb) between the lumen and the plaque, and the other is an adventitial boundary (ab) between the plaque and the vascular wall. many edge extraction methods have been proposed, most of which are mainly based on the gradient of image intensity. these gradient - based methods use the smoothing filter such as a gaussian filter for suppression of noise. an edge detection method by using a statistical discriminant measure called image separability has been proposed by fukui. this method has advantages over the gradient based methods, because the image separability has the following features : insensitivity to noisy and blurred edges, ability to differentiate the edges between texture regions. insensitivity to noisy and blurred edges, ability to differentiate the edges between texture regions. for this reason, to distinguish the difference between the plaque boundary and the speckle noise in an ivus image, the proposed method employs the image separability. figure 4 shows a local region which consists of two small regions a and b. the separability h for pixel h can be calculated by linear discriminant analysis with information from regions a and b as follows : (1)h = naiai2+nbibi2k=1siki2, where na and nb represent the numbers of the pixels in the regions of a and b, respectively. i - a and i - b represent the averages of intensities in the regions of a and b. i- stands for the average of the intensities in the combined regions of a and b. s and ik represent the number of the pixels and the intensity of the kth pixel in the combined regions of a and b. the weighted separability for pixel h, which is a modification of the original separability, is defined by (2)hw=himaxiaimaxibimax2, where imax is the maximum intensity in the whole of ivus image and h satisfies 0 h 1. the image separability takes a larger value when two regions are separated from each other. the weighted image separability detects the candidates of the inner and outer boundaries of plaque by considering the following two conditions peculiar to ivus images : intensity in the outside area of a luminal boundary (lb) tends to be stronger than that in the inside area of lb, intensity in the outside area of an adventitial boundary (ab) tends to be stronger than that in the inside area of ab. intensity in the outside area of a luminal boundary (lb) tends to be stronger than that in the inside area of lb, intensity in the outside area of an adventitial boundary (ab) tends to be stronger than that in the inside area of ab. we present in this paper a modified pmd filter to reduce speckle noise and enhance a coronary plaque boundary in ivus image. this chapter is divided into two sections which are (i) modified perona - malik diffusion (pmd) filter and (ii) boundary calculation procedure by fuzzy inference. perona and malik have proposed an anisotropic diffusion filter, which is known as perona - malik diffusion (pmd) filter, to filter noise and preserve the edges of an image. the basic idea of the pmd process is to get an increasingly smoothed image u(x, y, t) from an original image u0(x, y), indexed by diffusion parameter t. this process can be interpreted as an image convolution by a gaussian kernel g(x, y, t) with an increasing width as follows : (3)ix, y, t = i0x, ygx, y, t. ti = cx, y, ti+cx, y, ti, where (5)cx, y, t = gix, y, t is a diffusion coefficient. g() refers to an edge stopping function, which is a decreasing function of the gradient of image, which is defined by (6)gi=11+i / k2, respectively, where k is a parameter which controls the strength of diffusion. g() takes large values at the regions where the intensity gradients are low. on the contrary the initial condition is given by (7)ix, y,0=i0x, y. the discrete version of pmd process is defined as follows : (8)is(n+1)=is(n)+sgis, p(n)is, p(n), where s = (x, y) and p are the coordinates of the pixel of concern and its neighboring pixels, respectively. is is an intensity at s with an iteration count n. s represents the four neighboring pixels in north, west, south, and east diffusion directions. the structure of diffusion direction for the normal pmd filters is shown in figure 5(a). however, when the normal pmd filter is applied to ivus image, the coronary plaque boundary can not be preserved on several areas. by analyzing many experiments, we can conclude that the diffusion direction and its strength are very important factors in the pmd filter to enhance the edge of image and to reduce the noise. if the strength of diffusion is too large, the edge of the image tends to be lost. on the contrary, if the strength of diffusion is too small, the noise of image can not be reduced. when the diffusion direction and its strength are set properly therefore, the direction and strength of diffusion must be set properly for a good filtering performance. we propose here the modified direction and strength of diffusion of the pmd filter by considering the plaque boundary direction in the ivus image. from figure 4 it means that in order to preserve the plaque boundaries, the diffusion strength in horizontal direction should be smaller than that in other directions. in order to consider flexibly the direction of plaque boundary, we propose a new structure for diffusion directions as shown in figure 5(b). the modified pmd filter moves in eight directions with different strength in each direction. by modifying the diffusion process of the normal pmd filter of (8) based on the diffusion direction in figure 5(b), the proposed iteration formula for diffusion process of the modified pmd filter is given as follows : (9)is(n+1)=is(n)+1skgik, s(n)ik, s(n), where k = { nw, n, ne, e, se, s, sw, w}. nw, n through w represent the direction of northwest, north, northeast, east, southeast, south, southwest, and west, respectively. the boundary calculation procedure is briefly summarized as follows.a b - mode image is transform from cartesian coordinate shown in figure 6(a) into polar coordinate shown in figure 6(b).the b - mode image in polar coordinate is filtered by modified pmd filter of (9) whose filtering result is shown in figure 6(c).an image separability of figure 6(c), whose result is shown in figure 6(d), is calculated.seed points are roughly placed automatically on the b - mode image to obtain search areas as shown in figure 6(e) described in.the plaque boundary is inferred by using the takagi - sugeno (t - s) fuzzy model described in.the plaque boundary results inferred by t - s fuzzy model are shown in figure 6(f). a b - mode image is transform from cartesian coordinate shown in figure 6(a) into polar coordinate shown in figure 6(b). the b - mode image in polar coordinate is filtered by modified pmd filter of (9) whose filtering result is shown in figure 6(c). an image separability of figure 6(c), whose result is shown in figure 6(d), is calculated. seed points are roughly placed automatically on the b - mode image to obtain search areas as shown in figure 6(e) described in. the plaque boundary is inferred by using the takagi - sugeno (t - s) fuzzy model described in. the plaque boundary results inferred by t - s fuzzy model are shown in figure 6(f). in the experiments, we use three different ivus images which are shown in figure 7, and the proposed method is compared with the method using the normal pmd filter in. by analyzing for many ivus images, the parameters of the diffusion filter of (9) were set as n = s = 1 and w = e = nw = sw = se = ne = 1.2. figures 8(a), 9(a), and 10(a) show the filtering results by the normal pmd filter. figures 8(b), 9(b), and 10(b) show the filtering results by the modified pmd filter. the plaque boundaries by the modified pmd filter are more clearly enhanced than by the normal pmd filter. it is indicated that the modified pmd filter is better than the normal pmd filter in the plaque boundary enhancement. figures 11(a) and 11(b) show the weighted image separability of image 3 after applying the normal pmd filter and after applying the modified pmd filter, respectively. the red areas in figures 11(a) and 11(b) show the possible areas where the desired (true) boundaries exist. the desired (true) boundaries are detected by the experts, and they are indicated by the black solid lines in both figures. we can see that the red areas of figure 11(b), obtained by using the present modified pmd filter, catch the desired (true) boundaries in the center of red areas better than using the normal pmd filter. the root mean square errors (rmses) between the desired and the detected plaque boundaries are shown in table 1. the rmses of the proposed method are better than those of the method in using the normal pmd filter for the most parts of lb and all parts of ab. the comparisons of plaque boundary detection for image 1 by the method in with the normal pmd filter and by the proposed method are shown in figure 12. the detected boundary by the proposed method (red line) is closer to the desired boundary (white line) than that by the method in (green line). the accuracy of the proposed method is increased we have proposed a modified pmd filter to reduce speckle noise and to enhance the coronary plaque boundary in the ivus image. after applying the modified pmd filter, the results show that the proposed method is better than that of the method with the normal pmd filter. | we propose a modified perona - malik diffusion (pmd) filter to enhance a coronary plaque boundary by considering the conditions peculiar to an intravascular ultrasound (ivus) image. the ivus image is commonly used for a diagnosis of acute coronary syndrome (acs). the ivus image is however very grainy due to heavy speckle noise. when the normal pmd filter is applied for speckle noise reduction in the ivus image, the coronary plaque boundary becomes vague. for this problem, we propose a modified pmd filter which is designed in special reference to the coronary plaque boundary detection. it can then not only reduce the speckle noise but also enhance clearly the coronary plaque boundary. after applying the modified pmd filter to the ivus image, the coronary plaque boundaries are successfully detected further by applying the takagi - sugeno fuzzy model. the accuracy of the proposed method has been confirmed numerically by the experiments. |
dental bridges require additional reliance on the support teeth as they are individually solicitated by the occlusal forces. the removal of one root of a pluriradicular teeth through root amputation or root - coronary one reveals a delicate situation regarding the value of dental - periodontal support for the tooth. this forces us to reconsider the functional conditions of equilibrium for these teeth. by applying dental bridge, the support teeth will form a new functional complex, subject to other conditions of stress and strength than those exerted for unused teeth as support elements for bridges. this requires knowledge of two categories of factors : overload factors for the support teeth and factors that can enhance the resistance forces of these teeth. 23 patients were examined for the study aged between 23 - 47 years with low partial edentulous, interspersed mandibulary with prosthesis or without. patients had subjective complaints regarding their pluriradicular remaining teeth, which in turn were subjected to radiological examination (dental and periodontal wide).we studied the coronary condition of the periodontium, the degree of deployment and mobility of the remaining teeth, the precision or failure of the endodontic treatment. biometrics for the diagnosed models was performed to determine the value of local biological indices. the conservation of any given tooth from the side is a goal because it prevents the transformation of side edentulous into terminal edentulous, with all its difficulties for prosthesis. the preservation method used for the lateral molars in this study is called root - coronary amputation and consists of removing one of the affected roots with its coronal portion. the clinical case is representative for coronary - root amputation technique at the lower molars. patient aged 24 years, presents pulsatile pain during mastication at the level of 37. after performing a retroalveolar dental radiography, we found no endodontic treatment in the mesial root and the existence of a chronic apical periodontitis at this level (fig.1). the distal root is treated correctly from an endodontic point of view. in order to avoid extracting it, which would lead to the transformation of side edentulous into terminal edentulous, i decided to remove the mesial root using the root - coronary amputation and preserve the distal root in the form of a sustaining teeth. we explain to patient what represent the coronary - root amputation technique and he has accepted the remove of mesial root. in a first phase we performed the ablation of the old dental bridge and created a space of occlusion of several millimeters at the level of 37. this space has two purposes : to avoid overloading the remaining root and to prevent fracturing the remaining coronal portion for the root that is to be extracted. it is followed by sectioning the coronary portion which represents achieving a vestibulo - oral incision that must intersect the root network, but without being interested in the intraradicular bone septum, which is the main osteogenetic factor at this level (fig.2). root luxation for the root which is to be removed is made with vestibulo - oral movements and using curets, we removed all the chronic inflammatory tissue and we irrigated the socket with sterile saline solution. the sutured of alveolar wound was realized with two separated thread for ten days. after that, the stitches are removed and the coronal portion of the remaining root is prepared under the form of a bont (fig.3). modified of 37 form as premolar after surgery we applied at patient the follow - up procedures about six weeks for periodontal and endodontic treatment and than we made the final prosthetic restoration (fig. premature removal of contacts and occlusal interferences by selective grinding can be done both after the stage of temporary prosthesis or after final prosthesis. removing one of the roots of a tooth bridge sustainer raises two points of view regarding its prosthesis : a decrease in the dent- periodontal resistance of dental bridge sustainer and vulnerability of dental bridge balance and stability. in the process of mastication, through the process of jaw muscle contraction, occlusal forces can occur, which act on the teeth as individual chewing units and tend to move them in relation to the initial position of implantation in the dental alveoli. these occlusal forces have a relatively constant value during life, in condition of natural teeth, and their value is lower in patients carrying total or partial prothesis. the forces that oppose occlusal forces, which are meant to keep the natural teeth in position, after the cessation of occlusal forces, are called reactional forces. the balanced interaction between occlusal functional forces and resistance ones maintain the functional equilibrium position or kinematic one for the teeth. in case of partially reduced edentulous the prosthetic restoration of functional equilibrium is done using dental bridges, whose architectural and structural balance should harmonize with the functional balance of the support teeth. the existence of a low partial mandible edentulous, in which there is a single distal tooth for support which undergoes a prosthetic intervention, as coronary - root amputation is, without the possibility of supplementing the support teeth in the distal area, certain measures should be considered in order to prevent an overloading of the remaining distal root. thus, by provisional prosthesis, the practitioner obtains an immobilization of the remaining distal root and through the occlusal forces action an active response reaction of these root is also obtained. the architecture and the design of the dental bridge will be done in such a way as to avoid overloading the distal root, but respecting the occlusal morphology and reducing the vestibulo - oral size of this bridge. an important role has the occlusal equilibration step for removing premature contacts and occlusal interferences thus preventing the phenomenon of occlusal trauma. the overall goal of selective tooth grinding and fullmouth equilibration is to develop a state of homeostasis between the tissues that interact within the masticatory system and the local environmental factors. this modification process can help to establish a functional equilibrum between the teeth, supporting tissues, neuromuscular system and temporomandibular joint5. it is generally agreed that when occlusal adjustment is performed to treat abnormal tooth mobility, periodontal therapy has a more favorable outcome, with the clinical attachment level reportedly being improved in some cases in case of low bridges, the use of a single tooth as a distal support, with coronary - root amputation is done taking into account : root morphology, mobility, crown - root ratio and tooth position on the arch. use of the aggregation elements on both divergent or convergent roots, requires time to analyze possible effects on periodontal tooth support involved. project the body bridge which should be contained within the perimeter shape of the teeth horizontal bases of support. the occlusal equilibration phase is mandatory, it can be achieved both in the phase of provisional prosthesis or final prosthesis. | dental occlusion represent the function of dentomaxillary system at which realization participate all the components of that system holding the equilibrium between them. setting up the treatment with dental bridges in partial edentuluous is rule by some principles that have aims like restore the morphology and functions change by edentuluous condition and also prevention disorders of remaining teeth and other structures and functions of dentomaxillary system. during the prosthetic treatment with fixed bridgeworks in clinical cases presents in this study, we used also terapeutical techniques specifically other dental domains like endodontology and oral surgery. for avoid the appearance of occlusal dysfunction is necessary a good knowledge and respecting the biomorphocinematic behaviour of dentomaxillary system structure under action of occlusal force during mastication and also biomechanical rules that influence the functional equilibrium of teeth and dental bridges. |
biobanks, gradually begin to play an increasingly important role in studying the determinants of populations health and in shaping proper prevention programs and health policies. the expert group of the european commission defines biobanks as various types of biological samples themselves, plus the related databases (http://ec.europa.eu). the oganisation for economic co - operation and development (oecd) documents, on the other hand, characterize biobanks as structured resources that can be used for the purpose of genetic research and which include : human biological materials and/or information generated from the analysis of the same ; and extensive associated information they also emphasize that biobanks can be used in research on human genetics and the relationships between genetic, environmental and life - style factors in shaping population health (http://www.oecd.org). biobanks are diverse in terms of the purpose for which they collect the biological material. they exist for clinical purposes (transplants, blood transfusions, genetic diagnosis), research (e.g. research on neurodegenerative diseases, monozygotic siblings, isolated populations, pharmacogenetic studies), police and courts, and others (e.g. educational purpose). some of the projects related to biobanking are carried out on a large - scale, as exemplified by the genomic biobanks of entire populations, also called population biobanks (swede. 2007). public trust is the most important predictor of the publics intention to participate in biobank research (crithchley. 2010 ; kettis - lindblad. the most important hopes connected with the development of biobanks are : a better recognition of genetic and environmental factors of the hereditary diseases, examining people s susceptibility to specific diseases (frazier. 2008 ; wang. 2010 ; brozek. 2011 ; myszka. 2011) ; more effective personalized preventive care (e.g. the modern spectrometry allows detection of more than 30 metabolic disorders using the neonatal screening blood spot) (sharrard and pollitt 2007), adaptation of the treatment to the patient s individual genetic characteristics by development of pharmacogenetics (personalized medicine) (roses 2000 ; matimba. 2011) inventing innovative drug therapies and discovering new biomarkers of many diseases with the use of modern sequencing technologies (pareek. dna obtained from archived tissues, blood films and slides may be useful to establish the molecular diagnosis of a deceased patient and this information may be of value for relatives even after 1020 years (godard. population biobanks may also allow to trace the evolution of genetic changes in subsequent generations, allowing an improved understanding of pathogenic mechanisms leading to the development of many diseases caused by hereditary factors or the interaction of genetic and environmental factors (founti. the development of population biobanks will also isolate the group of patients unsusceptible to certain diseases, who will not require the unnecessary and expensive diagnostic, which in turn will allow for a rational management of financial resources in the health care system (roden. these opportunities contributed to the development of a new field within public health called community genetics or public health genetics / genomics (phg). phg is defined as the responsible and effective translation of genome - based knowledge and technologies for the benefit of population health (definition of bellagio group on public health genomics) (http://www.phgen.nrw.de). phd assesses the impact of genes and their interaction with behavior, diet (nutrigenomics) and the environment on the population s health (e.g. alspac the avon longitudinal study of pregnancy and childhood - a gene - environmental research focused on children s health) (www.alspac.bristol.ac.uk). there are many international initiatives in phg that promote the translation of genome - based science and technology into improvements in population health, e.g. hugenet (human genome epidemiology network) (www.cdc.gov/genomics/), p3 g consortium (public population project in genomics) (www.p3g.org/), graph - int (the genome - based research and population health international network) (www.graphint.org/) and grc (genomic resource centre) developed by who (www.graphint.org/). noteworthy is the project of the biobanking and biomolecular resources research infrastructure (bbmri) (www.who.int/genomics/en/). its main purpose is to connect, within the next few years, the existing genomic biobanks from different countries of the european union in one research network. this may become the basis for an integrated research, leading to a better understanding of factors affecting the health disparities in european countries, and to a more reasonable measure of improving the health of the european population. the objective of this article is to present organizational solutions to population biobanks in selected european countries. it appears that population biobanks are in fact diverse in this respect, and not only researchers and development centres possessing public finances are involved in the process of their creation, but in recent years, under the auspices of associations, foundations and private companies multiply the projects of gathering genetic materials. currently, population biobanks are experiencing a rapid development in european countries, which accelerated sharply after sequencing of the human genome in 2000. gradually, they become a part of the health care system not only by creating new opportunities for research, but also by giving the opportunity to implement effective measures to preserve the health of the european population (swede. we reviewed in pubmed database articles which concern population biobanks focusing on organizational models of these institutions and specific legal regulations regarding various ways of obtaining genetic data from the inhabitants, donors rights, organizational and legal aspects. we analysed more deeply uk biobank, spanish national biobank and estonian biobank because of their interesting and different organizational aspects that can be used in establishement of new population biobanks. model of a proposed polish national genomic biobank for research purposes was discussed regarding polish organizational and legal conditions. contemporary, an increasing number of projects on population biobanks, aimed at collecting biological samples and data on people belonging to a particular population appear. some countries establish national genomic biobanks (dna banks) in the hope of an advanced understanding and improved prevention of genetic diseases (hirtzlin. it is calculated that at the end of 2007 there were about 120 population biobanks worldwide, mainly in europe and north america but also in asia (e.g. china). in europe, most active countries in the creation of population biobanks are the countries of western europe and scandinavia. the countries of central and eastern europe, however, have already implemented such a project in estonia, and hungary and poland are in the process of its preparation (pawlikowski and sak 2010). below : there are three different organizational models of genomic biobanking : the british, spanish and estonian. the uk biobank was created in order to conduct research on new methods of prevention, diagnosis and treatment of the genetic and lifestyle diseases. the wellcome trust foundation, as well as public entities (medical research council, the scottish government, the british heart foundation, and the northwest regional development agency) played an important role in its creation. the scientific committee and the international bioethical committee supervized these activities. what is more, there were six regional centres working together and 22 british universities involved. first preparations for establishing of the uk biobank occurred in the late 1990s of the twentieth century. in 20002001 public consultations concerning the public perception of biobanking were conducted. in 2006, a pilot study on a group of 4000 residents of manchester was carried out and relevant legal regulations established (www.ukbiobank.ac.uk). in the next phase of creating the biobank, after planning of the recruitment process based on drawn conclusions from the pilot study, the uk biobank obtained samples from more than 500 thousand people aged 40 - 69. participants were voluntarily recruited based on the data contained in the databases of the national health service. founders of the biobank distributed to hundreds of thousands of potential donors an invitation to participate in the project. recipients, who agreed to participate in the study, gave blood, urine and saliva samples ; passed the basic diagnostic tests and filled out a questionnaire. acquired genomic samples are kept for a period of 20 - 30 years, and made available as anonymous only to researchers whose projects receive the approval of the ethics committee. gaining profits from the research may be allowed, e.g. by pharmaceutical companies which will conduct studies with the usage of these resources (yuille. the test results are not made available to donors ; however, they have a possibility of withdrawing data from the database. biobank is insured against any damage related to the donation or storage of samples from the participants of the study (http://www.ukbiobank.ac.uk/). an important feature of the uk biobank is its openness to the multicultural and multilingual potential donors living in the united kingdom. spanish national biobank (banco nacional de adn) was established in 2004 by the genome spain foundation (fundacion genome espana), in collaboration with the university of salamanca. the purpose of its creation was to support research on genetic diversity of the population living in spain, the development of emerging diseases in the process of human evolution and development of new treatments of lifestyle diseases, including pharmacogenetic research. the specific objectives of el banco nacional de adn include the study of determinants of susceptibility to certain diseases, early diagnosis, improvement of disease classification, the search for new prognostic factors, and optimization of individual treatment, monitoring of effects of treatment, and transferring the results of basic examinations to clinical reality. biobank centre is located in salamanca, at the local university, and the sanitary inspectorate of the castile and leon province, the founding the spanish genome foundation, an external scientific committee and a bioethical committee, exercises supervision. apart from the biobanks headquarters, since 2006 the biobank consists of four research institutes from other provinces of spain, which specialize in research on certain diseases. collecting samples is based on cooperation with the regional blood donation centres and blood banks. these institutions take samples of blood and are responsible for collecting data and completing the appropriate survey questionnaire containing questions directed to the donor about his lifestyle, diet, family medical history, etc. dna samples are sent to the headquarters in salamanca, where they are encoded, prepared and stored. access to the specimens is available for researchers from spain and those from abroad who collaborate with the spanish scientists an additional network of specialized centres and research networks on specific diseases : cardiovascular diseases (barcelona), metabolic (barcelona), neuropsychiatric (reus) and oncology (salamanca), was created. specialized centres collect, store and conduct tests on samples of biological material from persons suffering from specific types of diseases, e.g. research centre for cardiovascular diseases collects samples from individuals after myocardial infarction, coronary artery disease, hypertension, arrhythmia, etc. the donors have the opportunity to withdraw their consent and samples collected by the biobank, however they do not have access to individual test results, because samples sent to the researchers are anonymized, namely where the personal data and the genetic data of the donor are irreversibly separated (http://www.bancoadn.org/en/home.htm). available for researchers is therefore the sample with genomic data obtained concerning the state of health of the donor but without any possibility of restoring his identity. estonian genome project was the basis for the creation of the population genbank, whose goal is to gather samples from approximately 1 million inhabitants of estonia and latvia. the legal basis for its establishment was a relevant legal act (human genome research act), which came into force in 2001. it regulated the research on human genetics and the protection of the donors (confidentiality, non - discrimination by insurers and employers), and called into existence a national genomic biobank. participation in the project was voluntary and the participants had access to their results (the right to know), but also had a guaranteed right not to know. they could withdraw the previously granted consent and demand the destruction of data allowing for their identification. biobank is supervized by : a bioethical committee (28 members), a supervisory committee (consisting of nine members of parliament, government and the estonian academy of sciences). funding is based on the principles of public - private partnership. by agreement with the government, the project is pursued by a private non - profit foundation (estonian genome project foundation), which became the legal owner of the collected material and data. the right to access the data is given to the family doctors (who take care of the donors) and researchers working in the estonian research institutes ; however, the ethics committee must approve their project. intellectual property rights are shared between researchers and the foundation. in the first phase of the project (until 2003), about 100 family doctors were involved and samples were taken from about 10,000 people. in the second (main) phase, between the years 20032007, it was intended to gather samples from approximately three - quarters of the country s population (the population of estonia, according to data from 2010 was 1,340,000 people (http://www.stat.ee/main-indicators) engaging in it about 800 persons who collected these samples. this plan was not fully executed. in 2009, the biobank gathered samples and data from 40,000 donors, and at the end of 2010 had only 50,000 samples. each research participant gave about 50 ml of blood and filled out a questionnaire which included questions on personal data, environmental conditions, diet, workplace, lifestyle, clinical data (e.g. prior and current diseases), and family medical history to a third generation back. informed consent form was kept in the coding centre, which is also responsible for the anonymization of data or if necessary for the identification of donors (e.g. in the case of a request to withdraw data from the biobank) (http://www.geenivaramu.ee/index.php?id=462). population biobanks were organized in a different manners. for this purpose new, distinct from the existing institutions that collects samples of biological material from volunteers, or used existing samples and data after obtaining a new consent from the donors additional possibility was the creation of systems of cooperation between existing biobanking institutions (e.g. blood - donation centres.). individual countries have developed different organizational models of these institutions and specific methods of legal regulations (deschenes and sallke 2005). the british and estonian model seem quite expensive, what is more, the british model is based on specific, state system of national health service. the spanish model, in turn, is characterized by economy and simplicity of the organizational structure (using the existing organizational structure of regional blood donation centres) and at the same time is tailored to the population inhabiting a large area of varying population density. a valuable component of the collaboration with general practitioners included in the estonian solution, or the clear focus on selected donors in the british biobank (including those in a specific age group) should be noted. a good concept is to work with leading scientific institutions conducting research on specific hereditary diseases (as in the case of the spanish biobank) and medical universities (e.g. uk biobank), which will allow for a rational and effective management of accumulated scientific resources. it is extremely important to create an appropriate infrastructure and data protection system, which will allow for cooperation with other national and foreign scientific centres and within the bbmri network (kauffman and cambon - thomsen 2008 ; dhir. implementation of any new population biobank project should be preceded by a serious social debate, public opinion surveys and the introduction of the necessary legal changes governing the biobank in accordance with the recommendations of international organizations (crithchley. the process of establishing a new genomic biobanks (e.g. the polish genomic biobank), should rely on the selected items from other models and take into account local organizational, legal and social conditions. currently in poland, there is no nationwide organization obtaining genetic material for population studies (pawlikowski. there are only local (university or working at the regional oncology centres) facilities, which obtain genetic material for scientific research in a very limited extent. contemporarily in poland, discussion began on the subject of organizational, legal and ethical basis of the creation of a national genomic biobank. results of the expertise, completed in november 2009 suggest the possibility of creating such a biobank in polish conditions (pawlikowski and sak 2010). organizational structure of the polish genomic biobank should consist of two levels, i.e. composed of a superior national coordinating centre and subordinate regional, biobanking and specialized centres (targeted on research on specific disease entities). regional biobanking centres together with specialized biobanks should obtain samples and data from donors and transfer them to the national coordinating centre where samples and data should be encoded, processed and stored (fig. 1).fig. 1two - level structure of the project of the polish national genomic biobank and the system of supervision two - level structure of the project of the polish national genomic biobank and the system of supervision the establishment, from the first, of a population biobank in polish conditions would be a very expensive and time - consuming task. it would be more beneficial to base it on the already existing polish biobanking institutions, e.g. primarily on the blood donation centres and research centres. they have qualified staff and infrastructure, which after necessary modifications (and possibly a small expansion) could lead to their integration into the structure of a national genomic biobank. in organizational terms they could retain the availability of the realization of the existing tasks, and simultaneously make available facilities and personnel for the needs of population biobanking. established a few years ego, national centre for tissue and cell banking (krajowe centrum bankowania tkanek i komrek - kcbtik) could serve as a national coordinating centre. until now, the tasks undertaken by kcbtik was the coordination of the activities of other national and regional tissue and cell banks (biobanks created mainly for clinical and transplantation purposes), and acting as a place of reference, consultation and supervision in strictly medical terms. sectoral use of kcbtik as a national coordinating centre of the polish population biobank would be beneficial due to the possibility of inclusion into the newly formed biobank, the existing tissue and cells biobanks, which currently report their activities to the kcbtik (fig. 1). the regional biobanking centres could be based on the regional blood donation and haemotherapy centres in poland. the already existing research centres (medical universities, oncology centres, polish academy of sciences, genetic diagnostic centres) may fulfil the function of specialist biobanks directed at the diagnosis of certain types of diseases :. it is worthly to postulate for the establishment of an external scientific committee and ethics committee for the supervision on the correctness of the collection, storage and transfer of the genetic material and data for scientific research. in terms of polish legal solutions currently in force (ustawa z dnia 1 lipca 2005r) or a new law should be written which would autonomously and comprehensively regulate the issue of biobanking. in the case of a successful process of creating a national biobank in poland for the purpose of genomic research there is the possibility of incorporating it into other european biobanks and other entities conducting research within the confines of currently forming biobanking and biomolecular resources research infrastructure (bbmri) (http://www.bbmri.eu/). population biobanks are important for the development of the public health genomics and personalized medicine.some european countries have developed different organizational models of population biobanks and specific legal regulations.population biobanks can be created in collaboration with universities, general practitioners, blood donation centres and scientific research institutes.population biobanks can be founded basing on the public - privacy partnership. population biobanks are important for the development of the public health genomics and personalized medicine. some european countries have developed different organizational models of population biobanks and specific legal regulations. population biobanks can be created in collaboration with universities, general practitioners, blood donation centres and scientific research institutes. the uk biobank was created in order to conduct research on new methods of prevention, diagnosis and treatment of the genetic and lifestyle diseases. the wellcome trust foundation, as well as public entities (medical research council, the scottish government, the british heart foundation, and the northwest regional development agency) played an important role in its creation. what is more, there were six regional centres working together and 22 british universities involved. first preparations for establishing of the uk biobank occurred in the late 1990s of the twentieth century. in 20002001 public consultations concerning the public perception of biobanking were conducted. in 2006, a pilot study on a group of 4000 residents of manchester was carried out and relevant legal regulations established (www.ukbiobank.ac.uk). in the next phase of creating the biobank, after planning of the recruitment process based on drawn conclusions from the pilot study, the uk participants were voluntarily recruited based on the data contained in the databases of the national health service. founders of the biobank distributed to hundreds of thousands of potential donors an invitation to participate in the project. recipients, who agreed to participate in the study, gave blood, urine and saliva samples ; passed the basic diagnostic tests and filled out a questionnaire. acquired genomic samples are kept for a period of 20 - 30 years, and made available as anonymous only to researchers whose projects receive the approval of the ethics committee. gaining profits from the research may be allowed, e.g. by pharmaceutical companies which will conduct studies with the usage of these resources (yuille. the test results are not made available to donors ; however, they have a possibility of withdrawing data from the database. biobank is insured against any damage related to the donation or storage of samples from the participants of the study (http://www.ukbiobank.ac.uk/). an important feature of the uk biobank is its openness to the multicultural and multilingual potential donors living in the united kingdom. spanish national biobank (banco nacional de adn) was established in 2004 by the genome spain foundation (fundacion genome espana), in collaboration with the university of salamanca. the purpose of its creation was to support research on genetic diversity of the population living in spain, the development of emerging diseases in the process of human evolution and development of new treatments of lifestyle diseases, including pharmacogenetic research. the specific objectives of el banco nacional de adn include the study of determinants of susceptibility to certain diseases, early diagnosis, improvement of disease classification, the search for new prognostic factors, and optimization of individual treatment, monitoring of effects of treatment, and transferring the results of basic examinations to clinical reality. biobank centre is located in salamanca, at the local university, and the sanitary inspectorate of the castile and leon province, the founding the spanish genome foundation, an external scientific committee and a bioethical committee, exercises supervision. apart from the biobanks headquarters, since 2006 the biobank consists of four research institutes from other provinces of spain, which specialize in research on certain diseases. collecting samples is based on cooperation with the regional blood donation centres and blood banks. these institutions take samples of blood and are responsible for collecting data and completing the appropriate survey questionnaire containing questions directed to the donor about his lifestyle, diet, family medical history, etc. dna samples are sent to the headquarters in salamanca, where they are encoded, prepared and stored. access to the specimens is available for researchers from spain and those from abroad who collaborate with the spanish scientists. an additional network of specialized centres and research networks on specific diseases : cardiovascular diseases (barcelona), metabolic (barcelona), neuropsychiatric (reus) and oncology (salamanca), was created. specialized centres collect, store and conduct tests on samples of biological material from persons suffering from specific types of diseases, e.g. research centre for cardiovascular diseases collects samples from individuals after myocardial infarction, coronary artery disease, hypertension, arrhythmia, etc. the donors have the opportunity to withdraw their consent and samples collected by the biobank, however they do not have access to individual test results, because samples sent to the researchers are anonymized, namely where the personal data and the genetic data of the donor are irreversibly separated (http://www.bancoadn.org/en/home.htm). available for researchers is therefore the sample with genomic data obtained concerning the state of health of the donor but without any possibility of restoring his identity. estonian genome project was the basis for the creation of the population genbank, whose goal is to gather samples from approximately 1 million inhabitants of estonia and latvia. the legal basis for its establishment was a relevant legal act (human genome research act), which came into force in 2001. it regulated the research on human genetics and the protection of the donors (confidentiality, non - discrimination by insurers and employers), and called into existence a national genomic biobank. participation in the project was voluntary and the participants had access to their results (the right to know), but also had a guaranteed right not to know. they could withdraw the previously granted consent and demand the destruction of data allowing for their identification. biobank is supervized by : a bioethical committee (28 members), a supervisory committee (consisting of nine members of parliament, government and the estonian academy of sciences). funding is based on the principles of public - private partnership. by agreement with the government, the project is pursued by a private non - profit foundation (estonian genome project foundation), which became the legal owner of the collected material and data. the right to access the data is given to the family doctors (who take care of the donors) and researchers working in the estonian research institutes ; however, the ethics committee must approve their project. intellectual property rights are shared between researchers and the foundation. in the first phase of the project (until 2003), about 100 family doctors were involved and samples were taken from about 10,000 people. in the second (main) phase, between the years 20032007, it was intended to gather samples from approximately three - quarters of the country s population (the population of estonia, according to data from 2010 was 1,340,000 people (http://www.stat.ee/main-indicators) engaging in it about 800 persons who collected these samples. the biobank gathered samples and data from 40,000 donors, and at the end of 2010 had only 50,000 samples. each research participant gave about 50 ml of blood and filled out a questionnaire which included questions on personal data, environmental conditions, diet, workplace, lifestyle, clinical data (e.g. prior and current diseases), and family medical history to a third generation back. informed consent form was kept in the coding centre, which is also responsible for the anonymization of data or if necessary for the identification of donors (e.g. in the case of a request to withdraw data from the biobank) (http://www.geenivaramu.ee/index.php?id=462). population biobanks were organized in a different manners. for this purpose new, distinct from the existing institutions that collects samples of biological material from volunteers, or used existing samples and data after obtaining a new consent from the donors additional possibility was the creation of systems of cooperation between existing biobanking institutions (e.g. blood - donation centres.). individual countries have developed different organizational models of these institutions and specific methods of legal regulations (deschenes and sallke 2005). the british and estonian model seem quite expensive, what is more, the british model is based on specific, state system of national health service. the spanish model, in turn, is characterized by economy and simplicity of the organizational structure (using the existing organizational structure of regional blood donation centres) and at the same time is tailored to the population inhabiting a large area of varying population density. a valuable component of the collaboration with general practitioners included in the estonian solution, or the clear focus on selected donors in the british biobank (including those in a specific age group) should be noted. a good concept is to work with leading scientific institutions conducting research on specific hereditary diseases (as in the case of the spanish biobank) and medical universities (e.g. uk biobank), which will allow for a rational and effective management of accumulated scientific resources. it is extremely important to create an appropriate infrastructure and data protection system, which will allow for cooperation with other national and foreign scientific centres and within the bbmri network (kauffman and cambon - thomsen 2008 ; dhir. implementation of any new population biobank project should be preceded by a serious social debate, public opinion surveys and the introduction of the necessary legal changes governing the biobank in accordance with the recommendations of international organizations (crithchley. the process of establishing a new genomic biobanks (e.g. the polish genomic biobank), should rely on the selected items from other models and take into account local organizational, legal and social conditions. currently in poland, there is no nationwide organization obtaining genetic material for population studies (pawlikowski. there are only local (university or working at the regional oncology centres) facilities, which obtain genetic material for scientific research in a very limited extent. contemporarily in poland, discussion began on the subject of organizational, legal and ethical basis of the creation of a national genomic biobank. results of the expertise, completed in november 2009 suggest the possibility of creating such a biobank in polish conditions (pawlikowski and sak 2010). organizational structure of the polish genomic biobank should consist of two levels, i.e. composed of a superior national coordinating centre and subordinate regional, biobanking and specialized centres (targeted on research on specific disease entities). regional biobanking centres together with specialized biobanks should obtain samples and data from donors and transfer them to the national coordinating centre where samples and data should be encoded, processed and stored (fig. 1).fig. 1two - level structure of the project of the polish national genomic biobank and the system of supervision two - level structure of the project of the polish national genomic biobank and the system of supervision the establishment, from the first, of a population biobank in polish conditions would be a very expensive and time - consuming task. it would be more beneficial to base it on the already existing polish biobanking institutions, e.g. primarily on the blood donation centres and research centres. they have qualified staff and infrastructure, which after necessary modifications (and possibly a small expansion) could lead to their integration into the structure of a national genomic biobank. in organizational terms, it would be best to include the already existing biobanking institutions as they could retain the availability of the realization of the existing tasks, and simultaneously make available facilities and personnel for the needs of population biobanking. established a few years ego, national centre for tissue and cell banking (krajowe centrum bankowania tkanek i komrek - kcbtik) could serve as a national coordinating centre. until now, the tasks undertaken by kcbtik was the coordination of the activities of other national and regional tissue and cell banks (biobanks created mainly for clinical and transplantation purposes), and acting as a place of reference, consultation and supervision in strictly medical terms. sectoral use of kcbtik as a national coordinating centre of the polish population biobank would be beneficial due to the possibility of inclusion into the newly formed biobank, the existing tissue and cells biobanks, which currently report their activities to the kcbtik (fig. the regional biobanking centres could be based on the regional blood donation and haemotherapy centres in poland. the already existing research centres (medical universities, oncology centres, polish academy of sciences, genetic diagnostic centres) may fulfil the function of specialist biobanks directed at the diagnosis of certain types of diseases :. it is worthly to postulate for the establishment of an external scientific committee and ethics committee for the supervision on the correctness of the collection, storage and transfer of the genetic material and data for scientific research. in terms of polish legal solutions currently in force, there should be an amendment of the transplant act (ustawa z dnia 1 lipca 2005r) or a new law should be written which would autonomously and comprehensively regulate the issue of biobanking. in the case of a successful process of creating a national biobank in poland for the purpose of genomic research there is the possibility of incorporating it into other european biobanks and other entities conducting research within the confines of currently forming biobanking and biomolecular resources research infrastructure (bbmri) (http://www.bbmri.eu/). population biobanks are important for the development of the public health genomics and personalized medicine.some european countries have developed different organizational models of population biobanks and specific legal regulations.population biobanks can be created in collaboration with universities, general practitioners, blood donation centres and scientific research institutes.population biobanks can be founded basing on the public - privacy partnership. population biobanks are important for the development of the public health genomics and personalized medicine. some european countries have developed different organizational models of population biobanks and specific legal regulations. population biobanks can be created in collaboration with universities, general practitioners, blood donation centres and scientific research institutes. | population biobanks offer new opportunities for public health, are rudimentary for the development of its new branch called public health genomics, and are important for translational research. this article presents organizational models of population biobanks in selected european countries. review of bibliography and websites of european population biobanks (uk, spain, estonia). some countries establish national genomic biobanks (dna banks) in order to conduct research on new methods of prevention, diagnosis and treatment of the genetic and lifestyle diseases and on pharmacogenetic research. individual countries have developed different organizational models of these institutions and specific legal regulations regarding various ways of obtaining genetic data from the inhabitants, donors rights, organizational and legal aspects. population biobanks in european countries were funded in different manners. in light of these solutions, the authors discuss prospects of establishing a polish national genomic biobank for research purpose. they propose the creation of such an institution based on the existing network of blood - donation centres and clinical biobanks in poland. |
there are controversies among surgeons regarding deep vein thrombosis (dvt) prophylaxis in laparoscopic cholecystectomy. many surgeons have the same opinion for prophylaxis because of an increased intraperitoneal pressure caused by the laparoscopic surgery leading to an induced pneumoperitoneum. in addition, this condition reduces lower limb venous blood flow significantly (1), which is an extra risk factor for deep vein thrombosis.. however, some surgeons disagree based on clinical signs and symptoms which found it as a low risk procedure. the purpose of this study was to determine the incidence of clinically detectable dvt in patients undergoing laparoscopic cholecystectomy without a standard dvt prophylaxis regimen before surgery. in a prospective cross - sectional study, 100 candidates for laparoscopic cholecystectomy were studied. duplex scanning was performed by a group of experienced dedicated sonographers on the first day after operation. clinical follow up was done in the following two weeks after surgery for signs and symptoms of dvt. the quantitative data were expressed as mean standard deviation (sd), and frequency used for analysis of the qualitative data. ten male and 90 female patients with mean age of 48.68 (4.14) years were studied. all of the duplex scans were negative for dvt except in a 73-year old female who had partial thrombosis in the right femoral and popliteal veins. she did not have any dvt sign or symptom and received no treatment ; hense she was discharged in the second day of operation in a well condition. in the further 10 and 30 days clinical follow - up, no sign or symptom of deep vein thrombosis or pulmonary emboli was found. one to 2 weeks after operation there was not any sign or symptom of dvt in other patients. three deaths due to pulmonary embolismhave been reported in a retrospective survey of 77604 patients undergoing laparoscopic cholecystectomy (2). in another study, among 100 consecutive patients who intended to undergo cholecystectomy, two patients showed postoperative dvt including 1 patient after 59 laparoscopic and 41 mini - laparotomy cholecystectomy each, respectively. therefore, despite the theoretical risk of thromboembolic disease due to laparoscopic pneumoperitoneum, the frequency of dvt after either laparoscopic cholecystectomy or mini - laparotomy cholecystectomy was low. the low risk of thromboembolism occurs if adequate thromboprophylaxis regimen which is subcutaneous heparin 5000 units tds and intermittent pneumatic compression were administrated in the patients especially in the high risk ones (3). patel. reported 20 patients underwent elective or urgent laparoscopic cholecystectomy, in which all patients were given graduated compression stockings to wear and 16 received electrical stimulation of the calf during the operation. only 16 patients received pharmacological thromboprophylaxis before the operation, but all patients received the drug after the operation. eleven out of 19 patients completing all the required scans (venous duplex scan before the operation and on day 1, 7 and 30 after the operation) developed venous thrombosis (incidence, 55%) ; none of the dvts were suspected clinically (4). in a series of 438 laparoscopic cholecystectomies, thromboembolism has occurred in three : one dvt, one non - fatal pulmonary embolism, and one fatal pulmonary embolism. all three thrombotic cases were confirmed by phlebography or ventilation - perfusion scanning or after death (5). it is also reported that 114 patients were randomized into two groups, in which 58 and 56 patients underwent laparoscopic and open cholecystectomy, respectively. postoperative dvt developed in four (6.9%) and nine (16.07%) patients in the first and second groups, respectively (6). all the patients had received thromboembolism prophylaxis and dvt was detected by bilateral phlebography 7 - 11 days after surgery (7). it is import antfor a clinical practioner to consider the signs and symptoms further to paraclinic results. hense in addition to para clinic assessment of dvt by duplex scanning on the first day after operation, we set up a clinical follow up in the following two weeks after surgery for signs and symptoms of dvt. eighteen patients received some form of perioperative dvt prophylaxis, while 569 patients did not. in the course of 4 weeks clinical follow - up, none of the 587 patients had symptoms of dvt or pulmonary embolism (8). in one report, no postoperative thrombi was diagnosed with duplex scanning during unspecified laparoscopic procedures in 61 patients and none of them received prophylaxis (9). it is important to consider some risk factors like body mass index and duration of surgery in further researches. in this study, laparoscopic cholecystectomy may be considered as a low - risk procedure for thrombosis, and therefore routine use of prophylaxis is probably not justified for all patients. however, more clinical trials or further studies are crucial and recommended for an exact approval of this idea. | backgroundsthere are controversies among surgeons about prophylaxis of deep vein thrombosis (dvt) in laparoscopic cholecystectomy. the aim of this study was the assessment of patients condition after laparoscopic cholecystectomy without any prophylactic measure.methods100 cases of laparoscopic cholecystectomy without dvt prophylaxis were followed by duplex scanning in the first postoperative day and by physical examination and patient history at the first to second postoperative week however no clinical sign was found for dvt.resultsonly one case of partially thrombosis (1%) was found by duplex scanning which was managed conservatively.conclusionlaparoscopic cholecystectomy may consider as a low - risk procedure and routine prophylaxis may not be justified in the absence of other risk factor. |
the current study is part of an ongoing study of the etiology of ciap initiated in 2008 at the university medical center utrecht and was approved by the medical ethics committee. patients were referred to the university medical center utrecht neuromuscular outpatient clinic by their general practitioner or by a neurologist in a general hospital. all patients (n = 249) in whom ciap was diagnosed after evaluation in the university medical center utrecht between october 1, 2008 and december 1, 2011, were included in this study. patients were evaluated for their polyneuropathy in a standardized fashion by an experienced neuromuscular specialist. the clinical evaluation consisted of thorough history - taking (including questioning about disease course, neurologic symptoms, signs of autonomic dysfunction, comorbidity, intoxications, and medication use), anthropometric measurements, and neurologic examination. a detailed kinship history was taken and appropriate genetic testing was performed when necessary. if the possibility of a hereditary polyneuropathy could not be ruled out, then patients were excluded. the severity of the polyneuropathy was graded by calculation and summation of sensory and motor sum scores (6,12). the sensory sum score ranges from 0 to 28, and considers the anatomical distribution of sensory deficits bilaterally in the lower limbs regarding pinprick and light touch sensation (scored as : normal = 4 ; up to ankle abnormal = 3 ; up to distal half lower leg abnormal = 2 ; up to knee abnormal = 1 ; above knee abnormal = 0), vibration sense (evaluated with a graduated rydel - seiffer 64-hz tuning fork and scored as : normal = 4 ; decreased / absent at big toe = 3 ; decreased / absent at ankle = 2 ; decreased / absent at knee = 1 ; decreased / absent at crista iliaca = 0), and propriocepsis of distal interphalangeal joint of the big toe (scored as : normal / immediate perception of movement = 2 ; reduced / delayed perception of movement = 1 ; absent perception of movement = 0) (1315). for the motor sum score, manual muscle strength testing was scored according to the medical research council scale in four distal lower limb muscles (tibialis anterior, gastrocnemius, peroneal, toe extensors), resulting in a score from 0 to 40 for both lower limbs (12,15). those patients with ciap who had painful sensory symptoms and signs but no significant weakness (i.e., no weakness or only mild weakness of the toe extensors) were classified as having a painful predominantly sensory ciap, because this phenotype is most similar to diabetic polyneuropathy. waist circumference was measured in standing position in a horizontal plane midway between the inferior margin of the rib and the superior border of the iliac crest. blood pressure was measured sitting in the upright position using an omron intellisense 907 professional automatic blood pressure monitor. laboratory studies included complete blood count, erythrocyte sedimentation rate, glucose, hba1c, insulin, renal function, liver enzymes, creatine kinase, c - reactive protein, vitamin b1, vitamin b6, folic acid, vitamin b12, homocystein, cholesterol, triglycerides, hdl cholesterol, ldl cholesterol, thyroid - stimulating hormone, serum and urine m - protein, and serological screening for celiac disease (total iga level and iga antibodies against tissue transglutaminase and endomysium). an oral glucose tolerance test (ogtt) was performed in a selected group of patients with a fasting glucose 5.6 mmol / l. if indicated, then additional studies (e.g., a lumbar puncture, chest radiograph, magnetic resonance imaging of the spine, or additional laboratory studies such as immunoserology, genetic testing) were performed. the diagnosis of ciap was defined according to the following criteria (4,6) : presence of symmetrical distal sensory or sensorimotor symptoms such as numbness, pins and needles, tightness, coldness, unsteadiness, muscle cramps, and weakness with onset in the lower limbs, compatible with polyneuropathy ; presence of symmetrical distal sensory or sensorimotor signs with evidence of large nerve fiber involvement such as decreased sense of touch, vibration, and propriocepsis, usually in the presence of decreased pin prick / temperature sense, decreased / absent tendon reflexes, or slight muscle weakness on neurologic examination, compatible with polyneuropathy ; an insidious onset and slow or no progression of the polyneuropathy over the course of at least 6 months ; no identifiable cause for the polyneuropathy after thorough history - taking, clinical examination, and extensive laboratory testing ; no suggestion of a hereditary polyneuropathy based on detailed kinship history (i.e., one or more affected family member), neurologic examination, or confirmation by genetic analysis ; and nerve conduction studies excluding a demyelinating polyneuropathy and confirming large nerve fiber involvement if the findings on neurologic examination were equivocal considering the patient s age (1619). electrophysiological criteria for demyelination provided that distal compound motor action potential (cmap) baseline to negative peak amplitude > 1 mv were : reduction of motor nerve conduction velocity to 80% of lln, or to 125% of upper limit of normal value (uln) if cmap > 80% of lln, or to > 150% of uln if cmap 120% of uln if cmap > 80% of lln, or to > 150% of uln if cmap 50% area reduction or, in upper limb nerves, > 30% amplitude reduction in proximal cmap relative to distal ; and abnormal temporal dispersion in upper limb nerves > 30% duration increase or in lower limb nerves > 100% duration increase in proximal cmap relative to distal (20). we used participants in previously conducted population - based studies at the julius center for health sciences and primary care, university medical center utrecht, as controls for the current study. female controls were sampled from the prospect cohort, a dutch cohort participating in the european prospective investigation into cancer and nutrition (21). in short, a total of 17,395 healthy women presenting for breast cancer screening, aged 4970 years and living in utrecht and the surrounding area, were prospectively enrolled in prospect between 1993 and 1997. in 19992000, a subgroup of women (n = 533 from a total of 1,803) who had experienced natural menopause, had an intact uterus and at least one intact ovary, and had not used sex steroids after the reported date of last menstruation agreed to participate in a new study. women were considered sufficiently healthy if they were able to visit the study center without assistance (n = 402) ; in these women, fasting laboratory studies were performed, blood pressure and waist circumference were measured, and medication use was recorded. after exclusion of all female controls with (newly) diagnosed type 1 or type 2 diabetes or incomplete data (n = 27), 375 female controls were included in our study. as male controls, subjects from the hamlet study briefly, in the hamlet study, 400 independently living men aged 4080 years were prospectively enrolled between 2001 and 2002 to investigate the relationship between endogenous testosterone, sex hormone binding globulin, dehydroepiandrosterone sulfate, estradiol, and the metabolic syndrome. the subjects were recruited by asking female participants of other studies by letter whether they knew a possible interested male volunteer aged 4080 years ; 240 men volunteered to participate. in addition, a randomly selected male population aged 4080 years was drawn from the municipal register of utrecht and 1,230 invitation letters were sent. from this group, 390 men volunteered to participate. from the 630 volunteers, those who did not live independently and subjects who were not physically or mentally able to visit the study center independently (n = 16) all male controls had undergone fasting laboratory studies, had blood pressure and waist circumference measured, and medication use was recorded. after exclusion of all male controls with (newly) diagnosed type 1 or type 2 diabetes, or incomplete data (n = 66), 334 male controls were included in our study. the metabolic syndrome was diagnosed if three or more of the following adult treatment panel iii criteria were present : fasting glucose 5.6 mmol / l (100 mg / dl) ; systolic blood pressure 130 mmhg and/or diastolic blood pressure 85 mmhg ; waist circumference > 102 cm (> 40 in) in men or > 88 cm (> 35 in) in women ; hdl cholesterol 1 mv were : reduction of motor nerve conduction velocity to 80% of lln, or to 125% of upper limit of normal value (uln) if cmap > 80% of lln, or to > 150% of uln if cmap 120% of uln if cmap > 80% of lln, or to > 150% of uln if cmap 50% area reduction or, in upper limb nerves, > 30% amplitude reduction in proximal cmap relative to distal ; and abnormal temporal dispersion in upper limb nerves > 30% duration increase or in lower limb nerves > 100% duration increase in proximal cmap relative to distal (20). we used participants in previously conducted population - based studies at the julius center for health sciences and primary care, university medical center utrecht, as controls for the current study. female controls were sampled from the prospect cohort, a dutch cohort participating in the european prospective investigation into cancer and nutrition (21). in short, a total of 17,395 healthy women presenting for breast cancer screening, aged 4970 years and living in utrecht and the surrounding area, were prospectively enrolled in prospect between 1993 and 1997. in 19992000, a subgroup of women (n = 533 from a total of 1,803) who had experienced natural menopause, had an intact uterus and at least one intact ovary, and had not used sex steroids after the reported date of last menstruation agreed to participate in a new study. women were considered sufficiently healthy if they were able to visit the study center without assistance (n = 402) ; in these women, fasting laboratory studies were performed, blood pressure and waist circumference were measured, and medication use was recorded. after exclusion of all female controls with (newly) diagnosed type 1 or type 2 diabetes or incomplete data (n = 27), 375 female controls were included in our study. as male controls, subjects from the hamlet study were used (22). briefly, in the hamlet study, 400 independently living men aged 4080 years were prospectively enrolled between 2001 and 2002 to investigate the relationship between endogenous testosterone, sex hormone binding globulin, dehydroepiandrosterone sulfate, estradiol, and the metabolic syndrome. the subjects were recruited by asking female participants of other studies by letter whether they knew a possible interested male volunteer aged 4080 years ; 240 men volunteered to participate. in addition, a randomly selected male population aged 4080 years was drawn from the municipal register of utrecht and 1,230 invitation letters were sent. from this group, 390 men volunteered to participate. from the 630 volunteers, those who did not live independently and subjects who were not physically or mentally able to visit the study center independently (n = 16) all male controls had undergone fasting laboratory studies, had blood pressure and waist circumference measured, and medication use was recorded. after exclusion of all male controls with (newly) diagnosed type 1 or type 2 diabetes, or incomplete data (n = 66), 334 male controls were included in our study. the metabolic syndrome was diagnosed if three or more of the following adult treatment panel iii criteria were present : fasting glucose 5.6 mmol / l (100 mg / dl) ; systolic blood pressure 130 mmhg and/or diastolic blood pressure 85 mmhg ; waist circumference > 102 cm (> 40 in) in men or > 88 cm (> 35 in) in women ; hdl cholesterol < 1.03 mmol / l (< 40 mg / dl) in men or < 1.29 mmol / l (< 59 mg / dl) in women ; and triglycerides 1.7 mmol / l (150 mg / dl) (11). additionally, patients or controls using antihypertensive medication and/or lipid - lowering agents (fibrates, nicotinic acid, or statins) were considered to fulfill the criteria for hypertension, reduced hdl cholesterol, and hypertriglyceridemia, respectively. in the netherlands, the prevalence of the metabolic syndrome in the nondiabetic general population aged 40 years and older is at least 25% (23,24). if the true prevalence of the metabolic syndrome in patients with ciap is twice as high (i.e., 50%), then a power analysis (power 1 = 0.85 ; associated type i error probability = 0.05) to determine the sample size for a case - control study shows that 66 cases and 66 controls are needed to be able to reject the null hypothesis that the prevalence rates for cases and controls are equal (ps program version 2.1.31). the observed prevalence rates in patients and controls were used to calculate odds ratios (ors) with 95% confidence intervals (cis), and analysis (univariate) and binary logistic regression analysis (multivariate) were used to investigate the association between ciap and the metabolic syndrome or its individual components. the correlation between the severity of the polyneuropathy, based on the sum of the sensory and motor sum score, and the severity of the metabolic syndrome, based on the number of metabolic syndrome criteria that were met, was evaluated with the spearman correlation coefficient. all analyses were performed with adjustment for age, gender, and metabolic syndrome criteria if appropriate. statistical significance was set at p < 0.05. characteristics of the 249 included patients (32% women) and 709 controls (53% women) are outlined in table 1. all patients had sensory symptoms and/or signs, 48% of patients reported painful sensory symptoms, and 38% of patients were classified as having painful predominantly sensory ciap. characteristics of patients with ciap and controls table 2 details the prevalence of the metabolic syndrome and its individual components in patients and controls. of the patients, 55% fulfilled the metabolic syndrome criteria compared with 34% of the controls (or 2.2 [95% ci 1.73.0 ]). except for impaired fasting glucose, all metabolic syndrome criteria were significantly more prevalent in patients compared with controls when adjusted for age and gender. multivariate analysis, with adjustment for age, gender, and metabolic syndrome criteria, shows hypertension (2.9 [1.74.9 ]) and abdominal obesity (3.3 [2.44.6 ]) to be significantly more prevalent in patients than in controls. there was no correlation between the severity of the polyneuropathy and the severity of the metabolic syndrome (spearman correlation coefficient 0.068 ; p = 0.35). prevalence of the metabolic syndrome and its components in patients with ciap and controls of the patients classified as having a painful predominantly sensory ciap, 62% fulfilled the metabolic syndrome criteria (or 3.1 [95% ci 2.04.8 ]). in this subgroup, hypertension and abdominal obesity also were significantly more prevalent compared with controls (table 3). glucose levels in the 95 patients with painful predominantly sensory ciap (median, 5.4 ; range, 4.06.8) were comparable with glucose levels in the remaining 154 patients (median, 5.4 ; range, 3.86.9), and impaired fasting glucose was not more prevalent in either patient group compared with controls. prevalence of the metabolic syndrome and its components in patients with painful predominantly sensory ciap and controls the current study shows that the metabolic syndrome is significantly more prevalent in patients with ciap compared with controls from the general population, and that abdominal obesity and hypertension seem to be the most consistent contributing components. the prospective recruitment of patients with ciap and a control group derived from the general population in the netherlands distinguishes our study from previous studies. although controls were obtained from historical dutch cohorts, we had access to individual data ; this enabled appropriate direct comparisons between patients and controls. to retain the use of data from a large control group, all analyses were performed with adjustment for gender and age. the prevalence of the metabolic syndrome in the netherlands has not changed much in the past decade. a population - based study in 2006 found a prevalence of the metabolic syndrome of 29% in persons 4070 years old (23). in the most recent population - based study performed in 20092010, the prevalence of the metabolic syndrome was 35% in persons 4070 years old (25). these prevalence rates are comparable with the prevalence of 34% for the metabolic syndrome in the controls (data collection in 19992002) used in our study. it is therefore unlikely that the observed association between the metabolic syndrome and ciap can be largely explained by a change in prevalence of the metabolic syndrome (i.e., increased in patients with ciap) over time. controls in general (although not necessarily) may constitute a healthier representation of the population. however, except for being nondiabetic and sufficiently physically and mentally healthy to visit the study center, no other possibly relevant additional health - related eligibility criteria were used for our control groups. controls were not evaluated for polyneuropathy, but it is not to be expected that more than a few controls did have polyneuropathy after exclusion of patients with diabetes and given the low estimated prevalence of polyneuropathy (1,2). altogether, our findings were probably not influenced to an important degree by certain selective control characteristics. a previous case - control study observed a comparable association between hypertension and ciap (26). hypertension remained a risk factor for chronic symmetric polyneuropathy after adjustment for a few common causes of polyneuropathy in a population sample of italian subjects aged 55 years or older and screened for the presence of (sub)clinical signs of polyneuropathy (27). a cross - sectional study found a negative association between current blood pressure or history of hypertension and presence of (sub)clinical signs suggestive of polyneuropathy in subjects older than 65 years without a history of common diseases known to cause polyneuropathy, but this association was not observed when use of antihypertensive medication was taken into account (28). no studies have reported on the association between abdominal obesity as measured by waist circumference and ciap. the use of waist circumference instead of bmi is relevant because abdominal obesity appears a more robust risk factor for cardiovascular disease and type 2 diabetes than bmi (29). studies that used bmi as a measure of obesity vary considerably in mean bmi values and prevalence rates of obesity in the investigated study populations. a controlled study did not find a higher prevalence of an abnormal bmi in patients with ciap (26). another cohort study showed no association between bmi and the presence of (sub)clinical polyneuropathy in subjects without diseases commonly known to cause peripheral neuropathy when age and hypertension - related variables were taken into account (28). one small controlled study, however, found that patients with painful ciap had a higher mean bmi than healthy controls, but this was not observed for patients without pain (30). one case - control study reported hypercholesterolemia to be more prevalent among patients with ciap (26). another controlled study found no difference in fasting cholesterol levels in patients with ciap, but increasing fasting triglycerides level was significantly associated with the likelihood of ciap after adjusting for bmi, age, and gender (30). a recent, small, controlled study did not demonstrate a significantly higher frequency of dyslipidemia or fasting cholesterol level, but fasting triglycerides level was borderline nonsignificantly lower in patients with idiopathic neuropathy (31). the divergent observations could be explained by markedly higher fasting triglycerides levels in patients and lower levels in controls in the former study when compared with patients and controls in the latter study and our study (30,31). in the netherlands, treatment of dyslipidemia with fibrates or nicotinic acid is uncommon and only prescribed when statins are not tolerated or are insufficiently effective. because statins have an effect by lowering triglycerides and increasing hdl cholesterol, we chose to include statin treatment as a defining criterion in addition to the adult treatment panel iii metabolic syndrome criteria for dyslipidemia. the frequency of dyslipidemia thus could have been overestimated ; however, because this would equally concern patients and controls, we believe it did not skew our results. furthermore, when only considering hypertriglyceridemia and reduced hdl cholesterol without taking the use of statins into account, data analyses showed similar results (data not shown). previous north american studies have suggested an association between chronic polyneuropathy or, more specifically, idiopathic painful sensory neuropathy / small fiber neuropathy and prediabetes (3235). our study did not find ifg to be more prevalent in patients with ciap compared with controls. even subgroup analysis of patients with painful predominantly sensory ciap, a ciap phenotype most similar to diabetic polyneuropathy, demonstrated no association with ifg. meanwhile, the established associations of ciap with hypertension and abdominal obesity appear even stronger in this subgroup. the absence of an association with ifg is in accordance with a small controlled study, which did, however, find an elevated 2-h glucose level as determined by an ogtt suggestive of impaired glucose tolerance in patients with painful ciap, and a recently published prospective population - based study that showed a similar prevalence of polyneuropathy in subjects with normal and impaired glycemia (8,30). subjects in north american studies tend to have a higher bmi compared with those in european studies ; given the intricate association between overweight / obesity and prediabetes, this may distort the association between prediabetes and ciap. a routinely performed ogtt could have resulted in a higher percentage of patients and controls considered to have prediabetes in our study, but this would not have influenced our findings because impaired glucose tolerance is not a metabolic syndrome criterion. a systematic review showed that the reproducibility of the ogtt in prediabetes is, on average, only 49% compared with 73% in diabetes and 93% in normal glucose tolerance (36). thus, an ogtt as advocated by some investigators, and especially if only performed once, has limited usefulness and reliability to establish prediabetes and its association with idiopathic polyneuropathy. although our study was not designed to elucidate the precise pathofysiological mechanisms, the results support a microvascular hypothesis (10). for abdominal obesity (which probably plays a pivotal role among all metabolic syndrome factors), the mechanism is mainly related to a proinflammatory state at the level of the microvasculature, including endoneurial microvessels and endothelial cells, that could lead to microvascular changes, chronic ischemia, and consequently axonal injury. for hypertension, the mechanisms could be characterized by structural and functional changes in the microvasculature (3739). it shows there is a convincing association between ciap and the metabolic syndrome, which is determined mostly by metabolic syndrome components hypertension and abdominal obesity. considering these results, we advocate a proactive attitude to look for simultaneous occurrence of these conditions in patients and initiate appropriate treatment that could, perhaps, not only prevent but also alleviate progression of the polyneuropathy. | objectivethis study aims to investigate the association between chronic idiopathic axonal polyneuropathy (ciap) and the metabolic syndrome or its individual components.research design and methodsa total of 249 patients with ciap and 709 controls underwent fasting laboratory studies, and blood pressure and waist circumference were measured. the metabolic syndrome was diagnosed if three or more of the following adult treatment panel iii criteria were present : impaired fasting glucose, hypertension, abdominal obesity, reduced hdl cholesterol, and hypertriglyceridemia. subgroup analysis was performed for patients with a painful predominantly sensory ciap, because this phenotype is most similar to diabetic polyneuropathy. statistical analysis was performed with adjustment for age and gender.resultsfifty-five percent of all patients fulfilled the metabolic syndrome criteria compared with 34% of controls (odds ratio 2.2 [95% ci 1.73.0 ]). multivariate analysis shows hypertension (2.9 [1.74.9 ]) and abdominal obesity (3.3 [2.44.6 ]) to be significantly more prevalent in patients than in controls. of the patients classified as having a painful predominantly sensory ciap, 62% fulfilled the metabolic syndrome criteria (3.1 [2.04.8 ]). in this subgroup, hypertension and abdominal obesity also were significantly more prevalent compared with controls.conclusionsabdominal obesity and hypertension seem to be the most consistent contributing components of the metabolic syndrome in patients with ciap. evaluation and appropriate treatment of these risk factors in patients with ciap would be advocated. |
mitochondria are small membrane - enclosed organelles (from 0.5 to 1.0 m in diameter) found in most eukaryotic cells except mature red blood corpuscles. mitochondria are the powerhouses of eukaryotic cells and are involved in many cellular processes, including apoptosis ; ion homeostasis ; and the metabolism of glucose, lipids, and amino acids. atp, the energy currency of cell, is the final product of the respiratory chain / oxidative phosphorylation system, which consists of five protein complexes (complexes i v) localized to the inner mitochondrial membrane. mitochondrial defects including mitochondrial dna (mtdna) mutations, altered expression and activity of respiratory chain subunits and glycolytic enzymes, and decreased oxidation of nadh - linked substrates have been suspected to play an important role in the development and progression of diseases, such as certain neurodegenerative diseases, diabetes, leigh 's disease, and cancer [47 ]. mitochondrial genetic disorders are caused by defects in nuclear or mtdna that affect the expression of the mtdna - encoded mitochondrial respiratory complexes (mrcs) and the biosynthesis of the mtdna - encoded polypeptides. mutations in genes required for mtdna maintenance, expression, and replication regulate genetic disorders, indicating that differential expression of mrcs and related genes has a significant impact on mitochondrial dysfunction [7, 9 ]. therefore, systematic analysis of nuclear and mitochondrial gene expression in the context of well - defined disease models should provide insight into the interaction of gene regulatory networks with mrcs, improving our understating of mitochondrial disorders. coexpression analysis using transcriptome datasets generated by high - throughput microarray transcript profiling produces correlations that have often been considered to imply functional relationships [10, 11 ]. a strong correlation among transcripts for mrc components has been found by this type of coexpression analysis in plants. in the case of plant mrc genes, it has been shown that genes belonging to mrcs are clustered into the same coexpression group. similarly, several mtdna - encoded mitochondrial genes form a small cluster with a nuclear - encoded mitochondrial gene module and the glycolysis module. coexpression analysis has also implicated several unannotated genes in cancer and mitochondrial complex i disease, indicating that coexpression analysis is a useful tool not only for understanding many diseases at the molecular level but also for identification of novel candidate genes involved in mitochondria - related diseases. although several studies have demonstrated the power of coexpression analysis, few have exploited mrcs as an integrated component for analyzing the coexpression network. to further investigate the role of integrated mrcs in the whole coexpression network, we determined the coexpression networks of normal and chemically treated human tissues by analysis of pearson correlation factors. in the coexpression network under normal conditions this self - connection indicates that whole mrcs might play a role similar to that of single genes in the coexpression network. using the hypergeometric distribution, we considered genes with a p value less than 10 to be coexpression friends with mrcs. candidate functions for these friends were determined through enrichment analysis, using gene ontology (go) terms and kyoto encyclopedia of genes and genomes (kegg) pathways. we then explored the coexpression network between friends and mrcs in both normal and chemically treated tissues. the systematic coexpression network of genes interacting with whole mrcs identifies candidates that potentially participate in mitochondrial biogenesis and could serve as targets for future therapeutic interventions aimed at modulating mitochondrial function. to create coexpression friends with mrcs, we first constructed a genome - wide coexpression networks using two different microarray datasets. expression datasets for 65 human tissues were downloaded from the coxpresdb website (http://coxpresdb.jp/). transcription profiles (e - mtab-798) of human hepatocytes treated with 130 chemical compounds including drugs such as acetaminophen, aspirin, rifampicin, metformin hydrochloride, simvastatin, and tamoxifen citrate were obtained from embl - ebi (http://www.ebi.ac.uk/arrayexpress/). the spearman coexpression coefficient,, was calculated for each pair of genes, and all gene pairs with 0.3 were defined as gene - gene associations in the network. in the coexpression network, the nodes represent genes and the edges represent the connection with coefficient 0.3. the mrc is designated as nadh - coenzyme q reductase (complex i), succinate - coq reductase (complex ii), ubiquinol - cytochrome c reductase (complex iii), cytochrome c oxidase (complex iv), and atp synthase (complex v). complexes i, ii, iii, and iv play as the electron transfer complexes, whereas complex v is known as an enzyme - conserving complex. since different datasets contain different probes mapping to different gene symbols, we used gene symbols that are present in gene platform file containing 56 mrc genes (33 genes for complex i, 4 genes for complex ii, 8 genes for complex iii, and 23 genes for complex iv). to determine coexpression significance, the hypergeometric distribution was used to calculate the connection between the mrc genes and other genes in the whole coexpression network. this discrete probability distribution describes the probability of k successes in n draws, without replacement, from a finite population of size n containing k samples. for example, suppose that there are k (56) mrc genes among n (20,000) genes in the genome. we define genes with a p value less than 10 to be coexpression friends with mrcs. functional annotation of friends was carried out using the web based tool panther (http://www.pantherdb.org/). david was also used to analyze the functional annotations of the gene sets and modules. for pathway enrichment analysis of the mcr coexpressed genes, the p values and a modified fisher 's exact test were used to determine the enrichment of gene sets in ontology. two functional entities are involved in the generation of atp by a process called oxidative phosphorylation located in the mitochondrial inner membrane. the first entity is the electron transfer chain historically defined as four complexes (i, ii, iii, and iv), whereas the second entity is known as the system that phosphorylates adp to produce atp. among these complexes, complex i is the first and largest enzyme complex of the respiratory chain and is directly involved in maintaining cellular reduction - oxidation (redox ; nadh / nad) homeostasis. the mammalian complex i is composed of at least 45 subunits and is the main source of reactive oxygen species, which are implicated in cell signaling, disease, and aging. its deficiency is the most frequently encountered in mitochondrial disorders, and the large number of genes coding for complex i subunits might explain why complex i deficiency is characterized by marked clinical and genetic heterogeneity. complex ii is composed of four nuclear - encoded subunits, whereas complex iii is a complex of 11 subunits [19, 20 ]. complex ii receives electrons via fadh2 and transfers it to complex iii thought coenzyme q 10. then electrons are carried by cytochrome c to complex iv, which is composed of 19 subunits. this electron transport is required for the generation of the transmembrane proton gradient in inner mitochondrial membrane which is utilized by complex v to convert adp to atp. defect in any of mrcs leads to impaired atp production and results in a mitochondrial disease involving abnormality of the central nervous system and eyes, renal, muscle, heat, and haematological system, as well as diverse age - related disorders including cancer and degenerative diseases [2124 ]. this indicates that these complexes have a significant impact on mitochondrial function. to investigate the connectivity of expression between mrcs and other genes prior genome - wide expression analyses have demonstrated significant coexpression of mrc genes under various physiological conditions in several species. similarly, our large - scale analysis across different human tissues reveals a coexpression cluster (46 out of 56 mrc genes) significantly enriched in genes belonging to mitochondrial complexes i to iv (figure 1(a)). of these, mitochondrial complex i uses nadh as a cofactor for electron transfer and translocates protons across the inner mitochondrial membrane. the genes ndufb5 (p = 9.98e 55) and ndufa7 (p = 1.14e 54), two subunits of complex i, exhibited the lowest p values (supplementary table s1 available online at http://dx.doi.org/10.1155/2014/452891), indicating that these genes are significantly coexpressed with other mrcs. in many organisms, the complexes i, iii, and iv can associate into supercomplexes [2830 ]. among the various types of association, the i + iii2 + iv14 supercomplex or the respirasome is one of the most intriguing supercomplexes, because it considered the minimal unit to perform complete respiration from nadh to oxygen [29, 31 ]. this supercomplex has also been detected by inhibitor titration in bovine mitochondria, suggesting that the two mitochondrial electron transfer complexes specifically interact to form this supercomplex. in our coexpression network (figure 2(a)), ndufb5 is coexpressed with sdhc of complex ii and uqcrc2 of complex iii. in addition, uqcrc1 in mitochondrial complex iii is significantly coexpressed with the complex i subunits ndufa13, ndufaf1, and ndufs7. it has been shown that the absence in complex iii results in a dramatic loss of complex i in humans, and complex i is necessary for fully assembled complex iii [3234 ], indicating that supercomplex formation is necessary for assembly and stability of individual components. taken together, these results suggest that complex i is tightly coexpressed with complex iii compared to other complexes and that this coexpression might be required for maintaining the supercomplex. drugs can be metabolized into electrophilic chemicals or free radicals, which have direct effects upon mitochondrial proteins. damage to mitochondrial proteins decreases their affinity for substrates, resulting in mitochondrial dysfunction [36, 37 ]. therefore, understanding drug - induced mitochondrial toxicity is critical for the development of safe drugs. to investigate the effect of chemical toxicity on the coexpression network of mrc genes in liver, transcription profiles of human hepatocytes treated with 130 chemical compounds stress - related stimuli induce the remodeling of coexpression networks, resulting in the large - scale alteration of cellular function, involving a shift of resources from growth and metabolism to protection and maintenance [38, 39 ]. as shown in figure 1(b), coexpression of mrc genes in treated human hepatocytes was significantly lower than in nontreated human tissues. ndufaf4, a subunit of complex i, exhibited significant coexpression with other mrcs under normal conditions (p = 2.31e 54 ; supplementary table s1). however, the p value between ndufaf4 and other mrcs increased substantially (p = 0.209) after chemical treatment, indicating a major change in the coexpression network. uqcrc2, a complex iii subunit, is also tightly connected with other mrcs in normal tissue (p = 1.16e 52). a similar loss of coexpression was observed for 23 mrc genes, although the remaining 23 genes (19 from complex i, 2 from complex iii, and 2 from complex iv) were still highly coexpressed (figure 2(b)). one possible explanation for these changes in coexpression in response to chemical treatment is that these compounds directly or indirectly influence mrc gene expression. indeed, differential expression of mitochondrial genes has been induced by manipulating the agonal - ph state and through drug treatment [40, 41 ]. furthermore, some compounds might modulate cellular redox levels or dissipate the mitochondrial membrane gradient by facilitating anion flux across the mitochondrial inner membrane, as suggested by toogood, resulting in remodeling of the coexpression network. in normal tissue, of these, coexpression of 1,308 genes was observed in normal tissue but not in chemically treated tissue. the remaining 114 genes are coexpressed in both normal and treated tissues. to identify the function of these 114 genes, we analyzed their associated go terms using the online panther tool (http://www.pantherdb.org/genelistanalysis.do). a total of 51 (44.7%) genes were assigned to metabolic process (figure 3(a)), indicating that this process is closely related to mitochondrial function. immune system process and apoptosis were represented by 4.26% and 1.42% of these genes, respectively. the 1,308 genes that are coexpressed only in normal tissues were also categorized using panther (figure 3(b)). of these, immune system process and apoptosis were assigned to 4.84% and 2.50% of these genes, respectively. aifm1 (apoptosis - inducing factor, mitochondrion - associated 1) endog (endonuclease g) is involved in intrinsic (mitochondria - associated) pathway for cancer cell apoptosis, for example, tightly coexpressed with mrc genes (p = 9.09e 29 and 1.71e39, resp.) in normal tissue, but this coexpression disappeared in treated tissues (supplementary table s1). aifm1 is known to be important for the assembly and stability of complexes i and iii. in addition, the mutation or inhibition of mrc is widespread in cancer and intimately connected to apoptosis resistance, indicating that the mrc plays as a modulator of apoptosis for the treatment of cancer [45, 46 ]. coexpression among mrc genes and the mechanistic target of rapamycin (mtor) gene also disappeared after chemical treatment (p = 0.99 ; supplementary table s1). disruption of the mtor complex by treatment with the mtor inhibitor rapamycin reduces mitochondrial membrane potential, oxygen consumption, and atp synthetic capacity, indicating that formation of the mtor complex is required for overall mitochondrial activity. taken together, these findings indicate that coexpression of mrc genes with cellular genes such as aifm1 might be required to maintain the mitochondrial complexes. furthermore, the disruption of this coexpression by chemical treatment suggests that similar disruptions might be responsible for mitochondria - related side effects of pharmaceuticals. in contrast to the coexpression network in normal tissues, only 238 genes were significantly enriched in chemically treated tissues. of these, 114 are coexpressed in normal tissues, whereas 124 are coexpressed only in treated tissues. this finding suggests that chemical treatment alters the coexpression network between mrc genes and cellular genes. of the genes coexpressed only in treated tissues, almost half (48.91%) were associated with the term metabolic process (figure 3(c)). the gene for 2,4-dienoyl coa reductase 2 (decr2), an auxiliary enzyme in the mitochondrial beta - oxidation of unsaturated fatty acids, is coexpressed with a p value of 7.49e 8 (supplementary table s1). three acyl - coa thioesterases (acots), acot11 (p = 5.51e 42), acot13 (p = 1.14e 26), and acot2 (p = 1.83e 14), involved in peroxisomal lipid metabolism, were highly coexpressed with mrcs in normal tissue, whereas acot8 (p = 1.75e 08) is coexpressed with mrcs in treated tissues (supplementary table s1). coexpression network, which is the reconstruction of biological networks from high - throughput data, can be used to identify higher - level features of gene - gene relationships based on graph theoretic considerations such as clustering coefficient or node degree [49, 50 ]. however, large - scale analyses only provide clues that help in forming a hypothesis. although the differences among coexpression networks (supplementary table s1) should help identify and prioritize candidate genes to determine the effects of drugs on mitochondria, further study is required to determine the relationship between the coexpressed genes and specific mitochondrial functions. to investigate the biological processes represented by genes significantly coexpressed with mrc genes in normal tissue compared to treated tissues, we performed go term enrichment analyses using the functional annotation tool david (http://david.abcc.ncifcrf.gov/). for normal tissue, annotations for 1,308 genes were enriched in 17 terms, including muscle system process, cellular metabolic process, and carboxylic acid metabolic process for chemically treated tissues, go term enrichment of 124 genes coexpressed with mrc genes found only ribosome biogenesis terms, such as translational elongation and translation a sufficient supply of atp is required to maintain the contractile function of muscle, suggesting the importance of mitochondria during muscle contraction. myosin genes could be divided into several classifications, such as myosin heavy chain 1 (myh1), myosin light chain 3 (myl3), and myosin binding protein c2 (mybpc2). these findings indicate that muscle system closely interacts with mrc genes for improving mitochondrial function. for the go term, carnitine palmitoyltransferase 2 (cpt2) localizes to the inner leaflet of the inner mitochondrial membrane, where it oxidizes long - chain fatty acids to produce substrates for the mitochondrial fatty acid beta - oxidation pathway. most of the genes required for mitochondrial biogenesis are controlled by dna - binding transcription factors and coregulators. peroxisome proliferator - activated receptor gamma coactivator 1-alpha (ppargc1a) is a transcriptional coactivator that regulates various metabolic processes including mitochondrial biogenesis and respiration. therefore, coexpression of mrc genes with functional genes such as cpt2 and ppargc1a might be required for the function and maintenance of mitochondria. several molecular, cellular, biochemical, and animal model studies have suggested that mitochondrial dysfunction closely relates to the progression of several neurodegenerative diseases. using kegg pathway enrichment analysis, we found that 19 mrc genes (41% of mrc genes in the coexpression network), belonging to complexes i to iv, are coregulated with parkinson 's, alzheimer 's, and huntington 's disease pathways (table 3), indicating the importance of mrcs in neurodegenerative disease pathways. in parkinson 's disease, pten - induced putative kinase 1 (pink1), a mitochondrial serine / threonine - protein kinase, was found to be a coexpression friend with mrc genes in normal tissue (p = 1.28e 13) but not in treated tissues (p = 0.35) (supplementary table s1). in addition, the 1 subunit of dihydropyridine receptor (cacna1s), calmodulin - like 6 (calml6), presenilin protein 2 (psen2), bcl2-associated agonist of cell death (bad), and lipoprotein lipase (lpl) in alzheimer 's disease pathway are significantly coexpressed with mrc genes (table 3), whereas clathrin light chain b (cltb), clathrin heavy polypeptide - like 1 (cltcl1), dna - directed rna polymerase ii subunits (polr2e, polr2l), and ppargc1a were identified as coexpression these findings indicate that mrc genes are directly or indirectly linked with neurodegenerative disease pathways. genes were enriched in the hypertrophic and dilated cardiomyopathy pathways in normal tissue (table 3), whereas coexpression again disappeared after treatment with chemicals. kegg pathway enrichment analysis thus suggests that the coexpression network revealed by using mrc genes as seed genes provides a possible link between mitochondria and various disease pathways. in this study, we have compared the networks of genes coexpressed with mrc genes in normal and chemically treated tissues. these differences might be mediated by chemical - related stimuli, suggesting that coexpression network analysis can provide helpful information for understanding side effects of drugs on mitochondrial functions. | as energy producers, mitochondria play a pivotal role in multiple cellular processes. although several lines of evidence suggest that differential expression of mitochondrial respiratory complexes (mrcs) has a significant impact on mitochondrial function, the role of integrated mrcs in the whole coexpression network has yet to be revealed. in this study, we construct coexpression networks based on microarray datasets from different tissues and chemical treatments to explore the role of integrated mrcs in the coexpression network and the effects of different chemicals on the mitochondrial network. by grouping mrcs as one seed target, the hypergeometric distribution allowed us to identify genes that are significantly coexpress with whole mrcs. coexpression among 46 mrc genes (approximately 78% of mrc genes tested) was significant in the normal tissue transcriptome dataset. these mrc genes are coexpressed with genes involved in the categories muscle system process, metabolic process, and neurodegenerative disease pathways, whereas, in the chemically treated tissues, coexpression of these genes mostly disappeared. these results indicate that chemical stimuli alter the normal coexpression network of mrc genes. taken together, the datasets obtained from the different coexpression networks are informative about mitochondrial biogenesis and should contribute to understanding the side effects of drugs on mitochondrial function. |
spinal roots injuries constitute an important medical problem and usually affect the brachial plexus in consequence of high energy trauma, causing motor, sensibility, and autonomic loss [15 ]. the brachial plexus injury is extremely debilitating for the patient because, besides significant upper limb function loss, it causes an everyday tasks limitation, which can result in unemployment, depression, and in some cases even suicide. such medical problem is typically associated with young patients following motorcycle and radical sports accidents. root avulsion results in interruption of neurotrophic factors flow towards severed motoneurons, vascular trauma, and excitotoxicity, leading to drastic 80% death during the following 2 weeks after injury [79 ]. together with neurodegeneration, synaptic changes in the spinal cord microenvironment cause expressive reduction of complexity and decreased number of presynaptic boutons apposing to surviving motoneurons [1015 ]. astroglial and microglial reactivity, together with the presence of meningeal fibroblasts, contribute to the formation of the so - called glial scar, minimizing chances of target organ reinnervation, culminating with an irreversible state of paralysis. currently, the repair of spinal root injuries is a highly delicate challenge, being particularly uncertain because of the risk of additional damage to the spinal cord, since the procedure is based on pia mater suturing. nevertheless, the surgical reimplantation of injured ventral roots has been shown to be efficient and neuroprotective. in patients, it may be considered promising treatment to partially recover motor and autonomic functions, as well as reduce untreatable neuropathic pain [1, 19 ]. due to the fact that direct pial suture is not always possible because of tissue loss, alternative apposition and fixation of avulsed roots with surgical adhesives have been proposed. among available sealants, they are produced with constituents of human blood that, when combined, form a fibrin bioactive matrix with adhesive and hemostatic proprieties mimicking the final steps of the clotting cascade. the use of such sealants on clinical practice is already known and widely discussed. among many different applications, neurosurgery is of particular interest, due to the specific need of repair stability and absence of side effects. in order to avoid potential adverse effects of using human derived components, such as disease transmission, researchers from cevap, unesp, brazil, produced a new fibrin sealant, in which the thrombin fraction is substituted by a protein from crotalus durissus terrificus venom. the venom has thrombin - like fraction demonstrated by nahas. and isolated by raw. the thrombin - like fraction has the ability to transform the fibrinogen directly into fibrin, forming a stable clot [2529 ]. also, bubaline fibrinogen substitutes the human counterpart. as an alternative to commercial glues, such new biological product is more flexible in terms of formulation matching specific surgical needs, what can be an advantage to the commercial equivalent. additionally, its production costs are lower, allowing broader application [26, 29 ]. nevertheless, to date there is no comparative study, indicating that cevap 's sealant performs equally well to currently marketed similars. in this regard, the present study demonstrates that root implantation, on the site of injury, can be carried out in a reproducible way with both commercial and nonhuman derived sealants. the present study was carried out using ten - week - old adults female lewis rats (lew / hsdunib) weighting around 200 g. the animals were obtained from the multidisciplinary center for biologic research (cemib) at the university of campinas (unicamp), so paulo, brazil. the experiments were conducted according to the ethical standards for animal experimentation and were approved by the ethical committee for animal use (ceua, unicamp, protocol 3064 - 1). animals were subjected to unilateral ventral root avulsion and reimplantation at the lumbar intumescence (l4, l5, and l6 right side ; n = 5 for each experimental group and technique). the following experimental groups were constituted : (i) ventral root avulsion alone ; (ii) avulsion and reimplantation of ventral roots with fibrin sealant produced from the crotalus durissus terrificus venom (cevap) ; (iii) avulsion and reimplantation of ventral roots with commercial fibrin sealant tissucol (baxter ag, vienna, austria). the rats were anesthetized by a combination of xylazine chlorhydrate (anasedan, 10 mg / kg, sespo indstria e comrcio, paulnia / sp, brazil) and ketamine hydrochloride (dopalen, 50 mg / kg, sespo indstria e comrcio, paulnia / sp, brazil) and subjected to unilateral avulsion of the lumbar ventral roots as previously described [2, 3034 ]. unilateral avulsion was performed at the l4l6 lumbar ventral roots after unilateral laminectomy (right side). a longitudinal incision was made to open the dural sac, and the denticulate ligament was dissected. finally, the ventral and dorsal roots were carefully separated so that the ventral roots associated with the lumbar intumescence could be identified and avulsed with fine forceps (dumont, switzerland, part number 11242 - 40). after lesioning, the roots and spinal cord were returned to their original position, and the musculature, fascia, and skin were sutured in layers. chlorhydrate of tramadol (germed farmacutica ltda, hortolndia / sp, brazil) was administrated by gavage after the surgical procedures (20 mg / kg) and 2.5 mg / day soluble in water during 5 days. the animals were housed under a 12-hour light / dark cycle and controlled temperature (23c), with free access to food and water. in the vra + sealant groups, the roots were replaced at the exact point of detachment, on the ventral surface of the lumbar spinal cord at the avulsion site with the aid of fine forceps. the fibrin sealant derived from snake venom was kindly supplied by the center for the study of venoms and venomous animals (cevap) of unesp ; its constituents and instructions for use are stated in the respective patents (registration numbers br1020140114327 and br1020140114360). this sealant was composed of three separate solutions and homogenized immediately before use in a total final volume of 6 ml : (1) fibrinogen derived from bubaline blood (3 ml), (2) calcium chloride (2 ml), and (3) a thrombin - like enriched fraction (1 ml) [2529 ]. during surgical repair of the avulsed roots, the first two components were applied and the avulsed roots were returned to their original sites. the reimplanted roots were then gently pulled from the spinal cord, and the stability of the fixation was observed to evaluate the success of the repair, while the commercial fibrin sealant utilized was tissucol (baxter ag, vienna, austria). this sealant is a lyophilized concentrate of human proteins and composed by 4 components : (1) lyophilized fibrinogen ; (2) aprotinin solution ; (3) lyophilized thrombin ; and (4) calcium chloride solution. at the time of use, the components were previously thawed, reconstituted, mixed, and applied following the manufacturer instructions. for the gait recovery analysis, the catwalk system was used (catwalk, noldus inc., wageningen, netherlands ; http://www.noldus.com/animal-behavior-research/products/catwalk). in this setup, a green led illuminates the long edge of the floor so that the light highlights only the places where the animal 's paws touch the glass surface. by illuminating the footprints, the plantar surfaces were captured by a high - speed video camera (fujinon df6h-1b) equipped with a wide - angle lens (8.5 mm, fujicon corp., china) positioned underneath the walkway. the paw prints are automatically recorded and classified by the software and were recorded before and after the vra. the peroneal functional index (pfi) was calculated as the distance between the third toe and hind limb pads (print length) and the distance between the first and fifth toes (print width). measurements of these parameters were obtained from the right (lesioned) and left (unlesioned) paw prints, and the values were calculated using the following formula by bain. : pfi = 174.9 ((epl npl)/npl)) + 80.3 ((ets nts)) 13.4, where n is normal or nonoperated side ; e is experimental or operated side ; pl is print length ; ts is total toe spread or distance between first and fifth toe. the catwalk data from each day of evaluation were expressed as an ipsi / contralateral ratio. the animals were anaesthetized with an overdose of a combination of xylazine chlorhydrate (anasedan, 10 mg / kg, sespo indstria e comrcio, paulnia / sp, brazil) and ketamine hydrochloride (dopalen, 50 mg / kg, sespo indstria e comrcio, paulnia / sp, brazil) and the vascular system was rinsed by transcardial perfusion with 0.1 m saline phosphate buffer (ph 7.38) followed by fixative solution. for myelin fibers counting and morphometry (sciatic nerve regeneration analysis), the rats were killed and fixed by transcardial perfusion with 2,5% glutaraldehyde and 1% paraformaldehyde in 0,1 m phosphate buffer (ph 7,38). a 10 mm midthigh segment of the sciatic nerve (ipsi and contralateral) was exposed, dissected, and stored in the same fixative solution, for 24 hours at 22c. the fragments were washed with 0.1 m saline phosphate buffer (ph 7.38) and postfixed for 3 hours in osmium tetroxide solution 1% diluted in phosphate buffer (ph 7.38). after the postfixation the fragments were washed in distilled water and dehydrated through ethanol series and acetone (for 60 minutes) and embedded in durcupan acm (fluka, steinheim, switzerland). the specimens were trimmed and semithin sections (0.5 m) were obtained in ultramicrotome (leica ultracut uct ultramicrotome). for neuronal survival counting and immunohistochemical evaluation (n = 5 for each group) the rats were killed and fixed by transcardial perfusion with 10% formaldehyde in 0.1 m phosphate buffer (ph 7.38). the lumbar intumescence and sciatic nerves (ipsi and contralateral) were exposed, dissected out, postfixed for 24 hours at 22c in the same fixative solution, and then washed in 0.1 m phosphate buffer (ph 7.38) and subjected to 10, 20, and 30% sucrose in 0.1 m pb for 24 h each, before freezing. transverse sections (12 m thickness) of spinal cords and longitudinal sections (12 m thickness) of sciatic nerves were obtained in cryostat (microm hm 525, microm international gmbh - walldorf, germany) and transferred to gelatin - coated slides and dried at room temperature for 30 min before being stored at 20c until the moment of use. the sciatic nerves were processed for myelinated axon counting and morphometric evaluation by transverse semithin sections (0.5 m thick), stained in sudan black (0.7% in 70% alcohol) solution, and examined using a bright field microscope leica ctr 6500 (leica microsystems cms gmbh) and adobe photoshop cs5 (adobe systems) software. the analyses were performed by sampling at least 30% of each nerve cross section (magnification of 1,000x), generating approximately 12 images per animal. sampling bias was avoided by spreading the micrographs systematically over the entire cross section, according to the procedure proposed by mayhew and sharma. the images were used for counting the total number of myelinated axons in each specimen. for the morphometric analysis, the images were converted to black and white, so that any background artefacts would not be measured, including fibers that were partly captured. the myelinated axons were identified and the measurements were carried out automatically, after calibration. the measurements provided were myelin sheath 's area, perimeter, and smaller diameter of each myelinated fiber. these values were used to calculate the myelinated axons diameter, myelin thickness (fiber diameter axon diameter/2) and g ratio (axon diameter / fiber diameter). the data are represented as the mean standard error (se) for each group. transverse cryostat sections of the spinal cords were stained in aqueous toluidine blue (1 mg/100 ml) for 35 minutes at room temperature. the sections were washed with distilled water, dehydrated in crescent series of alcohol and xylene, and mounted with entellan (merck kgaa, darmstadt, germany). the motoneurons present in the lateral motor nucleus of the anterior horn on the ipsilateral side (injured) and contralateral side (not injured) were identified (based on their morphology, size, and location in the dorsolateral lamina ix) and counted in alternate sections of each specimen in about 12 sections with a 192 m space between them. only the cells with visible nucleus and nucleolus were counted. the percentage of surviving cells was analyzed by the ratio of absolute numbers of motoneurons, counted per section, on the lesioned and nonlesioned sides, respectively, and multiplying the result by 100. abercrombie 's formula was used to correct the duplicate counting of neurons : n = nt/(t + d), where n is the number of counted cells, t is the thickness of the sections, and d is the average diameter of neuronal nuclei. as the difference in the size affects the cells number significantly, value was calculated for each experimental group (ipsilateral and contralateral) specifically. in order to this, the nuclear diameter of 15 randomly chosen motoneurons from each group was measured (imagetool software, version 3.00, the university of texas health science center, tx) and the mean value calculated. the data are represented as the mean standard error (se) for each group. transverse cryostat sections of the spinal cords and longitudinal cryostat sections of the sciatic nerves were acclimatized, washed in 0.1 m phosphate buffer (ph 7.38, 3 times of 5 minutes each), and incubated for 45 min in a 3% bsa (bovine serum albumin) solution in 0.1 m phosphate buffer (ph 7.38) followed by incubation with the primary antibodies (table 1). the primary antibodies were diluted in a solution containing 1% bsa (bovine serum albumin) and 2% triton in 0.1 m phosphate buffer (ph 7.38). all sections were incubated for 4 hours at room temperature in a moist chamber. after rinsing in pb, the sections were incubated with a cy3-conjugated secondary antiserum (1 : 250, jackson immunoresearch, west grove, pa, usa) and diluted in 1% bsa and 0.2% triton in 0.1 m pb (ph 7.38) for 45 minutes in a moist chamber at room temperature. the sections were then rinsed in 0.1 m pb (ph 7.38) and mounted in a mixture of glycerol / pbs (3 : 1) and observed in fluorescence microscope leica dm5500b microscope coupled with a leica dfc345 fx camera (leica microsystems cms gmbh) utilizing rhodamine filters (cy3). for quantitative measurements, three representative images of the spinal cord (l4l6 at lamina ix, ventral horn) and sciatic nerve from each animal were captured at a final magnification of 200x. for the quantification, the integrated density of pixels, which represents the intensity of labeling, was measured utilizing the imagej 1.33u (national institutes of health, usa) software. for the analysis of anti - glial fibrillary acidic protein (gfap) and anti - ionized calcium binding adaptor molecule 1 (iba1) antibodies, the integrated density of pixels was measured in the lamina ix ventrolateral, as described by oliveira. and freria.. for analysis of synaptophysin immunolabeling, the integrated density of pixels was systematically measured in eight representative areas surrounding each motoneuron located at lamina ix, in the anterior horn of the spinal cord, according to oliveira.. the proportion of integrated density of pixels was calculated for each animal and then as the mean value for each spinal cord. the data are represented as the mean standard error (se) for each group. data from the functional analysis (walking track test) and morphometry of myelinated fibers were evaluated via two - way analysis of variance. the data are presented as the mean standard error (se) and the differences between groups were considered significant when the p value was > 0.05 (), > 0.01 (), and > 0.001 (). neuronal survival after vra and reimplantation was evaluated by the counting of motoneurons present at lamina ix of the ventral horn, in the ipsilateral side as compared to contralateral side, 4 and 12 weeks after lesion (figure 1). no statistical differences regarding motoneuron numbers in the contralateral side, in the different experimental conditions, were observed. based on that, all results were expressed as percentage of surviving motoneurons in comparison to the contralateral side. four weeks after lesion, a significant loss of motoneurons could be observed in the vra group, due to the injury (figure 1(a)). contrarily, the reimplanted groups displayed statistically higher number of surviving motoneurons (figures 1(e), 1(i), and 1(m)). also, no differences between the reimplanted groups were observed (vra only : 37.59 3.40 ; vra + venom glue : 66.27 5.63 ; vra + commercial glue : 70.93 5.21 ; mean ipsi / contralateral ratio se ; f2,12 = 13.86 ; p value = 0.0008 ; n = 5 per group). such neuroprotective effect remained up to 12 weeks after lesion, when the neuronal survival in the reimplanted groups was statistically superior to the vra only (figures 1(c), 1(g), and 1(k)). once again, no differences between the reimplanted groups were observed (vra only : 27.75 3.42 ; vra + venom glue : 53.64 6.43 ; vra + commercial glue : 51.57 7.24 ; mean ipsi / contralateral ratio se ; f2,12 = 5.883 ; p value = 0.016 ; n = 5 per group). synaptic activity changes after root avulsion and reimplantation were evaluated in the sciatic motor nucleus in the ventral horn of the spinal cord, by synaptophysin immunolabeling, 4 and 12 weeks after the injury (figure 2). in both experimental survival times analyzed, the synaptophysin immunoreactivity showed higher synaptic density in the contralateral side than the ipsilateral side of avulsed animals. such expression was drastically decreased in axotomized motoneurons surface in animals without reimplantation, indicating a significant decrease of complexity of propriospinal networks following lesion. in contrast, in the reimplanted groups, the repair resulted in preservation of synaptophysin immunoreactivity, particularly in the immediate vicinity of the motoneurons. statistical analysis revealed no differences between the reimplanted groups (4 weeks after lesion : vra only : 0.41 0.04 ; vra + venom glue : 0.68 0.06 ; vra + commercial glue : 0.72 0.04 ; mean ipsi / contralateral ratio se ; f2,12 = 13.47 ; p value = 0.0009 ; n = 5 per group) ; 12 weeks after lesion : vra only : 0.46 0.05 ; vra + venom glue : 0.72 0.02 ; vra + commercial glue : 0.77 0.03 ; mean ipsi / contralateral ratio se ; f2,6 = 16.13 ; p value = 0.0016 ; n = 3 per group). glial reactivity after ventral root avulsion and reimplantation was evaluated in the ventral horn of the spinal cord, by immunofluorescence analysis, 4 and 12 weeks after lesion. immunoreactivity against gfap was used to analyze the degree of astroglial reactivity after lesion (figure 3) while iba-1 immunoreactivity was used to assess the degree of microglial reactivity (figure 4). four weeks after lesion, the immunostaining showed increased astrocyte reactivity after vra as demonstrated by the presence of gfap - positive activated astrocytes, particularly concentrated in the vicinity of the avulsed motoneurons. however, the astroglial reactivity in reimplanted groups was not significantly different from the vra alone (vra only : 2.34 0.22 ; vra + venom glue : 2.08 0.30 ; vra + commercial glue : 2.26 0.21, mean ratio ipsi / contralateral se ; f2,12 = 0,30 ; p value = 0.74 ; n = 5 per group). similar results were observed for microglial reactivity, 4 weeks after lesion, where the immunostaining showed increased microglial reaction after vra, as demonstrated by the presence of iba1-positive activated microglia. in the same way, the microglial reactivity in the reimplanted groups was not significantly different from the vra alone (vra only : 4.13 0.55 ; vra + venom glue : 3.46 0.17 ; vra + commercial glue : 3.41 0.40 ; mean ratio ipsi / contralateral se ; f2,12 = 0.99 ; p value = 0.39 ; n = 5 per group). in contrast, twelve weeks after lesion, the immunostaining showed decreased glial reactivity in all experimental groups, in comparison with 4 weeks survival time. nevertheless, no significant difference between experimental groups was observed (astroglial reactivity : vra only : 2.40 0.27 ; vra + venom glue : 2.34 0.22 ; vra + commercial glue : 2.33 0.46 ; mean ratio ipsi / contralateral se ; f2,6 = 0,015 ; p value = 0.984 ; n = 3 per group ; microglial reactivity : vra only : 2.82 0.13 ; vra + venom glue : 2.29 0.16 ; vra + commercial glue : 2.33 0.11 ; mean ratio ipsi / contralateral se ; f2,6 = 4.639 ; p value = 0.046 ; n = 3 per group). therefore, the glial reactivity was not significantly further increased after reimplantation in both experimental times. schwann cell activity (s100 labeling) and p75 expression after ventral root avulsion and reimplantation were examined in the sciatic nerve by immunofluorescence, 4 weeks after lesion (figure 5). p75 is expressed on developing motoneurons and is de novo expressed by adult motoneurons under pathological conditions such as trauma or degeneration. in addition, roles of schwann cells on the regenerative process include phagocytosis of axon and myelin debris derived from wallerian degeneration, being responsible for the remyelination of axons. in this sense, it is important to identify the effects of reimplantation on p75 and s100 expression. the immunostaining revealed an increase of such neurotrophin receptor expression after vra as demonstrated in figure 5, being statistically higher in the vra + commercial glue group (vra only : 1,509 626.5 ; vra + venom glue : 2,286 788.1 ; vra + commercial glue : 3,266 403.9 ; control : 464.5 146.1 ; integrated density of pixels ; mean se ; f2,12 = 4.704 ; p value = 0.0154 ; n = 5 per group). in the same way, s100 labeling showed an increased schwann cell activity after injury / reimplantation in all experimental groups, being more expressive in the vra group (vra only : 8882 1362 ; vra + venom glue : 6237 754.8 ; vra + commercial glue : 4640 536.3 ; control : 3755 378.1 ; integrated density of pixels ; mean se ; f2,12 = 7.099 ; p value = 0.0030 ; n = 5 per group). the number of myelinated axons present in the sciatic nerve was estimated based on the total area of the nerve and on the fiber number in the evaluated fields. after 12 weeks after injury (figure 6), the number of myelinated axons was higher in reimplanted groups when compared to vra group, showing the capacity of regeneration of motor axons after reimplantation, allowing growth towards the target muscles (vra group 6,219 231.4 ; vra + snake venom sealant group 7,177 486.7 ; vra + commercial sealant group 7,508 151.5 ; in addition, morphometry revealed an increase of the myelinated fibers with thin myelin sheath in the animals from vra group (figures 6(m)6(p)). in the reimplanted groups it was possible to notice an increased number of myelinated fibers with thicker myelin sheath in comparison to the vra group, suggesting possible axonal regeneration. contralateral intact sciatic nerves showed normal distribution [4, 40 ] and greater number of fibers. the recovery of the functional recovery was evaluated using the catwalk system (noldus inc., netherlands, figure 7), from the 1st until the 12th week after injury. the preoperative peroneal functional index mean values did not significantly differ between groups. at the first postoperative week, all groups have shown drastic decrease in mean pfi, indicating functional loss. nevertheless, at the 12th postoperative week, the reimplanted groups presented significantly higher mean pfi compared to the vra group, according to the formula described by bain.. furthermore, these results are consistent with the footprint paw pressure data indicating that the reimplanted groups performed better in supporting their body weight on the injured limb (figure 8 ; see supplementary videos 14 in supplementary material available online at http://dx.doi.org/10.1155/2016/2932784). experimentally, it is possible to mimic human brachial plexus lesions by disconnecting ventral roots from the surface of the spinal cord. we have previously suggested a root reimplantation method, by apposing avulsed roots to the lesion site, stabilizing the repair by the application of a fibrin scaffold, derived from nonhuman blood. here we compare such restoration performance and functional recovery with a commercial fibrin sealant tissucol (baxter ag, vienna, austria), analyzing data 4 and 12 weeks after surgery. ventral root avulsion results in significant spinal motoneuron degeneration (mn), combined with glial reactivity and synaptic circuits shrinkage. it is known that such proximal lesion induces loss of about 80% of axotomized motoneurons during the first 2 weeks after the injury [7, 42, 43 ]. in this aspect, the present data allow concluding that reimplantation of avulsed roots is neuroprotective by itself. after 4 weeks of injury a lower rate of neuronal death further, reimplanting neuroprotective effects were long - lasting, as they were present at the same levels up to 12 weeks after injury. these findings are in line with hallin and contributors that suggested motoneuron survival after root repair is based on neurotrophic factor production by glial cells at the cns / pns interface. this has also been connected to the breakage of the blood - brain barrier, allowing neurotrophic factor producing immune cells to enter spinal cord gray matter environment [4547 ]. nevertheless, eggers. demonstrated that roots repair is unable to completely hampers neurodegeneration. besides neuronal death, a hallmark of vra is the reduction of presynaptic terminals to motoneurons. such process decreases or even abolishes synaptic transmission [1113 ]. in agreement with the literature, we observed extensive synaptic loss after avulsion without reimplantation. importantly, the groups that were subjected to root repair showed significant preservation of synapses when compared to the avulsion alone. therefore, it is possible that early reparation of lesion stabilizes spinal circuits in an inhibition / excitation proportion compatible with preservation of regenerating neurons. coupled with the above discussed synaptic changes, glial cells become reactive following vra. such reactive gliosis is characterized by hypertrophy of the cell body and processes of astrocytes and microglial hyperplasia [4951 ]. this in turn may result in scar formation, inhibiting axonal growth, or lead to neurotrophic support of the axotomized neurons, resulting in regeneration. reimplantation of ventral roots did not significantly decrease microglial and astroglial reactivity, four weeks after lesion. ohlsson. also did not observe differences in astroglial and microglial reaction, when comparing avulsed and reimplanted groups, at the same time point following lumbosacral ventral root avulsion injury. we believe that glial reaction following root repair did not hamper axonal sprouting, based on morphological evaluation of the sciatic nerve 12 weeks after lesion, as well as on behavioral assessment. further, in order to achieve such gliosis downregulation, association with pharmacological / cell therapy approaches seems to be necessary. the wallerian degeneration processes that follow proximal axotomy became clear at the ipsilateral sciatic nerve, already at 4 weeks after injury, by the significant decrease in number of myelinated axons, combined with diminished mean values of myelin sheath, compatible to what has been described previously [7, 54 ]. on the contrary, 12 weeks after repair revealed improved number of fibers, indicating regeneration, regardless the fibrin sealant used. in the same fashion, myelin thickness and g ratio reach close to normal values in reimplanted groups, similar to what has been shown in the literature [40, 55 ]. thus, root reimplantation has proven to be essential for the axon regrowth, allowing cns / pns reconnection towards target muscle fibers. importantly, the regeneration process followed by avulsed root repair must be evidenced via behavioral improvement by motor and gait recovery. in this sense, the use of a refined system to investigate the motor capacity can provide reliable data on animals ' functional recovery [56, 57 ]. the mostly automatic walking track analysis, by the catwalk system, has been recently introduced and proven to deliver highly precise data on mobility deficit / recovery detection, including the following parameters : footprint area, footprint pressure intensity, position, and footstep duration. our results demonstrated that motor function loss is evidenced immediately after injury (first week after operation), reflecting denervation of target muscles, corresponding to the territory of the sciatic nerve. since axonal regrowth is a slow process, observation of long - term survival times is of fundamental importance. such motor recovery possibly involved plasticity of the nervous system at different levels, including motor cortex and descending pathways as well as motor unit refinement. overall, the present work demonstrates that fibrin sealant based restoration of avulsed roots is efficient and results in neuroprotection of proximally axotomized motoneurons and motor recovery. also, our findings indicate similar regenerative performance of both biological adhesives employed, highlighting the significant potential of the nonhuman derived glue produced by cevap, brazil. the fibrin sealant obtained from the crotalus durissus terrificus venom as well as the commercial fibrin sealant tissucol (baxter ag, vienna, austria) performed equally well for the restoration of cns / pns interface, following ventral root avulsion lesion. the data herein indicate that such surgical approach is neuroprotective, but a critical recovery timeframe of at least 8 weeks after surgery is necessary, reaching stable motor function by 12 weeks after operation. | axonal injuries at the interface between central and peripheral nervous system, such as ventral root avulsion (vra), induce important degenerative processes, mostly resulting in neuronal and motor function loss. in the present work, we have compared two different fibrin sealants, one derived from human blood and another derived from animal blood and crotalus durissus terrificus venom, as a promising treatment for this type of injury. lewis rats were submitted to vra (l4l6) and had the avulsed roots reimplanted to the surface of the spinal cord, with the aid of fibrin sealant. the spinal cords were processed to evaluate neuronal survival, synaptic stability, and glial reactivity, 4 and 12 weeks after lesion. sciatic nerves were processed to investigate schwann cell activity by p75ntr expression (4 weeks after surgery) and to count myelinated axons and morphometric evaluation (12 weeks after surgery). walking track test was used to evaluate gait recovery, up to 12 weeks. the results indicate that both fibrin sealants are similarly efficient. however, the snake - derived fibrin glue is a potentially safer alternative for being a biological and biodegradable product which does not contain human blood derivatives. therefore, the venom glue can be a useful tool for the scientific community due to its advantages and variety of applications. |
during 20142015, we enrolled patients with acute febrile illness seeking treatment at garissa and wajir hospitals in northeastern kenya (figure) by using systematic sampling intervals based on previously documented proportions of febrile patients recorded at each hospital. the study protocol was approved by the scientific and ethics review committee of kenya medical research institute. we obtained serum samples and tested them for brucellosis by using the modified rose bengal plate test (rbpt) (vla weybridge, united kingdom) (12) and serion elisa classic brucella igm / igg kits (virion / serion, wurzburg, germany) according to the manufacturers instructions. we extracted dna from serum samples by using the high pure template kit (roche diagnostics, mannheim, germany). we performed quantitative real - time pcr (qpcr) assays for the detection of brucellosis and speciation of brucella species, as previously described (13) (technical appendix table 1). we classified patients as having brucellosis if they had positive qpcr results or had positive rbpt results confirmed by positive elisa results. we fitted multivariate logistic regression models to assess demographic, clinical features, and plausible risk factors associated with brucellosis seropositivity by using a stepwise backward analysis procedure. locations of the 2 hospitals in the northeastern province of kenya (dark gray shading) where human brucellosis was diagnosed in febrile patients seeking treatment, kenya, 20142015. the solid black area in northwestern kenya represents disputed territory among kenya, ethiopia, and south sudan. overall, 1,067 patients participated in the study ; 580 (54.4%) of participants were female, and 963 (90.3%) were of somali ethnicity (technical appendix table 2). of these, 29 (2.7%) had negative serologic test results for brucella infection. b. abortus was the only brucella species found using the brucella species specific qpcr. statistical analyses showed no significant differences in infection by ethnic group, county of residence, education status, or age group. men had a significantly higher probability (odds ratio [or ] 1.98, p = 0.001) for having brucellosis (table 1). na, not available ; or, odds ratio. considerable low sensitivity levels were found for clinical diagnosis of brucellosis in both hospitals (technical appendix table 3). patients with brucellosis were mainly diagnosed with typhoid fever (63 patients [43.2% ]), malaria (30 [20.5% ]), pneumonia (12 [8.2% ]), and other common tropical fevers or fevers of unknown origin (14 [9.6% ]) (table 1). in the final combined multivariate analyses, brucellosis was significantly associated (p 14 days (adjusted or [aor ] 2.86), contact with cattle (aor 6.50) or multiple animal species (aor 2.35), slaughtering of animals (aor 2.20), and consumption of raw cattle milk (aor 3.88). auc, area under the curve ; na, not available ; or, odds ratio ; roc, receiver operating characteristic. variables with p<0.20 (wald test) considered as potential risk and subsequently fitted in the multivariate analysis. this hospital - based study from a predominantly pastoral community in kenya indicated a high prevalence (13.7%) of brucellosis in febrile patients, highlighting brucellosis as an important cause of acute febrile illnesses in northeastern kenya. although brucellosis has previously been described to occur in hospital patients in kenya (1), it was not diagnosed by the treating hospital clinicians in 119/146 (81.5%) cases in our study. instead, these cases were mainly attributed to other causes of fevers or fevers of unknown origin. in addition, 29 (2.7%) patients who had negative serologic test results for brucella had positive results for b. abortus by qpcr. our findings strongly suggest that patients with brucellosis were likely to leave the hospital without the specific treatment for brucellosis. this agrees with recent findings that showed that clinicians in kenya continue to treat febrile patients for presumptive malaria, resulting in missed opportunities to accurately detect and treat other causes of fever (11,14). the results also highlight the usefulness of qpcr as a complementary assay to a combined elisa and rbpt diagnostic approach in diagnosis of acute brucellosis and the need to establish national and regional reference laboratories with facilities for performing qpcr assays. contact with cattle or multiple animal species and consumption of raw milk from cattle were significantly associated with brucellosis in our study (table 2). this association can be attributed to occupational and domestic contacts with livestock and social - cultural practices among communities in the study area that increase the risk for brucella transmission, including nomadic movements, taking care of animals during parturition, consumption of raw milk from cattle and camels, and household slaughter of animals during traditional and religious ceremonies (9,15). in this study, the only brucella species detected was b. abortus, strengthening the assumption that brucellosis might be highly linked to cattle more than other animal species ; however, further research is warranted. the lack of a clear clinical algorithm predictive of brucellosis supports the need for increasing clinician awareness of the disease and enhancing diagnostic capability for brucellosis in hospital settings. first, the study used acute - phase serum samples, making it difficult to demonstrate 4-fold titer rise. follow - up of patients to obtain a convalescent - phase serum sample was not feasible because of ongoing inter - clan conflicts and militia activities in the region. therefore, the possibility of patients who had previous exposure to brucella but had residual antibodies in circulation can not be ruled out. primers and probes for brucella genus and species - specific quantitative pcr on specimens from brucellosis patients, wajir and garissa hospitals, northeastern kenya, 20142015. characteristics of brucellosis patients. sensitivity and specificity of clinical diagnosis compared with laboratory - confirmed diagnosis of brucellosis. | during 20142015, patients in northeastern kenya were assessed for brucellosis and characteristics that might help clinicians identify brucellosis. among 146 confirmed brucellosis patients, 29 (20%) had negative serologic tests. no clinical feature was a good indicator of infection, which was associated with animal contact and drinking raw milk. |
in the last 20 years, several research groups have explored treating conventionally untreatable epilepsies with delivery of aeds directly into epileptogenic tissue, the cortical subarachnoid space, or the cerebral ventricles [126 ]. this emerging strategy of intracranial pharmacotherapy uses some type of drug delivery device placed inside the cranium. this distinguishes it from both systemic pharmacotherapy, which delivers drugs into the brain through the gastrointestinal, dermal, and/or cardiovascular systems, and from intrathecal pharmacotherapy, which delivers drugs through the theca of the spinal cord. the present article reviews the diverse origins, present state, main challenges, and future prospects for intracranial pharmacotherapy. we focus on our efforts to develop a feedback - controlled intracranial drug delivery device, the subdural hnp, for neocortical epilepsies. historically, four major strategies have been used for the treatment of epilepsies. these are dietary and behavioral therapy, systemic pharmacology, and neurosurgery. dietary therapy and behavioral therapy, the overwhelming majority of drug refractory epilepsies (dre) can not be controlled with these strategies. neurosurgical interventions reduce or eliminate seizures by either removing the epileptogenic zone (e.g., temporal lobectomy, hemispherectomy, neocortical tissue resection) or destroying the neural pathways of seizure propagation (e.g., by callosotomy, subpial resection). while these strategies can improve or cure many patients, both interventions are burdened with the risk of damaging normal neural tissue. however, during systemic aed intake the entire body is exposed to the compound, although the targeted epileptogenic zones occupy less than a thousandth of the body mass. indeed, a neocortical seizure focus with an average tissue volume of 7 cm is 10,000 less than the approx. 70,000 cm body volume of a 70 kg and 180 cm high person. the vagus nerve stimulator (vns) of cyberonics (houston, tx), approved by fda for dre in 1997, marked a new approach to epilepsy therapy. this device belongs to the family of neuroprostheses, which also includes the deep brain stimulator (dbs) [28, 29 ] and the responsive neurostimulation (rns) system by neuropace (mountain view, ca) [3032 ]. intracranial pharmacotherapy is the product of the same intellectual wave that, departing from the conventional therapies, produced the vns and other brain stimulation devices, as well as the ideas that intracerebral gene transfer [33, 34 ], cell transplantation [35, 36 ], or local cooling might also be used to treat focal epilepsies. approximately 30% of the epilepsy patient population will not achieve complete remission of seizures with standard aed therapy [38, 39 ]. this translates to about 600,000 people in our country and almost 15 million in the rest of the world with dre [8, 40, 41 ]. nearly a third of these patients suffer the severe condition of one or more seizures per month. many dre patients, especially those with mesial temporal lobe epilepsy (mtle), are candidates for neurosurgical intervention. however, about 90% of patients with severe dre are unsuitable for surgical tissue resection / lesion [42, 43 ], because the seizure - generating regions (a) overlap primary sensory, primary motor, or language (figure 1) areas, (b) occupy too large a tissue mass in one lobe or involve multiple foci which are multilobar and/or bihemispheric or (c) are nonlesional and difficult or impossible to localize. we estimate that there are about 140,000 dre patients in the us who might be considered as potential candidates for some form of nontraditional epilepsy treatment, including intracranial pharmacotherapy. the idea of treating epileptic seizures with drugs delivered directly into the brain is related to the paradigm shift in medicine that took place in the 1950s and 1960s, leading to the cardiac pacemaker, cochlear implant, and other implanted devices. thus, the pacemaker successfully implanted in the initial groups of patients could not be designed without the commercial availability of the transistor ; the vns could not be constructed without the microprocessor. the neurostimulators and other neuroprostheses designed to correct abnormal brain functions [4547 ] opened the eyes of the medical community to new possibilities in the treatment of neurological disorders. it has also become clear that with proper neurosurgical techniques and post - implantation care these devices cause no major damage in neural tissue, or at least such damage is not inherent to their use and does not carry substantially more risk than short - term intracranial electrode or catheter placement. the histopathology of brains of parkinson 's disease patients treated with dbs showed no differences in stimulated and nonstimulated tissues adjacent to the lead - track. in epilepsy clinical trials, no major side - effects were reported during the course of centromedian thalamic stimulation, just as no adverse stimulation - induced side effects were observed in epilepsy patients implanted with the rns device. thus, while these data obviously could not provide information on whether long - term intracranial drug applications would also be free of side - effects, they suggest that such interventions are not accompanied with prohibitive risks. neuropharmacological studies have shown that localized, intracerebral drug applications can modulate, prevent or stop epileptiform eeg and behavioral events. as early as 1970, collins reported that muscimol, applied topically on the neocortical surface, blocked focal seizures induced by penicillin, bicuculline and picrotoxin, in rats. muscimol could also suppress audiogenic seizures if injected into the inferior colliculus, a structure later proven to be the generator site of sound - induced eeg seizures. piredda and gale showed in rats that local application of muscimol into the deep prepiriform cortex can temporarily eliminate epileptogenicity in this region, concluding that this area may also represent a site at which gaba agonists could function therapeutically to control epileptogenesis. in smith. 's paper describing the antiepileptic effect of lidocaine injected into the deep prepiriform cortex, they suggest that microchip and implantable pump technology should make it possible to construct a system that would predict onset of a seizure and then inactivate the neurons in the focus before they could initiate an ictal event. shortly after, eder. reported that cortically delivered diazepam can attenuate bicuculline - induced local epileptiform eeg spikes, again suggesting the possible role for aed perfusion directly on seizure focus as a therapy for intractable partial seizures. seizures usually originate in discrete epileptogenic zones (figures 1(a) and 1(b)), while the rest of the brain may function normally until the electrophysiological seizure activity propagates to neighboring or even more distant structures. this has justified the search for ways to pharmacologically control cortical or subcortical epileptogenic zones, without the unnecessary and often harmful exposure of the body and the rest of the brain to drugs. advances in medical device manufacturing, neurostimulation research, intracerebral aed pharmacology and clinical epileptology were synthesized into specific engineering solutions for intracranial pharmacotherapy for focal epilepsy at the beginning of this decade. in 2000, a microcomputer - controlled intracerebrally implanted drug delivery device, in which the timing and duration of drug deliveries are determined by the implanted brain tissue 's own electrical activity was described. also in 2000, stein. published a study demonstrating the efficacy of an automated drug delivery system for focal epilepsy in rats, concluding that such therapy might avoid some of the problems inherent to systemic administration of antiepileptic drugs. in 2000 fischell. patented a responsive implantable system for the treatment of neurological disorders (us patent # 6,134,474), although these inventors focused on electrical stimulation and medication released into the cerebrospinal fluid of the human patient as the therapeutic interventions. the idea of focal methods of drug delivery tied to eeg activity was embraced by investigators at the national institutes of health and within a few years the development of intracranial pharmacotherapy for epilepsy has become the objective of several research teams in academia, in some cases closely collaborating with startup companies (e.g., sierra neuropharmaceuticals, medgenesis therapeutix, and others). the goal of treating focal epilepsy with intracranially delivered drugs is being pursued in diverse pathways. cortically implanted phenytoin ethylene - vinyl acetate (evac) controlled - release polymers have been demonstrated to reduce seizures in a cobalt - induced model of focal neocortical epilepsy. a second strategy aims to deliver aeds into the brain using a different approach : by utilizing fully implanted, responsive or nonresponsive, neuroprosthetic devices. these devices employ either intraparenchymal catheters or catheter / electrode units, or cannulas placed in the cerebral ventricles, or sealed, subdural fluid delivery / recording electrode units overlaying the neocortical epileptogenic zone(s). one promising technique for intraparenchymal drug administration into the seizure focus or foci, with or without electrophysiological recording capability, uses convection - enhanced delivery (ced), which seeks to distribute a therapeutic agent homogeneously throughout clinically significant volumes of brain parenchyma. the relative safety of this method was shown in nonhuman primates, while its efficacy to reduce the severity of amygdala - kindled seizures in rats was demonstrated by gasior., who administered n - type calcium blocker conotoxins into the amygdala via ced. however, this strategy addresses systemic, but not cns - related toxicity, at least with the devices and drug delivery protocols that have been tested in intracerebroventricular seizure - control studies. the anesthetic side - effect of intracerebroventricular pentobarbital administration in rats is a pentobarbital action that can be eliminated without decreasing its neocortical seizure - preventing potency by administering this compound transmeningeally into the cortex via a sealed device. this transmeningeal route offers another avenue for intracranial aed administration, with the assistance of the subdural / subarachnoid hnp device. the subdural hnp is a type of intracranial drug delivery device, which offers drug deliveries directly into epileptogenic brain tissue, via sealed, single or multiple, regularly flushed, subdural / subarachnoid units equipped with recording electrodes / sensors to provide feedback from the exposed neural tissue (figure 2). the basic concept and architecture of the device have been described [9, 17, 19, 55, 56 ]. its key distinguishing feature is the integration of both fluid exchange / drug delivery ports and recording electrodes / sensors into a silicone strip or grid that can be placed in the subarachnoid space (figure 3). thus, it is this subarachnoid space through which the device delivers aeds or other seizure - preventing therapeutic solutions into the underlying epileptogenic zone(s). consequently, the subdural hnp achieves pharmacological / therapeutic effects via drug diffusion through the cerebral arachnoid and pia maters, virtually eliminating the risk of damaging normal neocortical tissue. this transmeningeal pharmacotherapy is based on a known, albeit medically underutilized, physico - chemical property of the cerebral leptomeninges ; that is, their permeability to water - soluble molecules. this is why neurotransmitters released into the neocortical extracellular space can diffuse into a fluid collection device placed on the pia mater [57, 58 ]. thus, water - soluble small molecules, like n - methyl - d - aspartate (nmda) or methylene blue, penetrate through these membranes into the underlying cerebral cortex and stay close to the delivery area (figure 4) : findings consistent with prior autoradiographic studies. inclusion of recording electrodes (and in the future neurochemical sensors) in the subdural drug delivery unit gives potential for the device to execute three important functions. first, electrophysiological recordings can provide feed - back on the effects of the delivered drugs, so that their delivery parameters can be flexibly adjusted, eliminating the danger of applying too high, neurotoxic doses, while helping to avoid the application of too low, thus inefficient drug concentrations. second, these recordings can potentially provide information for the treating physician on the neurophysiological impact of the subdural implant, so that adverse reactions, if these occur, can be recognized and treated early. third, electrophysiological data acquisition may permit seizure prediction and seizure detection, thus allowing subdural drug delivery in a responsive, on - demand fashion, upon the occurrence of pre - seizure or seizure - onset signals. this set of three feed - back functions separates the subdural hnp from aed - releasing polymers, gene therapy and cell transplantation. multiple drug delivery / recording units, shaped either as strips or grids, can obviously also be used. thus, the device could apply treatment over large cortical areas, without the spatial limitations imposed upon devices using tissue - penetrating cannulas, catheters or tubes. this may allow the treatment of extended, multiple, and/or bilateral seizure foci, as well as diffusely distributed epileptogenic zones that are difficult to localize even with intracranial recordings. presently, muscimol is emerging as the choice of aed for the first generation subdural hnps [19, 20 ], because of the following reasons : (a) cortical seizure - preventing efficacy in low (1 mm ; figure 5) concentrations, (b) fast - developing (~30 seconds) pharmacological action, (c) high water - solubility, (d) long - term (~4 month) stability in solution, and (e) efficacy at neutral ph. another feature of the subdural hnp design is the sealing membrane around both the individual drug delivery ports (figures 2 and 3) and the entire subdural unit. this prevents significant drug spillover to neighboring, normal cortical areas and limits systemic exposure to the administered aed. during pentobarbital administration into the neocortex through a sealed epidural cup (as in rodents the thin, permeable dura mater allows transmeningeal drug application via such devices) focal seizures can be readily prevented, while the rat remains awake. the lack of significant drug spillover into the csf or the rest of the cerebral cortex is also demonstrated in figure 5(a). it illustrates that if one side of the frontal cortex is pretreated with transmeningeal saline, while the contralateral site is simultaneously pre - treated with muscimol in the same way, subsequent application of ach into both cortical areas leads to focal seizures in the saline - treated but not the muscimol - treated side. the hnp minipump is a dual, fully implantable, and transcutaneously refillable, miniature peristaltic device (figure 6). its design and two fluid reservoirs allow the hnp to execute, in an alternating fashion, two functions. one reservoir is for aed delivery, while the other one is for delivering a flushing solution (e.g., saline or artificial csf) or removing csf from the subarachnoid space to prevent pressure increase. in the experiment shown in figure 5(b), muscimol was delivered with this minipump into the cortical subarachnoid space of a freely moving rat : the apparatus was mounted on the head of the animal (figure 6). in addition to data generated in rats implanted with epidural drug delivery devices [1720, 22 ] (figures 46), monkey and human studies also suggest the therapeutic viability of the subdural hnp. in anthropoid new world monkeys (saimiri sciureus), muscimol delivery into the subarachnoid space prevented focal neocortical seizures. in patients with temporal lobe epilepsy (n = 3), in a neurosurgical setting prior to tissue resection, lidocaine (an irb - approved compound for the hnp project at the time) was applied to the pial surface overlaying the epileptogenic zone. within minutes, this local treatment markedly reduced the frequency of eeg spiking in the epileptogenic area. thus, seizure susceptibility in the primate cerebral cortex can be modulated by drugs delivered directly to the pial surface. the prospects of intracranial pharmacotherapy depend on how the intertwined scientific, engineering, clinical, and neurosurgical problems will be solved and the pertinent regulatory and commercial issues navigated. our initial aim is to investigate the most feasible simplest, nonresponsive version of the subdural hnp in phase i / ii clinical trials, after rat and monkey studies on safety and efficacy are completed. this can set the stage for testing the more complex, responsive hnp version [9, 17, 19 ]. the first main scientific challenge of hnp development is the thorough elaboration of the safety profile of the device. although subdural electrode grids and strips can be routinely kept over the neocortex for a few weeks with minimal or no complications for diagnostic purposes (figure 1), the safety of using the subdural unit of the hnp (figure 3) for months or years to deliver drug solutions must be independently investigated. second, the potential of tolerance to the applied hnp drugs and the possibility of withdrawal seizures upon the cessation of this treatment must be explored. that tolerance to antiepileptic drugs, including those acting on the gaba system, can develop during their long - term use has been demonstrated by lscher and schmidt, while the possibility of withdrawal seizures after the cessation of continuous, long - term intracortical gaba administration was shown by brailowsky.. since muscimol is a gaba - a receptor agonist, clarifying the tolerance - inducing and withdrawal - seizure - inducing potency of this drug is essential. the third main scientific challenge is to understand the cellular and neurochemical effects, as well as pharmacokinetics, of transmeningeally delivered aeds, so that these drugs can be rationally used with intracranial devices. figure 7 illustrates the complexity of the likely effects and fate of muscimol molecules upon their delivery into the cerebral subdural/ subarachnoid space. mapping of the diverse cellular actions of this molecule, including its heterogeneous effects on postsynaptic, extrasynaptic and presynaptic gaba - a receptors [62, 63 ], as well as its clearance pattern in the neocortex, require a major research effort. but the furnished information, along with corresponding data for other transmeningeal aeds, will help to elaborate the optimal delivery conditions for seizure - controlling drugs delivered with the subdural hnp. the engineering challenges are related to the complexity of the hnp, as it involves both pharmacological and electrophysiological components. this complexity is the product of the goals of (a) delivering drugs into the epileptogenic zones in a feedback - controlled manner, requiring electrophysiological monitoring of the drug - exposed tissue, (b) providing information on the recording and drug delivery functions of the device to the treating physician, with the option of modifying these functional parameters, if needed, requiring the use of a bidirectional rf communication module, and (c) supplying this apparatus with sufficient electrical power for years, requiring the integration of a battery rechargeable transcutaneously via electromagnetic coupling. the drug delivery minipump (figure 6) consumes about 8 mah energy, almost 10-times more than the rest of the device, and thus powering of the hnp needs a different engineering solution than what is adequate for the vns, dbs or rns. the complexity of the hnp as a seizure - preventing device mimics the evolutionary principle of implementing multiple (neural, endocrine and immune) control systems to maintain physiological functions and prevent disease. but complexity comes with a price, and this price for the subdural hnp is the increased likelihood of hardware errors : a risk less threatening for simpler drug delivery implants. yet the clinical challenges include the identification of the ideal candidates for intracranial pharmacotherapy and laying the groundwork for post - implantation patient care. within the population of focal dre patients who are not amenable to resective surgery the right subclass for the device needs to be further clarified. patients with nonlesional focal neocortical epilepsy, the most challenging group of surgical candidates, are likely candidates for subdural hnp treatment. but this is a heterogeneous group, including patients with frontal, parietal, extramesial temporal and occipital epileptogenic zones, some with involvement of mesial temporal lobe structures, and some with much less easily recognized influences from subcortical structures. the other challenge is to develop infrastructure for post - implantation care and identify protocols for (a) adjusting the right drug delivery parameters (concentration, volume, frequency) for the hnp, based on local recordings transmitted from the implant to the physician, (b) setting up the implant status indicators, and (c) confirming the integrity of the transcutaneous minipump - refilling and battery recharging modules. the main neurosurgical challenges are to determine (a) the optimal size and shape of the subdural / subarachnoid recording / drug delivery unit (figure 3), (b) the method for its safe placement over the epileptogenic zones in a way that assures fixed position, (c) the best technique to tunnel the wires and tubing from the hnp controller apparatus to the subdural implant, and (d) the optimal subcutaneous location for the minipump refilling ports and inner coil of the battery recharging circuit. whether the subdural hnp, along with other intracranial pharmacotherapy implants, will have a limited niche for the treatment of a quite specific class of patients or it may be useful for a larger patient population even beyond those afflicted by epilepsy, is difficult to predict. should the first generation of these devices prove to be safe and effective in the initial clinical trials, it will justify the development of the responsive version that could be used in patients with less frequent seizures. this device may well use microelectrodes to record multi - neuron activity, instead of conventional eeg electrodes, as monitoring cellular electrophysiological signals appears to be more suitable for early seizure detection / prediction and thus for activating aed delivery [6567 ]. newer hnps may also use a wide spectrum of drugs besides muscimol. since this device delivers drugs directly into the extracellular space, bypassing the blood - brain barrier (bbb), it can use bbb - impermeable compounds, such as neuroactive peptides and proteins, to achieve therapeutic action. investigators in other fields of neurology may adapt intracranial pharmacotherapy for maximizing stroke recovery after neocortical infarcts, treating cortical tumors, or improving the cognitive functions in alzheimer 's disease. orrin devinsky and ruben i. kuzniecky are consultants and members of the board of directors at cortica, inc., a company founded for the commercialization of the hnp. | intracranial pharmacotherapy is a novel strategy to treat drug refractory, localization - related epilepsies not amenable to resective surgery. the common feature of the method is the use of some type of antiepileptic drug (aed) delivery device placed inside the cranium to prevent or stop focal seizures. this distinguishes it from other nonconventional methods, such as intrathecal pharmacotherapy, electrical neurostimulation, gene therapy, cell transplantation, and local cooling. aed - delivery systems comprise drug releasing polymers and neuroprosthetic devices that can deliver aeds into the brain via intraparenchymal, ventricular, or transmeningeal routes. one such device is the subdural hybrid neuroprosthesis (hnp), designed to deliver aeds, such as muscimol, into the subdural / subarachnoid space overlaying neocortical epileptogenic zones, with electrophysiological feedback from the treated tissue. the idea of intracranial pharmacotherapy and hnp treatment for epilepsy originated from multiple sources, including the advent of implanted medical devices, safety data for intracranial electrodes and catheters, evidence for the seizure - controlling efficacy of intracerebral aeds, and further understanding of the pathophysiology of focal epilepsy. successful introduction of intracranial pharmacotherapy into clinical practice depends on how the intertwined scientific, engineering, clinical, neurosurgical and regulatory challenges will be met to produce an effective and commercially viable device. |
the danish national board of health issued a new guideline on prenatal screening in 2004.1 this guideline recommends that all pregnant women should be offered a first - trimester scan, including risk assessment for trisomy 21, based on a combination of maternal factors, ultrasound, and biochemical screening, and a second - trimester scan for fetal malformations. since june 2006, all obstetric departments in denmark have offered these two ultrasound screenings, and > 90% of danish women choose to have both first - trimester and second - trimester screening performed.2 the danish fetal medicine database was initiated by the danish fetal medicine specialists and established in 20082010 through collaboration between all obstetric departments in denmark. the aim of the danish fetal medicine database is to provide a tool for local and national quality assessment and research within prenatal screening in denmark, and to ensure uniform high screening quality by providing relevant and useful feedback on screening performance to all departments and regions on a regular basis. the danish fetal medicine database contains data from all pregnant women with prenatal screening results dating back to january 1, 2008, from all hospital departments of obstetrics and gynecology in denmark (bornholm hospital from january 1, 2011). in 2014, the database contained data about > 362,000 pregnancies, of which 353,049 are singleton pregnancies. of them, 359,058 have had first - trimester screening for trisomy 21 and/or second - trimester screening for fetal malformations. the danish fetal medicine database consists of data from the following four sources : the local astraia fetal medicine databases (astraia gmbh ; www.astraia.com) used in all departments of obstetrics and gynecology in denmark, the danish cytogenetic central register, the danish national patient register, and the danish national birth register (figure 1). the primary data source for the danish fetal medicine database is the local astraia databases, from where the following data are retrieved : data on maternal characteristics, first - trimester screening data, including risk assessments, fetal biometries, and registered prenatal malformations at any gestational age. these data have been recorded as part of routine obstetric practice at all departments in astraia in accordance with national standards since january 1, 2008, and the national database includes data on singleton and twin pregnancies. on a daily basis, data from all local astraia servers are automatically sent to the national database after encryption. before the national database was initiated, national standards on how the pregnancies were dated, how the first - trimester risk assessment was performed and handled, and use of specified biometric reference curves had been issued.3 the international classification of diseases, 10th revision code system (icd-10) is used to code malformations in the fetus and in the infant. pregnancy outcome data are collected from the danish national patient register (including spontaneous and induced abortions and information on congenital malformations), the danish national birth register (information about pregnancy complications, delivery, and the newborn), and the danish cytogenetic central register (results of pre- and postnatal chromosome analyses). information from these data sources is linked to the danish fetal medicine database using the unique personal identification number (cpr number), which everyone is given at birth or on immigration to denmark. algorithms have been developed to ensure that linking of data from different registries is pregnancy specific. for each pregnancy ultrasound scanning, information on one or more fetuses is linked to a karyotype result if performed during the pregnancy or just after birth of the fetus / infant. in addition, information on the outcome of pregnancy is available for all pregnancies, whether it is miscarriage, termination, stillbirth, or live birth. more than 95% of the pregnancies have an outcome registered. in some of the cases with unknown outcome, migration to other countries the high completeness of all variables and the outcome data is unique and internationally highly acknowledged. the clinicians have access to their locally collected data and selected quality indicators in comparison with national data through the web - based reporting system (in danish : analyseportalen) (figure 1). the quality indicators provide clinicians and administrators with information about the quality of the first - trimester screening for chromosomal abnormalities and the second - trimester screening for anomalies (table 2). a danish fetal medicine study group was established during implementation of the national database with one representative from all danish obstetric / fetal medicine departments joining the study group. this has proven to be essential in the process of cleaning up data, obtaining missing data, and maintenance of the local data collection system. a major upgrade of the fetal medicine database and the data collection system will be implemented in 2016. it includes an additional number of variables on prenatal ultrasound scanning data and a new function, which enables update of the national database when corrections in the local source data (astraia) are made. the database is updated annually with information on congenital malformations and postnatal karyotypes on all live born babies. audit of the data used to calculate the detection rates of trisomy 21, neural tube defects, and abdominal wall defects is performed yearly. the audit has recognized that data on fetal malformations retrieved from the danish fetal medicine database are less complete, especially for the years 20082010. in the planned upgrade of the database in 2016, data on fetal malformations will be entered by organ - specific tick boxes in addition to the recorded icd-10 codes, which is expected to improve the quality of the data substantially. the database serves as an important data source and has in total provided data for 44 research projects that have been presented at international conferences and/or published in peer - reviewed journals. the first - trimester screening results in denmark in 20082013 have been published in a paper which also provides more detailed information about the database establishment and organization and thus serves as a reference paper for future research based on the database data.4 due to the large amount of population - based data in the database, it has enabled us to study rare outcomes, such as rare chromosomal abnormalities and other adverse outcomes in both singleton and twin pregnancies.58 a recent editorial article in the scandinavian journal of obstetrics and gynecology complimented the danish fetal medicine collaboration and their efforts in the establishment of the danish fetal medicine database.9 the author hopes that the danish fetal medicine database can be used to identify associations between early fetal development, obstetric pathologies, and morbidities that are recognized in infancy, and then to use these data prospectively to improve perinatal and infant outcomes in the future. the danish fetal medicine database has an interdisciplinary steering committee with fetal medicine experts and sonographers from all five regions in denmark, as well as a clinical geneticist and a representative from the registry support centre of clinical quality and health informatics (east). this unit has supported the establishment of the database by hosting the servers and developing the software system that provides local and national access to data. after initial establishment of the database, the danish fetal medicine database was included as one of > 60 clinical databases funded, hosted, and supported by the danish clinical registries (rkkp) and financed and owned by the danish regions. the establishment of the danish fetal medicine database has had an important impact on the national fetal medicine collaboration. the local and national data are discussed at annual meetings and provided the information needed to discuss local differences and possible changes necessary to optimize the national screening program. within a few years, it has been possible to establish a national clinical database, including data regarding fetal screening, prenatal diagnostics, and pregnancy outcome. the primary data source is the astraia system, which is the local fetal medicine database and electronic health care record used at all obstetric / fetal medicine units in denmark. the quality and completeness of the entered data are extremely high due to the use of data entry validation and decision - aid support. furthermore, since all data are transferred electronically to the national database, no additional registration or data entry is necessary, thus, there is no extra workload for the clinicians and administrative staff when collecting data. the danish fetal medicine study group, with representatives from all departments, has proven to be advantageous in terms of management and maintenance of the data collection system, as well as solving practical and legal issues in the process of cleaning up and obtaining missing data. | aimthe aim of this study is to set up a database in order to monitor the detection rates and false - positive rates of first - trimester screening for chromosomal abnormalities and prenatal detection rates of fetal malformations in denmark.study populationpregnant women with a first or second trimester ultrasound scan performed at all public hospitals in denmark are registered in the database.main variables / descriptive datadata on maternal characteristics, ultrasonic, and biochemical variables are continuously sent from the fetal medicine units astraia databases to the central database via web service. information about outcome of pregnancy (miscarriage, termination, live birth, or stillbirth) is received from the national patient register and national birth register and linked via the danish unique personal registration number. furthermore, results of all pre- and postnatal chromosome analyses are sent to the database.conclusionit has been possible to establish a fetal medicine database, which monitors first - trimester screening for chromosomal abnormalities and second - trimester screening for major fetal malformations with the input from already collected data. the database is valuable to assess the performance at a regional level and to compare danish performance with international results at a national level. |
ectopic pregnancy is a recognized complication of in vitro fertilization (ivf) and embryo transfer. the most frequent implantation site of the ectopic pregnancy is in the fallopian tube, most commonly in its ampullary segment (80%). heterotopic pregnancy (hp) is defined as the presence of an intrauterine pregnancy (iup) coexisting with an ectopic pregnancy. it is a rare entity with an incidence of 1/10,0001/50,000 in natural conceptions. in pregnancies, resulting from assisted reproductive technology (art), the incidence is greater, ranging from 1/100 to 1/3,600, nearly as high as 1% in some series. the big difference in these percentages is attributed to the higher incidence of pelvic inflammatory disease (pid) observed currently resulting in tubal damage as well as ovarian stimulation and transfer of more number of embryos. with the increasing use of transvaginal sonography in art pregnancies, more cases of cervical ectopic pregnancy a recent review has identified only 37 cases reported in english literature, all but four of which are the result of art. so far, successful management resulting in healthy live birth of the iup was reported in very few cases of heterotopic cervical pregnancy (hcp). the aim of this paper is to emphasize the need for a high index of suspicion of this clinical entity during the routine first - trimester ultrasound examination, even in the presence of an intrauterine gestation, especially so in art conceptions. thirty - four - year - old female with primary infertility had presented to us following one failed ivf cycle done outside 2 years ago for tubal factor. she had a history of open myomectomy done several years ago, following which several cycles of ovulation induction (oi) + timed intercourse and oi + interuterine insemination were done. diagnostic laparoscopy done outside before the first ivf had shown an enlarged uterus, with multiple fibroids and dense adhesions. day 2 scan showed antral follicle count of 9/12 and anterior wall fibroid measuring 42 mm 38 mm and 16 mm 15 mm, posterior wall fibroid measuring 30 mm 30 mm, with a normal appearing cavity. husband 's semen analysis done at our institute showed severe oligo - astheno - teratozoospermia. the patient underwent a diagnostic hysteroscopy and endometrial scratching before intracytoplasmic sperm injection (icsi). the patient underwent controlled ovarian stimulation by long agonist protocol, 13 oocytes were retrieved, 11 m2 and 2 gv. icsi was done and it resulted in 11 grade a embryos, which were frozen at 8 cell stage. frozen embryo transfer (fet) was done after 8 weeks, 3 embryos 8 cells, grade a were transferred under ultrasound guidance with an endometrial thickness (et) of 11.6 mm, at a depth of 1.2 cm. luteal support was given and beta - human chorionic gonadotropin (beta - hcg) done on day 16 was 956 miu / ml, which showed a doubling in 48 h and scan done a week later showed an intrauterine gestation of 6 weeks + 6 days of gestation had shown an intrauterine live dichorionic diamniotic twin gestation of 6 weeks, with one sac ? close to the internal os. the patient was admitted at 8 weeks of gestation with a diagnosis of bleeding hcp. the beta - hcg on that day was 154,509 miu / ml and hemoglobin (hb) was 11.1 g / dl. on per speculum, cervix was ballooned up and blood was seen trickling through the external os. she continued to have mild bleeding overnight, and the hb done on the following morning was 9.6 g / dl. the patient was extensively counseled and management options were discussed and they opted for selective fetal reduction of the cervical pregnancy using potassium chloride (kcl). the patient started bleeding heavily on the subsequent day and her hb dropped to 7.5 g / dl. three units blood was transfused, and once the patient was hemodynamically stable, uterine artery embolization was done using polyvinyl alcohol particles after taking an informed consent. subsequently, injection methotrexate 50 mg i m was also given 24 h later, she started to expel the products of conception, emergency evacuation was done in view of increased bleeding per vaginum. she was finally discharged in a stable condition after a week, with a beta - hcg of 2244.8 miu / ml and hb of 9 g / dl. she was followed up with weekly beta - hcg, and it became negative after 4 weeks. she had a second fet after 8 months of the last methotrexate injection, at an et of 9 mm and a depth of 8 mm and had two embryos transferred in blastocyst stage. in both in art pregnancies, awareness of the possibility of a hp and its sonographic appearance plays an important role in accurate and timely diagnosis and successful management to help avert potentially fatal consequences. it is reported that approximately 70% of hps are diagnosed between 5 and 8 weeks of gestation, 20% are between 9 and 10 weeks, and the remaining 10% are after 11 weeks. the presence of an intrauterine gestation does not exclude the possibility of a concomitant extra - uterine pregnancy. careful evaluation of the cervical canal, uterine horns if any, and the adnexa should be a critical part of the early pregnancy ultrasound even after iup has been confirmed. this is true, especially when the blood hcg level is much higher than expected as was in our case. the cervical component of the hcp can be mistaken for other pathologies such as incomplete abortion, normal pregnancy with low uterine implantation, ep in a cesarean section scar, nabothian cyst, and cervical mass. the gestational sac in the cervix is typically eccentrically located and is usually round and similar to a normal pregnancy. it may, however, become elliptical or flattened, thus making diagnosis difficult, cardiac activity is basically pathognomonic. histologically, the diagnosis can be made by rubin 's criteria on the surgical specimen where cervical glands are opposite to the trophoblastic tissue, the trophoblastic attachment is below the entrance of the uterine vessels to the uterus or the anterior peritoneal reflection, and fetal elements are absent from the uterine corpus. as many pregnancies today are diagnosed early and no hysterectomy is performed, rubin 's criteria can often not be applied. other factors predisposing to hp are identical to those predisposing to ectopic pregnancy : factors related to ivf such as large number of transferred embryos, a transfer near the uterine horn, excessive pressure on the syringe and deep insertion of the catheter during transfer, the quality of the embryos, the hormonal milieu at the moment of transfer, the use of gonadotropins, the amount of fluid used as media for the embryos, and also adhesions related or not related to endometriosis and pid. for hps, transferring four or more embryos during art is a defined risk factor. however, no single risk factor, laboratory test, or combination of these is sensitive or specific enough to predict the occurrence of an hcp. theoretically, it is possible that after pushing the plunger during et, the existence of sticky mucus may cause one of the embryos to adhere and slide back to the canal ; thus, remnants of cervical mucus may be a risk factor. some authors have thus proposed that removing the cervical mucus effectively may be a preventive measure to try and avoid cervical pregnancy. the goals of treatment of hcp are the protection of a coexisting iup, the minimization of blood loss, and fertility preservation. since it is a rare entity, there are no standard protocols, but several options exist for the management. before routine ultrasound was introduced in obstetrics, cervical pregnancy was usually diagnosed during dilatation of the cervical canal and curettage and it usually resulted in life - threatening hemorrhage. as a result of this, most pregnancies of this kind ended in hysterectomy to save the patient 's life. the liberal use of high - resolution ultrasound as well as color doppler in obstetrics has enabled diagnosis of early cervical pregnancy. thus, various medical methods could be successfully applied in treating early cervical and hcp. the various medical therapeutic procedures which have been performed include selective fetal reduction procedure by ultrasound - guided kcl or methotrexate injections and systemic methotrexate in some cases. in the last two decades, cervical pregnancies most commonly have been treated by transvaginal ultrasound - guided selective fetal reduction procedure by injecting kcl into the ectopic component. systemic methotrexate is associated with adverse effects such as thrombocytopenia, leukopenia, elevated liver enzymes, and especially teratogenic effects, and thus, a treatment of choice only when our aim is not to preserve the simultaneous iup. our patient apart from the cervical pregnancy also had a viable iup because of which we did not resort to systemic methotrexate as the first line of treatment. recently, ligation of the hypogastric artery, embolization of the uterine arteries as well as cerclage of the cervix have also been performed to treat cervical pregnancy. in 2003, jozwiak. uterine artery ligation and embolization may be options when the iup is not of concern as latter may result in the radiation of the viable iup, and influence on endometrial receptivity, which could decrease future fertility. in our case, we did uterine artery embolization after extensive counseling as a life - saving measure. systemic methotrexate was also used in our patient, only once the decision to terminate the pregnancy was finally taken. other surgical treatments include suction evacuation, cervical curettage with or without cerclage, and foley catheter insertion. foley catheter insertion and cervical cerclage seem universal salvage maneuvers to stop early or late bleeding, whichever technique is used to terminate the cervically located gestation. the minimally invasive approach of embryo aspiration without complete evacuation of the conceptus in a hemodynamically stable patient can be the first treatment of choice in hcp if diagnosed early in pregnancy. however, before this procedure, the patient needs counseling about the risk of the potential complications including bleeding, abortion of the iup, cervical mass infection which may lead to premature rupture of the membrane, and postpartum bleeding and severe bleeding leading to potential need for emergency procedures including even hysterectomy. for isolated cervical pregnancies, complete evacuation of the pregnancy or systemic methotrexate administration seems a better choice. advancing technology and early diagnosis of hcp have led to a remarkable improvement in the prognosis of cervical gestations including hcp. due to the limited number of cases reported in literature, there is no single universally accepted management protocol for hcp. the therapeutic modality chosen should be able to successfully treat the hcp without causing threat to the concomitant iup and/or the patient 's life. | the wide use of assisted reproductive technologies has contributed to the increased risk of ectopic and subsequently heterotopic pregnancy (hp) rate. cervical ectopic pregnancy is a very rare and life - threatening form of ectopic pregnancy that can also present as hp. we are describing here a case of 34-year - old woman who presented with bleeding heterotopic cervical pregnancy (hcp). the concomitant viable cervical and intrauterine pregnancies were diagnosed at 8 weeks of gestation. selective fetal reduction was done for cervical pregnancy following which uterine artery embolization was done as a life - saving measure, and subsequently, injection methotrexate was also given. although cervical component of hcp has the potential for high morbidity due to massive hemorrhage, the mortality rate is low due to early ultrasonographic diagnosis. a high index of suspicion is mandatory for early diagnosis. |
a 70-year - old white male with a previous l3-s1 posterior decompression and fusion presented with pain radiating into the bilateral lower extremities, worse on the left than the right. magnetic resonance imaging revealed a disk bulge at l2 - 3 with associated canal stenosis. he was taken to the operating room for a lateral transpsoas approach to the l2 - 3 disk space with diskectomy and interbody cage placement. baseline s - emg discharges were recorded from the bilateral tibialis anterior electrodes (fig. once the diskectomy had been completed and the endplates were prepared, a trial spacer was inserted into the disk space causing distraction across the disk space. at this time, s - emg activity ceased. the interbody cage was then inserted into the disk space again causing distraction across the disk space and the s - emg discharges ceased (fig. the patient had immediate postoperative improvement in his radicular symptoms, although he had significant back pain attributed to presumed endplate injury during the procedure, which ultimately resolved. a 67-year - old african american male with a previous l3-s1 posterior decompression and fusion presented with left thigh pain, left foot pain, and left hip flexor weakness. workup revealed a herniated disk at l2 - 3 with canal stenosis and left neural foraminal stenosis. he was taken to the operating room for a lateral transpsoas approach to the l2 - 3 disk space with diskectomy and interbody cage placement. at the start of the procedure, s - emg discharges were noted from the left abductor hallucis electrode. as in case 1 s - emg discharges returned when the trial was removed, and then again ceased after cage placement. use of intraoperative emg monitoring has decreased postoperative parasthesias secondary to lumbosacral plexus injury from 30% to 0.7%. the monitoring modalities used during ltif typically include somatosensory evoked potentials, s - emg, and triggered emg. during placement of the retractor system, the dilator is rotated while stimulating directionally. response is typically monitored in the vastus medialis, tibialis anterior, biceps femoris, and medial gastrocnemius. depending upon the amplitude required to illicit a response, the surgeon can estimate his proximity to the nerves of interest to avoid injury. radiographically measured an average 41.9% increase in disk height, 13.5% increase in foraminal height, 24.7% increase in foraminal area, and 33.1% increase in central canal diameter in patients after surgical treatment via the ltif procedure. documented a 54% increase in the anterior - posterior plane and a 48% increase in the medial - lateral plane of thecal sac dimensions for a calculated canal area increase of 143% after ltif using magnetic resonance imaging. they concurrently measured statistically significant improvements in oswestry disability index scale and treatment intensity scale measurements. furthermore, using computed tomography scans, kepler. reported a 35% increase in neural foraminal area. disk height was increased by 58% when measured in the midline anteriorly and by 70% when measured in the midline posteriorly. they also found statistically significant improvements in oswestry disability index, short form-12 mental component summary, and physical component summary scores postoperatively. resolution of s - emg firing can be used to judge the adequacy of decompression during lumbar surgery. in a series of 120 cases of lumbar decompression via laminectomy, beatty. noted baseline s - emg firing in 18% of cases which was usually associated with clinical weakness (64%). after decompression of the l4 root, baseline quadriceps firing ceased in 84% of patients. after decompression of the l5 root, baseline tibialis anterior firing ceased in 97% of cases ; likewise decompression of the s1 nerve root alleviated gastrocnemius s - emg firing in 100% of cases. the 2 cases presented in this manuscript illustrate resolution of s - emg firing during ltif, which may represent decompression of the affected nerve roots. both of these cases were operated at the l2 - 3 level. in the first case resolution of emg firing may be explained by indirect decompression of the canal via ligamentotaxis as the l5 root traverses the l2 - 3 disk space. in the second case, s - emg firing was noted in the left abductor hallucis and resolved with distraction of the l2 - 3 disk space. again, this is may be explained by canal decompression via ligamentotaxis as the s1 root traverses the l2 - 3 disk space. in both cases, although s - emg discharges may be because of positioning or surgical manipulation, there are several reasons to believe that s - emg discharge in these 2 cases was related to pathological compression and resolved with indirect decompression. (1) s - emg firing was present immediately following induction of anesthesia, before positioning. (2) s - emg firing ceased with placement of the trial spacer and s - emg firing returned when the trial spacer was removed in both cases. (3) s - emg firing resolved with final placement of the interbody cage. therefore emg monitoring is employed routinely in the ltif approach to monitor and protect the lumbosacral plexus during retractor placement. unfortunately, only 18% of patients with lumbar radiculopathy have s - emg discharges at the time of surgery, usually patients who present with clinical weakness. in this subset of patients however, decompression of the involved root is associated with 84%, 97%, and 100% rates of s - emg resolution for the l4, l5, and s1 roots respectively. resolution of s - emg firing could potentially serve as an indicator of adequacy of indirect decompression in a subset of patients undergoing ltif. there is a growing body of evidence that indirect decompression of the spinal canal and neural foramina is achieved during ltif. previous authors have shown an association between resolution of s - emg firing and adequate nerve root decompression in open lumbar surgery. we have observed cessation of s - emg firing during ltif associated with distraction across the disk space that has correlated with clinical improvement in radicular symptoms in these 2 patients. emg monitoring is a necessary tool to perform the ltif procedure safely, to avoid injury to the lumbosacral plexus. there may be a subset of patients undergoing ltif in whom resolution of s - emg firing can be used as a marker for adequacy of indirect decompression intraoperatively. further study is needed to correlate s - emg cessation during ltif with clinical and radiographic evidence of indirect decompression. | purposethe lateral transpsoas interbody fusion (ltif) is an increasingly popular minimally invasive technique for lumbar interbody fusion. although a posterior approach to the lumbar spine has traditionally been favored for the treatment of canal stenosis and neural foraminal stenosis, a growing body of evidence suggests that indirect decompression of the spinal canal and neural foramen can be achieved using a lateral transpsoas approach to the lumbar spine. we present 2 cases that may suggest a role for spontaneous electromyography (s - emg) monitoring in assessing the adequacy of decompression during ltif.methodsthe 2 cases presented in this technical note illustrate resolution of s - emg firing during ltif, following distraction across the disk space. removal of the distracting device produced the return of s - emg firing. both of these cases were operated at the l2 - 3 level.resultsin the first case, s - emg firing was noted in the bilateral tibialis anterior leads. resolution of emg firing may suggest indirect decompression of the canal via ligamentotaxis as the l5 root traverses the l2 - 3 disk space. in the second case, s - emg firing was noted in the left abductor hallucis and resolved with distraction of the l2 - 3 disk space. again, this may be explained by canal decompression via ligamentotaxis as the s1 root traverses the l2 - 3 disk space.conclusionin both cases, distraction across the disk space resulted in resolution of s - emg discharges this correlated with an improvement in symptoms. these findings may suggest a role for s - emg as a marker for adequacy of decompression in a select subset of patients undergoing ltif. further study is needed to determine if resolution of s - emg is a useful measure of indirect decompression during ltif. |
adult male wistar rats were given 50 ppm cd in drinking water over a period of 1 - 24 weeks. the rats were killed and the cadmium concentration of whole blood, blood plasma red cells, liver and kidneys estimated. the plasma metallothionein concentration was measured by radioimmunoassay. kidney samples were taken for light, transmission and scanning electron microscopic examination. the accumulation of cadmium in the tissues was shown by a linear increase with time, after exposure for 12 weeks. plasma cd concentrations showed a clear increase after 3 weeks and preliminary investigation suggests that most is present as cd - thionein. early pathological changes in the rat kidney were seen around the 4 - 6 week period which coincided with the distinct rise in plasma cd. at 12 weeks, signs of tubular necrosis, interstitial fibrosis and glomerular epithelial cell hypertrophy were present in small areas of the cortex. by 24 weeks, the renal cortex showed clear evidence of tubulo - interstitial nephritis at a cd concentration of 60 micrograms cd / g wet weight.imagesfigure 4.figure 5.figure 6.figure 7.figure 8.figure 9.figure 10.figure 11.figure 12. |
|
for locally advanced cervical cancer, concurrent chemotherapy and radiation therapy (ccrt) has been recommended as a standard treatment [1, 2 ]. however, radiotherapy (rt) complications remain as clinical issues to be improved because of the vicinity of bone marrow and the small bowel, rectum, cervix uteri, and urinary bladder within rt fields [3, 4 ]. helical tomotherapy (ht), an advanced rt technique providing rotational beam delivery, has been shown to improve target conformality and to spare more normal tissue in comparison with intensity - modulated radiotherapy (imrt) [5, 6 ]. for the accurate delivery of rt to the target volumes, kilovoltage (kv) cone beam computed tomography (cbct) scanners were integrated into imrt machines to perform image - guided radiotherapy (igrt). the energy of cbct scanners equipped on ht machines is in megavolts (mv), which does not have the same performance characteristics as diagnostic computed tomography (ct) scanners and kv - cbct because of the lower contrast resolution. the inferior resolution of mv - cbct images without contrast medium enhancement usually results in an insufficient ability to differentiate tumor and surrounding tissues during the daily verification of patient positioning. the combination of variable factors, including tumor shrinkage, anatomical alteration, body contour change, and daily setup errors, renders the registration of initial planning ct to cbct images difficult. in clinical practice, radiation therapists commonly need to manually adjust the registered images according to bony markers, the most identifiable structure in cbct, to fit the initial planning target volumes. to validate the main tumor location before each high - precision ht fraction, the development of a more reliable marker or indicator is of clinical importance to avoid inadequate coverage of the main tumor. here, we present a case of locally advanced cervical cancer that received ht with the interdigitated combination of intracavitary brachytherapy. for the insertion of tandem of brachytherapy applicator, a silicone sleeve was placed into the endocervix before the beginning of brachytherapy. we found that the air cavity in the sleeve had a clear boundary and could serve as a reliable indicator for locating the main primary tumor during the daily verification of patient positioning in ht practice. a 61-year - old woman presented with post - menopause vaginal bleeding was diagnosed as having cervical squamous cell carcinoma by biopsy with parametrial involvement and paraaortic lymphadenopathy noted in ct images (figure 1a), figo stage ct2b2n1m1, ivb. external beam radiotherapy (ebrt) with concurrent chemotherapy, and the interdigitated delivery of intracavitary brachytherapy (high - dose - rate by using ir) after 34 gy of ebrt was performed from december 2015 to january 2016. external beam radiotherapy was planned for 60 gy for the main tumor and parametrium as well as 50 gy to the pelvic and paraaaortic lymph node areas in 30 fractions using the simultaneously integrated boost technique. helical tomotherapy (hi art ii with dynamic jaws) in conjunction with mv - cbct was used to administer ebrt in an image - guided mode. brachytherapy was designed for 30 gy in 6 fractions once a week during the ebrt course and twice a week after the ending of ebrt. the dose of brachytherapy was set 100% at point a. for external beam treatment, the dose to 95% volume of ptv for 50 gy and 60 gy was achieved. mean doses to surrounding normal organs were as follows : 53.0 gy for rectum, 46.0 gy for urinary bladder, 24.4 gy for small bowel, 25.6 gy for left femoral head, and 26.2 gy for right femoral head. external beam treatment was planned by using ct - based simulation (figure 2a) and the dose volume histogram of tomotherapy planning was demonstrated in figure 2d. for insertion of tandem of brachytherapy applicator, a silicone sleeve (manufactured by fortune medical instrument corp., new taipei city, taiwan) with a central hollow canal (figure 3a) was used. it was placed into the endocervical canal with the caudal end stopping at the outer surface of the cervical os, and making contact with the distal boundary of the cervical tumor during the entire brachytherapy course. subsequently, a planned kilovoltage ct scan was performed for visualization and validation of the sleeve insertion (figure 3b3d). a) before concurrent chemotherapy and radiation therapy (ccrt) treatment, b) after ccrt treatment. upper panel : renal vein level, arrow indicates enlarged paraaortic lymph node ; middle panel : external iliac vein level, arrow indicates enlarged right pelvic lymph node ; lower panel : acetabulum level, arrow indicates cervical tumor mass image registration of initial planning and cone beam computed tomography (ct). grey scale : initial planning ct image ; blue scale : cone beam ct image ; arrow : sleeve air cavity. d) dose volume histogram of tomotherapy planning demonstration of tandem insertion through sleeve and kilovoltage computed tomography scan images after sleeve insertion. a) white apparatus indicates sleeve, b) oblique coronal view, c) axial view, d) sagittal view in the remaining ebrt fractions overlapping the brachytherapy course, we found that the air cavity inside the central hollow canal of the sleeve could be clearly identified in the daily mv - cbct images (figure 2c). the radiation oncologists matched the bony markers to adjust the daily setup errors because the mv - cbct images could not provide a precise boundary for the soft tissue or the tumor, only for the bone (figure 2b). moreover, the main structure of sleeve was not visible in mv - cbct images due to lower contrast resolution. the visualization of sleeve by kilovoltage planning ct scan could verify the limitation of mv - cbct in this scenario. however, the sleeve air cavity had a clear boundary and could be used as a surrogate and reliable marker to guide the daily setup errors, and to demonstrate the primary tumor location before the delivery of each ht fraction. after the completion of the ht and brachytherapy course, the tumor and pelvic and paraaortic lymphadenopathy responded to ccrt (figure 1b) with acceptable toxicity. no specific complications were noted related to the insertion of the sleeve for 11 months. in summary, the application of the sleeve during the interdigitated course of ht and brachytherapy in this patient provided information for the feasibility of using the sleeve air cavity as a surrogate marker for the localization of the main primary tumor before the daily delivery of image - guided ht. this result may suggest the desirability of designing a clinical proposal for early integration of the sleeve during the ht course for a more precise target localization and validation. the possible limitation of using the sleeve air cavity as an indicator for the main tumor location remains to be carefully considered. for example, the insertion of the sleeve early in the ebrt course might be difficult due to the anatomical alteration caused by the bulky tumor at the cervical os [8, 9 ]. the relatively long term of insertion of the sleeve may increase the pain scale, vaginal edema, and the risk of infection. the unexpected scattering of the radiation dose from the sleeve needs further dose measurement in the phantom, especially when the scatter correction of cbct is concerned. taken together, the early use of the sleeve during the ebrt course, instead of the insertion of the sleeve in a standard interdigitated brachytherapy course, should be performed with caution and could be validated by further clinical trials. concerning the possible application of magnetic resonance imaging (mri) simulation in the future [12, 13 ], the scattering and artifacts surrounding the sleeve might be an issue that should be addressed. to enhance the applicability in both ct- and mri - based simulations, the modification of the sleeve component the application of ht in the treatment of cervical cancer is on the increase due to the concerns of normal tissue toxicity and quality of life. therefore, the efforts to further augment precision in the daily delivery of ebrt by ht is a crucial issue to be addressed. previous use of metal clips to indicate tumor at our center was not satisfactory, as the clips were not clearly visible on the cbct images of the ht machine. the adoption of the sleeve air cavity as a marker guiding ht is the first in the literature, so the clinical validation for this issue is therefore warranted. regardless of using tomotherapy or imrt, using sleeve may help image guidance during cbct. the application of the sleeve during the interdigitated course of ht and brachytherapy in this patient provided information for the feasibility of using the sleeve air cavity as a surrogate marker for the localization of the main primary tumor before the daily delivery of image - guided ht. | purposeradiotherapy with concurrent chemotherapy has been recommended as standard treatment for locally advanced cervical cancer. to validate the main tumor location before each high - precision helical tomotherapy (ht) fraction, the development of a more reliable marker or indicator is of clinical importance to avoid inadequate coverage of the main tumor.material and methodsa 61-year - old woman with cervical cancer, tmn stage ct2b2n1m1, figo stage ivb was presented. extended field external beam radiotherapy (ebrt) with concurrent chemotherapy and the interdigitated delivery of intracavitary brachytherapy was performed. helical tomotherapy equipped with megavoltage cone beam computed tomography (mv - cbct) was used for image - guided radiotherapy. for the insertion of tandem of brachytherapy applicator, a silicone sleeve with a central hollow canal was placed into the endocervical canal with the caudal end stopping at the outer surface of the cervical os, and making contact with the distal boundary of the cervical tumor during the entire brachytherapy course.resultsin the remaining ebrt fractions, we found that the air cavity inside the central hollow canal of the sleeve could be clearly identified in daily cbct images. the radiation oncologists matched the bony markers to adjust the daily setup errors because the megavoltage of the cbct images could not provide a precise boundary between the soft tissue and the tumor, but the sleeve air cavity, with a clear boundary, could be used as a surrogate and reliable marker to guide the daily setup errors, and to demonstrate the primary tumor location before delivery of each ht fraction.conclusionsthe application of the sleeve during the interdigitated course of ht and brachytherapy in this patient provided information for the feasibility of using the sleeve air cavity as a surrogate marker for the localization of the main primary tumor before the daily delivery of image - guided ht. |
an unexpected property of living matter was discovered about 50 years ago : eukaryotic cells are characterized by genomic chimerism because, in addition to the main nuclear genome, they possess additional genomes in the cytoplasm (ephrussi and slonimski 1955 ; chevremont 1960 ; nass mm and nass s 1962). the cytoplasm of all respiring cells contains organelles, called mitochondria, which are equipped with a specific genome (mtdna, mitochondrial dna) and a complete system for its replication and expression (for details, see box 1). box 1functional role of mitochondrial genomesthe most important biochemical process located in mitochondria is oxphos, the process by means of which aerobic eukaryotic cells synthesize atp with molecular oxygen as electron terminal acceptor. mitochondrial genomes encode for the three distinct gene classes involved in this process : ribosomal, transfer, and protein - coding genes. genes for small and large rrna (12 s and 16 s in mammals, for example) are found universally ; indeed, genes for trna vary greatly in terms of numbers, although a set of 2227 trnas is common in many eukaryotic groups. protein - coding genes are subdivided into two pools : ribosomal protein and bioenergetic. the former are involved in ribosomal subunit synthesis and mainly occur in protist and plant mitochondrial genomes (adams and palmer 2003). the latter are universal and encode for the protein subunits of the rc, the multienzymatic system which creates the proton gradient necessary for atp synthesis (or heat generation). although the bioenergetic gene repertoire varies among the several eukaryote domains (plants, fungi, protists, animals), two important ideas emerge from the accumulating sequence data. all respiring organisms always have a minimal set of bioenergetic genes (cytb and co1 are the most highly conserved), but, in all of them, the mitochondrial bioenergetic gene pool is never sufficient to encode for all the rc subunits (adams and palmer 2003).in metazoa, mitochondrial gene content is quite stable : there are 37 canonical mitochondrial genes encoding for two ribosomal rnas, 22 for trnas, and 13 for the rc subunits. complex i contains 7 of the 13 mitochondrially encoded proteins (nd1, nd2, nd3, nd4, nd4l, nd5, nd6) : nd2, nd4, and nd5 seem to work as electron transporters, whereas nd1 and nd2 play an important structural role between the membrane - embedded and peripheral arms of the complex (da fonseca. 2008). complex ii consists entirely of nuclear - encoded proteins ; cytb, having essentially catalytic activity (cytochrome c reduction), is the only mtdna - derived subunit of complex iii. in the co complex (complex iv), co1 protein catalyzes electron transfer to the ultimate acceptor, molecular oxygen ; co2 and co3 also belong to the catalytic core of the complex, in which nuclear subunits are mainly located externally. regarding atp synthase (complex v), atp6 is a key component of the proton channel (fo component) and atp8 seems to be a regulator of complex assembly (da fonseca. 2008).the nucleus regulates the production of all other rc proteins : about 39 for complex i, 4 for complex ii, 10 for complex iii, 10 for complex iv, and 15 for complex v (scarpulla 2008).however, the contribution of the nucleus to mitochondrial functionality is not limited to the 80-odd proteins (in mammals) directly involved in oxphos. it has been estimated that more than 1,500 genes regulate the varying aspects of mitochondrial activity (wallace 2005), such as dna replication and repair, gene expression and its modulation, complex assembly, etc. functional role of mitochondrial genomes the most important biochemical process located in mitochondria is oxphos, the process by means of which aerobic eukaryotic cells synthesize atp with molecular oxygen as electron terminal acceptor. mitochondrial genomes encode for the three distinct gene classes involved in this process : ribosomal, transfer, and protein - coding genes. genes for small and large rrna (12 s and 16 s in mammals, for example) are found universally ; indeed, genes for trna vary greatly in terms of numbers, although a set of 2227 trnas is common in many eukaryotic groups. protein - coding genes are subdivided into two pools : ribosomal protein and bioenergetic. the former are involved in ribosomal subunit synthesis and mainly occur in protist and plant mitochondrial genomes (adams and palmer 2003). the latter are universal and encode for the protein subunits of the rc, the multienzymatic system which creates the proton gradient necessary for atp synthesis (or heat generation). although the bioenergetic gene repertoire varies among the several eukaryote domains (plants, fungi, protists, animals), two important ideas emerge from the accumulating sequence data. all respiring organisms always have a minimal set of bioenergetic genes (cytb and co1 are the most highly conserved), but, in all of them, the mitochondrial bioenergetic gene pool is never sufficient to encode for all the rc subunits (adams and palmer 2003). in metazoa, mitochondrial gene content is quite stable : there are 37 canonical mitochondrial genes encoding for two ribosomal rnas, 22 for trnas, and 13 for the rc subunits. complex i contains 7 of the 13 mitochondrially encoded proteins (nd1, nd2, nd3, nd4, nd4l, nd5, nd6) : nd2, nd4, and nd5 seem to work as electron transporters, whereas nd1 and nd2 play an important structural role between the membrane - embedded and peripheral arms of the complex (da fonseca. complex ii consists entirely of nuclear - encoded proteins ; cytb, having essentially catalytic activity (cytochrome c reduction), is the only mtdna - derived subunit of complex iii. in the co complex (complex iv), co1 protein catalyzes electron transfer to the ultimate acceptor, molecular oxygen ; co2 and co3 also belong to the catalytic core of the complex, in which nuclear subunits are mainly located externally. regarding atp synthase (complex v), atp6 is a key component of the proton channel (fo component) and atp8 seems to be a regulator of complex assembly (da fonseca. the nucleus regulates the production of all other rc proteins : about 39 for complex i, 4 for complex ii, 10 for complex iii, 10 for complex iv, and 15 for complex v (scarpulla 2008). however, the contribution of the nucleus to mitochondrial functionality is not limited to the 80-odd proteins (in mammals) directly involved in oxphos. it has been estimated that more than 1,500 genes regulate the varying aspects of mitochondrial activity (wallace 2005), such as dna replication and repair, gene expression and its modulation, complex assembly, etc. more or less at the same time, the same situation was discovered in the plastids, which contain a specific plastidial genetic system (chiba 1951). important steps in study of mitochondrial genome note.table summarizes the main scientific contributions that have clarified some structural and metabolic features of mitochondrial genomes, until first sequencing experiments (1980s). it was in this scenario that the endosymbiotic theory that modern eukaryotes arose from an endosymbiotic event was later put forward (margulis 1970). this theory postulates that an alpha - proteobacterium gave rise to the mitochondrion, once it was engulfed in a heterotrophic host cell, probably an archaebacterium. various models regarding the precise nature of the endosymbiotic process from the archaebacterium to the eukaryotes are still being debated (gray. 1999 ; embley and martin 2006 ; poole and penny 2006). the main point of discussion is whether the endosymbiotic event directly promoted the development of eukaryotes in a single step or whether it is simply one of the evolutionary steps toward modern eukaryotes. indeed, in the case of the chloroplast, a cyanobacterium, engulfed by an ancestor of plants and algae, became a new photosynthetic organelle. the solution adopted by eukaryotes to preserve cytoplasmic genomes apparently has no economic value because many genes are necessary to replicate, transcribe, and translate the few genes still present today in cytoplasmic dnas, and it also poses serious constraints to the life cycle of eukaryotic cells (reinecke. the persistence of an auxiliary genome in the eukaryotic cell has been discussed at length : although the progenitor genome underwent reductive evolution (mainly in metazoans), the mitochondrial genome still survives and works in almost all eukaryotes. differences in genetic code (e.g., nuclear dna [ndna ] vs. mtdna code in metazoa) represent a strong barrier against complete absorption of mtdna into the nucleus (de grey 2005). the hydrophobicity hypothesis (von heijne 1986) emphasizes the concept that mitochondrially encoded respiratory chain (rc) subunits are too hydrophobic to be synthesized in the cytoplasm, requiring organellar dna in loco for their expression. according to the colocation for redox regulation (corr) hypothesis (allen 2003), auxiliary genomes still persist because organellar gene expression must be under direct redox control. lane and martin (2010) pointed out that it was the efficient redistribution of genetic information in eukaryotes (a few bioenergetically specialized genes in the organelles, with most of the information located in the nucleus) made an increase in genome size and complexity possible. basically, the endosymbiotic event was very advantageous and essential for creating a complex but efficient system of energy production (within organelles), enabling eukaryote genomes to increase their total protein production and complexity, something which was impossible in prokaryotes (lane and martin 2010). in our opinion, all these questions are connected with the role and evolution of the mitochondrial genome in the eukaryotic cell. in order to shed light on this problem, we need to identify the major driving force acting in cellular mtdna. we focus attention here on the role of drift, purifying, and positive selection on the evolution of mtdna in metazoa, a topic regarding which available data are sometimes inconsistent and highly debated. exactly how the mitochondrial genome evolved has been extensively debated : some details about how it is inherited and how it changes are shown in box 2. box 2inheritance patterns of mitochondrial genomesmitochondrial genomes are uniparentally transmitted and, in most cases, female individuals give their mitochondria to zygotes ; there are mechanisms which (almost entirely) prevent leakage of paternal cytoplasm in fertilization. in plants and fungi, mtdna is maternally inherited : however, in some species belonging to bivalvia, the paternal mitochondrial genome is transmitted in gonadal tissues and the maternal one is located in the somatic lines (double uniparental inheritance). in other groups, paternal leakage is an occasional event (barr. the main evolutionary consequence of this mode of inheritance is that mitochondrial genetic information is haploid : this means that, for every two copies (one paternal and one maternal) of any nuclear gene, only one copy (from the maternal line) of any mitochondrial gene exists. another important aspect of mtdna transmission is the absence of recombination (in metazoa) at significant levels, although piganeau. (2004) revealed the moderate occurrence of recombination in sexual and, although less so, also in asexual animal species. the extent of this phenomenon is still unclear.another important property of mitochondrial genomes in metazoa is the elevated mutational pressure which affects them. for 30 years (brown. high mutational rates in mtdna have been associated with an inaccurate dna repair system (bogenhagen 1999), the absence of histone - like protein, and the peculiar mitochondrial replication model, characterized by single - strand intermediates, which was found to be a convincing explanation for strand mutational bias (reyes. in addition, now nearly 40 years ago, harman (1972) was one of the first to propose that the mitochondrial rc was a major site of ros production in the cell, and that mitochondrial structures (also dna molecules) were primary targets for oxidative damage. however, the search for an aging - dependent accumulation of point mutations in mtdna has given often discordant results. this is probably due to the experimental methods adopted, which can differentially detect low frequency point mutations. in addition, cells which accumulate mutations are prone to apoptosis : thus, mutation accumulation during the life of an organism may be underestimated.in the case of mutator mice (trifunovic. 2005), no evidence of increased ros levels was detected in transgenic mice carrying a defective and error - prone polg, although the mice had a premature aging phenotype. again, experimental techniques can influence the detection of ros species (and ros levels) in a certain cellular context. some other factors, such as the effect of antioxidants, are debated in the literature, but go beyond the scope of this paper.in view of its reduced population size, absence of substantial recombination, and elevated mutational rate, the fate of mtdna seems to follow muller 's ratchet consequences (muller 1964 ; felsenstein 1974), which hypothesizes that small populations (just like mtdna) gradually tend to accumulate slightly deleterious mutations in the absence of a mechanism, like recombination, which could preserve the wild - type condition. this inexorable process would lead such populations to higher and higher mutational levels to the point of complete loss of functionality and subsequent extinction of the genome. inheritance patterns of mitochondrial genomes mitochondrial genomes are uniparentally transmitted and, in most cases, female individuals give their mitochondria to zygotes ; there are mechanisms which (almost entirely) prevent leakage of paternal cytoplasm in fertilization. in plants and fungi, biparental mtdna is maternally inherited : however, in some species belonging to bivalvia, the paternal mitochondrial genome is transmitted in gonadal tissues and the maternal one is located in the somatic lines (double uniparental inheritance). in other groups, paternal leakage is an occasional event (barr. the main evolutionary consequence of this mode of inheritance is that mitochondrial genetic information is haploid : this means that, for every two copies (one paternal and one maternal) of any nuclear gene, only one copy (from the maternal line) of any mitochondrial gene exists. another important aspect of mtdna transmission is the absence of recombination (in metazoa) at significant levels, although piganeau. (2004) revealed the moderate occurrence of recombination in sexual and, although less so, also in asexual animal species. another important property of mitochondrial genomes in metazoa is the elevated mutational pressure which affects them. for 30 years (brown. high mutational rates in mtdna have been associated with an inaccurate dna repair system (bogenhagen 1999), the absence of histone - like protein, and the peculiar mitochondrial replication model, characterized by single - strand intermediates, which was found to be a convincing explanation for strand mutational bias (reyes. in addition, now nearly 40 years ago, harman (1972) was one of the first to propose that the mitochondrial rc was a major site of ros production in the cell, and that mitochondrial structures (also dna molecules) were primary targets for oxidative damage. however, the search for an aging - dependent accumulation of point mutations in mtdna has given often discordant results. this is probably due to the experimental methods adopted, which can differentially detect low frequency point mutations. in addition, cells which accumulate mutations are prone to apoptosis : thus, mutation accumulation during the life of an organism may be underestimated. in the case of mutator mice (trifunovic. 2005), no evidence of increased ros levels was detected in transgenic mice carrying a defective and error - prone polg, although the mice had a premature aging phenotype. again, experimental techniques can influence the detection of ros species (and ros levels) in a certain cellular context. some other factors, such as the effect of antioxidants, are debated in the literature, but go beyond the scope of this paper. in view of its reduced population size, absence of substantial recombination, and elevated mutational rate, the fate of mtdna seems to follow muller 's ratchet consequences (muller 1964 ; felsenstein 1974), which hypothesizes that small populations (just like mtdna) gradually tend to accumulate slightly deleterious mutations in the absence of a mechanism, like recombination, which could preserve the wild - type condition. this inexorable process would lead such populations to higher and higher mutational levels to the point of complete loss of functionality and subsequent extinction of the genome. the role of purifying selection, necessary for maintaining mitochondrial gene functions, has been clarified in many papers based on in silico analysis, and also by in vivo experiments. rand and kann (rand and kann 1996 ; kann and rand 1998) used the neutrality index (ni ; rand and kann 1996) to determine the influence of selection on protein - coding genes in fruit fly and some mammalian species (mus musculus, homo sapiens). data analysis yielded an ni higher than 1, indicating the effect of purifying selection in removing disadvantageous mutations (most amino acid changes are deleterious). however, the strength of this selection depends on the ne parameter, that is, true population size, which varies according to species ecology. for instance, popadin. (2007) found an excess of amino acid substitutions in large - bodied mammals as opposed to smaller ones. body mass was considered a good approximation of ne : thus, species with small populations (e.g., elephants, humans) tend to fix more amino acid changes in mitochondrial genes than rodents (large ne). this problem becomes more obvious in species which have both sexual and asexual populations, as in the crustacean daphnia pulex, in which the populations adopting asexual reproduction are smaller and more prone to accumulating amino acid changes, whereas sexually reproducing populations are larger and selection is more efficient (reduced accumulation) (paland and lynch 2006). the great efficiency of purifying selection has been documented in vivo by sequence analysis of mutator mice (stewart. although the error - prone version of their gamma polymerase (polg) produces high mutational pressure, strong purifying selection removes nonsynonymous deleterious mutations during mtdna transmission from mother to offspring in the course of only a few generations. the above study reveals how purifying selection contrasts accelerated mutational meltdown, preventing the consequences of muller 's ratchet in mammalian mtdna. it should be noted that the very strong, very fast purifying selection observed in this experiment was not only the result of selection acting across individuals but also across a lower level of organization (cellular). in fact, neither mortality nor the differential in fertility allowed by the experiment could explain such extensive purging of accumulated mutations, although a different line of research suggests the action of selection across the oocytes of an individual and even across mtdna copies of single zygotes. it has been shown in metazoa that, during germ line development, there is a dramatic reduction in the number of mtdna copies for each germ cell. estimates of mtdna copy number variations during oogenesis and embryogenesis were recently reported by white. mtdna copies from mature oocytes are equally distributed within blastocyst cells ; later, a certain proportion of the total amount of mtdna (some thousands of copies per cell) is destined to the inner cell mass, which will constitute embryos and the remaining mtdna pool is shared among extraembryonic tissue cells. however, the above recent analysis indicates that primordial germ cells within the embryo contain only a few dozen mtdna molecules, signaling a dramatic reduction in the mtdna population. mtdna replication starts again during oogenesis, with the development of primary oocytes : in terms of copy number, mtdna expansion continues until oocyte maturation. mature mouse oocytes have been estimated to contain up to 200,000 mtdna molecules (white. there are several hypotheses about what the mitochondrial genetic bottleneck is exactly and what evolutionary force causes it. in mouse, the process was found to be random, in which many mtdna molecules are discarded and only a few are transmitted to primordial germ cells (jenuth. the scientific community is increasingly interested in the possible selective nature of the genetic bottleneck. krakauer and mira (1999) considered cellular atresia as a possible response to muller 's ratchet, particularly in birds and mammals. in their view, only a very small number of all primordial follicles (and the primary oocytes inside them) reach maturation during the first stages of fetal life ; the others undergo apoptosis. in this way, less functional mitochondrial genomes are removed. in particular, species which produce small numbers of offspring tend to undergo a more severe bottleneck event, in order to guarantee the survival and viability of the (few) members of the future generation. zhou. (2010) believe that selective bottleneck occurs in the maturation of oocytes. they hypothesize a role played by the mitochondrial cloud (mc), a particular cytoplasmic region which contains rna, organelles, and proteins and is later specifically transferred to the primordial germ cells of the future embryo. the above authors present some biochemical assays (based on estimates of the mitochondrial inner membrane potential of mature zebra fish oocytes), showing how the mc possesses the most efficient mitochondria. although these mechanisms have been described in several organisms for example, mc structures have been observed in danio rerio, xenopus laevis, m. musculus, and drosophila melanogaster selection operating on intraindividual mtdna (at the level of oogenesis or fulliculogenesis) needs to be supported by more precise physiological tests, which should consider a wider range of biochemical parameters (such as membrane potential variations). this would be very important for better understanding of the transmission of mitochondrial diseases in the light of improved artificial reproductive techniques (poulton. these putative internal selection mechanisms do exist (stewart. 2008) but are far from being faultless, as human mitochondrial diseases clearly demonstrate. these are caused partly by mitonuclear interactions which may mask the effect of deleterious mutations (for a deeper discussion, see below positive selection of mitochondrial genomes). conversely, the internal quality control of mtdna takes place in specific physiological conditions (maturation of oocytes) and does not act in all possible situations of energy requirements of the organism (embryo, fetus, adult, restricted diet, cold environment, etc.). energy demands do differ and are contradictory, imposing specific trade - offs for each functional state of the oxphos (oxidative phosphorylation) machinery (das 2006). positive selection appears to be rampant in insects (species with large ne), which have ni indexes well below 1, and mitochondrial genetic diversity was found to be independent of ne. the results of bazin. gave rise to extensive debate : some authors criticized the manipulation and interpretation of the data (e.g., meikeljohn. 2007) ; others are still convinced of the usefulness of the mtdna for demography and conservation studies, even with draft (mulligan. (2008) explored the possibility of adaptive evolution in 12 of 13 mitochondrial rc subunits in 40 mammalian species. by detecting radical amino acid changes (i.e., changes between two amino acids with differing chemical and physical properties) and their functional role in predicted 3d structures, the above authors proposed a correlation between the physiological consequences of those changes and some life cycle traits in the species examined. further studies have shed light on the possible role of mtdna in adapting to new environments. ballard. (2007) demonstrated the role of amino acid polymorphisms in the three cytochrome oxidase (co) mitochondrial subunits between sympatric populations of drosophila simulans, a pan - african species. fly populations with a particular mtdna haplotype were found to be more tolerant to cold and could even colonize temperate regions, whereas others lived prevalently in loco. adaptation of human mtdna to changes in climatic conditions has been shown by balloux. two nonsynonymous substitutions in genes atp6 and nd3 turned out to be significantly associated with indigenous populations living in cold regions. the ancestral alleles were found to be located in africa and in warm regions very recently colonized by humans, although no neutral population markers from ndna were significantly associated with decreasing temperature. in a large - scale human mtdna sequence analysis, (2004) showed that some human haplotypes are good candidates for adaptation to colder environments (less efficient cell respiration, more heat production, more tolerance to starvation) with respect to those more frequently found in warmer regions. coevolution studies on nuclear and mtdna provide further information on this topic. as shown in box 1, the mammalian nuclear genome encodes for about 80 subunits of the rc complexes : they interact with each other and with mitochondrial subunits, enabling correct assembly of the rc. thus, different mitochondrial phenotypes derive from different cytogenotypes, that is, varying allele combinations between diploid nuclear oxphos and haploid mitochondrial genes. coevolution between ndna and mtdna may occur by means of a two - step procedure : according to this hypothesis, the faster evolutionary genome (mtdna) drives the slower one to fix mutations in such a way that mitochondrial functions are guaranteed. even in the case of negatively selected mitochondrial mutations, other mutations may occur in interacting sites located in the same protein or in another interacting one (both mitochondrial and nuclear encoded). these secondary mutations are compensatory in a way that restores the functionality of the mutated protein domain, thus facilitating its normal activity. coevolution also implies interplay between the evolutionary dynamics of interacting genes : in the case of oxphos, this coevolution involves cooperation between entire genomes (rand. 2004). an interesting view of coevolving sites in rc subunits is provided by schmidt. the former authors studied the evolution of interacting amino acid residues among co nuclear and mitochondrial subunits in several mammalian species. they generated a model in which mitochondrial interacting surfaces were fast evolving with respect to non - interacting parts, with the aim of exploring new possibilities of optimizing interactions ; the nuclear counterparts are slow evolving (with respect to the rest of the protein) in order to respond to mitochondrial novelties. the latter authors, after detecting various mutant and compensatory sites in mammalian mitochondrial oxphos subunits by in - depth structural analysis, presented two possible scenarios of coevolution in mtdna : the quasi - simultaneous occurrence of deleterious amino acid changes and compensatory mutations or the occurrence of a deleterious mutation in a genetic environment characterized by a preexisting compensatory background. this may be the case of a homoplasmic mitochondrial transfer rna (trna) gene mutation which has caused severe pathologic phenotypes in several human individuals but not in their mothers, who probably possess a compensatory nuclear genetic background (mcfarland. mtdna coevolution comes from biochemical tests on hybrid organisms, characterized by diverging nuclear mitochondrial genetic backgrounds. in marine copepods (tigriopus californicus), coevolution seems to be very intense : functional assays of hybrids derived from several geographically separated populations show that cell respiration is considerably altered (rawson and burton 2002). in addition, copepod populations may evolve in order to optimize mitonuclear genetic interactions and, when back crossed, the new cytonuclear genotypes are incapable of guaranteeing efficient oxphos. (1998) carried out physiological tests on xenomitochondrial cybrids (i.e., cell lines with a human nuclear background and extraspecific mitochondria) and confirmed the importance of coevolution, revealing the increase in evolutionary divergence among primate species. primate cybrids can perform respiration, although mitochondrial functionality is partially compromised : analyzing the functional consequences on each hybrid complex, the above authors found that complex i activity is defective, whereas other hybrid rc complexes appear to have normal activity. in mouse cell lines, a near - linear relationship between mitochondrial dysfunction and divergence among various murid species (m. musculus, mus spretus, etc.) is observed in cybrids : this dysfunction is not observed in complex ii activity, which is entirely encoded by the nucleus (mckenzie. however, the drosophilids remain the favorite phylogenetic group in which to study gene and genome interactions because programmed crossings and fitness assays are possible relatively quickly and cheaply. (2003) showed the disruption effect on co activity when lines of d. simulans and drosophila mauritiana were crossed together ; the causes of incompatibilities were attributed to nuclear loci. selection operates on a single unit, given by the interaction of all the nuclear and mitochondrial encoded oxphos subunits. each subunit may tolerate mutations differentially because of the mutation position, protein function, and presence of compensatory mutations in other sites. mtdna gene evolutionary rates, being lineage specific, may drive oxphos nuclear genes to coevolve closely in some lineages (as in copepod populational hybrids) rather than others (as cited in montooth. 2010). in conclusion, the probability of mitonuclear coadaptation depends on standing variation, modulated by mutational rates, and intensity of purifying selection. some years ago, we examined the evolutionary properties of mtdnas in metazoans, focusing on the asymmetrical mutational pressure occurring in mitochondrial genes. in 1998, our group explored the mutational pressure occurring in mammalian mtdnas, showing that base composition is asymmetrical between the two strands and along the genome (reyes. 1998). asymmetry (or bias) between base pairs was practically evaluated by the gc and at skew indexes (perna and kocher 1995), where gc skew = (g c)/tot(gc) and at skew = (a t)/tot(at). reyes. (1998) presented a model in which mammalian mtdna compositional bias was positively correlated with the duration time of the single - strand state of h strand genes. the more a mitochondrial gene persists in an impaired state (i.e., the complementary strand is displaced), the longer the time of exposure to mutation (e.g., spontaneous cytosine deamination and conversion in uracil). according to this view, the molecular basis of the difference of base composition between mtdna strands is caused by the replication system of this genome. today, accumulating genome sequence data depict a scenario in which the compositional properties of metazoan mtdnas are quite variable : arthropoda have the lowest gc content and chordata are the most gc - rich group (in particular, birds). when analysis is extended to several animal phyla (see table 2), gc skew is significant in most of the groups, whereas at asymmetry is generally less pronounced, with the sole exception of flatworms (platyhelminthes). the directionality of these compositional asymmetries is also variable : in most groups, gc skew is negative (i.e., g is preferentially located in the minor coding strand), at skew is positive (i.e., more a in h strand, more t in l strand). again, platyhelminthes shows the precisely opposite behavior : skews are remarkably high, but indices are positive for the at pair and negative for the gc one. in some taxa, such as molluscs, echinoderms, and arachnids, the indices show great within - group variability and their mean compositional skews are close to 0. compositional properties of most frequently sampled metazoan phyla note.data have been downloaded from mitozoa database vrs. 6 (lupi. 2010) and contain reference and nonreference complete mtdna sequences. again in the late 1990s, we focused attention on substitution rates within mammalian mitochondrial genomes (pesole. they were found to be gene specific : nonsynonymous sites of protein - coding genes and large rrna genes turned out to be slow - evolving, whereas synonymous sites, small and trna genes, and the d - loop (excluding the conserved central domain) were classified as fast - evolving sites. as shown in table 3, when compared with nuclear analogues, synonymous mitochondrial sites and small rrna genes sequence divergence (% substitutions / site) calculated for specific mtdna sites and their nuclear analogues (from pesole. 1999) more recently, we decided to examine the relative effect of mutation and selection in the evolution of metazoan mitochondrial genomes according to two different approaches, studying codon usage and the synonymous / nonsynonymous substitution rates of the 13 mitochondrial protein coding genes. apart from its great influence on nonsynonymous variability, purifying selection has also been demonstrated to affect synonymous variability in prokaryotic and eukaryotic genomes. according to the efficiency hypothesis (bulmer 1991), in highly expressed genes, synonymous codon usage (scu) evolved in order to recognize the most abundant isoacceptor trnas, guaranteeing efficient translation. akashi (1994) presented a model (the accuracy hypothesis), in which genes tended to use the most efficient codons (within a certain codon family) in the trna anticodon recognition process. this would prevent the incorporation of the wrong amino acids during translation and the consequent production of cytotoxic misfolded proteins (see fig. (a) canonical translation of protein (yellow polygon) : optimal codon (yellow vertical bars) efficiently recognizes trna (in yellow) with correct amino acid (yellow circle). right polypeptide is produced (yellow stripe) and properly folded (yellow polygon). (b) codon is not optimal (yellow vertical bars plus red bar) ; then wrong trna (in red) can be recruited ; an uncorrected amino acid (red cross) is inserted in nascent polypeptide (chain in yellow and gray). a misfolded protein is produced (gray and red polygon) and may undergo degradation (in gray) but, if it does not, it can interact with cell structures. drummond and wilke (2008) supported the accuracy hypothesis by finding a positive correlation between nonsynonymous and synonymous variability in genomes of several model organisms, from escherichia coli to nuclear h. sapiens. the effect of selection on scu was found to be more intense for highly expressed genes and for tissues which are believed to be more susceptible to protein misfolding. thus, this kind of selection, called mistranslation - induced protein misfolding (mim), is a genome - wide constraint for coding sequence evolution in both prokaryotic and eukaryotic domains. in view of the extreme economy of the mitochondrial translational process (a single trna recognizes an entire codon family), we do not expect the efficiency hypothesis to occur in mitochondrial genes. in the search for support for the accuracy hypothesis, jia and higgs (2008) demonstrated that mitochondrial scu in mammals and fish is caused by the significant contribution of mutational input, with a certain effect of base composition in the close proximity of synonymous sites. thus, at first, we decided to evaluate the contribution of mutation and nonmutational factors in shaping synonymous variability by using classical codon usage indexes. in a large genomic data set, consisting of 138 reference genome sequences for the class insecta and 1173 for the subphylum vertebrata (see table 4), we calculated a codon usage bias index, the effective number of codons or enc (wright 1990) and the mutational enc (or expected enc), according to the base composition of the third codon positions of 4-fold degenerate codon families taxonomic description of insect and vertebrate genomic data set note.table describes classification for the 138 and 1173 insect and vertebrate genomes (respectively) for which codon usage analysis has been performed (for details, see text). for insects, the enc gives a measure of scu preferentiality and ranges from 20 (only one codon is used for a given amino acid) to 60 (all encoding codons are used). the enc index is not based on knowledge of which codons are optimal (i.e., the codons used in highly expressed genes), and this is an advantage for the genetic context we studied (mtdna), in which there are no sufficiently clear and complete gene expression data available. mutational enc may be considered as the codon usage preferentiality which would occur in mtdnas if mutational pressure were the sole evolutionary force governing such preferentiality. the mutational enc index was calculated on quartets (encoding for val, ala, pro, thr, arg, and gly amino acids in vertebrate and insect mitochondrial genetic codes) because the corresponding third positions are presumed to be the freest evolving sites in protein - coding genes. we analyzed enc variability for the 13 genes of each genome, evaluating the significance of the contribution of the following predictors : species (138 and 1173 for insects and vertebrates, respectively) ; gene (the 13 mitochondrial genes, although seven species have no canonical gene content) ; position (the middle gene position from the major coding replication origin, normalized for genome length) ; strand ; and mutational enc. we used linear modeling to find that mutation (as exemplified by the mutational enc) greatly contributes to enc variability, incorporating the contribution of the position and strand predictors. however, a significant part (about 15% in both groups) is associated with the nonmutational factors gene and species, whereas 25% and 35% of enc variability (insect and vertebrate data sets, respectively) remain unexplained. we then decided to study codon usage bias by directly observing 4-fold degenerate codon frequencies, gene by gene and genome by genome. this enabled us to overcome the limitations of an index - based approach, which compresses multidimensional codon usage variability into a 1d parameter and suffers in cases of poorly represented amino acid families and short genes. at first, we considered base composition on the third codon positions of the val, ala, pro, thr, arg, and gly codon families. we assumed that base frequencies were uniform along each genome (the simplex model, with only 4 free parameters, corresponding to the 4 base frequencies) or, alternatively, different from gene to gene (the most complex model, with 13 4 parameters). m was calculated as follows : where x is the count for each of the four b codons of the a codon family within the g gene and n is the total count within each family of each gene. the observed frequency of codons within each defined group was used as the maximum likelihood estimator for the parameters the likelihood ratio test was used to find that base composition was gene specific for all the genomes in question. regarding scu, we evaluated whether it was uniform along the genome (the simplest model, with 4 parameters), different from gene to gene (52 parameters), or dependent on the mean genomic use of each synonymous codon (6 4 parameters, where 6 refers to the six codon families considered). all these models were compared with the saturated likelihood model with 13 6 4 parameters, in which each synonymous codon has its own occurrence probability. the second model aimed at demonstrating the role of gene - specific mutational input in shaping scu and the third was based on the consideration that synonymous codon frequencies could be determined by their optimality in the translation process (and, because of this, they would be uniform among the genes). we found that the simple codon usage model was rejected with respect to the saturated model. using bonferroni sequential correction for multiple testing, we evaluated in which species simple models were rejected (species influencing the results of global likelihood comparisons). in particular, in only 20 of 138 insect species (20%) and 469 of 1173 vertebrate species (40%) was the hypothesis that gene - specific mutational input is the sole evolutionary force governing codon usage bias rejected. they confirm that base composition along the genome is not uniform ; the molecular mechanism which causes this bias may be the asymmetrical replication of vertebrate and insect mtdnas (berk and clayton 1974 ; goddard and wolstenholme 1978), which would give rise to a different probability of acquiring mutations (reyes. 1998). scu is thus prevalently determined by gene - specific base composition, but a certain selective effect on it may coexist. in this case, it would be very difficult to detect because it may be very subtle (occurring only in certain genes or codon families) or go in the same direction of mutation (i.e., the optimal codon for translation is the same one preferred by mutational input). in our second approach, we tried to verify the existence of selection on scu, considering the relationship between two nucleotide substitution rates : nonsynonymous, related to nonsynonymous (amino acid change) variability and synonymous, due to silent synonymous codon sites. we emulated the approach of drummond and wilke (2008) : regression analysis between these two estimates enabled us to evaluate the presence of selection in our second genomic data set. this was built by collecting about 350 complete genomes belonging to 45 vertebrate genera, for which at least 4 complete mtdnas (belonging to at least two different cogeneric species) were available in the public databases (see table 5). taxonomic description of second vertebrate mitochondrial genomic data set note.table describes taxonomic classification for the second vertebrate genomic data set, used to estimate gene - specific nonsynonymous and synonymous substitution rates (for details, see text). with respect to the first data set, the second one did not consist solely of reference sequences and was checked for the presence of appropriate annotations. we decided to focus on genus level in order to expand our investigation as much as possible. however, at species level, only very few vertebrate species have a large number of complete published mtdnas (including h. sapiens, m. musculus, canis domesticus, rattus norvegicus, and bos taurus) with which inferences can be made. we extracted nucleotide fasta sequences for all 350 13 genes and made nucleotide multialignments with protein multialignments as guides. we then inferred phylogenetic relationships among gene sequences with each multialignment by bayesian analysis : consensus tree topologies served as input for subsequent parameter calculation. that is, we calculated nonsynonymous and synonymous substitution rates (log dn, log ds) for the 45 13 gene alignments which were constructed by the codeml program (paml package ; yang 2007). we imposed a single dn / ds ratio (, omega) for each gene multialignment, hypothesizing that selective processes were constant along each gene tree ; lastly, we obtained about 550 dn, ds, and estimates. in linear model analysis, synonymous variability was set as the dependent variable because we considered the selection which would eventually operate on it as a minor component of the purifying selection which affects whole gene sequences. the model which best fitted our data (goodness of fit = 0.87) wasin which 0.03 is the slope value (p value = 0.058) and ki the intercept for group relationship among log ds and the log dn and genus predictors note.logarithmic synonymous and nonsynonymous substitution rates were calculated for genes belonging to the second genomic data set (table 5). according this linear model (log ds log dn + genus, described in the text) intercept values (fourth column) are significantly different among genera (p values near 0, fifth column). horizontal lines represent genus - specific intercepts and p values for the remaining 42 genera. the categorical predictor genera has a significant addictive effect on the dependent variable (linear model intercepts are significantly different from genus to genus). the parameter established the role of positive and negative selection affecting gene sequences : an estimate of dn higher than that for ds means that nonsynonymous mutations are fixed at a higher rate than synonymous ones and that positive selection is at work. when is less than 1, purifying selection is evidenced, whereas neutral evolution affects the sequence in the case of = 1. in our data set, we showed the great influence of purifying selection on all mitochondrial genes (log well below 0). co and cytochrome b genes revealed the most efficient selection, although this was more relaxed for atp synthase and some nadh dehydrogenase complex genes (see fig. 2). log estimates for vertebrate genome data set (see text and table 5), divided by 13 mitochondrial genes. horizontal black lines : mean values ; black points : outliers. genes encoding for proteins of same rc complex grouped together by black horizontal lines. horizontal dotted line : threshold by which positive or negative selection affecting sequences can be determined. in particular, the gene order obtained by log mean values was co1 < co2 < co3 < cytb nd1 < nd5 < atp6, nd2, nd3, nd4, nd4l, nd6 < atp8. synonymous variation estimates were quite uniform among the 13 genes : gene - specific log ds values ranged within four orders of magnitude for all groups. nonsynonymous variability estimates were in fact responsible for the significantly different values among the mitochondrial genes. thus, the dn parameter turns out to be a good proxy for selection efficiency in mitochondrial vertebrate genes. our results clearly consolidate a scenario in which mutation and drift govern synonymous changes in mitochondrial protein coding genes, while gene function is strongly preserved by purifying selection. it efficiently removes mutations which cause amino acid changes in mitochondrial rc subunits possessing crucial functions, such as co1, co2, co3, cytb (see box 1). thus, the mitochondrial genome is not characterized by purifying selection on scu, in contrast to the nuclear one. protein misfolding, perhaps caused by inefficient codon versus anticodon recognition (according to the accuracy hypothesis), is probably not a major problem in organelles. we would like to argue that mutational load, that is, decrease in fitness due to mutation, at individual level is much lower in mtdna than in nuclear dna. 1999) that mutational rates are estimated to be higher in mtdna than in the nuclear locus, and this apparently indicates that mutational load is higher in mtdna. however, in the same work, we showed that this discrepancy is caused only by synonymous sites, whereas nonsynonymous variation is comparable between the two genomes. we also show here that synonymous variation can not affect fitness, as in the case of the nucleus, greatly reducing the possible appearance of slightly deleterious mutations. in addition, intraindividual selection, probably due to atresia and the mc, purges nonsynonymous deleterious mutations without evolutionary cost for the individual. this makes the mitochondrion a very efficient engine for evolution, dedicated to a small but key set of genes connected with energy production. it is still unclear whether this purging mechanism can also detect slightly deleterious mutations but, if so, we should expect that, in a given population, there would be fewer segregating deleterious mutations in the mtdna than in an equivalent portion of the nuclear genome. this would mean that a proportion of standing polymorphisms (greater than the proportion in the nucleus) would have the opportunity to go to fixation because they are beneficial, slightly beneficial, or neutral. hitch - hiking events by the fact that the decrease would only affect the deleterious mutation. the dn / ds estimator is generally used to evaluate and compare the strength of selection. this is in fact the statistic according to which the common view that mutational load is higher in mtdna is based (neiman and taylor 2009 and lynch and blanchard 1998 therein). we believe that a straight comparison of dn / ds between nucleus and mitochondria would be misleading, due to the different dynamics of ds and dn in the two compartments. synonymous changes are truly neutral only in mtdna, whereas nonsynonymous changes in mtdna, when they appear in a population, have very probably already passed some kind of quality control. it is therefore not surprising that a greater proportion of nonsynonymous mutations than in the nucleus would be accepted. for this reason, we propose comparing more systematically absolute rates of dn in mtdna versus nuclear dna and expect, as found in mammals (pesole. this higher efficiency in exploring mutational space would allow mtdna to adapt fast : consequently, the well - known bigenomic cooperation for the oxphos function should work, the adaptation appearing first in mtdna and then in the nuclear genome. in order to verify the population consequences of intraindividual purifying selection, population genetic data in nucleus and mtdna must be compared with the same set of individuals. but in order to distinguish among the different coevolutionary scenarios, the nuclear data must include the nuclear oxphos genes, and this has not yet been done. the hapmap project (international hapmap consortium 2003) may have been one opportunity, but unfortunately none of the 5,000 and more individuals for which complete mitochondrial genome information was obtained was used in this project. | the mitochondrial genome is a fundamental component of the eukaryotic domain of life, encoding for several important subunits of the respiratory chain, the main energy production system in cells. the processes by means of which mitochondrial dna (mtdna) replicates, expresses itself and evolves have been explored over the years, although various aspects are still debated. in this review, we present several key points in modern research on the role of evolutionary forces in affecting mitochondrial genomes in metazoa. in particular, we assemble the main data on their evolution, describing the contributions of mutational pressure, purifying, and adaptive selection, and how they are related. we also provide data on the evolutionary fate of the mitochondrial synonymous variation, related to the nonsynonymous variation, in comparison with the pattern detected in the nucleus.elevated mutational pressure characterizes the evolution of the mitochondrial synonymous variation, whereas purging selection, physiologically due to phenomena such as cell atresia and intracellular mtdna selection, guarantees coding sequence functionality. this enables mitochondrial adaptive mutations to emerge and fix in the population, promoting mitonuclear coevolution. |
three groups of adult heterozygous aldh1l1egfp - l10a mice were used (n = 6/group) : awake mice (w) were collected during the dark phase (~ 35 am) at the end of a long period of wake (> 1 h, interrupted by periods of sleep of 45 min, interrupted by periods of wake of 2 and p 1 h, interrupted by periods of sleep of 45 min, interrupted by periods of wake of 2 and p < 0.01 were considered enriched, while probesets with ip / ub ratio < 2 and p < 0.01 were considered depleted. finally, to independently verify the validity of the trap method, ip / ub ratios for 200 top genes previously found to be enriched in astrocytes, oligodendrocytes, and neurons were calculated (fig. | transcriptomic studies revealed that hundreds of mrnas show differential expression in the brains of sleeping relative to awake rats, mice, flies, and sparrows. although these results have offered clues regarding the molecular consequences of sleep and sleep loss, their functional significance thus far has been limited. this is probably because the previous studies pooled transcripts from all brain cells, including neurons and glia. in bellesi. (2015) [1 ], we used the translating ribosome affinity purification technology (trap) and microarray analysis to obtain a genome - wide mrna profiling of astrocytes as a function of sleep and wake. we used bacterial artificial chromosome (bac) transgenic mice expressing egfp tagged ribosomal protein l10a under the promoter of the aldh1l1 gene, a highly expressed astrocytic gene. using this approach, we could extract only the astrocytic mrnas, and only those already committed to be translated into proteins (l10a is part of the translational machinery).here, we report a detailed description of the protocol used in the study (bellesi., 2015 [1 ]). array data have been submitted to ncbi geo under accession number (gse69079). |
micrornas (mirnas) are approximately 22-nucleotide - long non - coding rna molecules that were first discovered in caenorhabditis elegans but are now known to be expressed in all metazoans. mirna genes are present singly or in clusters, often with their individual promoters and regulatory elements, while some mirna genes are also present within the introns of other protein coding genes. mirnas are transcribed as long primary transcripts and are processed first in the nucleus by the microprocessor drsh-1/drosha and then in the cytoplasm by the dcr-1/dicer complex to generate the active 21 to 23-nucleotide rna molecules. these are recruited by the rna - induced silencing complex (risc) and guided to the 3 ' untranslated regions of target messenger rnas (mrnas) with complete or incomplete sequence complementarity, resulting in either degradation of the target mrna or inhibition of its translation. a single mirna can target multiple mrnas and this ability to control the expression of numerous genes makes mirnas pivotal in regulating various life processes, from development through metabolism to senescence, aging and death (reviewed in). while animals like c. elegans contain hundreds of mirnas, little is known about their functions. moreover, with the advent of deep sequencing technology, novel mirnas are being discovered in different model organisms, requiring faster and more convenient methodologies to study their functional importance through inhibiting their activity in vivo. a recent report by zheng and colleagues now demonstrates the efficient and specific inhibition of mirnas in c. elegans by dextran - conjugated modified antisense oligonucleotides. mirnas were first identified in c. elegans in 1993 and since then this elegant model system has been extensively utilized for functional analysis of mirnas. c. elegans is so useful for these analyses because of its easy genetics, completely sequenced genome and simple anatomy. to study the role of individual mirnas in different cellular pathways, forward genetics approaches have yielded many ' loss of function ' (lf) mutant strains for various mirnas. although such mutant strains have been extensively used for functional studies of the target mirnas, their generation is both time consuming and labor intensive. further, it is tedious to generate knockout alleles for mirnas that are essential for survival and development, and in cases where the mirnas are located in the intronic sequences of protein coding genes, it is possible that their knockout will perturb expression of the protein coding gene. it is also difficult to specifically knock out a single mirna from a mirna gene cluster without affecting the expression of the remaining mirnas in the cluster. many groups have tried using reverse genetics approaches to inhibit specific mirna function transiently in different model systems. the most popular tool of choice is differently modified antisense oligonucleotides, which are easy to synthesize and deliver. several research groups have demonstrated inhibition of mirna function with limited success using antisense oligonucleotides such as locked nucleic acids (modified rna nucleotides) or morpholinos (nucleic acid analogs) in drosophila, zebrafish, and mice [8 - 10 ]. c. elegans has been widely used as the model system to study the biological role of small non - coding rnas and yet, to date, no standard techniques or protocols are available to effectively and conveniently knockdown mirna function transiently. to inhibit mirna expression in c. elegans, zamore 's group injected 2'-oxy - methyl oligonucleotides into developing embryos. their embryo injection technique is technically difficult and, therefore, has not been used extensively. furthermore, the primary drawback of using these modified oligonucleotides is the incumbent toxicity caused by poor solubility and inadequate cytoplasmic retention and tissue distribution. to address these issues, zheng and colleagues conjugated 2'-oxy - methyl antisense oligonucleotides complementary to the target mirnas with the polysaccharide dextran, which has high solubility in water and shows increased cellular uptake and availability. the authors also modified these oligonucleotides by conjugating one to three molecules of rhodamine per molecule of dextran. these modified antisense oligonucleotides were injected into the syncytial gonads of adult worms and embryos were selected based on the presence of rhodamine (figure 1). schematic representation of the technique elaborated in the study by zheng and colleagues. in this technique, a dextran - conjugated rhodamine - labeled antisense oligonucleotide complementary to the target microrna are selected by the presence of rhodamine. in these progeny the antisense oligonucleotides bind to and deplete the available pool of target mirna, thus inhibiting mirna function in the animal. zheng and colleagues used antisense oligonucleotides complementary to lin-4 and let-7 and comprehensively demonstrated robust knockout phenotypes similar to those seen in the respective loss of function mutant strains of lin-4 and let-7. for example, progeny of animals injected with antisense oligonucleotides against lin-4 show delayed exit in differentiation of seam cells and lack of vulva formation, known phenotypes for lin-4(lf). the progeny of worms injected with antisense oligonucleotides complementary to let-7 demonstrated a bursting vulva phenotype with lack of alae (longitudinal ridge) formation in 100% of animals, consistent with the phenotype shown by let-7 knockout mutant animals. by contrast, progeny of animals treated with antisense oligonucleotides complementary to mir-84, a let-7 family member, exhibited no visible phenotype, consistent with the known phenotype of mir-84(lf) mutants. mir-84 has a sequence that is highly homologous to let-7 sequence, yet the phenotype observed in the mir-84 knockdown animals was completely unlike the let-7 knockdown phenotype therefore confirming the specificity of inhibition. using a similar strategy, the authors could demonstrate specific inhibition of lys-6 mirna in the asel neurons, where it functions to maintain left - right symmetry. this technique also successfully demonstrated the inhibition in activity of mir-42, which is active only during embryonic development. in addition, the authors demonstrate that this new class of antisense oligonucleotides could be utilized for specific and simultaneous inhibition of two mirnas, in this case lin-4 and lys-6. as with all antisense techniques, the dextran - conjugated antisense 2'-oxy - methyl oligonucleotides have a limitation, in this case with respect to the period of efficacy of inhibition against target mirnas in the animal. the inhibition was robust until 15 - 20 hours after the l4 molt with higher doses of injection, after which the effect diminished, most probably due to the dilution of the antisense oligonucleotides inside the animal. for this reason this method of mirna inhibition might not be the most effective for long - term functional assays, such as aging assays, where the mirna in question needs to be inhibited throughout the life span of the animal. overall, the data presented in the report by zheng and colleagues convincingly suggest that this new class of antisense oligonucleotides could prove to be a very powerful and effective tool for the robust and specific inhibition of mirna function in vivo. this technique would be very useful for studying the different biological functions of essential and novel mirnas in c. elegans. while it remains to be tested, this technology does not appear to be limited to just c. elegans, and could be applicable in other organisms as well. | micrornas (mirnas) are small non - coding rnas that regulate the expression of numerous target genes. yet, while hundreds of mirnas have been identified, little is known about their functions. in a recent report published in silence, zheng and colleagues demonstrate a technique for robust and specific knockdown of mirna expression in caenorhabditis elegans using modified antisense oligonucleotides, which could be utilized as a powerful tool for the study of regulation and function of mirnas in vivo.see research article http://www.silencejournal.com/content/1/1/9 |
we present the case of a 19-year - old african american female who presented with left - sided parastheia, weakness, and headache for 2 days duration. her past medical history was significant for four syncopal episodes lasting less than 30 sec, for which the patient did not seek medical attention. laboratory test including complete blood count (cbc), basic metabolic profile, urine drug screen, and thyroid functions were within normal limits. computerized tomography of head showed multiple small foci of hypoattenuation in the right periventricular white matter. contrast magnetic resonance imaging of the brain showed scattered areas of t2 hyper intense signal within the white matter of the right cerebral hemisphere in the watershed areas (fig. additional laboratory tests including erythrocyte sedimentation rate (esr), c - reactive protein (crp), antinuclear antibodies (ana), cytoplasmic antineutrophil cytoplasmic antibodies (c - anca), perinuclear antineutrophil cytoplasmic antibodies (p - anca), protein c and s, antithrombin iii, homocysteine, factor v leiden, coagulation profile, rapid plasma reagin (rpr), hiv, vitamin b12, folate, and a lumbar puncture were all within normal limits. contrast magnetic resonance imaging of the brain showed scattered areas of t2 hyper intense signal within the white matter of the right cerebral hemisphere in the watershed areas. magnetic resonance angiography of the brain revealed near occlusion of the distal supraclinoid segment of the right internal carotid artery (ica) with extensive collateral augmentation (fig. 2). conventional angiography of the head and neck showing near occlusion of the distal supraclinoid segment of the right internal carotid artery.. also revealed was a moyamoya - like conversion of the lenticulostriate arteries giving a classic puff of smoke appearance (fig. she then underwent neurosurgical anastomosis of the right superficial temporal artery to the right middle cerebral artery. conventional angiography of the head and neck showing moyamoya - like conversion of the lenticulostriate arteries giving a classic puff of smoke appearance. moyamoya disease (mmd) is a chronic idiopathic neurovascular occlusive disorder that was first described in 1957 by takeuchi and shimizu (1). it is characterized by progressive occlusion of the ica and circle of willis, leading to formation of collateral small vessels at the base of the brain to compensate the reduced blood flow from arterial stenosis (1). puff of smoke in japanese, describes the hazy appearance of the abnormal vascular network seen on cerebral angiography (2). the etiology of mmd is unknown ; however, it has been suggested that the onset of the disease can be contributed to genetic, ethnic, and environmental factors (3). there has been a strong link to mmd in japan and korea with rnf213, which is a mmd susceptible gene found on chromosome 17q25.3, and mutation p.r4859k (46). mmd is more commonly seen in east asia, particularly japan, than europe and the americas (7, 8). it has been noted that there is a female predominance (9, 10) with a bimodal age distribution involving the first and fourth decade of life (1013). other symptoms seen in pediatric patients can include episodes of seizures, headaches, strokes, or focal neurological deficits (14). in adults, cerebral hemorrhage, in the intraventricular region, is a common symptom while it is uncommon in children (15). cerebral ischemia is another presentation seen in adult patients and has been noted to affect the the main diagnostic imaging studies for mmd include magnetic resonance imaging (mri), magnetic resonance angiography (mra), and cerebral angiography. the mri and mra are noninvasive imaging studies in mmd that demonstrate steno - occlusive disease of the terminal ica, the proximal portions of the anterior and middle cerebral arteries, and basal moyamoya vessels, which is the abnormal vascular network in the vicinity of the stenosis or occlusion (16). when the described findings are seen bilaterally on mri and mra, it is a definitive diagnosis of mmd and does not require cerebral angiography, while probable diagnosis of mmd is noted if the findings are seen unilaterally (1). it demonstrates fine basal collaterals giving the classic puff of smoke appearance (2). in our case, the mri showed watershed areas having hyper tense signal within the white matter demonstrating cerebral ischemia, and the conventional angiography of the head and neck in our patient revealed the classic puff of smoke from the conversion of the lenticulostriate arteries. direct revascularization has proven to be more effective, while in the pediatric patients indirect approach is more successful (1). in adults with mmd having cerebral ischemia, it is recommended that direct arterial bypass with possible indirect surgical techniques is carried out (1). moyamoya disease is a rare progressive neurologic condition characterized by occlusion of the cerebral circulation with extensive collaterals recruitment in children and adults. early recognition and swift institution of therapy is vital in order to minimize neurological deficits. this case emphasizes that moyamoya, despite being extremely rare, even more so in african american 's, should be considered in any patient with unexplained neurological symptom indicative of cerebral ischemia. the author has not received any funding or benefits from industry or elsewhere to conduct this study. | moyamoya disease is a rare neurological condition that affects children and adults of all ages. it is characterized by chronic, progressive stenosis of the circle of willis that ultimately leads to the development of extensive collateral vessels. presenting symptoms are usually due to cerebral ischemia or hemorrhage. the japanese term moyamoya (meaning puffy or obscure) was coined to describe the characteristic smoke in the air appearance of these vessels on cerebral angiography. moyamoya has the highest recorded incidence in japan (0.28 per 100,000). in the west it is an extremely rare condition with an overall incidence of (0.086 per 100,000) in the western united states. etiology for the most part is unknown ; however, genetic susceptibility related to rnf213 gene on chromosome 17q25.3 has been suggested. moyamoya is being diagnosed more frequently in all races with varying clinical manifestations. moyamoya disease is a rare progressive neurologic condition characterized by occlusion of the cerebral circulation with extensive collaterals recruitment in children and adults. distinguished radiological findings confirm the diagnosis. early recognition and swift institution of therapy is vital in order to minimize neurological deficits. we present the case of a 19-year - old african american female who presented with left - sided parastheia, weakness, and headache for 2 days duration. |
don nelson first described a syndrome which consisted of visual field defects, hyperpigmentation, elevated plasma acth levels and a large sellar mass which is now named after him. nelson 's syndrome (ns) refers to a clinical spectrum arising from progressive enlargement of pituitary adenoma and elevated adrenocorticotrophic hormone after total bilateral adrenalectomy for cushing 's disease (cd) and can be life threatening. we report here a 50-year male presenting with rapid onset of ns with an unusual finding of bilateral oculomotor palsy and the management strategies. a 50-year male presented initially seven years back with right renal calculi, multiple dorso - lumbar vertebral fractures, progressive centripetal obesity, wide violaceous striae over abdomen and axilla, proximal myopathy, diabetes mellitus and hypertension. evaluation was suggestive of cd [overnight dexamethasone suppression test : serum cortisol 23 g / dl ; low dose dst 16 g / dl ; morning plasma adrenocorticotrophic hormone (acth) of 123 pg / ml ]. magnetic resonant imaging (mri) of sella showed a pituitary macroadenoma measuring 141518 mm with minimal suprasellar extension. post - operative evaluation showed persistent hypercortisolism and a residual lesion in sella for which he received fractionated conventional radiotherapy of 5400 gy and initiated on ketoconazole. he again reported 30 months back with worsening of his initial symptoms ; however he had noticed improvement in his symptomatology three years following radiotherapy. he noticed increasing abdominal girth, generalised weakness, easy fatigability, proximal muscle weakness, decreased libido, erectile dysfunction, loss of pubic and axillary hairs and worsening of proximal muscle weakness, diabetes and hypertension. evaluation at this stage revealed recurrence of hypercortisolism, secondary hypothyroidism, secondary hypogonadism and secondary osteoporosis. mri sella showed a recurrent pituitary macroadenoma with minimal suprasellar extension [figure 1a and b ]. computed tomography (ct) showed bilateral enlarged adrenals. the patient underwent total bilateral adrenalectomy (tba) with prophylactic gamma knife irradiation for the pituitary macroadenoma. post - operatively he was started on hydrocortisone, fluodrocortisone, thyroxin and testosterone replacement. ten months after tba he presented to a local clinician with complains of progressive drooping of right eye. ocular myasthenia was made and treated with pyridostigmine. with no relief of symptoms he reported to us two months later. he again complained of reappearance of his initial symptoms, worsening of proximal muscle weakness, drooping of eyelids, visual field defects and darkening of skin. examination revealed cushingoid habitus, hyperpigmented tba scar, bilateral ptosis [right more than left, figure 2 ], restriction of extra - ocular movements, bitemporal hemianopia and proximal muscle weakness. his 0800 hours cortisol was 36 g / dl and plasma acth was 774 pg / ml. mri sella showed enlargement of macroadenoma - measuring 263029 mm extending into bilateral cavernous sinus, completely encasing right internal carotid artery (ica) and abutting left ica [figure 1b and d ]. histopathology was consistent with acth secreting pituitary adenoma. comparing the ki-67 labelling index (li), it was 4% after first transsphenoidal excision and 8% after second transsphenoidal excision. t1 weighted magnetic resonant imaging of sellar region. (a) and (c) are pre - total bilateral adrenalectomy (tba) images showing pituitary macroadenoma with minimal suprasellar extension. (b) and (d) are post- tba images showing asymmetrical suprasellar enlargement of the tumor with invasion into bilateral cavernous sinus (a) photograph showing the hyperpigmented scar mark of total bilateral adrenalectomy (tba), also note the hyperpigmentation of upper limb ; (b) bilateral ptosis of both eyes ; right more than left ten months after tba ; (c) complete ptosis of both eyes 12 months after tba, note the rapid progression of hyperpigmentation over two months (a and b) computed to mography of abdomen showing bilateral adrenal rest enlargement post - operative hypercortisolism in cd is managed with redo excision of pituitary adenoma, radiotherapy, medical therapy or at times tba. ns occurs after cd in about 8 - 43% adult cd patient and is more frequent in children. a case series from india found ns in two out of their 16 patients and was suspected in eight other patients. our patient presented about 10 months after tba, which is very short duration for development of ns. ns can appear as early as 2 months or as late as 24 years after tba with an average of about 6 years. hyperpigmentation is seen in about 50% of patients with ns in present series and is no longer an essential feature of ns. our patient had an unusual presentation in the form of bilateral progressive oculomotor palsy presenting as ptosis. however, the diagnosis was delayed due the presentation of the patient with ptosis to neurologist and being treated as ocular myasthenia. those who receive prophylactic conventional radiotherapy 25% go on to develop ns while those who do nt about 50% develop. about 65% of the patients who received prophylactic gamma knife radiosurgery (gks) showed decreasing trends of plasma acth, however 35% had increasing trend of acth. though our patient received prophylactic gks he still went on to develop ns over a very short period of time. this could have been due to stimulation by acth of some adrenal tissue left during surgery or more likely due to hyperplasia of adrenal rest tissue. adrenal rest tissue can be seen in the adrenal bed or in para - testicular and para - ovarian tissue. however scrotal ultrasound performed did nt show any testicular adrenal rest tissue (tart). there are many postulated predictors of ns development - young age at tba, pregnancy, insufficient steroid replacement, fulminant course of cd and more recently ki-67 li. ki-67 li is marker for proliferation which gives an estimate for aggressiveness of the tumor. ki-67 li of > 3% is associated with aggressive tumors and which in turn predict the development of ns. ki-67 li after the first tumor excision was 4% while that after the second debulking surgery was 8%. the patient has progressive course he is planned for alternate forms of therapy like dopamine agonist, somatostatin analogues, sodium valproate or ppar agonists. approach to patient being considered for tba for cd : prophylactic radiotherapy to pituitary.optimal steroid replacement.visual field charting once in 6 months and subsequently decrease the frequency.periodic plasma acth levels - once in 3 months in the first year and subsequently decrease the frequency.mri sella once in 6 months in the first year then annually.evaluation for testicular adrenal rest tissue - testicular ultrasound.. optimal steroid replacement. visual field charting once in 6 months and subsequently decrease the frequency. periodic plasma acth levels - once in 3 months in the first year and subsequently decrease the frequency. recently a new diagnostic criteria for ns has been proposed which should satisfy either of the following components : an expanding pituitary mass lesion post - tba surgery (shown on mri or ct scan) compared with mri of the pituitary prior to tba surgery.an elevated level of acth to > 500 ng / l from a single plasma sample taken at 0800 h prior to steroid administration and post - tba surgery, in addition to progressive elevations of acth levels from plasma samples taken on at least three consecutive occasions at different time - points post - tba surgery (a rise of acth by > 30% of the initial post - tba sample). an expanding pituitary mass lesion post - tba surgery (shown on mri or ct scan) compared with mri of the pituitary prior to tba surgery. an elevated level of acth to > 500 ng / l from a single plasma sample taken at 0800 h prior to steroid administration and post - tba surgery, in addition to progressive elevations of acth levels from plasma samples taken on at least three consecutive occasions at different time - points post - tba surgery (a rise of acth by > 30% of the initial post - tba sample). | nelson 's syndrome refers to a clinical spectrum arising from progressive enlargement of pituitary adenoma and elevated adrenocorticotrophic hormone after total bilateral adrenalectomy for cushing 's disease comprising of hyperpigmentation, visual field defects which can be life threatening. we report here a 50-year male presenting with rapid onset of nelson 's syndrome with an unusual finding of bilateral oculomotor palsy mistakenly treated as ocular myasthenia. |
it is recommended that the cuff pressure be maintained between 20 - 30 cm of h2o. if it is below the recommended cuff pressures the glottis seal is inadequate and it may lead to micro aspiration. increased cuff pressure leads to tracheal complications, though often associated with long procedures, these complications may occur even after a short duration of anesthesia. although cuff pressures are checked initially after intubation, continuous or intermittent measurement of endotracheal tube cuff pressures in the mechanically ventilated patients during anaesthesia and maintaining it within the recommended range is not routinely done. neurosurgical procedures are usually long and involve positioning the head in different positions. to our knowledge, there are no studies highlighting the effect of positioning on the changes in the cuff pressure. this study is intended to highlight the cuff pressure changes in the neurosurgical patients at different positions and to look for complications due to either excessive or decreased cuff pressure. a prospective observational study was conducted at christian medical college, vellore, india in patients undergoing elective neurosurgical procedures for a period of 2 months after getting the ethical clearance. seventy asa 1and 2 patients within the age group of 11 - 77 posted for elective neurosurgical procedures were included. patients coming for emergency procedures, asa > 3, pregnant patients, patients with known laryngeal or tracheal pathology, patients intubated with uncuffed tubes and those on tracheostomy were excluded. on arrival of the patient to operating room, intravenous (iv) cannula was secured on the hand and iv fluid was started. the intraoperative parameters monitored include : noninvasive blood pressure (nibp), electrocardiogram (ecg), oxygen saturation (spo2), end tidal co2 (etco2) and temperature monitoring. after preoxygenation for 3 minutes, anesthesia was induced with fentanyl 2 mcg / kg, propofol 1 - 2 mg / kg. the trachea was intubated with a high volume low pressure cuffed oral endotracheal tube of appropriate size (male-8 mm i d and female -7 mm i d portex). air was inflated through the pilot balloon and the cuff pressure was checked with endotest (teleflex medical, rush) and regulated to be between 20 - 30 cms h2o. the cuff pressure was checked by the anesthesia provider who was blinded to the study. the volume of air required was noted. after intubation, the presence of equal air entry on both the sides were checked with the neck flexion and extension. the ventilatory parameters were set at a respiratory rate of 10 - 12/minute, tidal volume of 8 - 10 ml / kg to maintain the end tidal carbondioxide concentration between 28- 32 mmhg. patients were maintained with 0.8 mac of isoflurane with 40% oxygen in air and boluses of intravenous fentanyl was given intraoperatively as needed. neuromuscular blockade was maintained with a vecuronium infusion until the end of the procedure maintaining two twitches. the cuff pressure was checked again after achieving the final position with the head on pins (b), at the end of the procedure on the same position (c) and after achieving the supine position, before extubation (d). if there was a significant reduction in the cuff pressure after final positioning of the head on pins, which is below 20 cmh2o, then the cuff is inflated again such that the cuff pressure lies between 20 - 30 cms of h2o. data parameters collected were entered into the excel sheet and transferred into the spss software (statistical package for social sciences version 19) for the statistical analysis. paired sample t - test was used to analyze the differences in cuff pressure within the group at different positions. data parameters collected were entered into the excel sheet and transferred into the spss software (statistical package for social sciences version 19) for the statistical analysis. repeated measures anova paired sample t - test was used to analyze the differences in cuff pressure within the group at different positions. the repeated measures anova (rmanova) was initially done to find the existence of significance between the four time positions for the corresponding groups. we found a significant difference for the supine (p <.022) and the prone groups (p <.001). a paired sample t - test was conducted between the baseline and the other three time points for each group. we observed a significant difference in the cuff pressures between the baseline and before extubation with a p value of 0.000 in the supine group. we also observed a highly significant difference in the cuff pressures between the baseline and final positioning, end of procedure, and before extubation with p values less than. tables 2 and 3 shows the t - test results for the supine and prone population. none of the complications such as sore throat, stridor and hoarseness was noted postoperatively in any patient. p<.05 is considered statistically significant descriptive statistics and t - test results for the neurosurgical procedures in prone position. our study shows a significant reduction in the endotracheal cuff pressures from the initial supine position to the further end points in the study in the patients undergoing neurosurgical procedures [figure 1 ]. it is more obvious in the patient population who underwent procedures in supine and prone [figures 2 and 3 ]. we could nt comment on the patient population who underwent procedures in the lateral and sitting position as the numbers were insufficient. line graph showing mean cuff pressure against four different time points during two positions namely supine and prone changes in cuff pressure at four different time points in the supine group. the values are represented as mean and 95% confidence interval changes in cuff pressure at four different time points in the prone group. the values are represented as mean and 95% confidence interval once the head is supported on pins, it is likely that the neck is no longer held firmly against the back of the table but is free. the tension on the posterior laryngeal structures is reduced hence the drop in the cuff pressure. the reduction in the cuff pressure might have probably occurred the moment the neck was freed, even before our first measured time point which is after final positioning of the patient. we could nt appreciate that as we measured the cuff pressure only at four time points since recurrent intermittent measurements of the cuff pressure by itself reduces the cuff pressure. there are several factors responsible for the changes in the cuff pressures, which are time, positive pressure ventilation, use of nitrous oxide, altitude, measurement of the cuff pressure, muscle relaxation, sedation, hypothermia, different neck and body positions, endotracheal tube positions, duration of intubation, pathologic factors such as laryngeal edema, bronchoconstriction and etc. sole., have showed that the cuff pressure over time results in the reduction of cuff pressure and our study agrees with their findings. skeletal muscle tone and the consciousness in the patients are responsible in maintaining the upper airway structures and the laryngeal dimensions in position. in the supine position, during the induction of anesthesia, loss of consciousness is associated with loss of the tonicity of the muscles around the neck which results in the posterior displacement of upper airway structures, gravity in part also plays a role in the posterior displacement of the upper airway structures by the loss of activity of the muscles during induction of anesthesia, this might have resulted in the reduction of the cuff pressures initially and the continuous level of unconsciousness provided by the adequate level of depth of anesthesia and paralysis over time would have resulted in the reduction of cuff pressures over a period of time. studies have shown that the use of muscle relaxant results in the reduction of the cuff pressure. induction of anesthesia and neuromuscular blockade results in the reduction of oropharyngeal dimension and increase in the laryngeal dimensions because of the loss of the muscular tension around the neck. anatomic relationship between the endotracheal tube and the larynx constantly fluctuates because of the changing levels of consciousness and muscle relaxation. this causes an undue strain on the laryngeal structures which might result in the variations in the cuff pressure. in our case, since we maintained a mac of 0.8 and a continuous infusion of muscle relaxant, the patient was kept at an adequate level of anesthesia at all times and a continuous drop in cuff pressures was noticed. patients inter individual variability such as the anatomy of trachea, cuff positioning over trachea and cuff physical characteristics such as material, diameter, thickness, compliance, geometry are also the factors which influence the cuff pressure. different body positions results in wide variations in cuff pressure, there are conflicting results observed by different authors in the measurement of the cuff pressures in different positions. our study showed a significant reduction of cuff pressure over a period of time in both the supine and prone population. few authors have explained the importance of endotracheal tube positioning and the variations of cuff pressures. lower cuff pressures have been observed in the patients frequently when the endotracheal tubes have gone deeper into the trachea. during extension of the head in neurosurgical procedures, positioning often involves some element of flexion and extension along with mild rotation of the neck, which may further add on to the decrease in cuff pressures. multiple mechanical factors such as a defect in the inflation valve, pilot balloon and the cuff itself can result in the decrease in cuff pressure ; in our case we checked all the endotracheal tubes for cuff leak before intubating the patient. there are a few limitations to our study ; continuous measurement of the cuff pressures was not done. we could not comment on the cuff pressure changes in the patients who underwent procedures in lateral and sitting position as the sample size was insufficient. the relationship between the duration of surgery and decrease in cuff pressure was not studied. long term assessment for the postoperative complications such as sore throat, stridor and hoarseness was not been assessed. the effect of this decrease in cuff pressure during surgery is probably more significant in the sub group of patients who undergo emergency surgery rather than elective surgery where the chances of aspiration are much higher and the need for cuff pressure to be regulated between 20 - 30 cms of h2o is mandatory. to conclude, cuff pressure has to be checked after achieving the final position with the head on mayfield clamp and adjusted to the prescribed limits to prevent micro aspiration. whenever the patient position is changed, care must be taken to adjust the cuff pressure within the prescribed limits. frequent intermittent measurement or continuous measurement of endotracheal tube cuff pressures where available is warranted in all the neurosurgical patients. the clinical significance of these cuff pressure variations in neurosurgical population needs to be further studied in a larger study groups in all positions. | background : placement of a cuffed endotracheal tube for the administration of general anesthesia is routine. the cuff of the endotracheal tube is inflated with air to achieve an adequate seal to prevent micro - aspiration. over inflation of the cuff can decrease the mucosal perfusion, leading to pressure necrosis and nerve palsies. inadequate seal can lead to micro aspiration. so the cuff pressure has to be monitored and kept within the prescribed limits of 20 - 30 cms of water.aim of the study : to observe the effect of different positions on the endotracheal cuff pressure in patients undergoing neurosurgical procedures.materials and methods : this is an observational study conducted on 70 patients undergoing neurosurgical procedures in various positions. after intubation, the cuff pressure was checked with a cuff pressure manometer, endotest (teleflex medical, rush) and adjusted to be within the allowable pressure limits as is the routine practice. the cuff pressure was checked again at three time points after achieving the final position with the head on pins, at the end of the procedure and before extubation. various factors such as the age, position, duration of surgery were studied. there were no major complications like aspiration, stridor or hoarseness of voice post extubation in any of the patients.results:a significant decline in the cuff pressures were noted from the initial supine position to extubation (p <.001) in the supine group. also a significant decline in the cuff pressures were found in the prone group from their initial intubated supine position to all the other three corresponding time points namely after final positioning (p <.001), at the end of the procedure (p <.001) and before extubation (p <.001).conclusion : cuff pressure has to be checked after achieving the final positioning of the patient and adjusted to the prescribed limits to prevent micro aspiration. |
case 1. a 31-year - old man presented with left periorbital swelling and diplopia after a blunt orbital trauma (fig. the man reported being hit by a lump of iron at work the previous day. the patient complained of diplopia in the primary position, left gaze, and downgaze. initial orbital computed tomography (ct) showed a large left inferior and medial orbital wall fracture extending posteriorly (fig. 1c), and soft tissue was found to be entrapped at the orbital floor fracture site. incarcerated soft tissue was released from the fracture site and a 26161 mm sized medpor barrier sheet implant (porex surgical products group, newnan, ga, u.s.a.) was placed over the defect. one month after surgery, the diplopia disappeared in the primary position but remained in downgaze, especially during adduction. in addition, we detected newly developed left upper eyelid retraction and lid lag on downgaze (fig. an orbital ct scan revealed adhesion between the superior rectus and superior oblique muscle (fig. the patient was followed for 10 months and the left upper eyelid retraction persisted without improvement. this case was very similar to the first case, with the exception of the latent period. a 24-year - old man presented with left upper eyelid swelling and diplopia after falling down the previous day. orbital ct showed a large left medial wall blow - out fracture and no specific findings regarding extraocular muscles or orbital soft tissue (fig. the surgical outcome was satisfactory without any ocular motility disorder except left upper eyelid retraction that developed at the time of trauma (fig. the patient strongly denied preexisting upper eyelid retraction and refused a secondary orbital ct, therefore we could not pursue further evaluation of other possible causes of the eyelid retraction. case 3. a 44-year - old woman presented with left upper eyelid retraction following periorbital contusion one week prior to initial presentation. upon examination we noted upper eyelid retraction with a 1 mm scleral show and a lid lag of her left eye (fig. thyroid function testing was performed to exclude thyroid ophthalmopathy, and an orbital ct scan was performed. the thyroid function test included t3, t4, free t4, tsh, tsh receptor antibodies, and antithyroglobulin antibodies. the laboratory results were normal and ct showed mild and diffuse thickening between the left superior rectus muscle and the levator complex (fig. a 56-year - old man visited our clinic for a left orbital contusion and lower canalicular laceration sustained in a traffic accident. his initial orbital ct scan revealed no specific findings, so the following day we repaired the canalicular laceration and inserted a crawford tube. however, one month after surgery, even though the sutured wound was in good condition, he presented with upper eyelid retraction and a slight lid lag on the left eye (fig. a second orbital ct scan with enhancement was performed, but no noticeable findings resulted, unlike in cases 1 and 3 (fig. he was followed for 3 months and did not return for subsequent follow - up. ptosis is a common complication of orbital trauma, and is caused by damage of the levator muscle and aponeurosis.8 for that reason, upper eyelid retraction is a paradoxical response. there are few reports on posttraumatic upper eyelid retraction with latency periods ranging from several months to years.4 - 6 putterman.4 reported upper eyelid retraction after a blow - out fracture, and hypothesized that lid retraction occurs secondary to overaction of mller 's muscle. this argument was supported by the fact that two patients were successfully treated with excision of mller 's muscle. in contrast, conway5 reported upper eyelid retraction following a blow - out fracture of the orbit with enophthalmos and postulated that this was due to traction on the connective tissue sheath of the levator palpebrae muscle through its connective tissue interconnections with other extraocular muscles. hatt6 also described three cases of posttraumatic upper eyelid retraction without apparent involvement of the superior rectus muscle after latency periods ranging from several months to years ; however, he was uncommitted regarding the mechanism of eyelid retraction. in our study, none of the patients had any systemic diseases and they all denied the possibility of preexisting retraction of the upper eyelid prior to trauma. if upper eyelid retraction is caused by overstimulation of the superior rectus and levator, lid lag should improve in downgaze, when innervation to these muscles is minimal.5 in our reported cases, patients had constant lid retraction in all fields of gaze and a significant lid lag in downgaze. these observations suggest a mechanical cause for lid retraction rather than a neuromuscular cause, such as levator overaction. because thyroid eye disease may occur in the absence of clinical or biochemical evidence of thyroid dysfunction, thyroid orbitopathy can not be easily excluded.9 however, sudden onset immediately after trauma and only affecting the traumatized eye support the role of adhesion rather than thyroid orbitopathy as the underlying mechanism of eyelid retraction. in the first case, since no limitation was noted at elevation and we relieved all of the incarcerated soft tissue during surgery, this diplopia may have been induced by involvement of the superior oblique muscle, as indicated by the ct scan (fig. this hypothesis is similar to that proposed by conway,5 who suggested that upper eyelid retraction is brought on by adhesion of the levator complex to adjacent connective tissue after a blow - out fracture. in the last two cases of our study, upper eyelid retractions were present without fractures. in case 3, considering the patient 's history of trauma, we believe that an organizing hematoma or granulation tissue caused an adhesion to form around the levator complex, and this adhesion contributed to the upper eyelid retraction. in case 4, ct and laboratory tests showed no abnormalities, and we speculate that a slight adhesion not visualized by ct might have caused the upper eyelid retraction. cases 2 and 3 complained of eyelid retraction immediately after periorbital contusion without any latency periods. case 2 especially differs from those previously described as there was no fracture or latency period. conway5 reported lid retraction 2 months after a blow - out fracture and hatt6 stated that it took several months to years before lid retraction occurs following periorbital trauma. in our study, it took one to two months to show lid retraction after trauma in the first and fourth cases, but retraction was immediate in cases 2 and 3. we are unable to propose a mechanism underlying this latency, but our cases demonstrate that it can vary greatly. thus, when taking a history in a patient with upper eyelid retraction, a protracted trauma history should be considered. case 3 is very concerned about the eyelid retraction, and cosmetic surgery will be undertaken in the absence of improvement over the next 6 months. in summary, eyelid retraction due to superior complex adhesion can occur after orbital trauma, and should be considered one of the complications of trauma. moreover, adhesions that cause eyelid retraction are likely to be visualized by ct, but they may not be observed in all cases. | we report four unusual cases of upper eyelid retraction following periorbital trauma. four previously healthy patients were evaluated for unilateral upper eyelid retraction following periorbital trauma. a 31-year - old man (case 1) and a 24-year - old man (case 2) presented with left upper eyelid retraction which developed after blow - out fractures, a 44-year - old woman (case 3) presented with left upper eyelid retraction secondary to a periorbital contusion that occurred one week prior, and a 56-year - old man (case 4) presented with left upper eyelid retraction that developed 1 month after a lower canalicular laceration was sustained during a traffic accident. the authors performed a thyroid function test and orbital computed tomography (ct) in all cases. thyroid function was normal in all patients, ct showed an adhesion of the superior rectus muscle and superior oblique muscle in the first case and diffuse thickening of the superior rectus muscle and levator complex in the third case. ct showed no specific findings in the second or fourth cases. upper eyelid retraction due to superior complex adhesion can be considered one of the complications of periorbital trauma. |
aminosalicylates are key drugs in the treatment of mild - to - moderate active ulcerative colitis (uc). previous systematic reviews and meta - analyses have suggested that these drugs are effective in both inducing remission of mild - to - moderate active disease and in preventing relapse [13 ]. mesalazine, a 5-aminosalicylic acid (5-asa) compound, was developed as a better tolerated alternative to sulphasalazine and is now used throughout the world for the treatment of uc. the efficacy of mesalazine in maintaining remission in patients with uc is well established [19 ]. paoluzi. demonstrated that patients taking 2.4 g of oral mesalazine remained in remission longer than those taking a 1.2 g dose.. also demonstrated that a 3.0 g dose of oral mesalazine is more effective in the prevention of uc relapse than a 1.5 g dose.. demonstrated that a significantly greater number of patients maintained remission with a 4 g / day dose of mesalazine than with placebo, and the frequency of adverse events was not increased. although schroeder. demonstrated that 4.8 g / day mesalazine is effective in inducing remission and in the short - term (6 weeks) for mildly to moderately active ulcerative colitis, the efficacy in the long - term for remission maintenance has not been demonstrated. the efficacy of high - dose (4.0 g / day) mesalazine as maintenance therapy remains to be determined. therefore, we focused on the prevention of relapse in patients that showed improvement or achieved clinical remission, and conducted a multicenter retrospective study to investigate the efficacy of continuous treatment with 4.0 g / day mesalazine in patients with uc. japanese patients with uc were identified from 14 hospitals and 6 clinics associated with the nagasaki ibd study group. diagnosis was based on clinical, endoscopic, and histological features. from january 2008 to may 2012, patients who clinically improved or who achieved clinical remission after acute induction therapy with 4.0 g / day mesalazine were selected. the median duration of treatment with 4.0 g / day mesalazine (from date of initiation of 4.0 g / day mesalazine to the date to discontinuation of 4.0 g / day mesalazine, or from the date of initiation of 4.0 g / day mesalazine to september 1, 2012, if 4.0 g / day mesalazine was continued) was calculated. patients were then categorized into 2 sub - groups according to the median duration of treatment with 4.0 g / day mesalazine : a short - term treatment group and a long - term treatment group. written consent was not sought for this retrospective study, as it did not affect the management of its subjects. however, patients who underwent continuous 4.0 g / day mesalazine therapy agreed to the treatment protocol. approval for analysis of outcomes in this study was obtained from the nagasaki ibd study group. data were obtained from medical records from january 2008 to september 2012. the clinical relapse frequency and the time to relapse from the date of initiation of 4.0 g / day mesalazine relapse was defined as the occurrence of any clinical symptoms of uc requiring further rescue therapy. fisher s exact test and student s t - test were used to compare data from the 2 patient groups. survival times were evaluated using the kaplan - meier method, while the variability of these measurements was compared using the log - rank test. a p - value 105 days, n=57). the characteristics of the patients in the 2 groups at baseline are shown in table 1. the mean exposure period to 4.0 g of mesalazine in the long - term and short - term treatment groups was 426 and 55 days, respectively. no significant differences were observed with regard to demographic or disease - specific parameters between the treatment groups. overall, 45 (39.1%) patients relapsed : 28 (48.3%) in the short - duration treatment group and 17 (29.8%) in the long - duration treatment group and this difference was statistically significant (fisher s exact test, p 2 years). this further suggests that a higher luminal concentration of 5-asa may be necessary in the early stages of remission. in the present study, the mean time to relapse in the short - term treatment group was 282.9217.2 days, and most relapses occurred within 12 months of the start of acute induction therapy. although good adherence may be more important than high mucosal concentrations of 5-asa for long - term maintenance of remission, high - dose mesalazine (4.0 g / day) may be necessary in the early stages of remission maintenance. safety and tolerance are important aspects of chronic maintenance treatment. in this analysis, 4.0 g in conclusion, the results emerging the present study showed that long - term continuous treatment with high - dose mesalazine (4.0 g / day) may be more effective than short - term treatment for maintenance of remission in uc patients. first, the retrospective and nonrandomized nature of the design entailed an inherent bias in patient selection and confounding. second, our population included only patients who clinically improved or who achieved clinical remission. therefore, this population may have consisted of patients who had not achieved full mucosal healing, as well as those who had achieved complete clinical and endoscopic remission. to confirm our findings, prospective randomized controlled trials are necessary | backgroundhigh - dose (4.0 g / day) mesalazine is typically used for induction therapy, but its efficacy as maintenance therapy remains to be determined. we conducted a multicenter retrospective study to investigate the efficacy of continuous treatment with 4.0 g / day of mesalazine.material/methodsjapanese ulcerative colitis (uc) patients receiving acute induction therapy with 4.0 g / day mesalazine were enrolled and followed. those who clinically improved or who achieved clinical remission were categorized into 2 sub - groups according to the median duration of treatment with 4.0 g / day of mesalazine. the clinical relapse frequency and the time to relapse were analyzed.resultswe enrolled 180 patients with active uc, and then 115 patients who clinically improved or who achieved clinical remission after treatment with 4.0 g / day mesalazine were categorized into 2 sub - groups according to the median of treatment duration : a short - term treatment group (105 days, n=58) and a long - term treatment group (> 105 days, n=57). overall, 45 (39.1%) patients relapsed : 28 (48.3%) in the short - term treatment group and 17 (29.8%) in the long - term treatment group. this difference was statistically significant (p<0.05). the relapse - free rate in the long - term treatment group was significantly higher than that in the short - term treatment group (p<0.05). the mean time to relapse in the long - term treatment group was significantly longer than that in the short - term treatment group (425.6243.8 days vs. 277.4224.5 days ; p<0.05).conclusionslong - term continuous treatment with high - dose mesalazine (4.0 g / day) may be more effective than short - term treatment for maintenance of remission in uc patients. |
lung cancer is the most common cause of cancer - related deaths worldwide, with more than 1.6 million new cancer cases and 1.4 million deaths in the year 2008. more than 80% of patients with primary lung cancer were pathologically diagnosed with non - small cell lung cancer (nsclc). approximately 55% of patients with nsclc suffer from distant metastases, including to bone, liver, lung, and brain. it has been reported that 3065% of patients with nsclc will develop bone metastases, with a median survival of less than 8 months. bone metastases can result in significant morbidity and severely decreased quality of life [911 ] due to various skeletal complications such as pathological fractures, serious bone pain, spinal cord compression, and hypercalcemia. to date, the exact molecular and cellular mechanisms of the development of nsclc and bone metastasis in nsclc are still unclear. many studies [1417 ] have recently reported that genes, including osteopontin, ccr2 - 64i, xrcc1, and il-1 gene, may play roles in the emergence and progression of nsclc. the cd44 gene, located on chromosome 11p13, encodes a cell surface glycoprotein cd44 [1921 ]. as a cell - surface glycoprotein, cd44 is widely known as a cell - surface receptor for osteopontin and hyaluronate and it mediates cellular adhesion to the cell - extracellular matrix (ecm), which is correlated with tumor cell migration. besides its roles in many cellular processes, cd44 plays a crucial role in tumor cell differentiation, invasion, and metastasis. overexpression of cd44 reportedly leads to poor clinical outcomes and poor prognosis for human tumors [2628 ]. recent studies have shown that the cd44 gene interacts with proliferation, migration, and invasion of tumor cells, and that genetic variants of cd44 are associated with the survival, risk prediction, and prognosis of patient with nsclc. as the most common type of dna sequence variation, single - nucleotide polymorphisms (snps), may affect the expression of certain genes. previous studies have reported that snps rs13347 and rs187115 in cd44 gene were significantly associated with the risk of human tumors [32,3537 ]. however, few studies have investigated the association of cd44 polymorphisms with survival of nsclc and risk of bone metastasis. therefore, we conducted the present study to investigate whether the 2 snps (rs13347 and rs187115) in the cd44 gene have roles on the survival of patients with nsclc and to explore whether they are associated with incidence of bone metastasis development. this study was approved by the ethics committee of the 307 pla hospital. written informed consent was obtained from all enrolled participants. in this study, we recruited a total of 234 patients (156 males and 78 females) diagnosed with nsclc between july 2003 and september 2010, 65 of which had bone metastasis and the remaining 169 were without bone metastasis. matched - control subjects were 468 unrelated healthy individuals (308 males and 160 females) during the same period, based on sex and age. the tnm stage of the patients and differentiation system were determined according to international association of lung cancer. the characteristics of the patients from medical records, including age, sex, pathological tumor stage, histopathological diagnosis, and the follow - up, are displayed in table 1. all patients in this study were assessed for bone metastasis according to methods described in a previous study. blood samples were collected for genomic dna extraction from patients and healthy controls in vacutainer tubes containing heparin as an anticoagulant. genomic dna was prepared using the qiaamp dna mini kit (qiagen, hilden, germany) according to the manufacturer s instructions. snp rs13347 and rs187115 in cd44 gene were genotyped using the taqman allelic discrimination method (applied biosystems, foster city, ca, usa). the taqman assays were conducted in a final reaction volume of 5 ml containing 0.25 ml primer, 0.125 ml probe, 2 ml pcr mixture reagent, and 25 ng dna. the polymerase chain reaction (pcr) consisted of an initial step at 95c for 10 min followed by 55 cycles of denaturing at 95c for 15 s and annealing at 60c for 1 min. all statistical analyses were performed using spss19.0 software (spss company, chicago, il). the differences in distributions of the variables between patients and healthy controls were evaluated by mann - whitney u test and fisher s exact test. the comparison of allele type and genotype distributions in the patients and healthy controls was assessed by computing the odds ratio (or) and 95% confidence intervals (95% ci) from logistic regression analyses. survival was calculated from the date of surgery to the time of death using the kaplan - meier method. genomic dna was prepared using the qiaamp dna mini kit (qiagen, hilden, germany) according to the manufacturer s instructions. snp rs13347 and rs187115 in cd44 gene were genotyped using the taqman allelic discrimination method (applied biosystems, foster city, ca, usa). the taqman assays were conducted in a final reaction volume of 5 ml containing 0.25 ml primer, 0.125 ml probe, 2 ml pcr mixture reagent, and 25 ng dna. the polymerase chain reaction (pcr) consisted of an initial step at 95c for 10 min followed by 55 cycles of denaturing at 95c for 15 s and annealing at 60c for 1 min. all statistical analyses were performed using spss19.0 software (spss company, chicago, il). the differences in distributions of the variables between patients and healthy controls were evaluated by mann - whitney u test and fisher s exact test. the comparison of allele type and genotype distributions in the patients and healthy controls was assessed by computing the odds ratio (or) and 95% confidence intervals (95% ci) from logistic regression analyses. survival was calculated from the date of surgery to the time of death using the kaplan - meier method. among a total of 702 subjects of this study, the mean age was 62.219.8 years in cases and 61.8710.75 in matched - pair controls. there were no significant differences in mean age and sex distributions between cases and controls. distribution of genotypes of the 2 polymorphisms in healthy control group was in accordance with hardy - weinberg equilibrium in our study. of the 234 nsclc patients, 65 (28%) were diagnosed with bone metastasis. the follow - up period ranged from 23 to 117 months (median, 60 months ; mean, 71 months). among the 234 patients, 171 patients survived less than 5 years and 63 patients survived over 5 years (5-year survival rate of 27%). characteristics of nsclc patients and age- and sex - matched controls are shown in table 1. table 2 shows the association of each of the snps in cd44 gene with the risk of nsclc. there were no significant differences in the genotypic and allelic distributions of rs13347 polymorphisms between nsclc patients and healthy controls. however, significant differences were observed in the relationship between frequency distributions of rs187115 and the nsclc risk. as shown in table 3, further analysis based on stratification of clinicopathological features revealed significant correlations of frequency distributions of rs187115 snp with nsclc risk in tumor stage (or=2.6, 95%ci : 1.504.45, p=0.001) and bone metastasis (or=0.4, 95%ci : 0.200.64, p t found that polymorphism may affect breast cancer development and prognosis by increasing cd44 expression.. also found that ct and tt genotypes of rs13347 in cd44 gene were associated with increased risk of nasopharyngeal carcinoma. did not observe any significant differences in the frequency distribution of the genetic variants cd44 rs13347 between cases and healthy controls. in our study, we found that rs187115 was associated with nsclc ; however, the relationship between rs13347 and nsclc was not observed. snp rs187115 might become a potential prognostic marker for nsclc patients. some limitations in this study design must be noted. first, selection bias may have occurred because the cases were enrolled from hospital but the healthy controls were chosen from the community. in summary, our study indicated that cd44 rs187115 variant genotypes (ag + gg) could increase the risk of nsclc and decrease survival time compared with aa genotype, and rs187115 was correlated with bone metastasis and tumor stage. however, well - designed, population - based, case - control studies with lager sample sizes are required to confirm these results. | backgroundprevious studies have reported cd44 expression influenced the development and progression of tumors. the aim of this study was to investigate whether single - nucleotide polymorphisms (snps) of the cd44 gene are associated with survival of non - small cell lung cancer (nsclc) and occurrence rate of bone metastasis.material/methodsa total of 234 patients with nsclc between 2003 and 2010 were enrolled in this study and 468 healthy persons were used as controls. two polymorphisms, rs13347 and rs187115, in the cd44 gene were genotyped using dna from blood lymphocytes. for statistical analysis we used the chi - square test, fisher s exact test, kaplan - meier method, and log - rank test.resultscd44 gene rs13347 polymorphism was not associated with nsclc risk. for rs187115, the association with nsclc risk was observed (p<0.001). allele g carriers had significantly higher occurrence rates of bone metastasis (or=0.4, 95%ci : 0.200.64, p<0.001) and more advanced tumor stage (or=2.6, 95%ci : 1.504.45, p=0.001) compared to carriers of allele a. the survival rates for patients with aa genotype were significantly higher than for patients with the ag+gg genotypes (p<0.001). in multivariate analysis of survival in nsclc patients, significant predictors were cd44 gene (ag+gg) (rr=0.48, 95%ci : 0.340.68, p<0.001), tumor stage (rr=0.45, 95%ci : 0. 0.310.65, p<0.001), and bone metastasis (rr=1.52, 95%ci : 1.052.21, p=0.027).conclusionscd44 gene rs187115 polymorphism is a potential predictive marker of survival in nsclc patients, and is significantly correlated with bone metastasis and tumor stage. |
pathologic studies1)2)3) have shown plaque ruptures in 55 - 70% of patients and plaque erosion in 25 - 40% of patients with sudden coronary death. however, the mechanisms of plaque erosion remain unclear and the clinical importance and impact have been underestimated due to limited resolution of intravascular ultrasound (ivus) and computed tomography.4) in addition, small plaque ruptures and unruptured thin - capped fibroatheromas (tcfa) with superimposed thrombosis have not been fully evaluated.5)6) optical coherence tomography (oct) provides high resolution cross - sectional imaging of atheromatous plaques in vivo. previous studies have shown that oct can detect tcfas, plaque ruptures, plaque erosions, and intracoronary thrombus with greater accuracy than ivus, although the limited penetration of oct especially in the setting of high - risk plaques with superimposed red thrombus can affect the quantitative assessment of lipid and remodeling.7)8)9)10)11)12)13) we hypothesized that combined imaging with oct and radiofrequency (virtual histology [vh])-ivus would overcome the limitation of each imaging modality. therefore, we conducted a multicenter study using both oct and vh - ivus to assess culprit lesion morphology of patients presenting acute coronary syndromes (acs). a total of 133 consecutive patients underwent percutaneous coronary intervention with both oct and vh - ivus imaging to assess the culprit lesion at five centers participating in korea cardiovascular imaging oct registry. patients with bifurcation lesions, ostial lesions, vein graft lesions, multiple culprit lesions, arteries with previous stent placement, debulking or plaque modification procedures before intravascular imaging, prominent red thrombus, end - stage renal disease, and severe left ventricular dysfunction were excluded. thrombus aspiration was performed prior to oct and ivus imaging using an aspiration catheter (thrombuster, kaneka co., osaka, japan) according to operator discretion, but typically for large thrombi in the setting of st - segment elevation myocardial infarction (stemi) because such thrombi would affect the penetration of oct and the accuracy of vh - ivus. stemi was diagnosed based on continuous chest pain for at least 30 min, arrival at the hospital within 6 hour from the onset of symptoms, st - segment elevation>0.1 mv in two or more contiguous leads or newly found left bundle - branch block on the 12-lead electrocardiogram (ecg), and elevated cardiac markers (plasma creatine kinase - myocardial band or troponin i). st - segment elevation myocardial infarction (nstemi) was defined as ischemic symptoms with elevated cardiac markers in the absence of st - segment elevation on ecg. unstable angina pectoris was defined as new onset / accelerating chest symptoms on exertion or rest angina within two weeks. culprit lesion was identified based on coronary angiogram, stress test, ecg, and/or echocardiogram. hypertension was defined as systolic blood pressure140 mmhg or diastolic blood pressure90 mmhg or current use of antihypertensive treatment. diabetes mellitus was defined as hemoglobin a1c6.5 mg / dl or taking medication for diabetes mellitus. dyslipidemia was classified as total cholesterol level220 mg / dl, low - density lipoprotein cholesterol level140 mg / dl, high - density lipoprotein cholesterol level 10% confluent necrotic core (nc) with > 30 nc abutting the lumen in 3 consecutive frames without evidence of fibrous cap. thcfa was a fibroatheroma (> 10% of confluent nc in 3 consecutive frames) with a definable fibrous cap.16) positive remodeling was defined as a remodeling index (lesion / reference external elastic membrane [eem ] area)>1.05. intermediate remodeling was defined as a remodeling index between 0.95 and 1.05, and negative remodeling as a remodeling index0.1 mv in two or more contiguous leads or newly found left bundle - branch block on the 12-lead electrocardiogram (ecg), and elevated cardiac markers (plasma creatine kinase - myocardial band or troponin i). st - segment elevation myocardial infarction (nstemi) was defined as ischemic symptoms with elevated cardiac markers in the absence of st - segment elevation on ecg. unstable angina pectoris was defined as new onset / accelerating chest symptoms on exertion or rest angina within two weeks. culprit lesion was identified based on coronary angiogram, stress test, ecg, and/or echocardiogram. hypertension was defined as systolic blood pressure140 mmhg or diastolic blood pressure90 mmhg or current use of antihypertensive treatment. diabetes mellitus was defined as hemoglobin a1c6.5 mg / dl or taking medication for diabetes mellitus. dyslipidemia was classified as total cholesterol level220 mg / dl, low - density lipoprotein cholesterol level140 mg / dl, high - density lipoprotein cholesterol level 10% confluent necrotic core (nc) with > 30 nc abutting the lumen in 3 consecutive frames without evidence of fibrous cap. thcfa was a fibroatheroma (> 10% of confluent nc in 3 consecutive frames) with a definable fibrous cap.16) positive remodeling was defined as a remodeling index (lesion / reference external elastic membrane [eem ] area)>1.05. intermediate remodeling was defined as a remodeling index between 0.95 and 1.05, and negative remodeling as a remodeling index<0.95. culprit lesions on vh - ivus and oct studies were compared using reproducible axial landmarks (usually the aorto - ostial junction, a large proximal side branch, and/or reference segment calcific deposits) and know pullback speeds. student t test was performed to compare the difference between measurements, and categorical variables were compared using chi - square test. all analyses were performed using standard statistical software (spss version 18.0, ibm, chicago, il, usa). all p values were two - tailed, and statistical significance was considered when p value was less than 0.05. a total of 133 consecutive patients had oct and vh - ivus studies that were amenable to analysis, including 90 patients who had oct - plaque rupture and 43 patients who had oct - plaque erosion (36 definite and 7 probable). patient age, gender, and clinical risk factors in the plaque rupture group were similar to those in the plaque erosion group. however, the prevalence of plaque rupture was significantly higher in stemi, 71% (64/90) (p<0.0001) compared to the prevalence of plaque erosion, while the prevalence of plaque erosion was significantly higher in unstable angina / nstemi (68% [28/43 ]) (p<0.0001) compared to that of plaque rupture. quantitative angiographic analysis showed that plaque erosion lesions were significantly shorter than plaque ruptures(p<0.0001), had significantly smaller reference vessel diameter (p=0.0047), less severe diameter stenosis (p<0.0001), and higher rate of thrombolysis in myocardial infarction (timi) grade 3. eem area was larger in plaque ruptures (p=0.0359) with larger plaque burden (p<0.0001), more positive remodeling (p=0.029) and larger nc (p<0.0001). conversely, 58.1% (25 of 43) definite / probable plaque erosions showed negative remodeling. based on vh - ivus, 83% (36 of 43) of plaque erosions were fibrotic, and 17% (7 of 43) were fibrocalcific. compared to plaque erosions, 43.3% (39 of 90) of plaque ruptures had the appearance of vh - tcfa, 12.2% (11 of 90) were pit, 17.7% (16 of 90) were fibrotic, 20% (18 of 90) were fibrocalcific, and 6.6% (6 of 90) were thick cap fibroatheromas (p<0.0001, table 2). the oct findings of plaque erosion are shown in fig. 1 and table 3. based on oct, 60% (26 of 43) of definite / probable plaque erosions were fibrotic, 16% (7 of 43) were fibrocalcific, 11.6% (5 of 43) were lipidic, and 11.6% (5 of 43) had thick capped fibroatheroma. none of the plaque erosion had the appearance of oct - tcfa, and three of 43 (7%) definite / probable plaque erosions contained calcific nodule. conversely, 52% (47 of 90) of ruptured plaques had the appearance of oct - tcfa with a fibrous cap thickness of 517.05 m. approximately 4.4% (4 of 90) of ruptured plaques were fibrotic, 28.8% (26 of 90) were lipidic, 13.3% (12 of 90) were fibrocalcific, and 1.1% (1 of 90) had the appearance of a thick cap fibroatheroma (p<0.0001 vs. plaque erosions). intracoronary thrombus was found in 83.7% (36/43) of plaque erosion ; white thrombus in 55.8% (24/43) with red thrombus in 27.9% (12/43), and no detectable thrombus in 16.3% (7/43) of plaque erosion. conversely, intracoronary thrombus was observed in all cases of plaque rupture which was mostly red thrombus (92% [83/90 ]) (p<0.0001 vs. plaque erosions). overall, 46.5% (20/43) of plaque erosions were located proximal to the minimal lumen area (mla) site while 27.9% (12/43) were distal to the mla site and 25.6% (11/43) were at the mla site. conversely, 57.7% (52/90) of plaque ruptures were located proximal to mla site, 42.3% (38/90) were distal to the mla site, and none was located at the mla site (p=0.002 vs. plaque erosions). the distance from the coronary ostium to the site of plaque rupture was 23.5418.42 mm, similar to the distance from the coronary artery ostium to the site of plaque erosion (23.3717.82 mm, p=0.96). overall, 65% (28/43) of plaque erosions were located in the proximal 30 mm of a culprit vessel, similar to plaque ruptures (72%, 65 of 90, p=0.29) (fig. 2), although erosions in right coronary artery (rca) appeared to be more diffusely distributed compared to erosions in left anterior descending coronary artery or left circumflex coronary artery and also compared to ruptures in rca. a total of 133 consecutive patients had oct and vh - ivus studies that were amenable to analysis, including 90 patients who had oct - plaque rupture and 43 patients who had oct - plaque erosion (36 definite and 7 probable). patient age, gender, and clinical risk factors in the plaque rupture group were similar to those in the plaque erosion group. however, the prevalence of plaque rupture was significantly higher in stemi, 71% (64/90) (p<0.0001) compared to the prevalence of plaque erosion, while the prevalence of plaque erosion was significantly higher in unstable angina / nstemi (68% [28/43 ]) (p<0.0001) compared to that of plaque rupture. quantitative angiographic analysis showed that plaque erosion lesions were significantly shorter than plaque ruptures(p<0.0001), had significantly smaller reference vessel diameter (p=0.0047), less severe diameter stenosis (p<0.0001), and higher rate of thrombolysis in myocardial infarction (timi) grade 3. at the site of plaque erosion or plaque rupture, eem area was larger in plaque ruptures (p=0.0359) with larger plaque burden (p<0.0001), more positive remodeling (p=0.029) and larger nc (p<0.0001). conversely, 58.1% (25 of 43) definite / probable plaque erosions showed negative remodeling. based on vh - ivus, 83% (36 of 43) of plaque erosions were fibrotic, and 17% (7 of 43) were fibrocalcific. compared to plaque erosions, 43.3% (39 of 90) of plaque ruptures had the appearance of vh - tcfa, 12.2% (11 of 90) were pit, 17.7% (16 of 90) were fibrotic, 20% (18 of 90) were fibrocalcific, and 6.6% (6 of 90) were thick cap fibroatheromas (p<0.0001, table 2). the oct findings of plaque erosion are shown in fig. 1 and table 3. based on oct, 60% (26 of 43) of definite / probable plaque erosions were fibrotic, 16% (7 of 43) were fibrocalcific, 11.6% (5 of 43) were lipidic, and 11.6% (5 of 43) had thick capped fibroatheroma. none of the plaque erosion had the appearance of oct - tcfa, and three of 43 (7%) definite / probable plaque erosions contained calcific nodule. conversely, 52% (47 of 90) of ruptured plaques had the appearance of oct - tcfa with a fibrous cap thickness of 517.05 m. approximately 4.4% (4 of 90) of ruptured plaques were fibrotic, 28.8% (26 of 90) were lipidic, 13.3% (12 of 90) were fibrocalcific, and 1.1% (1 of 90) had the appearance of a thick cap fibroatheroma (p<0.0001 vs. plaque erosions). intracoronary thrombus was found in 83.7% (36/43) of plaque erosion ; white thrombus in 55.8% (24/43) with red thrombus in 27.9% (12/43), and no detectable thrombus in 16.3% (7/43) of plaque erosion. conversely, intracoronary thrombus was observed in all cases of plaque rupture which was mostly red thrombus (92% [83/90 ]) (p<0.0001 vs. plaque erosions). overall, 46.5% (20/43) of plaque erosions were located proximal to the minimal lumen area (mla) site while 27.9% (12/43) were distal to the mla site and 25.6% (11/43) were at the mla site. conversely, 57.7% (52/90) of plaque ruptures were located proximal to mla site, 42.3% (38/90) were distal to the mla site, and none was located at the mla site (p=0.002 vs. plaque erosions). the distance from the coronary ostium to the site of plaque rupture was 23.5418.42 mm, similar to the distance from the coronary artery ostium to the site of plaque erosion (23.3717.82 mm, p=0.96). overall, 65% (28/43) of plaque erosions were located in the proximal 30 mm of a culprit vessel, similar to plaque ruptures (72%, 65 of 90, p=0.29) (fig. 2), although erosions in right coronary artery (rca) appeared to be more diffusely distributed compared to erosions in left anterior descending coronary artery or left circumflex coronary artery and also compared to ruptures in rca. the main findings of the present study were as follows : (1) plaque rupture was often associated with positive remodeling, large plaque burden, and red thrombus. (2) plaque erosion was associated with negative remodeling, a modest plaque burden, white thrombus, uncommon features of a fibroatheroma, and proximal distribution. plaque rupture has been reported to be the main cause of acute coronary syndrome (acs) with plaque erosion responsible for most of other cases.1)2)3) necrotic cores were uncommon in erosion lesions and a thick fibrous cap separated the lumen from the necrotic core when it exists. erosions occurred on top of lesions which were rich in smooth muscle cells and proteoglycans.2) plaque erosion is prevalent in female patients less than 50 years old1) and is associated with smoking.3) in this current study, patients with plaque erosions vs plaque ruptures had similar age and gender profiles. however, unstable angina and nstemi were found to be more frequent in plaque erosions while plaque rupture seen in 71% of patients with stemi. these findings were consistent with results of previous studies that plaque erosions are infrequent in stemi patients on admission.2)11)17) the low resolution of ivus precludes the evaluation of erosions. however, a limited number of imaging studies have used oct to evaluate the role of plaque erosion in the pathophysiology of acs in vivo. oct was superior to coronary angioscopy and ivus in the detection of fibrous cap erosion, and fibrotic plaque was most frequent in plaque erosion. however, coronary artery remodeling was not defined due to limited number and heterogenous clinical entities.9)10)11) ruptured plaques of culprit lesion were predominately located in the proximal segments of the coronary arteries. the proximal 30 mm was associated with plaque rupture, tcfa, and lower minimal cap thickness.18)19)20)21) in this study, the longitudinal distribution of plaque erosions was identical to plaque rupture mostly located in proximal portion of the coronary artery except the rca with less plaque area and negative remodeling. positive remodeling is typically considered to be indicative of an unstable lesion, while negative remodeling is indicative of stable coronary artery disease.22) however, the current study suggests that negative remodeling could be a feature of plaque erosions and therefore, it is not always benign, consistent with recent data from providing regional observations to study predictors of events in the coronary tree (prospect) study.23) more than half of plaque erosions in the current study appeared to be fibrotic plaque rather than tcfa. plaque erosions is frequently found with white thrombus compared to plaque rupture in patients with acs associated with red thrombus.11) autopsy studies24) have reported that more than 88% of coronary thrombi overlying plaque erosions showed later stages of healing that are characterized by invasion of organized layers of smooth muscle cells and endothelial cells with various degrees of platelet / fibrin layering. however, only 50% of thrombi showed evidence of healing in patients with plaque rupture.24) fibrin rich red thrombus was frequently found over ruptured plaque, whereas platelet rich white thrombus was the predominant type of thrombus formed over plaque erosion and calcified nodule. in contrast, plaque erosion seems to result in less thrombus burden, preserved vascular structure, and larger lumen.1)24) in current study, 93% of plaque erosions showed < 50% diameter stenosis. more importantly, it might provoke a misdiagnosis in acs patient who showed near normal or normal coronary angiogram. the accuracy of oct and vh - ivus analysis was reduced in the setting of intracoronary thrombus. although patients with culprit lesion with prominent thrombi burden were excluded, the presence of thrombus overlying the culprit lesion might reduce the ability to assess underlying plaque characteristics by oct. in addition, we excluded ambiguous plaque ruptures / erosion with prominent thrombus. plaque erosion and calcified nodule as detected by oct were not validated by autopsy study. patients with large mi and heart failure patients were less likely to undergo pre - intervention oct imaging. although the complication rate was very low, we could not exclude the possibility that plaque erosion might be developed during contrast injection for oct. although plaque erosion shows a near normal coronary angiogram, a modest plaque burden with negative remodeling and uncommon fibroatheroma might be the nature of plaque erosion, which should not be misdiagnosed. multimodality intravascular imaging with oct and vh - ivus showed fundamentally different pathoanatomic substrates underlying plaque rupture and erosion, indicating that they are distinct lesion phenotypes. although plaque erosion shows a near normal coronary angiogram, a modest plaque burden with negative remodeling and uncommon fibroatheroma might be the nature of plaque erosion, which should not be misdiagnosed. multimodality intravascular imaging with oct and vh - ivus showed fundamentally different pathoanatomic substrates underlying plaque rupture and erosion, indicating that they are distinct lesion phenotypes. | background and objectiveswe assessed plaque erosion of culprit lesions in patients with acute coronary syndrome in real world practice.subjects and methodsculprit lesion plaque rupture or plaque erosion was diagnosed with optical coherence tomography (oct). intravascular ultrasound (ivus) was used to determine arterial remodeling. positive remodeling was defined as a remodeling index (lesion / reference eem [external elastic membrane area) > 1.05.resultsa total of 90 patients who had plaque rupture showing fibrous - cap discontinuity and ruptured cavity were enrolled. 36 patients showed definite oct - plaque erosion, while 7 patients had probable oct - plaque erosion. overall, 26% (11/43) of definite / probable plaque erosion had non - st elevation myocardial infarction (nstemi) while 35% (15/43) had st elevation myocardial infarction (stemi). conversely, 14.5% (13/90) of plaque rupture had nstemi while 71% (64/90) had stemi (p<0.0001). among plaque erosion, white thrombus was seen in 55.8% (24/43) of patients and red thrombus in 27.9% (12/43) of patients. compared to plaque erosion, plaque rupture more often showed positive remodeling (p=0.003) with a larger necrotic core area examined by virtual histology (vh)-ivus, while negative remodeling was prominent in plaque erosion. overall, 65% 28/43 of plaque erosions were located in the proximal 30 mm of a culprit vessel - similar to plaque ruptures (72%, 65/90, p=0.29).conclusionalthough most of plaque erosions show nearly normal coronary angiogram, modest plaque burden with negative remodeling and an uncommon fibroatheroma might be the nature of plaque erosion. multimodality intravascular imaging with oct and vh - ivus showed fundamentally different pathoanatomic substrates underlying plaque rupture and erosion. |
in recent times, there has been an increase in the number of people with diabetes. india currently has 62.4 million people with diabetes.1 by 2030, this number is expected to increase to over 100 million.2 the majority of people with diabetes (> 90%) have type 2 diabetes. type 2 diabetes predominantly affects older individuals in developed countries, but in developing nations it also affects the younger population. therefore, the proportion of patients developing cataracts associated with diabetes will be on the rise. are important factors in cataract development.36 cataracts occurring in diabetic patients can be due to the diabetes itself or due to an accelerated age - related cataract, in which case the cataracts occur earlier than normal.7 developments in self - sealing clear corneal incision and topical anesthesia have made fast postoperative functional recovery possible. the challenges encountered by surgeons during phacoemulsification of diabetic cataracts include poor pupillary dilatation, capsulocortical and nuclear cortical adhesions leading to difficult hydroprocedure and nucleus rotation, the sticky and leathery nature of the nucleus, and pupillary constriction during the procedure.811 phacoemulsification is the preferred method for the treatment of diabetic cataract.914 it minimally affects the blood aqueous barrier, thus making it the procedure of choice.15 topical anesthesia is the safe and effective alternative to peribulbar and retrobulbar anesthesia for phacoemulsification.16,17 adequate mydriasis is a prerequisite for cataract surgery, which is achieved by repeated instillation of sympathomimetic and anticholinergic eyedrops. systemic absorption of these drugs may lead to side effects, such as rise in blood pressure, which could be of great concern in patients with a cardiovascular disorder. acute psychotic reactions have been observed after topical instillation of 1% cyclopentolate.18 superior safety and efficacy of intracameral mydriatic solution (cyclopentolate 0.1% and phenylephrine 1.5%) over topical mydriatic drops have been proved in various studies.1921 however, intracameral mydriatic solution needs to be prepared from commercially available eyedrops before cataract surgery. intracameral phenylephrine has a weaker mydriatic effect than topical mydriatics, rendering small pupillary size during cataract surgery.22 this may be disadvantageous in diabetic patients with cataract. the safety and efficacy of 1% intracameral lignocaine hydrochloride during routine phacoemulsification surgery have been shown in various studies.2327 it not only produces a mydriatic effect but also causes anesthesia of the iris. we evaluated the role of preservative - free intracameral 1% lignocaine in initiating and maintaining pupillary dilatation during phacoemulsification of diabetic cataract without the use of preoperative mydriatics or nonsteroidal anti - inflammatory drugs. secondary aims were to see the effect of the chop technique, nucleus density, and the duration of diabetes on sustainability of mydriasis during the phacoemulsification. this prospective interventional case series comprised patients with visually significant cataracts and type 2 diabetes of variable duration scheduled for phacoemulsification and intraocular lens implantation. after dilatation of pupil (tropicamide 0.8% and phenylephrine hydrochloride 5%), the patient was taken to the operation theater for measurement of pupillary size. the grade of cataract was determined with lens - opacity classification system iii on slit lamp.28 patients with pseudoexfoliation, subluxated lens, pupillary size of 4 mm and less after dilatation, postvitrectomized eyes, just one eye, and visible iris deformity were excluded from the study. patients with intraoperative iris trauma and posterior capsular rupture were also excluded from the study. the ethical committee of government medical college and hospital, yavatmal, maharashtra, india approved the study, and all patients signed an informed consent form to participate in the study. a single surgeon performed all surgeries. proparacaine hydrochloride 0.5% (paracain ; sunways ltd, mumbai, india) drops were instilled every 5 minutes, twice before the start of the surgery. preservative - free lignocaine (1%, 0.3 ml ; oculigno ; entod pharmaceuticals, mumbai, india) was injected into the anterior chamber. phacoemulsification was accomplished in the capsular bag by the vertical chop technique (galaxy pro phacoemulsifier ; appasamy associates, chennai, india).29 subsequently, cortical cleanup was completed, followed by implantation of a single - piece hydrophilic intraocular lens in the bag. the horizontal diameter of the pupil was measured under an operating microscope with a caliper. the intensity of the microscope light was kept constant throughout the procedure. at 90 seconds after injection of 1% lignocaine in the anterior chamber, the horizontal diameter of the pupil was measured again. at the termination of the surgical procedure and after the wound - hydration diameter of the pupil was measured, the patient s comfort, painful intraoperative sensations perceived by the patient, supplemental anesthesia, complications, and surgeon discomfort were noted. data were entered into an excel spreadsheet (version 14.1.0 ; microsoft corporation, redmond, wa, usa), and statistical analysis was performed with spss version 20.0 (ibm, armonk, ny, usa). proparacaine hydrochloride 0.5% (paracain ; sunways ltd, mumbai, india) drops were instilled every 5 minutes, twice before the start of the surgery. preservative - free lignocaine (1%, 0.3 ml ; oculigno ; entod pharmaceuticals, mumbai, india) was injected into the anterior chamber. phacoemulsification was accomplished in the capsular bag by the vertical chop technique (galaxy pro phacoemulsifier ; appasamy associates, chennai, india).29 subsequently, cortical cleanup was completed, followed by implantation of a single - piece hydrophilic intraocular lens in the bag. the horizontal diameter of the pupil was measured under an operating microscope with a caliper. the intensity of the microscope light was kept constant throughout the procedure. at 90 seconds after injection of 1% lignocaine in the anterior chamber, the horizontal diameter of the pupil was measured again. at the termination of the surgical procedure and after the wound - hydration diameter of the pupil was measured, the patient s comfort, painful intraoperative sensations perceived by the patient, supplemental anesthesia, complications, and surgeon discomfort were noted. data were entered into an excel spreadsheet (version 14.1.0 ; microsoft corporation, redmond, wa, usa), and statistical analysis was performed with spss version 20.0 (ibm, armonk, ny, usa). the study included 21 males and eleven females with a mean age of 543 years. the average size of the pupil under microscopy without intracameral injection of 1% lignocaine was 20.5 (range 1.53) mm. the average size of the pupil after intracameral injection of 1% lignocaine was 50.5 (range 2.58.5), which took 90 (0.9) seconds. at the end of the surgical procedure, the mean size of the pupil was 5.50.5 (range 2.55.8) mm (p=0.45). there was no difference in dilatation from preoperative dilating drops (5.20.5 mm, range 38.3 mm) and intracameral 1% lignocaine during the surgical procedure (p=0.63). there was a negative correlation (r=0.92) between duration of diabetes and pupil dilatation of with dilating drops and intracameral lignocaine (figure 1). with an increase in the duration of diabetes the average duration of the surgical procedure was 18 (5.3) minutes, and the average effective phacoemulsification time was 38 (15.11) seconds. intracameral supplementation of adrenaline was necessitated in three patients, as the pupil became extremely small. in one patient, topical 0.5% proparacaine drops were instilled and 1 ml subconjunctival injection of 2% lignocaine hydrochloride was given. the effect of mydriatic and intracameral lignocaine on blood pressure and pulse is illustrated in table 2. though there was an increase in blood pressure and pulse from baseline, it was not statistically significant after instillation of mydriatics. however, with intracameral lignocaine, there was no significant rise in blood pressure or pulse. there were no surgical complications that could have compromised the visual outcome, and none of the patients had developed macular edema after a follow - up period of 3 months. a total of 28 patients (87.5%) had best - corrected visual acuity from 20/30 to 20/20. four patients (12.5%) had visual acuity ranging from 20/60 to 20/30 due to disc pallor. mydriasis before cataract surgery is achieved by repeated instillation of topical anticholinergic and sympathomimetic mydriatic agents, such as tropicamide 1%, cyclopentolate 1%, and phenylephrine 2.5% or 10%. this increases the preoperative waiting period and has systemic and ocular adverse effects.1823 ocular surface problems are known in diabetic patients.30,31 the presence of preservatives in the formulations may precipitate surface ocular problems. to overcome these problems, 1% intracameral lignocaine, combined 0.5% lignocaine + 0.001% epinephrine, and intracameral mydriatic solution containing lignocaine and phenylephrine have been tried in routine cataract surgery for the initiation and maintenance of pupillary dilatation.18,19,3034 intracameral preservative - free lignocaine has been tried in routine cataract and trabeculectomy surgery to relieve pain and discomfort.35 lincoff noted that mydriasis after accidental injection of lignocaine resulted in complete recovery of retinal functions after 4 hours and both retinal and pupillary function by 16 hours.36 the safety of intracameral lignocaine on the endothelium has been shown in various studies.24,37,38 it has been shown by iradier that it does not cause inflammation of ocular tissues on intraocular use.27 none of the patients in our study developed corneal complications in the form of prolonged corneal edema or decompensation. nikeghbali studied the role of intracameral lignocaine in cataract surgery.23 however, only four patients with diabetes were included, and the duration of diabetes was not mentioned in the study. this study shows that phacoemulsification can be performed safely with intracameral injection of 1% preservative - free lignocaine without preoperative instillation of mydriatics in diabetic patients with variable duration. average pupillary size was 5 mm with preoperative mydriatics and intracameral lignocaine, suggesting that mydriatics can be avoided preoperatively. the pupillary size noted by nikeghbali after intracameral injection of lignocaine was 7.1 mm.23 the pupillary size noted by gupta was 6.9 mm with 0.5% lignocaine and 0.001% epinephrine in routine cataract surgery.33 we did not use epinephrine in irrigating solution. bozkurt have shown that intracameral injection of 0.2 ml epinephrine (1:5,000) did not increase the risk of central macular edema in eyes with no risk factors that had uneventful phacoemulsification with intraocular lens implantation.39 however, patients with diabetes are prone to the development of macular edema postoperatively with the use of epinephrine.40,41 none of the patients in our study had developed macular edema at the 3-month follow - up. autonomic dysfunction in diabetes has been shown to cause small pupils and reduced dilatation with mydriatics.42 we have noted a negative correlation between the duration of diabetes and pupil size. a number of previous studies have noted that patients with a long duration of diabetes have small pupils and that there is a statistically significant association between small pupils and the severity of diabetes.4345 the average duration of diabetes in our study was 11.2 years. with an increase in the duration of diabetes one patient needed a pupil - expanding device, as the pupil failed to dilate with epinephrine. the average surgical time in our study was 18 minutes, which is more than that of the studies by nikeghbali, gupta, and joshi.23,29,33 the average surgical time noted by joshi in age - related cataract was 6.9134.45 minutes, wherein patients with nondilating pupils and diabetic cataracts were excluded. we could not compare our surgical time, as there is no literature available on the use of only intracameral lignocaine in patients with cataract with type 2 diabetes. the lower surgical times in nikeghbali (11.673.05 minutes) and gupta (13 minutes) could have been due to the inclusion of nondiabetic patients and the use of epinephrine in the irrigation. the extended surgical time in our study could have been related to peculiarities in cataract associated with diabetes of longer duration. it took an average of 90 seconds to dilate pupils to the level of preoperative mydriatics. none of the patients developed glaucoma postoperatively. with the hard, tenacious fibers of diabetic cataract, the phacoemulsification energy used to separate and emulsify the fragments is more than that for routine cataract. visual outcome was not compromised : 28 (87.5%) patients had best - corrected visual acuity from 20/30 to 20/20. this correlates with lundberg and behndig s findings.19 the safety of intracameral lignocaine was also proved by the fact that no remarkable rise of pulse and blood pressure was observed throughout the surgical procedure. there are a few limitations to this study, predominantly the lack of a control arm using intracameral mydriatic solution or epinephrine in the irrigation. however, the study intended to validate the use of intracameral lignocaine, rather than a comparison with the various formulations. we did not consider the magnification factor of the cornea while calculating actual pupillary diameter. the study was carried out at a single center and a single surgeon was involved, which omits the comparison in skills and surgical techniques. intracameral lignocaine is a safe and efficient alternative to traditional topical and intracameral mydriatics during phacoemulsification surgery in patients with cataracts and type 2 diabetes of variable duration. though local mydriatics are available cheaply, the use of a preoperative intracameral dilating solution before cataract surgery avoids the preoperative waiting period, surface ocular problems as intracameral mydriatics are preservative free, systemic rise of blood pressure and precipitation of cardiovascular problems known with diabetics. | aimto study the effect of intracameral injection of preservative - free lignocaine to induce pupil dilatation, without using any preoperative dilating eyedrops or intraoperative mydriatics in patients with age - related cataract associated with type 2 diabetes mellitus.designthis was a prospective, observational, and interventional case series conducted at a tertiary eyecare center in rural india.materials and methodsa total of 32 patients underwent phacoemulsification under topical anesthesia for visually significant cataract. preoperative pupillary diameter was measured 3 days prior to surgical procedure under mydriatics (tropicamide 0.8%, phenylephrine hydrochloride 5%). intraoperative pupillary dilatation was achieved by 1% intracameral lignocaine solution alone. effective phacoemulsification time (ept), total surgical time, and final pupillary diameter were recorded at the conclusion of surgery.resultsthe average duration of diabetes was 11.2 (range 525) years. there was no difference in dilatation by preoperative pupil - dilating drops (5.20.5 mm, range 38.3 mm) and intracameral 1% lignocaine during the surgical procedure (p=0.63). there was a negative correlation (r=0.92) between diabetes duration and dilatation of pupils with dilating drops and intracameral lignocaine. the duration of the surgery, ept, and phacoemulsification chop had statistically insignificant effects on mydriasis, while the grade of the nucleus had a statistically significant effect on mydriasis. intracameral lignocaine had no significant effect on blood pressure or pulse. there were no surgical complications that could have compromised the visual outcome. none of the patients developed macular edema in a follow - up period of 3 months ; 28 patients (87.5%) had best - corrected visual acuity from 20/30 to 20/20.conclusionintracameral lignocaine 1% provides sufficient mydriasis for the safe phacoemulsification of cataract in patients with type 2 diabetes of variable duration. |
accurate segregation of the genetic material during cell division requires that sister kinetochores attach to microtubules emanating from opposite spindle poles. merotelic kinetochore orientation is an error in which a single kinetochore is attached to microtubules emanating from both spindle poles. if a merotelically attached kinetochore remains uncorrected, it causes the chromatid to lag on the anaphase spindle, hindering its poleward segregation (figure 1). it is important to understand how cells prevent and correct merotelic kinetochore attachments because merotely represents a major mechanism of aneuploidy in mitotic cells and is the primary mechanism of chromosomal instability in cancer cells [38 ]. several proteins have been implicated in correcting or preventing merotelic attachments, including condensin and the fission yeast pcs1/mde4 complex, a homolog of the budding yeast monopolin complex [913 ]. two recent studies provide important insights into how pcs1/mde4 and condensin prevent merotelic kinetochore attachments. previous studies suggested that both the csm1/lrs4 monopolin subcomplex in the budding yeast saccharomyces cerevisiae and its counterpart pcs1/mde4 in the fission yeast schizosaccharomyces pombe act at kinetochores as molecular clamps which lock together microtubule attachment sites. while the pcs1/mde4 complex clamps together microtubule attachment sites on a single kinetochore in order to prevent merotelic attachments, the csm1/lrs4 complex clamps together microtubule binding sites from sister kinetochores during meiosis i in order to establish mono - orientation (attachment of sister kinetochores to microtubules emanating from the same pole). although this model was consistent with the experimental data and nicely explained the mutant phenotype observed in cells lacking csm1/lrs4 or pcs1/mde4, it was rather speculative. a strong argument in favour of the. showed that the csm1/lrs4 complex has a distinctive v - shaped structure, with two pairs of kinetochore - binding domains positioned about 10 nm apart. thus, a plausible model for csm1/lrs4-mediated mono - orientation of sister kinetochores is that these two pairs of kinetochore - binding domains bind across sister kinetochores, bringing them so close together that they effectively prevent bi - orientation (attachment of sister kinetochores to microtubules emanating from the opposite poles).. showed that the s. pombe pcs1/mde4 complex has the same general architecture as csm1/lrs4, suggesting that both csm1/lrs4 and its s. pombe counterpart pcs1/lrs4 may function as molecular clamps or crosslinkers at kinetochores. although the clamp model is now supported by structure function analyses, it has been challenged by a recent study from tada., who showed that the role of pcs1 and mde4 is to recruit condensin to kinetochores and proposed that condensin at kinetochores clamps together microtubule attachment sites. chromatin immunoprecipitation experiments clearly showed that kinetochore condensin localization is diminished in pcs1 cells and, notably, artificial targeting of condensin to kinetochores largely suppressed the growth defect and halved the incidence of lagging chromosomes in pcs1 cells. these observations are consistent with previous studies showing that both pcs1/mde4 and condensin are important for preventing merotelic attachments and that in budding yeast, monopolin proteins csm1 and lrs4 are required for recruitment of condensin to ribosomal dna. however, other studies showed that condensin associates with kinetochores independently of csm1 and lrs4 and that condensin is not an obligate component of a system preventing merotelic attachments in vertebrate kinetochores. these apparent discrepancies in the literature further underscore the importance of the tada. study. does the pcs1/mde4 complex act as a microtubule site clamp (figure 2a), or does it prevent merotelic attachments solely by recruiting condensin to kinetochores (figure 2b) ? although tada. nicely showed that pcs1 and mde4 act as a condensin recruiter at kinetochores and that this is an important mechanism for preventing merotelic attachments, further experiments are needed to establish whether condensin recruitment is the only role of the pcs1/mde4 complex in preventing merotely, or whether pcs1/mde4 also functions as a microtubule site clamp, as suggested by previous studies (figure 2). elegant experiments in which kinetochore condensin was specifically inactivated by proteolytic cleavage showed that this disturbed the structure of centromeric chromatin, and frequent separation of core and pericentromeric domains was observed. it is likely that this defect contributes to the high incidence of merotelic attachments observed in condensin mutant cells ; therefore, it will be important to determine whether the absence of pcs1 or mde4 leads to a similar phenotype. moreover, the distinct v - shape structure of the monopolin complex makes important predictions about its putative clamping function. using this structure as a guide, mutations that prevent the clamping ability should be designed and tested in vivo. finally, in order to extend the current studies to other organisms, it will be important to identify counterparts of the fission yeast pcs1/mde4 complex in higher eukaryotes. although structural and sequence analyses showed that the pcs1/mde4 complex shares similar features with the conserved kinetochore complex spc24/spc25, it is not known whether in higher eukaryotes the spc24/spc25 complex took over the function of the pcs1/mde4 or whether there are true homologs of pcs1/mde4 which have not been identified yet. in summary, mounting evidence suggests that both the pcs1/mde4 and condensin complexes are required to prevent merotely. whereas the clamp model suggests that pcs1/mde4 complex itself acts as a molecular clamp which locks together microtubule attachment sites (figure 2a), an alternative model suggests that the pcs1/mde4 complex prevents merotely indirectly by recruiting condensin to kinetochores and that condensin acts as a molecular clamp which locks together microtubule attachment sites (figure 2b). these two models are not mutually exclusive and it is possible that both pcs1/mde4 's clamping activity and its role as a condensin recruiter are required to efficiently prevent merotelic kinetochore attachments. further studies are needed to unveil molecular details of how kinetochore pools of pcs1/mde4 and condensin complexes prevent merotely. given the importance of this process for our understanding of how cells ensure faithful segregation of chromosomes, it is likely that this will continue to be an area of intense research in the future. | summaryto segregate chromosomes properly, the cell must prevent merotely, an error that occurs when a single kinetochore is attached to microtubules emanating from both spindle poles. recent evidence suggests that cooperation between pcs1/mde4 and condensin complexes plays an important role in preventing merotely. |
neurothekeomas are divided into myxoid, intermediate, or cellular types based on the amount of the myxoid matrix present. although the myxoid variants of neurothekeomas are likely of neural sheath origin, cellular neurothekeoma is thought to be of different origin. we present this case of cellular neurothekeoma in a patient with a history of guillain - barr syndrome and suggest that when a cellular neurothekeoma is found in a patient, the existence of concurrent or preceding guillain - barr syndrome should be ascertained from the history or medical records. we could find no prior association in the literature between neurothekeoma and guillain - barr syndrome. a 68-year - old female with a history of left nasal alar basal cell carcinoma (bcc) presented to the clinic for her yearly skin examination. she complained of a persistent bump on the left shoulder that she felt had been slowly growing over the preceding 3 months. her past medical history included hypertension, hyperlipidemia, and monoclonal gammopathy of undetermined significance ; she had a history of bcc and, most notably, a history of guillain - barr syndrome that was treated with intravenous immunoglobulin about 2 years prior to the presentation to our clinic. her family history was significant for melanoma in her mother. her medications included hydrochlorothiazide, benazepril, simvastatin, multivitamin, and calcium. on physical examination, histopathologic examination along with immunochemistry of the lesion was consistent with cellular neurothekeoma with fascicles of plump spindle cells in the dermis, containing abundant pale cytoplasm with well - defined cellular membranes (fig. the lesion exhibited positive staining for microphthalmia transcription factor (mitf) and nki - c3 (fig. was then excised with 3-mm margins, and at follow - up 1 year later, there was no recurrence of the tumor. cellular neurothekeoma is a rare benign cutaneous tumor that was described by rosati. in 1986 as well as by barnhill and mihm in 1990. this tumor is more prominent in females, affecting twice as many women as men. cellular neurothekeoma is present in a wide age group of individuals but strongly affects those in the second decade of life, which is another reason why this patient 's case was quite unique. most of the lesions typically present as asymptomatic single slow - growing flesh - colored dome - shaped nodules or papules, as in our patient. the tumors range in size, with a mean size of 1.1 cm, and with the majority of tumors being < 2 cm. the majority of the tumors occur in the upper extremities or in the head and neck area. histologically, most cellular neurothekeomas have a poorly defined micronodular architecture with nests of epithelioid to spindle cells that are often separated by collagen. the cells exhibit abundant eosinophilic or pale cytoplasm, with many cells having well - defined cellular membranes. some cellular neurothekeomas exhibit atypical histological features such as a high mitotic rate, pleomorphism, and fat infiltration. these atypical characteristics do not appear to correlate with increased recurrence or have any clinical importance, and thus, cellular neurothekeomas are considered benign. cellular neurothekeomas occasionally recur and are mainly seen to recur in the case of incompletely excised facial lesions. no definite associations have been reported between systemic diseases and cellular neurothekeomas in our literature search. however, cellular neurothekeoma may possibly be associated with estrogen production, as demonstrated by the case of a young female who presented with a nodular erythematous lesion that appeared after menarche and displayed worsening erythema during menstruation ; this nodule was histologically diagnosed as a cellular neurothekeoma. because the tumor appeared a few months after menarche (when estrogen is produced in more substantial amounts) and because there was worsening erythema around the lesion during every menstrual cycle (with the start of the follicular phase), the authors suggested a possible link between estrogen production and neurothekeoma. thus, cellular neurothekeoma may be associated with other medical conditions that have a high estrogen production. there is a case report of orf, a cutaneous zoonotic viral infection, associated with guillain - barr syndrome as it was found to precede the onset of this syndrome by 2 weeks. guillain - barr syndrome may be associated with pyogenic granulomas and beau 's lines as both these cutaneous manifestations have been reported to occur at the same time following the onset of this syndrome. a cellular neurothekeoma can be difficult to diagnose clinically as it often appears similar to other common skin lesions, especially bcc. both frequently present as a papule with overlying telangiectasias. cellular neurothekeoma has been reported to exhibit overlying blood vessels arranged in a branch - like pattern under dermoscopy, similar to bcc, but these lesions can be differentiated based on histopathologic examination with immunochemistry. histologically, cellular neurothekeomas can be confused with melanocytic tumors like the spitz nevus, dermal nerve sheath myxoma, pilar leiomyoma, plexiform fibrohistiocytic tumor (pfh), and cellular dermatofibroma. a dermal nerve sheath myxoma can be differentiated from a cellular neurothekeoma as it mostly occurs on distal extremities, has an abundant myxoid matrix on histology, is positive for schwann cell markers s-100 protein and glial fibrillary acidic protein (gfap), and has a higher recurrence rate. spitz nevi can also be differentiated from cellular neurothekeoma as they are positive for s-100 and mart-1, which are negative in cellular neurothekeoma. pilar leiomyomas can be differentiated from cellular neurothekeomas as they often have longer nuclei and brighter eosinophilic cytoplasm and are almost always positive for desmin. pfh can be differentiated from cellular neurothekeoma because the cells it contains are less epithelioid and, histologically, it often exhibits a prominent fibroblastic component. it has been found that the expression of mitf, which is a transcription factor involved in melanocyte development in histiocytoid cells, may help differentiate between histiocyte - predominant pfh and cellular neurothekeoma, as it was found to be diffusely positive in the latter and negative in pfh. in addition, cellular neurothekeomas have been described at times to have a mainly fascicular growth pattern with associated collagen at the periphery, similar to cellular dermatofibroma. however, cellular neurothekeomas can be differentiated from cellular dermatofibromas because they contain a focal area of lobulated / nested architecture, the presence of epithelioid cells with abundant cytoplasm, and a lack of factor xiiia expression. the origin of cellular neurothekeomas is currently undetermined. in a study involving an analysis of 178 cellular neurothekeomas, they typically stained positive for nki - c3 and mitf and were negative for s-100 protein, gfap, and melan - a. the absence of gfap and s-100 protein supports a lack of peripheral nerve sheath origin (unlike myxoid neurothekeomas). in contrast, it has been shown in a gene expression array study that nerve sheath myxomas likely have a peripheral nerve sheath origin because they mainly had upregulated genes that are involved in the development of the peripheral nerve sheath. the most statistically significant factor between the two groups was the differential expression of the s100b gene. the main genes that were found to be differentially expressed and upregulated in cellular neurothekeomas in comparison to nerve sheath myxomas and schwannomas included those involved in extracellular matrix remodeling and growth. therefore, it is currently believed that cellular neurothekeomas are not derived from the neural sheath. instead, cellular neurothekeomas have a similar gene expression profile to fibrous histiocytomas, suggesting that these two entities may be related. cellular neurothekeomas may have a neuroectodermal origin, as supported by positive staining with nki - c3, mitf, and pgp 9.5. although cellular neurothekeomas have been shown to stain positive for the melanocytic markers nki - c3 and mitf, these markers are not specific for melanocytes. and, because cellular neurothekeomas have been found to be negative for melanocytic markers including s100, hmb-45, tyrosinase, and melan - a, it can be argued that they are not of melanocytic origin. in addition, myofibroblastic differentiation of the tumor cells in cellular neurothekeoma has been suggested because about 60% of the lesions exhibited focal staining for smooth muscle actin in a study involving the detailed characterization of 133 cellular neurothekeomas. it has also been implied that the tumor cells of cellular neurothekeomas have a neuroendocrine differentiation as they were found to stain positive for neuroendocrine markers including neuron - specific enolase, chromogranin, synaptophysin, and cd56. thus, it is possible that cellular neurothekeomas originate from undifferentiated mesenchymal cells that exhibit characteristics of both neuroendocrine and myofibroblastic differentiation or can be derived from either. mohs micrographic surgery has also been recommended for cellular neurothekeomas, primarily because of their poorly defined clinical margins, their ability to recur if not completely excised, and their primary location on cosmetically appealing areas such as the head and neck region. furthermore, no recurrence has been noted for the 4 cases of cellular neurothekeoma that have been treated with mohs micrographic surgery. this case report is unique because cellular neurothekeoma is a rare tumor, and no prior association could be found between guillain - barr syndrome and neurothekeoma in our literature search. thus, the question arises whether this is in fact truly a rare association or whether clinicians need to be further educated to be aware of this potential relationship. the authors declare that there are no conflicts of interest regarding the publication of this paper. | cellular neurothekeoma is a rare cutaneous tumor that occurs more frequently in women. a 68-year - old female with a history of left nasal alar basal cell carcinoma and guillain - barr syndrome presented to the clinic with a 3-mm firm skin - colored papule with scattered telangiectasias. histopathologic examination with immunochemistry of the lesion was consistent with cellular neurothekeoma. it stained positive for microphthalmia transcription factor and nki - c3 and negative for hmb-45 and s-100. the lesion was excised with 3-mm margins, and no recurrence was noted within 1 year of follow - up. we present a case of cellular neurothekeoma in a patient with a history of guillain - barr syndrome as well as a review of the literature. our case report is unique in that no prior association has been found in the literature between cellular neurothekeoma and guillain - barr syndrome. |
unfortunately, in some individuals, the diagnosis remains difficult, particularly in the preschool wheezer. this leads to widespread underdiagnosis, which negatively affects the quality of life of asthmatic children. in an attempt to provide insight into the wheezy infant, much research has been conducted in order to provide markers, that may help predict and aid in the diagnosis of asthma in young individuals. despite this, the epidemiology and disease expression of asthma and other allergic diseases still remain poorly understood. in the northern hemisphere, the relationship between asthma and atopy has been clearly shown [1, 2 ]. for this reason, the presence of atopy in children is often used as a surrogate marker to assist in making the diagnosis of asthma. in the developing world and particularly in the south african context, the relationship between atopy and asthma may not be as clear. since 2005 the atopic status of asthmatic children attending the steve biko academic hospital paediatric asthma clinic has been investigated. results have demonstrated that only 49% of the children with asthma had one or more positive skin prick tests to common aero - allergens. this is much lower than the atopic rate of asthmatics reported in the northern hemisphere. this suggests that these children had a lower rate of an atopic diathesis than other groups reported, suggesting that using atopy as a diagnostic tool in our context may not be a perfect marker for asthma. while the genetic basis for asthma remains unclear, the interaction between genetics and the environment is even more illusive. the role of the environment has been postulated to explain the increase in the prevalence of asthma [711 ], as well as other allergic diseases. some of these environmental factors include urbanisation, dietary changes, vitamin and micronutrient insufficiency, changes in microbial burden, and industrial pollution. it is possible that these environmental factors play a greater role in the aetiology and expression of asthma and other allergic diseases in our context. it has important implications because identification of atopy in relatives of asthmatic children may be unhelpful in the diagnosis of childhood asthma, especially in our setting, since fewer asthmatic patients are atopic. it is important to provide practitioners with diagnostic tools to aid in making a diagnosis, so as not to waste time asking questions that do not provide information. in the context of this study, the following definitions are used. family history of allergy : family member with symptoms suggestive of an allergic disease or a known allergic disease, for example, allergic rhinitis, asthma, and food allergy. atopy : positive skin prick test to common aero - allergens and/or foods, in the context of an allergic disease. allergic mom : presence of a positive skin prick test, or a history of allergic symptoms or disease. family history of allergy : family member with symptoms suggestive of an allergic disease or a known allergic disease, for example, allergic rhinitis, asthma, and food allergy. atopy : positive skin prick test to common aero - allergens and/or foods, in the context of an allergic disease. allergic mom : presence of a positive skin prick test, or a history of allergic symptoms or disease. to determine if a maternal history of allergic disease or symptoms of such a disease and/or atopy is a useful predictor of the allergic basis of her child 's asthma. this was performed by determiningthe association of atopy in mothers with atopic asthma in their children, the association of asthma in mothers with atopic asthma in their children, andthe association of maternal history of allergic symptoms in mothers with atopic asthma in their children. the association of atopy in mothers with atopic asthma in their children, the association of asthma in mothers with atopic asthma in their children, and the association of maternal history of allergic symptoms in mothers with atopic asthma in their children. a random sample of children and their mothers attending the children 's chest and allergy clinic at steve biko academic hospital, in pretoria, south africa, were enrolled. children diagnosed with asthma and known to the clinic for longer than six months were considered. a diagnosis of asthma was made by all of the following : a history of respiratory symptoms which respond to bronchodilators, a history of respiratory symptoms which respond to inhaled or oral corticosteroids, andairway hyper responsiveness, as demonstrated by reversible spirometry. a history of respiratory symptoms which respond to bronchodilators, a history of respiratory symptoms which respond to inhaled or oral corticosteroids, and airway hyper responsiveness, as demonstrated by reversible spirometry. the study was approved by the research ethics committee of the faculty of health sciences of the university of pretoria. skin - prick testing (spt) or immunocap (thermofisher) test results, to common aeroallergens and foods, of the children were obtained from the child 's hospital records. mothers completed a detailed questionnaire which included demographic details, birth history, occupation, habits, present environment, medical history, symptomatology of atopy, and a history of symptoms suggestive of allergic diseases. these were subdivided into skin, upper and lower respiratory tract symptoms, such as allergic rhinitis, sinusitis, eczema and asthma, as well as any history of proven asthma. spt using the alk - abell allergen extracts (laboratory specialities) and negative (0.5% phenol) and positive (1% histamine) controls were performed on the mothers. the aero - allergen extracts used were bermuda grass, corn pollen, 5 grass mix, tree mix, candida albicans, aspergillus fumigatus, cat - hair epithelium, dog - hair dander, feather mix, house - dust mix, and standardised dermatophagoides pteronyssinus. each extract was applied to the volar surface of the forearm with a sterile prick lancette. reactions were read at 10 minutes, any wheal 3 mm or greater than the negative control was regarded as positive. the results were captured and the proportions were compared using the fisher's - exact test. test characteristics were evaluated for sensitivity, specificity, and predictive values using a standard two by two table. the outcome of interest was asthmatic children with a positive spt (used to distinguish between atopic and nonatopic asthma). the age range was 112 years (mean 6 years). along with their mothers, the proportion of children with atopic asthma does not differ significantly for mothers with and without a positive spt (p = 0.836 ; 0.625 (30/48) versus 0.654 (34/52)). the proportion of children with atopic asthma does not differ significantly for mothers with and without a history of doctor diagnosed asthma (p = 0.045 ; 0.875 (14/16) versus 0.595 (50/84)). the proportion of children with atopic asthma does not differ significantly for mothers with and without symptoms suggestive of an allergic disease (p = 1.000 ; 0.643 (45/70) versus 0.633 (19/30)). the proportion of children with atopic asthma does not differ significantly for mothers who were considered to be allergic (presence of a positive skin prick test, or a history of allergic symptoms) or not (p = 0.806 ; 0.649 (50/77) versus 0.608 (14/23)). the diagnostic variables (sensitivity, specificity, and predictive values) are reflected in table 1. the relative risk (odds ratio) of a child having atopic asthma when having a mother with or without the exposure variable is reflected in table 2. the data in table 1 demonstrates that all maternal allergic or asthmatic associations are poor predictors of atopic asthma in their children. only the association of maternal doctor diagnosed asthma reached statistical significance (p = 0.045) but this may have limited clinical significance. despite the increased risk of atopic asthma in a child of a mother that has a doctor diagnosis of asthma (or 4.76 p = 0.045), a mother with a doctor diagnosis of asthma is, nevertheless, a poor predictor of a child with atopic asthma (sensitivity 21.9%). the findings in this study are important because the literature suggests that the most reliable way to demonstrate the inherited tendency of asthma is to demonstrate a positive family history of atopy. this is why atopy is used as a surrogate marker to aid in the diagnosis of childhood asthma. but it is clear here, that a history of maternal atopy or allergic diseases is not a good predictor of childhood asthma in our cohort of patients. it has previously been suggested that the reason for the poor association between family history of allergic disease and childhood asthma may be due to lack of diagnosis of allergic diseases in family members. this is because of underreporting of these symptoms by patients, or failure of doctors to recognise and diagnose allergic diseases. parents should rather be asked about specific symptoms suggestive of asthma, allergic rhinitis, and other allergic diseases. however, this study demonstrates that this can not be the whole explanation for a poor family history, since mothers were specifically questioned on symptomatology suggestive of allergic diseases perhaps the relationship between asthma and atopy has been overestimated in the first world. or perhaps this relationship, which we rely upon in everyday practice to aid in the diagnosis of childhood asthma, can not be extrapolated to the third world. atopic disease expression carries an inherited or genetic component. despite this, in our study, maternal atopy (positive spt and an allergic disease process), or a history of symptoms suggestive of allergic diseases were not good predictors of atopic asthma in children. even previously doctor diagnosed maternal asthma may not be a useful predictor of atopic asthma in her child. this supports previous local studies that have demonstrated that atopy occurs in fewer asthmatic children, at best in south africa, than previously thought. this raises questions about the uniform association between allergy and asthma, especially in africa and suggests that asthma may be associated with other aetiological factors. some environmental factors postulated are urbanisation, dietary changes, vitamin and micronutrient insufficiency, changes in microbial burden, and industrial pollution. the data obtained are the result of a pilot study based on a limited number of patients. also, reliance on maternal history to differentiate allergic from nonallergic mothers is another important limitation. families should also be stratified according to race, to determine whether the results are influenced. the information obtained however, together with other local studies, has shown the emergence of a new picture of childhood asthma in the third world context. currently, the diagnosis of asthma in young children is aided by a family history of atopy or allergic diseases. but this may not be appropriate in the south african setting. this provides the motivation for further studies to be conducted among different ethnic groups, especially in south africa. this will hopefully shed more light on the complexity of the epidemiology and aetiology of childhood asthma in the third world. | introduction. asthma is the commonest chronic condition of children. diagnosis of this condition remains difficult. many surrogate markers are used, such as documenting evidence of atopy. method. a random sample of asthmatic children and their mothers attending the children 's chest and allergy clinic at steve biko academic hospital were enrolled. children were classified as having atopic or nonatopic asthma. mothers completed a questionnaire to uncover atopic features. results. along with their mothers, 64 children with atopic asthma and 36 with nonatopic asthma were studied. the proportion of children with atopic asthma does not differ for mothers with and without a positive spt (p = 0.836), a history of asthma (p = 0.045), symptoms suggestive of an allergic disease (p = 1.000), or who were considered to be allergic (p = 0.806). the odds ratio of a child having atopic asthma when having a mother with a doctor diagnosed history of asthma is 4.76, but the sensitivity is low (21.9%). conclusion. the data demonstrates that all maternal allergic or asthmatic associations are poor predictors of childhood atopic asthma. despite the increased risk of atopic asthma in a child to a mother that has a doctor diagnosis of asthma (or 4.76 p = 0.045), this is a poor predictor of atopic asthma (sensitivity 21.9%). |
percutaneous vertebroplasty (pvp) or kyphoplasty (pkp) have been shown to be safe and effective procedures for the treatment of both primary and glucocorticosteroid - induced osteoporosis (giop). nevertheless, there are only a few reports on the application of pvp or pkp for the treatment of glucocorticosteroid - induced osteoporotic vertebral compression fractures (giopvcfs) along the entire thoracic - lumbar length of the vertebral column. here, we present a rare case where a giop patient, who suffered from a cluster of spontaneous vertebral fractures in a 27-month period, was relieved of sustained pain and had an improved quality of life following four successive rounds of pvp operations with 17 cement augmented vertebral bodies. a 67-year - old man had been living with rheumatoid arthritis and pulmonary fibrosis and regularly took glucocorticoids for more than 20 years. he was subsequently diagnosed with giop with a hip bone mineral density test score of -4.0. alendronate, supplemental calcium, and vitamin d were prescribed as prophylactic treatment for giop. this patient has suffered three rounds of giopvcfs and experienced significant pain relief and improvements in his daily life following three rounds of pvp with 12 augmented vertebral bodies from the sixth thoracic to the fifth lumbar vertebrae since april 2008. in july 2010, he returned to our hospital presenting with serious chest and back pain confining him to bed, with no significant reasons for the pain. radiography and magnetic resonance imaging (mri) were performed to assess the recurrence of severe pain and revealed stable vertebroplasty changes within augmented vertebral bodies from the sixth thoracic to the fifth lumbar vertebral bodies and new osteoporotic vertebral compression fractures on the first to fifth thoracic vertebral bodies. the patient was treated conservatively with medication and bed rest. however, the patient 's symptoms failed to subside during the hospitalization. because we were concerned about both his chronic pulmonary fibrosis and the likelihood of delayed side effects if conservative management did not work, we reluctantly decided that pvp was the treatment of choice. after written informed consent was obtained from the patient and his family, a fourth round of pvp was performed on the first to fifth thoracic vertebral bodies using 11-gauge bone puncture needles (cook, inc., bloomington, in, usa) under digital subtraction angiography (axiom zee biplane, siemens healthcare, erlangen, germany) guidance. the volumes of bone cement (polymethyl methacrylate, pmma) (simplex p ; howmedica osteonics, kalamazoo, mi, usa) used were as follows : 1.5 ml were injected at the first thoracic vertebra, 2 ml were injected at the second thoracic vertebra, 1.5 ml were injected at the third thoracic vertebra, 1 ml was injected at the fourth thoracic vertebra, and 1.5 ml at the fifth thoracic vertebra. from april 2008 to may 2008, the patient had experienced multiple vertebral compression fractures on three separate occasions : 1) the sixth thoracic and twelfth thoracic vertebral bodies ; volumes of bone cement used were 1.5 ml for the sixth thoracic body and 3.5 ml for the twelfth thoracic vertebral body ; 2) first lumbar and third to fifth lumbar vertebral bodies ; volumes of bone cement used were 3 ml, 3 ml, 2.5 ml, and 3.5 ml, respectively ; and 3) seventh to eleventh thoracic and second lumbar vertebral bodies ; volumes of bone cement used were 2.5 ml, 2 ml, 2.5 ml, 2.5 ml, 3 ml, and 3.5 ml, respectively. within a two - year period he underwent four pvp operations with 17 cement augmented vertebral bodies from the first thoracic to fifth lumbar vertebrae (fig. 1, table 1). the average interval between each pvp operation was 206.7 days (range, 20 - 791 days). as the population ages, an increasing proportion of people are suffering from primary osteoporosis and giop that commonly results in vertebral fractures. these fractures not only reduce the patient 's quality of life, but can also lead to multiple comorbidities such as functional limitations, depression, disability, height loss, spinal instability, and kyphotic deformity associated with impaired lung capacity. unfortunately, conservative treatment strategies, such as analgesia, bed rest, and physical therapy, and other surgical options do not appear to effectively manage the disease (1, 2). although the surgical treatment of spine deformities can be very challenging due to the frequently poor bone quality and to the patient 's expectations regarding the improvement of chronic pain, which need to be clarified upfront, percutaneous vertebroplasty has become an interesting treatment option to improve functionality and quality of life and for the management of acute pain resulting from vertebral fractures, based on recent clinical results, especially for elderly patients (3, 4, 5). a review of the literature from january 1, 1995, to december 1, 2013, was conducted for relevant studies about pvp or pkp regarding glucocorticosteroid - induced osteoporotic vertebral compression fractures in the pubmed database. one hundred and seventeen patients with giopvcfs treated by pvp or pkp were included in the 11 eligible studies (3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13). fifty of 117 (42.8%) patients sustained refractures after vertebropalsty and received repeated vertebroplasty procedures, of which 24 and 26 patients underwent pkp and pvp procedures, respectively. almost all the refractures occurred within one year after the pkp or pvp procedure and 41.8% were adjacent level refractures while 58.2% were remote level refractures. therefore, studies have debated whether there is a correlation between the use of the vertebroplasty technique and the recurrence of new fractures in giop or primary osteoporosis patients (3, 8, 9, 14, 15). although the mechanism by which future fractures may occur after the procedure is unclear, it is postulated that vertebroplasty and kyphoplasty might shift the normal load transmission through the spine, predisposing it to fracture (7). (8) reported that patients presenting on oral steroid therapy at their initial vertebroplasty are almost twice as likely to have symptomatic refractures as primary osteoporosis patients are during follow - up. however, others have suggested that the refractures were simply the result of the natural progression of osteoporosis and vertebroplasty did not increase the risk of new fractures (16, 17, 18). lindsay. (16) found the annual incidence of a vertebral compression fracture (vcf) in postmenopausal women without prior vcf was 3.6%. if the patient presented with one vcf on baseline imaging studies, then there was a 19.2% incidence rate of subsequent vertebral compression over the ensuing year. this incidence increased to 24% if there were two or more vcfs present on the initial radiograph (16). recent data from the vertos ii trial (17), randomizing painful vcfs to conservative treatment or pvp, showed the incidence of new vcf between the two groups was not different at 12 months follow - up, and the only risk factor identified for new vcfs was the number of baseline vcfs. although the principle of applying pvp or pkp to not more than three levels or not more than 8 ml of bone cement at a time was adhered to in order to prevent complications resulting from cement, air, and fat emboli due to too many levels of cement being injected at a time (19, 20, 21, 22). pvp was performed on the first to fifth thoracic vertebra bodies in a single session with a total of 8 ml bone cement, as the smaller upper thoracic vertebrae require less bone cement. as consequence of the more serious degree of osteoporosis in giop patients, more prudence is needed during the puncture process of pvp for giopvcfs, and the puncture needle can be placed into the target vertebra with merely a small amount of thrust. in view of the findings presented here, we demonstrate that, in specific cases, repeated pvp can be an effective treatment modality for recurring osteoporotic vertebral compression fractures and giopvcfs, although it may be necessary to perform multiple rounds of pvp, provided successive surgeries are beneficial to the patient. there is increasing evidence to suggest the possibility of pmma chemotoxic accumulation inside the patient 's body, whether from successive treatment via pvp or extensive vertebral restoration in a single pvp procedure. (24) both found that pao2 decreases and paco2 increases as the number of treated vertebral bodies in single pvp increases. in our patient, we compared the major clinical biochemical indicators before and 48 hours after surgery, and identified no significant changes in this patient 's electrolyte, hepatorenal, and hematopoietic functions ; indicating that the accumulated pmma does not cause a negative effect in this patient. however, we did observe both pao2 and sao2 increases to almost the normal range after pvp, when the operation was performed on the thoracic vertebrae. this change may be related to the pain relief and pulmonary improvement after pvp. from the examination of this unique case study and the review of the literature, we conclude that pvp is a safe, effective, and feasible procedure for selected patients subjected to repeat and/or multiple thoracic - lumbar giopvcfs, bringing significant pain relief and improvement in the quality of life for the patient. however, long - term, large cohort, and randomized controlled studies are required to conclusively define the clinical application of vertebroplasty for serious multiple glucocorticosteroid - induced osteoporotic vertebral compression fractures. | glucocorticosteroid - induced osteoporosis is the most frequent of all secondary types of osteoporosis, and can increase the risk of vertebral compression fractures (vcfs). there are promising additions to current medical treatment for appropriately selected osteoporotic patients. few studies have reported on the efficiency of percutaneous vertebroplasty (pvp) or kyphoplasty for whole thoracic and lumbar glucocorticosteroid - induced osteoporotic vertebral compression fractures. we report a case of a 67-year - old man with intractable pain caused by successional vcfs treated by pvp. |
the clinical expression of hypertrophic cardiomyopathy (hcmp) has proved to be particularly heterogeneous with a diverse disease spectrum.1)2) the end stage of hcmp, characterized by a left ventricular (lv) ejection fraction (ef) 2 mm on ii, iii, and avf, and st elevation on avr and v1 during exercise. during the recovery phase, electrocardiography showed down - sloping st depression and t wave inversions on ii, iii, and avf. to evaluate the underlying cause of the regional wall motion abnormality, cag was performed but showed a normal coronary artery. cardiac mri revealed multifocal transmural and subepicardial delayed enhancing areas at the anteroseptal, septal and inferoseptal lv wall with wall thinning and decreased motion of the anteroseptal lv wall (fig. echocardiographic findings from several examinations during the follow - up period are shown in table 1. hcmp is widely regarded as a disease predominantly associated with hyperdynamic lv systolic function in the absence of another cardiac or systemic disease (e.g., hypertension or aortic stenosis) capable of producing the magnitude of hypertrophy evident.4) hcmp is not rare and is the most common genetic cardiovascular disease.1) remodeling of the lv chamber in hcmp has been shown to occur in several clinical circumstances, including progression of lv hypertrophy during adolescence. 5) a small distinctive subset of hcmp patients (approximately 510%) show the evolution into the end stage (or " burned - out " phase), characterized by lv wall thinning, cavity enlargement and systolic dysfunction that resembles dilated cmp and produces a relentlessly progressive and irreversible heart failure.1)6) in 1998, chang.7) demonstrated two cases of hcmp progressing to dilated cmp - like features after a follow - up period of more than 15 years with echocardiographic evidence, but there was no cardiac mri evaluation. chang.7) concluded that pathologic study of more cases is necessary to reveal the pathogenesis of the functional and morphological changes of the myocardium. in the previous study, end stage of hcmp among 44 cases was identified approximately after 14 18 years.3) this case that we encountered in 1999, diagnosed on echocardiography and cag, had hypertrophied lv walls but a non - dilated lv chamber. after a follow - up period of more than 16 years, echocardiographic findings from several examination were akinesis at the basal to mid anteroseptal, basal to mid septal, and basal to mid inferior, and basal to mid anterior lv wall motion with decreased lv systolic function (ef = 45%) but a normal chamber size. we considered the possibility of hcmp progressing to ischemic cmp - like features after a follow - up period of more than 16 years with echocardiographic evidence. however, it is unclear what progressed the end stage of hcmp by the cause of the pathologic mechanism. we consider that microvascular dysfunction is a common feature of hcmp and reflects the interplay of a variety of mechanisms, including reduced arteriolar density, fibrosis, and myocyte disarray.8) microvascular dysfunction promotes blunted myocardial blood flow, leading to recurrent myocardial ischemia, replacement fibrosis, and possibly adverse lv remodeling.9) in patients with hcmp, the degree of microvascular dysfunction is a strong, independent predictor of clinical deterioration and death.10) similar to that observed in this patient, end - stage hcmp is an unfavorable complication with a mortality rate of 11% per year and a risk of sudden death.3) therefore, patients with marked disease progression and lv remodeling eventually become candidates for primary prevention of sudden death with implantable defibrillators and evaluation for heart transplantation. the reason cardiac mri is essential is that it provides contrast visualization of late gadolinium enhancement (lge), generally considered indicative of myocardial fibrosis.11) maron and spirito6) said that lge, presumably representing the consequences of longstanding microvascular ischemia, and resulting in myocyte death and ultimately replacement fibrosis as a repair process, to be evident predominantly in thinner segment of lv and associated with abnormalities of wall motion.12) there is also an inverse relationship between systolic function and lge on cardiac mri.2)12) in patients with hcmp, a subset of patients with low - normal ef values (5065%) are identified on contrast - enhanced cardiac mri as having substantial degrees of lge, suggesting a transition phase, potentially heralding the beginning of advanced lv remodeling and systolic dysfunction. in conclusion, the present case demonstrated non - obstructive hcmp that progressed to ischemic cmp - like features diagnosed on echocardiography and cardiac mri after a follow - up period of more than 16 years. this case was confirmed as the end stage of hcmp with fibrosis on cardiac mri. therefore, we may consider the end stage of hcmp in case of systolic and/or diastolic dysfunction with a variety of wall motion abnormality on echocardiography but normal coronary artery. | a 58-year - old man had been diagnosed with non - obstructive hypertrophic cardiomyopathy (hcmp) according to echocardiography findings 16 years ago. echocardiography showed ischemic cardiomyopathy (cmp)-like features with decreased systolic function but a non - dilated chamber. coronary angiography was performed but showed a normal coronary artery. cardiac magnetic resonance imaging (mri) revealed multifocal transmural and subepicardial delayed - enhancing areas at the anteroseptal, septal, and inferoseptal left ventricular (lv) wall, and wall thinning and decreased motion of the anteroseptal lv wall. findings of ischemic cmp - like features by echocardiography suggested microvascular dysfunction. this late stage of hcmp carries a high risk of sudden death. cardiac mri evaluation may be necessary in cases of ischemic cmp - like features in hcmp. in this case, the diagnosis of end - stage hcmp with microvascular dysfunction was confirmed by using cardiac mri after a follow - up period of more than 16 years. |
insulin resistance (ir) is associated with obesity and characterized by its metabolic consequences, including hyperglycemia, dyslipidemia, and hypertension. these metabolic consequences are known collectively as metabolic syndrome (mets) and are potent risk factors for adverse clinical outcomes. ir has been found to be associated, directly and indirectly, with cardiovascular complications, including atherosclerosis that may lead to myocardial infarction and stroke. moreover, obesity is associated with increased production of proinflammatory adipokines, including monocyte chemoattractant protein-1, interleukin-6 (il-6), and tumor necrosis factor- (tnf-). they contribute to chronic, low - grade inflammation and play a pivotal role in the development of insulin resistance [4, 5 ]. fructose intake has been directly linked to hyperuricemia, which may result in obesity and mets [7, 8 ]. the mechanism by which uric acid (ua) induces the development of mets may be due to ua inhibition of nitric oxide synthase and induction of endothelial dysfunction [9, 10 ]. recent evidence supports the concept that hyperuricemia itself can be a significant and independent cardiovascular risk factor. lowering ua in fructose - induced mets has been found to be associated with lowering bp, improving insulin sensitivity, and reducing hypertriglyceridemia, through improving endothelial and adipocyte dysfunction. therefore, the aim of the current study was to evaluate the effect of allopurinol, as a xanthine oxidase enzyme inhibitor reducing the level of ua, on insulin resistance induced experimentally in rats and its possible protective effect on the cardiovascular complications. the following drugs and chemicals were used : allopurinol and urethane (sigma - aldrich, st. male wistar rats weighing 120140 g (king fahd center for medical research, king abdulaziz university, saudi arabia) were housed in clear polypropylene cages (four rats per cage) and kept on equal durations of the dark - light cycle, under constant environmental conditions. experimental protocol was approved by the research ethics committee of the faculty of medicine, king abdulaziz university. rats were randomly divided into four experimental groups (eight animals each) as follows : control, insulin resistant (ir), allopurinol - treated insulin resistance (ir - allo), and allopurinol - treated control (allo). ir was induced by adding fructose (10%) to everyday drinking water and feeding rats on high fat, high salt diet (16% crude protein, 28.2% crude fat, 2.8% crude fiber, 4.8% ash, and 3.4% salt) for 12 weeks, while control animals receive tap water and standard diet (20% crude protein, 4% crude fat, 3.5% crude fiber, 6% ash, and 0.5% salt). allopurinol (20 mgkgday) was daily administered by dissolving in drinking water (90110 mg / l) depending upon water consumption. drinking water was measured every week and allopurinol concentration in drinking water was readjusted. at the end of the study then the rats were anesthetized by intraperitoneal injection of urethane (1.5 gkg) and the invasive bp was recorded. finally, venous blood was withdrawn and allowed to coagulate for 30 min at 4c and then was centrifuged (3000 g, 4c, 20 min) to separate serum. serum was divided into aliquots and stored at 20c till being analyzed for insulin, tnf-, adiponectin, and lipid profile. glucose was determined in tail blood by a glucose meter (bionime gmbh) using noble metal electrode strips. serum insulin level as well as fructosamine was measured by enzyme - linked immunosorbent assay (elisa, millipore, billerica, ma, usa) that uses a plate coated with monoclonal anti - rat insulin antibodies. the homeostasis model assessment of insulin resistance (homa - ir) (ir index) was calculated using the serum nonfasting glucose and insulin levels according to the equation of matthews. : homa - ir = glucose concentration (mmol / l) insulin (u / l)/22.5. serum levels of ua, triglycerides (tg), total cholesterol (tc), and high density lipoprotein cholesterol (hdl - c) were determined using the elitech assay kit (elitech, laindon, essex, france). the low density lipoprotein cholesterol (ldl - cholesterol) was computed using the friedewald equation : total cholesterol (hdl - c + 1/5tg). invasive bp was continuously recorded for 10 minutes by microtip catheter (millar, bella vista, australia) inserted in the thoracic aorta through the right carotid artery. the microtip catheter was connected to power lab data interface module connected to a pc running labchart professional software (v7.3, adi instruments, bella vista, australia) containing bp module. the bp module detects and calculates different bp parameters like systolic bp, diastolic bp, mean bp, notch pressure, heart rate, cycle duration, ejection duration, and diastolic duration. serum tnf- and adiponectin levels were determined by elisa using quantikine kit (r&d systems, minneapolis, mn, usa) using rat tnf- or rat adiponectin and antibodies raised against the rat tnf- or rat adiponectin, respectively. immunofluorescence staining of 4-hydroxy-2-nonenal (4-hne), ang ii, ang r1, and collagen protein expression in rat paraffin embedded aortic sections (5 m) was carried out according to the method used in our previous works [1618 ]. fixed aortic tissue section slides were deparaffinized in xylene and rehydrated in ethanol and distilled water. then, perforation is carried out by incubation with methanol at 20c for 30 min followed by washing with distilled water. epitopes were retrieved (antigen retrieval) in citrate buffer for 30 min at 95c followed by washing with pbs. nonspecific binding sites were blocked (pbs containing 5% ngs, 1% bsa, and 0.1% triton) at room temperature for 1 h. after the blocking, sections were washed (3 5 min) with pbs. aortic sections were then incubated with the intended primary antibody diluted in blocking buffer at 4c overnight. the sections were then washed (3 5 min) with pbs followed by incubation with a fluorescent conjugated secondary antibody (dilution 1 : 200 in blocking buffer) for 1 h in dark. then sections were washed (3 5 min) with pbs and slides were dried and mounted with prolong lasting mounting media. the slides were stored in dark overnight before examination with zeiss lsm 780 confocal microscope (carl zeiss, gottingen, germany) at excitations (488 and 561 nm) and filters (497542 and 596655 nm). quantitative comparisons of images fluorescence were made with imagej software (national institute of health, bethesda, md, usa). for printing purposes, the level of the confocal images was equally adjusted after the fluorescence quantifications were carried out on unmanipulated images. sections treated with the secondary antibody alone did not show specific staining while incubating the primary antibody with the blocking peptide significantly reduced the signal. the used primary antibodies were rabbit polyclonal anti - ang ii (1 : 2000, phoenix pharmaceuticals inc., burlingame, ca, usa), mouse monoclonal anti - angiotensin ii type 1 receptor (1 : 133, abcam, cambridge, ma, usa), mouse monoclonal anti - collagen type i (1 : 1000, abcam), and rabbit polyclonal anti-4hne (1 : 250, millipore, billerica, ma, usa). the used secondary antibodies were alexa fluor (ex = 488) conjugated goat anti - mouse and alexa fluor (ex = 594) conjugated goat anti - rabbit (1 : 200, life technologies, grand island, ny, usa). statistical analysis was performed by analysis of variance (anova) followed by newman - keuls ' post hoc test using a computer based fitness program (prism 5, graphpad, ca, usa). body weight gain of the ir group was significantly increased (p < 0.001) compared to control group. this increase was ameliorated in ir - allo group but still significantly higher than the control group (p < 0.01). body weight gain was also higher in allo group (p < 0.01) (table 1). treatment with allo has decreased the level of ua significantly (p < 0.01) in ir - allo group, while there was no effect in allo group. fructosamine, fasting blood glucose, and insulin were significantly increased in ir group (p < 0.01, p < 0.001, and p < 0.01, resp.). allo showed no effect on fructosamine or fasting blood glucose in allo group but showed significant increase (p < 0.01) in insulin level compared to control group (table 1). this increase was significant in ir and ir - allo groups (p < 0.05) (figure 1). table 2 revealed that ir model in the current study failed to show any significant changes regarding the fasting lipoprotein profile. however, there was tendency to increase both tg and hdl - c, but this increase was nonsignificant. allo treatment has decreased nonsignificantly the tg level and increased nonsignificantly the ldl - c level with no effect on tc or hdl - c in either ir - allo group or allo group. bp tracing showed normal bp values including systolic pressure, diastolic pressure, mean arterial pressure, and dicrotic notch pressure as well as normal heart rate in both control group and allo group (figures 2 and 3). moreover, cycle duration in all rats in both control and allo groups was found to be normal (table 3). ir has significantly increased systolic (p < 0.01), diastolic (p < 0.01), mean (p < 0.01), pulse (p < 0.01), and dicrotic notch (p < 0.01) pressures. treatment of ir with allo has significantly decreased the elevated systolic (p < 0.05), diastolic (p < 0.05), mean (p < 0.05), and dicrotic notch (p < 0.05) pressures with no effect on pulse pressure. moreover, allo has decreased the heart rate compared to control and ir groups (figures 2 and 3). regarding the cycle duration ir has no significant changes, including total cycle duration, ejection duration, diastolic duration, and time to peak. in addition, allo has no effect in all cycle durations except for the ejection duration where it has significantly (p < 0.05) increased compared to the control group. allo treatment was significantly (p < 0.01) able to decrease this increased level to the nearly normal control value, while allo treatment has no effect on allo group (figure 4(a)). regarding adiponectin, ir showed no effect on its level and the treatment with allo has no effect also in ir - allo group. moreover, allo treatment in allo group revealed no effect on the level of adiponectin (figure 4(b)). ir group showed significant increase in 4hne (p < 0.05) compared to control group. allo treatment significantly (p < 0.01) ameliorated this increased level to the normal control values (figure 5). on the other hand, ir caused no significant changes in ang ii or ang r1 but allo treatment caused significant decrease (p < 0.05) of both even when compared with normal control values (figures 6 and 7). regarding the aortic collagen, no significant changes were observed between all groups (figure 8). in the present study, rats fed on fructose and high fat and high salt diet revealed ir evidenced by an increase in both insulin and glucose levels. ir is an important predisposing factor for several clinical disorders, including type 2 diabetes, obesity, dyslipidemia, and hypertension. although dyslipidemia was not observed in the current study, obesity, increased levels of both glucose and insulin, and hypertension were clearly evident. moreover, fructosamine, which is an indicator of the average blood glucose concentration over a short - medium period, has increased significantly in the ir model. the prevalence of hyperinsulinemia and ir rise with increasing body mass index and obesity, but whether insulin causes these phenomena or is a compensatory response has remained unsettled for decades. the current study showed that allo has increased the level of insulin without effect on glucose level and associated with increased body weight in control groups. this increase in the body weight may be explained by the allopurinol - induced secretion of insulin observed in the current study with subsequent increase in the glucose uptake in muscles and adipose tissue leading to weight gain with no effect on insulin resistance as the level of glucose was comparable to the normal control group. regarding dyslipidemia, a possible explanation for insignificant change in the lipid profile is that fructose - induced hyperuricemia is maximal in the first few hours following ingestion of fructose and is also dose - dependent. moreover, the small sample size used in the present study may be another explanation for this insignificant change in the lipid profile. clinical and experimental studies have assessed the relationship between hyperuricemia and hypertension and found that hyperuricemia might play a double role as a risk factor for hypertension and as a pathological condition enhanced by hypertension itself. moreover, many studies revealed that ir is a risk factor for cardiovascular disease [3, 23 ]. despite the fact that it was nonsignificant, the results of the present study demonstrated that ir caused an increase of the ua level with significant vascular complications including the systolic, diastolic, mean, pulse, and dicrotic notch pressure with no effect on the cycle durations or heart rate. previous studies found that xo activity is increased by various cytokines, including tnf- [24, 25 ], which is significantly increased in the current study. moreover, xo was found to be significantly elevated in a variety of vascular diseases including limb ischemia, coronary artery disease, and heart failure. circulating xo binds to glycosaminoglycans on the surface of endothelial cells where it is involved in the pathogenesis of endothelial injury [29, 30 ]. in addition, our previous study demonstrated that tnf- has an important role in ir and vascular impairment. in contrast, the nonsignificant elevation of ua in the current study may be attributed to the fact that the present model is not the ideal model representing the hyperuricemia using 2% oxanic acid. this may pay attention to the possibility that the effect of allo is not dependant on decreasing the ua level, but due to effect on other byproducts of purine metabolism by xo as superoxide. moreover, hyperglycemia and hyperinsulinemia in ir may reduce arterial wall compliance by promoting plaque growth, vascular smooth muscle cell proliferation, and nonenzymatic glycosylation of vessel wall proteins [32, 33 ]. it was reported that the vascular endothelium expresses membrane insulin receptors and is a target for the biologic effects of insulin, which stimulate production of nitric oxide (no) leading to vasodilatation. moreover, hyperglycemia itself increases the release of the vasoconstricting peptide endothelin-1 from cultured endothelial cells, offsetting the vasodilating actions of no. the results of the current study showed that allo has alleviated the vascular complications of ir including systolic, diastolic, mean, and dicrotic notch pressures compared to the untreated ir group. the elevations in diastolic and notch bp are largely attributed to the increased peripheral arterial resistance, whereas pulse pressure reflects stiffening of large arteries. pulse pressure was not significantly changed in ir as a result of significant increase in both systolic and diastolic bp in ir group. the present study may provide an explanation for these palliative effects which may be related to the effect of allo on increasing insulin secretion observed in the allo control group which in turn have a vasodilator effect on blood vessels. moreover, previous studies found that allo improves endothelial dysfunction and prevents the development of arteriosclerosis. moreover, inhibition of xo by allo was found to improve endothelium - dependent dilation and reduced superoxide production in isolated coronary arterioles following ischemia reperfusion. another explanation may be related to the effect of allo on renal function improvement and increasing glomerular filtration rate as shown by neal.. on the other hand, it was found that allo treatment decreased the heart rate significantly even when compared to the normal controlled group which is in accordance with the results of sakabe. which revealed that allo has a protective effect on atrial fibrillation in dogs associated with tachycardia - induced cardiomyopathy. despite the fact that it was nonsignificant, this bradycardia was associated with increase in the total cycle duration. moreover, the current study showed an increased level of tnf- which is an important inflammatory cytokine that is overexpressed in adipose and other tissues in cases of ir and obesity. this is in accordance with many studies which revealed the role of tnf- in the induction of ir and type 2 diabetes [43, 44 ]. although there was no significant change in the lipid profile as mentioned before, this elevated tnf- may be responsible for the mild increase in the tg and hdl - c and decreased level of ldl. previous studies showed that hepatic tg and vldl production are increased in both human and murine studies after tnf- administration [45, 46 ]. this may be due to increased hepatic levels of citrate, the rate - limiting enzyme in free fatty acid synthesis, or through inhibition of lipoprotein lipase activity. whereas tnf--induced changes in tg metabolism are similar in all species, its effect on cholesterol metabolism differs between rodents and primates. whereas the administration of tnf- in rodents is followed by increase in hepatic cholesterol synthesis and ldl [45, 49 ], nonhuman primates and humans show either no change or a decrease in serum cholesterol and ldl levels. the mechanisms underlying this species difference are not known. regarding the elevated hdl - c, it may be related to the inhibitory effect of tnf- on cholesteryl ester transfer protein (cetp) which transfers cholesteryl esters from hdl to tg - rich lipoproteins leading to high levels of hdl - c. allo treatment significantly alleviated this increased tnf- level nearly to the normal control levels which is in accordance with previous studies that showed the reducing effect of allo on tnf- [52, 53 ]. a novel explanation of this decrease in the tnf- evidenced by the results of the current study is that it may not be due to direct effect, but may be due to secondary to the ability of the allo to increase the level of insulin. beyond its classic metabolic actions, insulin has also anti - inflammatory effect, decreasing the activity of proinflammatory cytokines including tnf-. unlike many studies which revealed that, in ir, the levels of leptin and resistin increase while adiponectin, which has insulin - sensitizing and anti - inflammatory effect, decreases [5557 ], the current study revealed no significant change in adiponectin level this may be explained by the finding of shapiro. who reported that rats fed fructose ad libitum chronically become leptin resistant with a compensatory increase in adiponectin. in addition, the results of the present study showed that treatment with allo has ameliorated and decreased the increased level of 4hne that was observed in ir group. it was found that 4hne is a biomarker aldehyde of oxidative stress which readily binds covalently to nucleophilic residues of proteins, peptides, phospholipids, and nucleic acids and thereby exhibits cytotoxic effects. 4hne can also modulate signaling pathways involved in cell proliferation, fibrosis, apoptosis, and inflammation, which are all hallmarks of cardiovascular diseases [5961 ]. moreover, the current study revealed that allo treatment decreased significantly ang ii and ang r1. angiotensin ii directly causes vasoconstriction, increases the release of antidiuretic hormone via release of vasopressin, thus causing the reabsorption of water at the level of the kidneys, and triggers release of aldosterone from the adrenal cortex, causing the kidney to reabsorb sodium. it is assumed that the current study reveals that the ameliorative effect of allo treatment is not related to its effect on ua and may be related to other mechanisms as the ua in the present model of ir was not significantly increased compared to the normal control group. in conclusion, the results of the current study show that allo has a protective effect on vascular complications of ir which may be attributed to the effect of allo on decreasing the level of tnf-, 4hne, ang ii, and ang r1 as well as increasing the level of insulin secretion regardless of the effect of associated dyslipidemia. | the aim of the current study was to evaluate the possible protective effect of allopurinol (allo) on experimentally induced insulin resistance (ir) and vascular complications. rats were divided into four groups : control, ir, allopurinol - treated ir (ir - allo), and allopurinol - treated control (allo). ir was induced by adding fructose and high fat, high salt diet for 12 weeks. the results showed that allo has alleviated the increased level of tnf- and the systolic, diastolic, mean, and notch pressure observed in ir with no change in pulse pressure. in addition, allo decreased the heart rate in the treated group compared to ir rats. on the other hand, it has no effect on increased levels of insulin, glucose, fructosamine, or body weight gain compared to ir group, while it increased significantly the insulin level and body weight without hyperglycemia in the control group. moreover, allo treatment ameliorated increased level of 4hne, ang ii, and ang r1. in conclusion, the results of the current study show that allo has a protective effect on vascular complications of ir which may be attributed to the effect of allo on decreasing the tnf-, 4hne, ang ii, and ang r1 as well as increasing the level of insulin secretion. |
isolated right ventricular noncompaction (irvnc) is a very rare disorder electrocardiographic (ekg) changes associated with it have never been described before. noncompaction of the ventricular myocardium (ncvm) is a rare disorder caused by the intrauterine arrest of endomyocardial development and affects both children and adults. world health organization / international and federation of cardiology task force categorized ncvm as a unclassified cardiomyopathy in 1995. american heart association in its scientific statement in 2006 classified it under the genetic section of primary cardiomyopathies. the exact prevalence of ncvm in the adult population is unknown, but has been estimated to be between 0.014% and 0.045% of all patients referred for echocardiography left ventricle (lv) is the usual site of ncvm, with / without involvement of right ventricle (rv) and other congenital heart disease. however, irvnc has been rarely reported. a 51-year - old gentleman presented to our clinic for cardiovascular evaluation after an abnormal ekg at his primary care physician 's office. his family history was unremarkable. on examination, his heart rate (hr) was regular, with normal heart sounds and without any murmurs, rubs or gallops. initial ekg showed normal sinus rhythm, bradycardia with hr 59 beats / min, normal qrs duration, with poor r - wave progression and st segment elevation in leads v1v3 [figure 1 ]. two - dimensional transthoracic echocardiography (tte) showed spongy rv with hypertrophied trabeculae and deep inter - trabecular recesses in rv without any signs of hypokinesia. transesophageal echocardiography was done for further visualization which revealed spongy appearance of the rv with trabeculations forming deep fissures and grooves, located in the rv apical wall consistent with apical noncompaction in rv [figure 2 ]. initial electrocardiography revealing st segment elevation in leads v1v3 transesophageal echocardiography image revealing spongy appearance of the right ventricle with trabeculations forming deep fissures and grooves, located in the right ventricle apical wall no other cardiovascular abnormality was present. contrast echo demonstrated flow from rv cavity into the trabecular recesses [figure 3 ]. a follow - up tte was done a year later and no changes were noticed. noncompaction of ventricular myocardium is a rare disorder now classified as a genetic primary cardiomyopathy. during normal fetal development, ventricles initially are a meshwork of interwoven fibers and between 5 and 8 week, ventricular compaction occurs from the base towards the apex and from epicardium to endocardium. during compaction, inter - trabecular spaces in ventricular myocardium are obliterated and the recesses in the trabecular network are reduced to capillaries. the exact pathophysiology of ventricular dysfunction is unclear, but it has been suggested that subendocardial hypoperfusion and coronary microcirculatory dysfunction are a cause of arrhythmogenesis and fibrosis. this, to the best of our knowledge, is the first ever reported case with asymptomatic patient and with ekg changes of st elevation in v1v3 leads. the previous reported cases of irvnc have described it with symptoms of right heart dysfunction, palpitations, and neurological symptoms. the following diagnostic criteria for isolated left ventricular noncompaction (ilvnc) have been used in literature : (i) the absence of coexisting cardiac anomalies ; (ii) a two - layered structure of lv wall, with the end - systolic ratio of the noncompacted to compacted myocardial layer > 2 ; (iii) finding this structure predominantly in the apical and mid - ventricular areas ; and (iv) blood flow directly from the ventricular cavity into deep inter - trabecular recesses as assessed by doppler echocardiography. in the absence of clear cut criterion, the above mentioned criteria had been used earlier by authors, and similarly by us for this case. the ratio of noncompacted to compacted myocardium > 2.3, as measured at end diastole in the long axis views was used by zhang., and chiribiri., and presence of marked trabeculations and inter - trabecular recess was used by ying., our patient was diagnosed on the basis of transesophageal echocardiography. in our patient, all four echocardiographic criteria of irvnc were present. kohli., has classified noncompaction into three morphological categories namely ; spongy, meshwork, and prominent trabeculations only. in our case, noncompaction belongs to prominent trabeculations only type. to better define the noncompaction, other diagnosing modalities such as contrast ventriculography, computed tomography and magnetic resonance imaging (mri) mri has been shown to provide good correlation with echocardiogram for localization and extent of noncompaction and has been found to be useful in cases with poor echocardiography quality. the demonstration of differences in mri signal intensity in ncvm may also help identify substrate for potentially legal arrhythmia. in our patient, both familial and nonfamilial cases ncvm have been described. in the isolated form of ncvm, zasp (z - line) and mitochondrial mutations and x - linked inheritance resulting from mutations in the g4.5 gene encoding tafazzin (including association with barth syndrome in neonates) noncompaction associated with congenital heart disease has been shown to result from mutations in the -dystrobrevin gene and transcription factor nkx2.5. because of the risk of familial occurrence, a few authors have recommended screening of first - degree relatives by echocardiography to identify asymptomatic patients. although ekg is found to be abnormal in about 8794% of patients with ivnc, none of these changes are specific. however, other abnormalities including left ventricular hypertrophy changes, repolarization changes, wolff parkinson white syndrome and atrial fibrillation have been reported. in patients with irvnc, ekg changes including normal sinus rhythm and atrial fibrillation with complete right bundle branch has been reported. there has been a single case of reported of ncvm with st elevation changes in v1v3 leads. there are no long term follow - up studies to date of patients with irvnc. in a long - term follow - up of 34 patients with ilvnc by oechslin., features of noncompaction were found predominantly in apical and mid - ventricular segments of both inferior and lateral walls in > 80% of the patients. endomyocardial morphology in ncvm is responsible for the development of mural thrombi within the inter - trabecular spaces. recommended that all adult patients with ilvnc be on oral anticoagulation irrespective of ventricular functions. ilvnc patients had variable prognosis, ranging from prolonged asymptomatic course to severe cardiac disability, leading to heart transplantation and death. the worse prognostic indicators include patients with heart failure new york heart association classes iii iv, the lv end diastolic diameter > 60 mm, the left bundle branch block, chronic atrial fibrillation, a ratio of noncompacted to compacted myocardium > 3 and involvement of three or more segments. our knowledge on patients with irvnc is still benign and based on a few case reports. in the absence of studies on irvnc, our understanding of etiopathogenesis and however, definite studies might be needed before prophylactic anticoagulation is initiated for irvnc cases as recommended for ilvnc patients. to date this, to the best of our knowledge is first ever reported case of asymptomatic patient with ekg changes and irvnc. | non - compaction of ventricular myocardium (ncvm) is a rare genetic disorder caused by intrauterine arrest of endomyocardial development. left ventricle is the usual site of ncvm with very rare reports of isolated right ventricular non - compaction (irvnc). we describe a case of asymptomatic irvnc with unique ekg changes. |
vitamin d (vitd) classically recognized for its role in the musculoskeletal system, has been implicated in myriad of conditions such as diabetes, immune dysfunction, cancers, heart disease, metabolic syndrome, etc. we studied the role of vitd in acute care setting and its correlation with mortality. a total of 85 consecutive consenting patients admitted in medical intensive care unit of tertiary care hospital who fulfilled the inclusion criteria were included. all patients were evaluated clinically, and blood samples were collected for hemogram, biochemical investigations including serum calcium, phosphorus, alkaline phosphatase, magnesium, along with 25(oh) vitd, 1,25(oh) vitd and intact parathormone levels. simplified acute physiology score (saps ii) was calculated for all patients. vitd was deficient (< 30 ng / ml) in 27 patients (32%). the overall mortality was more in vitd deficient group as compared to vitd sufficient group (74 vs. 41% ; p < 0.05). the actual mortality in vitd deficient group was higher than the mortality predicted by saps ii score (50 vs. 74% ; p < 0.0507). vitd deficiency was also associated with more mortality among those requiring ventilator support (95% vs. 40% ; p < 0.05) as well as with higher blood glucose (124.5 29.7 vs. 94.8 19.8 : p < 0.01) levels. vitd deficiency was associated with increased mortality, poor ventilator outcomes, and increased blood glucose in critically ill patients. vitamin d (vitd) is associated with both classical (actions on the musculoskeletal system) and non - classical actions (which are related to the role in the immune system, cell growth and differentiation and regulation of hormone secretion). the role of vitd and impact of vitd deficiency has been widely studied in chronic conditions like cardiovascular disorders, diabetes mellitus, polycystic ovaries, autoimmune disorders, etc. a limited number of studies have previously dealt with the subject, which had shown increased mortality, increased infections and more likelihood of renal impairment. no study from india until date deals with the topic to the best of our knowledge. the aim of the current study was to study the impact of vitd deficiency on outcomes in critically ill patients. the study was conducted at a large tertiary care center hospital run by the municipal corporation in mumbai, india (latitude 1855n longitude 7254e). after institutional ethics committee approval consecutive consenting patients (consent of legally accepted representative in case of patients who can not give valid consent) admitted to the critical care unit of the institute were included in the study excluding those with tuberculosis, chronic kidney disease, chronic liver disease, acute coronary syndromes, known malignancy, known diabetes mellitus, immediate post - operative (surgery within last 14 days), pregnancy (currently pregnant or pregnancy in last 2 years) and those on anti - epileptic medications or corticosteroids prior to sample collection. simplified acute physiology score ii (saps ii) and predicted mortality rate (pmr) calculated at 24 h of admission to intensive care unit from age, heart rate, systolic blood pressure, temperature, need for mechanical ventilation, arterial oxygen partial pressure (by arterial blood gas analysis), fraction of inspired oxygen, urine output, blood urea nitrogen, white blood cell count, sodium, potassium, bicarbonate, bilirubin, glasgow coma scale and presence of chronic diseases. samples for analytes mentioned were collected at the time of admission to the critical care unit. indicated samples were repeated as directed by the intensive care unit team protocol as per case to case basis. an additional fasting blood glucose sample was collected if the initial sample collection was a non - fasting. calcium metabolism parameters (serum calcium, magnesium, phosphorus, albumin, alkaline phosphatase, serum creatinine, 25(oh) vitd3 [radioimmunoassay, by biosource [analytical sensitivity ; as-0.5 ng / ml ] and intact parathyroid hormone [pth ] ] by immunoradiometric assay by immunotech ; as < 0.2 pg / ml) were assayed from the blood sample collected within 3 h of admission to intensive care unit. vitd deficiency was defined as level below 20 ng / ml, insufficiency as level between 20 and 30 ng / ml and sufficiency as level above 30 ng / ml. statistical analysis was performed by spss version 19 manufactured by ibm, armonk, newyork, us. mean standard deviation (standard error of mean or quartiles wherever applicable) were given as descriptive statistics. log transformations were applied to highly skewed variables. chi - square test of independence or fisher 's exact test was used to test the distribution of discrete variables. the mann - whitney rank sum test was used to test the difference among groups in continuous variables at baseline. a linear regression analysis was done to assess the individual effect of various parameters on mortality. out of the 85 patients included 64 (75%) were males and 21 (25%) were females. the mean age was 42.4 16.9 years (range 19 - 75 years). 16 (19%) of cases had some other chronic medical illness apart from acute medical condition (12 were known hypertensive, two had chronic obstructive airway disease one had a past history of ischemic heart disease and 1 had cardiomyopathy). mean vitd was 41.5 24 ng / ml (range 4.8 - 142.1 ng / ml). 27 (32%) cases had vitd deficiency / insufficiency, while remaining 58 (68%) were vitd sufficient. the average saps ii score was 48 (q1 = 31 ; q3 = 64) and pmr was 47 30.61 (range 4.2 - 98.5). the actual observed mortality was 44/85 (52%). there was no difference in age, sex distribution and corrected calcium levels amongst the vitd deficient / insufficient and sufficient groups. the pth levels were higher and 1.25 vitd levels lower in vitd deficient / insufficient group ; however, the differences did not reach statistical significance. in our study, we found higher blood glucose levels in vitd deficient / insufficient cases as compared vitd sufficient group (124.5 29.7 vs. 94.8 19.8 ; p < 0.01). saps score in vitd deficient / insufficient group was 53 (q1 = 32 ; q3 = 62), where as in vitd sufficient group it was 46 (q1 = 30 ; q3 = 66) (p = 0.47). the differences in parameters mentioned among two groups are summarized in table 1. the pmr was 50% in vitd deficient / insufficient group whereas it was 44% in vitd sufficient group (p = 0.43). the actual mortality was 20/27 (74%) in vitd deficient / insufficient group and 24/58 (41%) in vitd sufficient group. the difference between predicted and actual mortality neared, but did not reach statistical significance in vitd deficient / insufficient group (p = 0.0507). thus, vitd deficiency / insufficiency was associated with higher mortality when compared to vitd sufficient population. the mortality was more than what was predicted by saps ii score in vitd deficient / insufficient groups. there was no difference in mortality in vitd deficient (9/12 died ; mortality 75%) and vitd insufficient (11/15 died ; mortality 73.4%) groups (p = 0.73). there was no difference in number of patients with vitd deficiency / insufficiency (21/27) and sufficiency (40/54) who required ventilator support during the critical care unit stay, but the mortality rate among patients requiring ventilator support was more in vitd deficient / insufficient (20/21 died) group when compared to vitd sufficient (16/40 died) group (p = 0.0001). the number of patients requiring ventilator support and mortality rates among them is shown in figure 2. comparison of vitamin d deficient / insufficient and vitamin d sufficient groups comparison of predicted and actual mortality (expressed as percentage) amongst 25(oh) vitamin d (vitd) deficient / insufficient versus sufficient groups. there was no difference in predicted mortality by simplified acute physiology score ii score among the 2 groups (p = 0.43), but the actual mortality was much more than predicted mortality in 25(oh) vitd deficient / insufficient groups. pmr = predicted mortality rate outcomes of patients requiring ventilator support amongst vitamin d (vitd) deficient / insufficient and vitd sufficient groups (p = 0.0001) logistic regression showed that vitd deficiency / insufficiency was significantly associated with mortality with and odds ratio of 4.125 (confidence interval = 1.56:11.06). the result of logistic regression and mortality risk associated is shown in table 2. logistic regression analysis for mortality risk using possible responsible variables vitd is classically known for its actions on the musculoskeletal system. however, more and more focus is being shifted on non - classical actions of vitd, which include hormone secretion, cell cycle regulation and immunomodulation. vitd deficiency has been independently proven to increase the risk of all - cause mortality in the general population. the study focuses on the role of vitd in acute care setting and tries to understand the impact of vitd deficiency in critically ill patients. this is the first study in indian subcontinent trying to focus on the role of vitd in acute care setting to the best of our knowledge. we found the prevalence of vitd deficiency to be 27% which is similar to reports from previous studies (around 20 - 40%), though some authorities have reported higher prevalence. shivane. in their study among healthy volunteers in western india found much higher prevalence of vitd deficiency (76%). previous studies have looked at vitd deficiency / insufficiency and mortality in critically ill patients. paul lee first reported increased mortality among vitd deficient / insufficient groups and stated that vitd deficiency was the second most important association with mortality after saps score. hospital mortality was higher amongst vitd deficient patients (p < 0.001), but there was no difference in hospital stay and ventilator days. the present study also found increased mortality in vitd deficiency / insufficiency, but the ventilator outcomes were extremely poor in vitd deficiency / insufficiency group, though there was no significant difference in ventilator requirement in two groups. respiratory muscle weakness attributable to vitd deficiency may result in poor ventalitory outcomes in vitd deficient group. previous studies in hospitalized patients have correlated vitd deficiency with renal impairment, more severity of illness, more blood culture positive infections (odds ratio = 1.64) and mortality among critically ill (odds ratio = 1.69). vitd has also been attributed in sepsis as it influences local immune responses and systemic inflammatory process as suggested by kempker. vitd deficiency is associated with increases incidence of diabetes, poor glycemic control in diabetics as well as more micro and macrovascular complications of diabetes. in our study, we found higher blood glucose levels in vitd deficient / insufficient cases as compared vitd sufficient group (124.5 29.7 vs. 94.8 19.8 ; p < 0.01). to the best of our knowledge, no study previously has reported relation of increased blood glucose with vitd deficiency in critical care setting. vitd is important for secretion of insulin as well as vitd deficiency is associated with insulin resistance, and these could be the possible reasons for high blood glucose in vitd deficient group. the patients who were known diabetic were excluded from this subset analysis (n = 4 ; two each in deficient and sufficient groups). vitd deficiency is associated with myopathy that might also be associated with diaphragmatic and respiratory muscle weakness. the high mortality in vitd deficient / insufficient group who required ventilator support may be due to this and also due to the association of vitd deficiency / insufficiency with a wide range of pulmonary diseases including viral and bacterial respiratory infection, asthma and chronic obstructive pulmonary disease. critical illness is associated with severe bone resorption as pointed out by nierman. whereas van den berghe tried vitd supplementation in critically ill. this failed to normalize vitd levels in most and no significant decrease was recorded in bone resorption markers. however the doses used by van den berghe were much less (200 - 500 iu / day). critically ill patients usually have multisystem involvement and role of vitd is being implicated in many systems beyond the musculoskeletal. there is plenty of data about vitd in chronic diseases, but there are very few studies about vitd in acute care setting. the principle of critical care is to reversible factors that may benefit the patient. there is no denial of the fact that primary medical illness and underlying comorbidities are going to be the most important factors affecting survival and outcome in medical intensive care. correction of vitd deficiency / insufficiency may at least improve ventilatory outcomes, reduce stress induced hyperglycemia, reduce renal dysfunction and may protect against bone resorption in critically ill patients. more studies regarding this as well as a therapeutic intervention trials regarding same may further clarify about the role of vitd in critical care setting. lack of intervention and treatment of vitd deficiency can be one of the major limitation as well as future study direction from the study. a smaller sample size in the present study is not enough for the drawn conclusion and a larger sample size and involvement of multicentric trials preferably involving wider spectrum of critically ill patients can probably widen our knowledge horizon regarding the crucial role of vitd. | introduction : vitamin d (vitd) classically recognized for its role in the musculoskeletal system, has been implicated in myriad of conditions such as diabetes, immune dysfunction, cancers, heart disease, metabolic syndrome, etc. we studied the role of vitd in acute care setting and its correlation with mortality.materials and methods : a total of 85 consecutive consenting patients admitted in medical intensive care unit of tertiary care hospital who fulfilled the inclusion criteria were included. all patients were evaluated clinically, and blood samples were collected for hemogram, biochemical investigations including serum calcium, phosphorus, alkaline phosphatase, magnesium, along with 25(oh) vitd, 1,25(oh) vitd and intact parathormone levels. simplified acute physiology score (saps ii) was calculated for all patients.results:vitd was deficient (< 30 ng / ml) in 27 patients (32%). the overall mortality was more in vitd deficient group as compared to vitd sufficient group (74 vs. 41% ; p < 0.05). the actual mortality in vitd deficient group was higher than the mortality predicted by saps ii score (50 vs. 74% ; p < 0.0507). vitd deficiency was also associated with more mortality among those requiring ventilator support (95% vs. 40% ; p < 0.05) as well as with higher blood glucose (124.5 29.7 vs. 94.8 19.8 : p < 0.01) levels.conclusion:vitd deficiency was associated with increased mortality, poor ventilator outcomes, and increased blood glucose in critically ill patients. |
research inspired by black holes has dominated several areas of gravitational physics since the early seventies. laws of black hole mechanics brought out deep, unsuspected connections between classical general relativity, quantum physics, and statistical mechanics [35, 44, 45, 46 ]. in particular, they provided a concrete challenge to quantum gravity which became a driving force for progress in that area. on the classical front, black hole uniqueness theorems [69, 119 ] took the community by surprise. the subsequent analysis of the detailed properties of kerr - newman solutions and perturbations thereof constituted a large fraction of research in mathematical general relativity in the seventies and eighties. just as the community had come to terms with the uniqueness results, it was surprised yet again by the discovery of hairy black holes [38, 49 ]. much of the research in this area has been driven by problems related to black holes, particularly their formation through gravitational collapse, the associated critical phenomenon [67, 105 ], and the dynamics leading to their coalescence (see, e.g., [1, 126, 160, 58, 2, 138 ]). finally, black holes now play a major role in relativistic astrophysics, providing mechanisms to fuel the most powerful engines in the cosmos. they are also among the most promising sources of gravitational waves, leading to new avenues to confront theory with experiments. thus there has been truly remarkable progress on many different fronts. yet, as the subject matured, it became apparent that the basic theoretical framework has certain undesirable features from both conceptual and practical viewpoints. dynamical situations for fully dynamical black holes, apart from the topological censorship results which restrict the horizon topology [110, 90 ], there has essentially been only one major result in exact general relativity. this is the celebrated area theorem proved by hawking in the early seventies [111, 113 ] : if matter satisfies the null energy condition, the area of the black hole event horizon can never decrease. this theorem has been extremely influential because of its similarity with the second law of thermodynamics. however, it is a qualitative result ; it does not provide an explicit formula for the amount by which the area increases in physical processes. now, for a black hole of mass m, angular momentum j, area a, surface gravity, and angular velocity, the first law of black hole mechanics, 1\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\delta m = { \kappa \over { 8\pi g}}\delta a + \omega \delta j,$$\end{document } does relate the change in the horizon area to that in the energy and angular momentum, as the black hole makes a transition from one equilibrium state to a nearby one [35, 182 ]. this suggests that there may well be a fully dynamical version of equation (1) which relates the change in the black hole area to the energy and angular momentum it absorbs in fully dynamical processes in which the black hole makes a transition from a given state to one which is far removed. indeed, without such a formula, one would have no quantitative control on how black holes grow in exact general relativity. note however that the event horizons can form and grow even in a flat region of space - time (see figure 4 and section 2.2.2 for illustrations). during this phase, the area grows in spite of the fact that there is no flux of energy or angular momentum across the event horizon. hence, in the standard framework where the surface of the black hole is represented by an event horizon, it is impossible to obtain the desired formula. equilibrium situations the zeroth and first laws of black hole mechanics refer to equilibrium situations and small departures therefrom. therefore, in this context, it is natural to focus on isolated black holes. it was customary to represent them by stationary solutions of field equations, i.e, solutions which admit a time - translational killing vector field everywhere, not just in a small neighborhood of the black hole. while this simple idealization was natural as a starting point, it is overly restrictive. physically, it should be sufficient to impose boundary conditions at the horizon which ensure only that the black hole itself is isolated. that is, it should suffice to demand only that the intrinsic geometry of the horizon be time independent, whereas the geometry outside may be dynamical and admit gravitational and other radiation. indeed, we adopt a similar viewpoint in ordinary thermodynamics ; while studying systems such as a classical gas in a box, one usually assumes that only the system under consideration is in equilibrium, not the whole world. in realistic situations, one is typically interested in the final stages of collapse where the black hole has formed and settled down or in situations in which an already formed black hole is isolated for the duration of the experiment (see figure 1). in such cases, there is likely to be gravitational radiation and non - stationary matter far away from the black hole. thus, from a physical perspective, a framework which demands global stationarity is too restrictive.even if one were to ignore these conceptual considerations and focus just on results, the framework has certain unsatisfactory features. consider the central result, the first law of equation (1). here, the angular momentum j and the mass m are defined at infinity while the angular velocity and surface gravity are defined at the horizon. because one has to go back and forth between the horizon and infinity, the physical meaning of the first law is not transparent1. for instance, there may be matter rings around the black hole which contribute to the angular momentum and mass at infinity. should nt only the angular momentum and mass of the black hole feature in the first law ? thus, one is led to ask : is there a more suitable paradigm which can replace frameworks based on event horizons in stationary space - times ? entropy calculations the first and the second laws suggest that one should assign to a black hole an entropy which is proportional to its area. this poses a concrete challenge to candidate theories of quantum gravity : account for this entropy from fundamental, statistical mechanical considerations. string theory has had a remarkable success in meeting this challenge in detail for a subclass of extremal, stationary black holes whose charge equals mass (the so - called bps states). however, for realistic black holes the charge to mass ratio is less than 10. it has not been possible to extend the detailed calculation to realistic cases where charge is negligible and matter rings may distort the black hole horizon. from a mathematical physics perspective, the entropy calculation should also encompass hairy black holes whose equilibrium states can not be characterized just by specifying the mass, angular momentum and charges at infinity, as well as non - minimal gravitational couplings, in presence of which the entropy is no longer a function just of the horizon area. one may therefore ask if other avenues are available. a natural strategy is to consider the sector of general relativity containing an isolated black hole and carry out its quantization systematically. a pre - requisite for such a program is the availability of a manageable action principle and/or hamiltonian framework. unfortunately, however, if one attempts to construct these within the classical frameworks traditionally used to describe black holes, one runs into two difficulties. first, because the event horizon is such a global notion, no action principle is known for the sector of general relativity containing geometries which admit an event horizon as an internal boundary. second, if one restricts oneself to globally stationary solutions, the phase space has only a finite number of true degrees of freedom and is thus too small to adequately incorporate all quantum fluctuations. thus, again, we are led to ask : is there a more satisfactory framework which can serve as the point of departure for a non - perturbative quantization to address this problem ? global nature of event horizons the future event horizon is defined as the future boundary of the causal past of future null infinity. while this definition neatly encodes the idea that an outside observer can not look into a black hole, it is too global for many applications. first, since it refers to null infinity, it can not be used in spatially compact space - times. surely, one should be able to analyze black hole dynamics also in these space - times. more importantly, the notion is teleological ; it lets us speak of a black hole only after we have constructed the entire space - time. thus, for example, an event horizon may well be developing in the room you are now sitting in anticipation of a gravitational collapse that may occur in this region of our galaxy a million years from now. when astrophysicists say that they have discovered a black hole in the center of our galaxy, they are referring to something much more concrete and quasi - local than an event horizon. is there a satisfactory notion that captures what they are referring to?the teleological nature of event horizons is also an obstruction to extending black hole mechanics in certain physical situations. consider for example, figure 2 in which a spherical star of mass m undergoes a gravitational collapse. the singularity is hidden inside the null surface 1 at r = 2 m which is foliated by a family of marginally trapped surfaces and would be a part of the event horizon if nothing further happens. suppose instead, after a million years, a thin spherical shell of mass m collapses. then 1 would not be a part of the event horizon which would actually lie slightly outside 1 and coincide with the surface r = 2(m + m) in the distant future. on physical grounds surely one should be able to establish the standard laws of mechanics not only for the equilibrium portion of the event horizon but also for 1.next, let us consider numerical simulations of binary black holes. here thus, one needs to identify initial data containing black holes without the knowledge of the entire space - time and evolve them step by step. one may then ask : can we use apparent horizons instead of event horizons in other contexts as well ? unfortunately, it has not been possible to derive the laws of black hole mechanics using apparent horizons. furthermore, as discussed in section 2, while apparent horizons are local in time they are still global notions, tied too rigidly to the choice of a space - like 3-surface to be directly useful in all contexts. is there a truly quasi - local notion which can be useful in all these contexts ? disparate paradigms in different communities within gravitational physics, the intended meaning of the term thus, in a string theory seminar, the term fundamental black holes without further qualification generally refers to the bps states referred to above a sub - class of stationary, extremal black holes. in a mathematical physics talk on black holes, the fundamental objects of interest are stationary solutions to, say, the einstein - higgs - yang - mills equations for which the uniqueness theorem fails. in a numerical relativity lecture, both these classes of objects are considered to be so exotic that they are excluded from discussion without comment. the focus is primarily on the dynamics of apparent horizons in general relativity. in astrophysically interesting situations, the distortion of black holes by external matter rings, magnetic fields and other black holes is often non - negligible [87, 98, 88 ].. laws of black hole mechanics and the statistical mechanical derivation of entropy should go through for all black holes in equilibrium. laws dictating the dynamics of apparent horizons should predict that the final equilibrium states are those represented by the stable stationary solutions of the theory. is there a paradigm that can serve as an unified framework to establish such results in all these disparate situations ? physically, one would expect the first law to apply to even though the entire space - time is not stationary because of the presence of gravitational radiation in the exterior region. right panel : space - time diagram of a black hole which is initially in equilibrium, absorbs a finite amount of radiation, and again settles down to equilibrium. one would expect the first law to hold on both portions although the space - time is not stationary. much later, a spherical shell of mass m the resulting black hole. while 1 and 2 are both isolated horizons, only 2 is part of the event horizon. dynamical situations for fully dynamical black holes, apart from the topological censorship results which restrict the horizon topology [110, 90 ], there has essentially been only one major result in exact general relativity. this is the celebrated area theorem proved by hawking in the early seventies [111, 113 ] : if matter satisfies the null energy condition, the area of the black hole event horizon can never decrease. this theorem has been extremely influential because of its similarity with the second law of thermodynamics. however, it is a qualitative result ; it does not provide an explicit formula for the amount by which the area increases in physical processes. now, for a black hole of mass m, angular momentum j, area a, surface gravity, and angular velocity, the first law of black hole mechanics, 1\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\delta m = { \kappa \over { 8\pi g}}\delta a + \omega \delta j,$$\end{document } does relate the change in the horizon area to that in the energy and angular momentum, as the black hole makes a transition from one equilibrium state to a nearby one [35, 182 ]. this suggests that there may well be a fully dynamical version of equation (1) which relates the change in the black hole area to the energy and angular momentum it absorbs in fully dynamical processes in which the black hole makes a transition from a given state to one which is far removed. indeed, without such a formula, one would have no quantitative control on how black holes grow in exact general relativity. note however that the event horizons can form and grow even in a flat region of space - time (see figure 4 and section 2.2.2 for illustrations). during this phase, the area grows in spite of the fact that there is no flux of energy or angular momentum across the event horizon. hence, in the standard framework where the surface of the black hole is represented by an event horizon, it is impossible to obtain the desired formula. equilibrium situations the zeroth and first laws of black hole mechanics refer to equilibrium situations and small departures therefrom. therefore, in this context, it is natural to focus on isolated black holes. it was customary to represent them by stationary solutions of field equations, i.e, solutions which admit a time - translational killing vector field everywhere, not just in a small neighborhood of the black hole. while this simple idealization was natural as a starting point, it is overly restrictive. physically, it should be sufficient to impose boundary conditions at the horizon which ensure only that the black hole itself is isolated. that is, it should suffice to demand only that the intrinsic geometry of the horizon be time independent, whereas the geometry outside may be dynamical and admit gravitational and other radiation. indeed, we adopt a similar viewpoint in ordinary thermodynamics ; while studying systems such as a classical gas in a box, one usually assumes that only the system under consideration is in equilibrium, not the whole world. in realistic situations, one is typically interested in the final stages of collapse where the black hole has formed and settled down or in situations in which an already formed black hole is isolated for the duration of the experiment (see figure 1). in such cases, there is likely to be gravitational radiation and non - stationary matter far away from the black hole. thus, from a physical perspective, a framework which demands global stationarity is too restrictive. even if one were to ignore these conceptual considerations and focus just on results, the framework has certain unsatisfactory features. consider the central result, the first law of equation (1). here, the angular momentum j and the mass m are defined at infinity while the angular velocity and surface gravity are defined at the horizon. because one has to go back and forth between the horizon and infinity, the physical meaning of the first law is not transparent1. for instance, there may be matter rings around the black hole which contribute to the angular momentum and mass at infinity. should nt only the angular momentum and mass of the black hole feature in the first law ? thus, one is led to ask : is there a more suitable paradigm which can replace frameworks based on event horizons in stationary space - times ? entropy calculations the first and the second laws suggest that one should assign to a black hole an entropy which is proportional to its area. this poses a concrete challenge to candidate theories of quantum gravity : account for this entropy from fundamental, statistical mechanical considerations. string theory has had a remarkable success in meeting this challenge in detail for a subclass of extremal, stationary black holes whose charge equals mass (the so - called bps states). however, for realistic black holes the charge to mass ratio is less than 10. it has not been possible to extend the detailed calculation to realistic cases where charge is negligible and matter rings may distort the black hole horizon. from a mathematical physics perspective, the entropy calculation should also encompass hairy black holes whose equilibrium states can not be characterized just by specifying the mass, angular momentum and charges at infinity, as well as non - minimal gravitational couplings, in presence of which the entropy is no longer a function just of the horizon area. a natural strategy is to consider the sector of general relativity containing an isolated black hole and carry out its quantization systematically. a pre - requisite for such a program is the availability of a manageable action principle and/or hamiltonian framework. unfortunately, however, if one attempts to construct these within the classical frameworks traditionally used to describe black holes, one runs into two difficulties. first, because the event horizon is such a global notion, no action principle is known for the sector of general relativity containing geometries which admit an event horizon as an internal boundary. second, if one restricts oneself to globally stationary solutions, the phase space has only a finite number of true degrees of freedom and is thus too small to adequately incorporate all quantum fluctuations. thus, again, we are led to ask : is there a more satisfactory framework which can serve as the point of departure for a non - perturbative quantization to address this problem ? global nature of event horizons the future event horizon is defined as the future boundary of the causal past of future null infinity. while this definition neatly encodes the idea that an outside observer can not look into a black hole, it is too global for many applications. first, since it refers to null infinity, it can not be used in spatially compact space - times. surely, one should be able to analyze black hole dynamics also in these space - times. more importantly, the notion is teleological ; it lets us speak of a black hole only after we have constructed the entire space - time. thus, for example, an event horizon may well be developing in the room you are now sitting in anticipation of a gravitational collapse that may occur in this region of our galaxy a million years from now. when astrophysicists say that they have discovered a black hole in the center of our galaxy, they are referring to something much more concrete and quasi - local than an event horizon. the teleological nature of event horizons is also an obstruction to extending black hole mechanics in certain physical situations. consider for example, figure 2 in which a spherical star of mass m undergoes a gravitational collapse. the singularity is hidden inside the null surface 1 at r = 2 m which is foliated by a family of marginally trapped surfaces and would be a part of the event horizon if nothing further happens. suppose instead, after a million years, a thin spherical shell of mass m collapses. then 1 would not be a part of the event horizon which would actually lie slightly outside 1 and coincide with the surface r = 2(m + m) in the distant future. on physical grounds surely one should be able to establish the standard laws of mechanics not only for the equilibrium portion of the event horizon but also for 1. thus, one needs to identify initial data containing black holes without the knowledge of the entire space - time and evolve them step by step. one may then ask : can we use apparent horizons instead of event horizons in other contexts as well ? unfortunately, it has not been possible to derive the laws of black hole mechanics using apparent horizons. furthermore, as discussed in section 2, while apparent horizons are local in time they are still global notions, tied too rigidly to the choice of a space - like 3-surface to be directly useful in all contexts. is there a truly quasi - local notion which can be useful in all these contexts ? disparate paradigms in different communities within gravitational physics, the intended meaning of the term thus, in a string theory seminar, the term fundamental black holes without further qualification generally refers to the bps states referred to above a sub - class of stationary, extremal black holes. in a mathematical physics talk on black holes, the fundamental objects of interest are stationary solutions to, say, the einstein - higgs - yang - mills equations for which the uniqueness theorem fails. in a numerical relativity lecture, both these classes of objects are considered to be so exotic that they are excluded from discussion without comment. the focus is primarily on the dynamics of apparent horizons in general relativity. in astrophysically interesting situations, the distortion of black holes by external matter rings, magnetic fields and other black holes is often non - negligible [87, 98, 88 ].. laws of black hole mechanics and the statistical mechanical derivation of entropy should go through for all black holes in equilibrium. laws dictating the dynamics of apparent horizons should predict that the final equilibrium states are those represented by the stable stationary solutions of the theory. is there a paradigm that can serve as an unified framework to establish such results in all these disparate situations ? left panel : a typical gravitational collapse. physically, one would expect the first law to apply to even though the entire space - time is not stationary because of the presence of gravitational radiation in the exterior region. right panel : space - time diagram of a black hole which is initially in equilibrium, absorbs a finite amount of radiation, and again settles down to equilibrium. one would expect the first law to hold on both portions although the space - time is not stationary. a spherical star of mass m undergoes collapse. much later, a spherical shell of mass m the resulting black hole. while 1 and 2 are both isolated horizons, only 2 is part of the event horizon. these considerations led to the development of a new, quasi - local paradigm to describe black holes. this framework was inspired by certain seminal ideas introduced by hayward [117, 118, 115, 116 ] in the mid - nineties and has been systematically developed over the past five years or so. evolving black holes are modelled by dynamical horizons while those in equilibrium are modelled by isolated horizons. in contrast to event horizons, neither notion requires the knowledge of space - time as a whole or refers to asymptotic flatness. therefore, in contrast to apparent horizons, they are not tied to the choice of a partial cauchy slice. this framework provides a new perspective encompassing all areas in which black holes feature : quantum gravity, mathematical physics, numerical relativity, and gravitational wave phenomenology. more importantly, it has overcome some of the limitations of the older frameworks and also led to new results of direct physical interest. many of the key issues are still open and new results are likely to emerge in the coming years. nonetheless, as the editors pointed out, there is now a core of results of general interest and, thanks to the innovative style of living reviews, we will be able to incorporate new results through periodic updates. applications of the quasi - local framework can be summarized as follows : black hole mechanics isolated horizons extract from the notion of killing horizons, just those conditions which ensure that the horizon geometry is time independent ; there may be matter and radiation even nearby. yet, it has been possible to extend the zeroth and first laws of black hole mechanics to isolated horizons [26, 75, 15 ]. recall that, in presence of internal boundaries, time evolution need not be hamiltonian (i.e., need not preserve the symplectic structure). if the inner boundary is an isolated horizon, a necessary and sufficient condition for evolution to be hamiltonian turns out to be precisely the first law ! finally, while the first law has the same form as before (equation (1)), all quantities which enter the statement of the law now refer to the horizon itself. this is the case even when non - abelian gauge fields are included.dynamical horizons allow for the horizon geometry to be time dependent. this framework has led to a quantitative relation between the growth of the horizon area and the flux of energy and angular momentum across it [30, 31 ]. the processes can be in the non - linear regime of exact general relativity, without any approximations. thus, the second law is generalized and the generalization also represents an integral version of the first law (1), applicable also when the black hole makes a transition from one state to another, which may be far removed. the isolated horizon framework provides an action principle and a hamiltonian theory which serves as a stepping stone to non - perturbative quantization. using the quantum geometry framework, the horizon states are then counted to show that the statistical mechanical black hole entropy is indeed proportional to the area [10, 11, 84, 149, 25 ]. this derivation is applicable to ordinary, astrophysical black holes which may be distorted and far from extremality. it also encompasses cosmological horizons to which thermodynamical considerations are known to apply. finally, the arena for this derivation is the curved black hole geometry, rather than a system in flat space - time which has the same number of states as the black hole [174, 145 ]. therefore, this approach has a greater potential for analyzing physical processes associated with the black hole.the dynamical horizon framework has raised some intriguing questions about the relation between black hole mechanics and thermodynamics in fully dynamical situations. in particular, they provide seeds for further investigations of the notion of entropy in non - equilibrium situations. mathematical physics the isolated horizon framework has led to a phenomenological model to understand properties of hairy black holes [21, 20 ]. in this model, the hairy black hole can be regarded as a bound state of an ordinary black hole and a soliton. a large number of facts about hairy black holes had accumulated through semi - analytical and numerical studies. their qualitative features are explained by the model.the dynamical horizon framework also provides the groundwork for a new approach to penrose inequalities which relate the area of cross - sections of the event horizon ae on a cauchy surface with the adm mass madm at infinity : \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\sqrt { { a_e}/16\pi } \leq { m_{{\rm{adm}}}}$\end{document}. relatively recently, the conjecture has been proved in time symmetric situations. the basic monotonicity formula of the dynamical horizon framework could provide a new avenue to extend the current proofs to non - time - symmetric situations. it may also lead to a stronger version of the conjecture where the adm mass is replaced by the bondi mass. numerical relativity the framework has provided a number of tools to extract physics from numerical simulations in the near - horizon, strong field regime. first, there exist expressions for mass and angular momentum of dynamical and isolated horizons which enable one to monitor dynamical processes occurring in the simulations and extract properties of the final equilibrium state [15, 85 ]. these quantities can be calculated knowing only the horizon geometry and do not pre - suppose that the equilibrium state is a kerr horizon. the computational resources required in these calculations are comparable to those employed by simulations using cruder techniques, but the results are now invariant and interpretation is free from ambiguities. recent work has shown that these methods are also numerically more accurate and robust than older ones.surprisingly, there are simple local criteria to decide whether the geometry of an isolated horizon is that of the kerr horizon. the isolated horizon framework also provides invariant, practical criteria to compare near - horizon geometries of different simulations and leads to a new approach to the problem of extracting wave - forms in a gauge invariant fashion. finally, the framework provides natural boundary conditions for the initial value problem for black holes in quasi - equilibrium [72, 124, 81 ], and to interpret certain initial data sets. many of these ideas have already been implemented in some binary black hole codes [85, 34, 48 ] and the process is continuing. gravitational wave phenomenology the isolated horizon framework has led to a notion of horizon multipole moments. they are distinct from the hansen multipoles in stationary space - times normally used in the analysis of equations of motion because they depend only on the isolated horizon geometry and do not require global stationarity. they represent source multipoles rather than hansen s field multipoles. in kerr space - time, while the mass and angular momenta agree in the two regimes, quadrupole moments do not ; the difference becomes significant when a m, i.e., in the fully relativistic regime. in much of the literature on equations of motion of black holes, the distinction is glossed over largely because only field multipoles have been available in the literature. however, in applications to equations of motion, it is the source multipoles that are more relevant, whence the isolated horizon multipoles are likely to play a significant role.the dynamical horizon framework enables one to calculate mass and angular momentum of the black hole as it evolves. in particular, one can now ask if the black hole can be first formed violating the kerr bound a m but then eventually settle down in the kerr regime. preliminary considerations fail to rule out this possibility, although the issue is still open. the possibility that the bound can indeed be violated initially has interesting astrophysical implications. black hole mechanics isolated horizons extract from the notion of killing horizons, just those conditions which ensure that the horizon geometry is time independent ; there may be matter and radiation even nearby. yet, it has been possible to extend the zeroth and first laws of black hole mechanics to isolated horizons [26, 75, 15 ]. recall that, in presence of internal boundaries, time evolution need not be hamiltonian (i.e., need not preserve the symplectic structure). if the inner boundary is an isolated horizon, a necessary and sufficient condition for evolution to be hamiltonian turns out to be precisely the first law ! finally, while the first law has the same form as before (equation (1)), all quantities which enter the statement of the law now refer to the horizon itself. this framework has led to a quantitative relation between the growth of the horizon area and the flux of energy and angular momentum across it [30, 31 ]. the processes can be in the non - linear regime of exact general relativity, without any approximations. thus, the second law is generalized and the generalization also represents an integral version of the first law (1), applicable also when the black hole makes a transition from one state to another, which may be far removed. the isolated horizon framework provides an action principle and a hamiltonian theory which serves as a stepping stone to non - perturbative quantization. using the quantum geometry framework, the horizon states are then counted to show that the statistical mechanical black hole entropy is indeed proportional to the area [10, 11, 84, 149, 25 ]. this derivation is applicable to ordinary, astrophysical black holes which may be distorted and far from extremality. it also encompasses cosmological horizons to which thermodynamical considerations are known to apply. finally, the arena for this derivation is the curved black hole geometry, rather than a system in flat space - time which has the same number of states as the black hole [174, 145 ]. therefore, this approach has a greater potential for analyzing physical processes associated with the black hole. the dynamical horizon framework has raised some intriguing questions about the relation between black hole mechanics and thermodynamics in fully dynamical situations. in particular, they provide seeds for further investigations of the notion of entropy in non - equilibrium situations. mathematical physics the isolated horizon framework has led to a phenomenological model to understand properties of hairy black holes [21, 20 ]. in this model, the hairy black hole can be regarded as a bound state of an ordinary black hole and a soliton. a large number of facts about hairy black holes had accumulated through semi - analytical and numerical studies. the dynamical horizon framework also provides the groundwork for a new approach to penrose inequalities which relate the area of cross - sections of the event horizon ae on a cauchy surface with the adm mass madm at infinity : \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\sqrt { { a_e}/16\pi } \leq { m_{{\rm{adm}}}}$\end{document}. relatively recently, the conjecture has been proved in time symmetric situations. the basic monotonicity formula of the dynamical horizon framework could provide a new avenue to extend the current proofs to non - time - symmetric situations. it may also lead to a stronger version of the conjecture where the adm mass is replaced by the bondi mass. numerical relativity the framework has provided a number of tools to extract physics from numerical simulations in the near - horizon, strong field regime. first, there exist expressions for mass and angular momentum of dynamical and isolated horizons which enable one to monitor dynamical processes occurring in the simulations and extract properties of the final equilibrium state [15, 85 ]. these quantities can be calculated knowing only the horizon geometry and do not pre - suppose that the equilibrium state is a kerr horizon. the computational resources required in these calculations are comparable to those employed by simulations using cruder techniques, but the results are now invariant and interpretation is free from ambiguities. recent work has shown that these methods are also numerically more accurate and robust than older ones. surprisingly, there are simple local criteria to decide whether the geometry of an isolated horizon is that of the kerr horizon. the isolated horizon framework also provides invariant, practical criteria to compare near - horizon geometries of different simulations and leads to a new approach to the problem of extracting wave - forms in a gauge invariant fashion. finally, the framework provides natural boundary conditions for the initial value problem for black holes in quasi - equilibrium [72, 124, 81 ], and to interpret certain initial data sets. many of these ideas have already been implemented in some binary black hole codes [85, 34, 48 ] and the process is continuing. gravitational wave phenomenology the isolated horizon framework has led to a notion of horizon multipole moments. they are distinct from the hansen multipoles in stationary space - times normally used in the analysis of equations of motion because they depend only on the isolated horizon geometry and do not require global stationarity. they represent source multipoles rather than hansen s field multipoles. in kerr space - time, while the mass and angular momenta agree in the two regimes, quadrupole moments do not ; the difference becomes significant when a m, i.e., in the fully relativistic regime. in much of the literature on equations of motion of black holes, the distinction is glossed over largely because only field multipoles have been available in the literature. however, in applications to equations of motion, it is the source multipoles that are more relevant, whence the isolated horizon multipoles are likely to play a significant role. the dynamical horizon framework enables one to calculate mass and angular momentum of the black hole as it evolves. in particular, one can now ask if the black hole can be first formed violating the kerr bound a m but then eventually settle down in the kerr regime. preliminary considerations fail to rule out this possibility, although the issue is still open. the possibility that the bound can indeed be violated initially has interesting astrophysical implications. in this review, we will outline the basic ideas underlying dynamical and isolated horizon frameworks and summarize their applications listed above. we recall the basic definitions, motivate the assumptions and summarize their implications. in section 3 we discuss the area increase theorem for dynamical horizons and show how it naturally leads to an expression for the flux of gravitational energy crossing dynamical horizons. we outline the main ideas using both isolated and dynamical horizons. in the next three sections we review applications. section 5 summarizes applications to numerical relativity, section 6 to black holes with hair, and section 7 to the quantum entropy calculation. having read section 2, sections 3, 4, 5, 6, and 7 are fairly self contained and the three applications can be read independently of each other. all manifolds will be assumed to be c (with k 3) and orientable, the space - time metric will be c, and matter fields c. for simplicity we will restrict ourselves to 4-dimensional space - time manifolds \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\mathcal m}$\end{document } (although most of the classical results on isolated horizons have been extended to 3-dimensions space - times, as well as higher dimensional ones). the space - time metric gab has signature (, +, +, +) and its derivative operator will be denoted by. the riemann tensor is defined by \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${r_{abc}}^d{w_d } : = 2{\nabla _ { \left [a \right.}}{\nabla _ { \left. b \right]}}{w_c}$\end{document }, the ricci tensor by \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${r_{ab } } : = { r_{acb}}^c$\end{document }, and the scalar curvature by \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$r:{g^{ab}}{r_{ab}}$\end{document}. we will assume the field equations 2\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${r_{ab } } - { 1 \over 2}r{g_{ab } } + \lambda { g_{ab } } = 8\pi g{t_{ab}}.$$\end{document } (with these conventions, de sitter space - time has positive cosmological constant.) we assume that tab satisfies the dominant energy condition (although, as the reader can easily tell, several of the results will hold under weaker restrictions.) cauchy (and partial cauchy) surfaces will be denoted by m, isolated horizons by, and dynamical horizons by h., we provide the basic definitions and discuss geometrical properties of non - expanding, weakly isolated, and isolated horizons which describe black holes which are in equilibrium in an increasingly stronger sense. these horizons model black holes which are themselves in equilibrium, but in possibly dynamical space - times [13, 14, 26, 16 ]. for early references with similar ideas, a useful example is provided by the late stage of a gravitational collapse shown in figure 1. in such physical situations, one expects the back - scattered radiation falling into the black hole to become negligible at late times so that the end portion of the event horizon (labelled by in the figure) can be regarded as isolated to an excellent approximation. this expectation is borne out in numerical simulations where the backscattering effects typically become smaller than the numerical errors rather quickly after the formation of the black hole (see, e.g., [34, 48 ]). the key idea is to extract from the notion of a killing horizon the minimal conditions which are necessary to define physical quantities such as the mass and angular momentum of the black hole and to establish the zeroth and the first laws of black hole mechanics. like killing horizons, isolated horizons are null, 3-dimensional sub - manifolds of space - time. let us therefore begin by recalling some essential features of such sub - manifolds, which we will denote by. the intrinsic metric qab on has signature (0,+,+), and is simply the pull - back of the space - time metric to, \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${q_{ab } } = \underleftarrow { { g_{ab}}}$\end{document }, where an underarrow indicates the pullback to. since qab is degenerate, it does not have an inverse in the standard sense. however, it does admit an inverse in a weaker sense : q will be said to be an inverse of qab if it satisfies qamqbnq = qab. as one would expect, the inverse is not unique : we can always add to q a term of the type \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\ell ^{{(^a}{v^b})}}$\end{document }, where is a null normal to and v any vector field tangential to. all our constructions will be insensitive to this ambiguity. given a null normal to, the expansion () is defined as 3\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${\theta _ { (\ell) } } : = { q^{ab}}{\nabla _ a}{\ell _ b}.$$\end{document } (throughout this review, we will assume that is future directed.) we can now state the first definition : definition 1 : a sub - manifold of a space - time (\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\mathcal m},{g_{ab}}$\end{document }) is said to be a non - expanding horizon (neh) if is topologically s and null;any null normal of has vanishing expansion, () = 0 ; andall equations of motion hold at and the stress energy tensor tab is such that \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$ - t_b^a{\ell ^b}$\end{document } is future - causal for any future directed null normal. is topologically s and null ; any null normal of has vanishing expansion, () = 0 ; and all equations of motion hold at and the stress energy tensor tab is such that \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$ - t_b^a{\ell ^b}$\end{document } is future - causal for any future directed null normal. the motivation behind this definition can be summarized as follows. condition 1 is imposed for definiteness ; while most geometric results are insensitive to topology, the s case is physically the most relevant one. the key condition in the above definition is condition 2 which is equivalent to requiring that every cross - section of be marginally trapped. (note incidentally that if () vanishes for one null normal to, it vanishes for all.) condition 2 is equivalent to requiring that the infinitesimal area element is lie dragged by the null normal. in particular, then, condition 2 implies that the horizon area is constant in time. we will denote the area of any cross section of by a and define the horizon radius as \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${r_\delta}:\sqrt { { a_\delta}/4\pi}$\end{document}. because of the raychaudhuri equation, condition 2 also implies 4\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${r_{ab}}{\ell ^a}{\ell ^b } = { \sigma _ { ab}}{\sigma ^{ab } } = 0,$$\end{document } where is the shear of a, defined by \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\sigma _ { ab } } : = \underrightarrow \nabla ({ a^\ell}b) - { 1 \over 2}\theta (\ell){q_{ab}}$\end{document }, where the underarrow denotes pull - back to a. now the energy condition 3 implies that rab is non - negative, whence we conclude that each of the two terms in the last equation vanishes. this in turn implies that \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\underleftarrow { { t_{ab}}{\ell ^b } } = 0$\end{document } and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\underleftarrow { { \nabla _ { ({ a^\ell}b) } } } = 0$\end{document } on. the first of these equations constrains the matter fields on in an interesting way, while the second is equivalent to \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${{\mathcal l}_\ell}{q_{ab } } = 0$\end{document } on. thus, the intrinsic metric on an neh is time - independent ; this is the sense in which an neh is in equilibrium. the zeroth and first laws of black hole mechanics require an additional structure, which is provided by the concept of a weakly isolated horizon. to arrive at this concept, let us first introduce a derivative operator \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\mathcal d}$\end{document } on. because qab is degenerate, there is an infinite number of (torsion - free) derivative operators which are compatible with it. however, on an neh, the property \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\underleftarrow { { \nabla _ { ({ a^\ell}b) } } } = 0$\end{document } implies that the space - time connection induces a unique (torsion - free) derivative operator \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\mathcal d}$\end{document } on which is compatible with qab [26, 136 ]. weakly isolated horizons are characterized by the property that, in addition to the metric qab, the connection component \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${{\mathcal d}_a}{\ell ^b}$\end{document } is also time independent. two null normals and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${{\tilde \ell}^a}$\end{document } to an neh are said to belong to the same equivalence class [] if \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${{\tilde \ell}^a } = c{\ell ^a}$\end{document } for some positive constant c. then, weakly isolated horizons are defined as follows : definition 2 : the pair (, []) is said to constitute a weakly isolated horizon (wih) provided is an neh and each null normal in [] satisfies 5\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$({{\mathcal l}_\ell}{{\mathcal d}_a } - { { \mathcal d}_a}{{\mathcal l}_\ell}){\ell ^b } = 0.$$\end{document } it is easy to verify that every neh admits null normals satisfying equation (5), i.e., can be made a wih with a suitable choice of []. however the required equivalence class is not unique, whence an neh admits distinct wih structures. compared to conditions required of a killing horizon, conditions in this definition are very weak. nonetheless, it turns out that they are strong enough to capture the notion of a black hole in equilibrium in applications ranging from black hole mechanics to numerical relativity. (in fact, many of the basic notions such as the mass and angular momentum are well - defined already on nehs although intermediate steps in derivations use a wih structure.) this is quite surprising at first because the laws of black hole mechanics were traditionally proved for globally stationary black holes, and the definitions of mass and angular momentum of a black hole first used in numerical relativity implicitly assumed that the near horizon geometry is isometric to kerr. although the notion of a wih is sufficient for most applications, from a geometric viewpoint, a stronger notion of isolation is more natural : the full connection \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\mathcal d}$\end{document } should be time - independent. definition 3 : a wih (, []) is said to constitute an isolated horizon (ih) if 6\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$({{\mathcal l}_\ell}{{\mathcal d}_a } - { { \mathcal d}_a}{{\mathcal l}_\ell}){v^b } = 0$$\end{document } for arbitrary vector fields v tangential to. while an neh can always be given a wih structure simply by making a suitable choice of the null normal, not every wih admits an ih structure. however, even for this stronger notion of isolation, conditions in the definition are local to. furthermore, the definition only uses quantities intrinsic to ; there are no restrictions on components of any fields transverse to. (even the full 4-metric gab need not be time independent on the horizon.) robinson - trautman solutions provide explicit examples of isolated horizons which do not admit a stationary killing field even in an arbitrarily small neighborhood of the horizon. in this sense one expects them to be met to an excellent degree of approximation in a wide variety of situations representing late stages of gravitational collapse and black hole mergers2. the class of space - times admitting nehs, wihs, and ihs is quite rich. first, it is trivial to verify that any killing horizon which is topologically s is also an isolated horizon. this in particular implies that the event horizons of all globally stationary black holes, such as the kerr - newman solutions (including a possible cosmological constant), are isolated horizons. these arise because the notion is quasi - local, referring only to fields defined intrinsically on the horizon. first, let us consider the sub - family of kastor - traschen solutions [125, 152 ] which are asymptotically de sitter and admit event horizons. they are interpreted as containing multiple charged, dynamical black holes in presence of a positive cosmological constant. since these solutions do not appear to admit any stationary killing fields, no killing horizons are known to exist. nonetheless, the event horizons of individual black holes are wihs. however, to our knowledge, no one has checked if they are ihs. a more striking example is provided by a sub - family of robinson - trautman solutions, analyzed by chrusciel. these space - times admit ihs whose intrinsic geometry is isomorphic to that of the schwarzschild isolated horizons but in which there is radiation arbitrarily close to. more generally, using the characteristic initial value formulation [92, 161 ], lewandowski has constructed an infinite dimensional set of local examples. here, one considers two null surfaces and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\mathcal n}$\end{document } intersecting in a 2-sphere s (see figure 3). one can freely specify certain data on these two surfaces which then determines a solution to the vacuum einstein equations in a neighborhood of s bounded by and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\mathcal n}$\end{document }, in which is an isolated horizon. the weyl tensor component 0 on the null surface is part of the free data which vanishes if is an ih. the weyl tensor component 0 on the null surface is part of the free data which vanishes if is an ih. as we remarked in section 2.1.1, there is a functional rescaling freedom in the choice of a null normal on an neh and, while the choice of null normals is restricted by the weakly isolated horizon condition (5), considerable freedom still remains. that is, a given neh admits an infinite number of wih structures (, []). on ihs, by contrast, the situation is dramatically different. given an ih (, []), generically the condition (6) in definition 3 can not be satisfied by a distinct equivalence class of null normals []. thus on a generic ih, the only freedom in the choice of the null normal is that of a rescaling by a positive constant. this freedom mimics the properties of a killing horizon since one can also rescale the killing vector by an arbitrary constant. the triplet (\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${q_{ab}},{{\mathcal d}_a},[{\ell ^a}]$\end{document }) is said to constitute the geometry of the isolated horizon. first note that, by definition 1, is expansion free and shear free. this means that the contraction of ab with any two vectors tangent to is identically zero, whence there must exist a 1-form a on such that for any v tangent to, 7\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${v^a}{\nabla _ a}{\ell ^b } = { v^a}{\omega _ a}{\ell ^b}.$$\end{document } note that the wih condition (5) requires simply that a be time independent, \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${{\mathcal l}_\ell}{w_a } = 0$\end{document}. given a, the surface gravity () associated with a null normal is defined as 8\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${\kappa _ { (\ell) } } : = { \ell ^a}{\omega _ a}.$$\end{document } thus, () is simply the acceleration of. note that the surface gravity is not an intrinsic property of a wih (, []). rather, it is a property of a null normal to : (c) = c(). an isolated horizon with () = 0 is said to be an extremal isolated horizon. note that while the value of surface gravity refers to a specific null normal, whether a given wih is extremal or not is insensitive to the permissible rescaling of the normal. consider any (space - time) null tetrad (\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\ell ^a},{n^a},{m^a},{{\bar m}^a}$\end{document }) on such that is a null normal to. then, it follows from definition 1 that two of the newman - penrose weyl components vanish on \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\delta:{\psi _ 0 } : = { c_{abcd}}{\ell ^a}{m^b}{\ell ^c}{m^d } = 0$\end{document } and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\psi _ 1 } : = { c_{abcd}}{\ell ^a}{m^b}{\ell ^c}{n^d } = 0$\end{document}. this in turn implies that \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\psi _ 2 } : = { c_{abcd}}{\ell ^a}{m^b}{{\bar m}^c}{n^d } = 0$\end{document } is gauge invariant (i.e., does not depend on the specific choice of the null tetrad satisfying the condition stated above.) the imaginary part of 2 is related to the curl of a, 9\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$d\omega = 2({{\rm i m } } { \psi _ 2})\epsilon,$$\end{document } where ab is the natural area 2-form on. horizons on which i m 2 vanishes are said to be non - rotating : on these horizons all angular momentum multipoles vanish. therefore, im2 is sometimes referred to as the rotational scalar and a as the rotation 1-form of the horizon. on any neh we have : \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\phi _ { 00 } } : = { 1 \over 2}{r_{ab}}{\ell ^a}{\ell ^b } : = 0,{\phi _ { 01 } } : = { 1 \over 2}{r_{ab}}{\ell ^a}{m^b } = 0$\end{document}. in the einstein - maxwell theory, one further has : on, \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\delta,{\phi _ { 02 } } : = { 1 \over 2}{r_{ab}}{m^a}{m^b } = 0$\end{document } and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\phi _ { 20 } } : = { 1 \over 2}{r_{ab}}{{\bar m}^a}{{\bar m}^b } = 0$\end{document}. given the geometry (\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${q_{ab}},{\mathcal d},[\ell ] $ \end{document }) of an ih, it is natural to ask for the minimum amount of information, i.e., the free data, required to construct it. let s be a spherical cross section of. the degenerate metric qab naturally projects to a riemannian metric \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${q_{ab}}$\end{document } on s, and similarly the 1-form a of equation (7) projects to a 1-form \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${{\tilde w}_a}$\end{document } on s. if the vacuum equations hold on, then given (\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${{\tilde q}_{ab}},{{\tilde w}_a}$\end{document }) on s, there is, up to diffeomorphisms, a unique non - extremal isolated horizon geometry (\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${q_{ab}},{\mathcal d},[\ell ] $ \end{document }) such that \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${{\tilde q}_{ab}}$\end{document } is the projection of qab, \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${{\tilde w}_a}$\end{document } is the projection of the a constructed from \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\mathcal d}$\end{document }, and = a 0. (if the vacuum equations do not hold, the additional data required is the projection on s of the space - time ricci tensor.) first, since qab is degenerate along, its non - trivial part is just its projection \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${{\tilde q}_{ab}}$\end{document}. second, \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${{\tilde q}_{ab}}$\end{document } fixes the connection on s ; it is only the quantity \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${s_{ab } } : = { { \mathcal d}_a}{n_b}$\end{document } that is not constrained by \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${{\tilde q}_{ab}}$\end{document }, where na is a 1-form on orthogonal to s, normalized so that na = 1. it is easy to show that sab is symmetric and the contraction of one of its indices with gives a : sab = a. furthermore, it turns out that if a 0, the field equations completely determine the angular part of sab in terms of \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${{\tilde w}_a}$\end{document } and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${{\tilde q}_{ab}}$\end{document}. finally, recall that the surface gravity is not fixed on because of the rescaling freedom in ; thus the -component of a is not part of the free data. putting all these facts together, we see that the pair (\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${{\tilde q}_{ab}},{{\tilde w}_a}$\end{document }) enables us to reconstruct the isolated horizon geometry uniquely up to diffeomorphisms. as at null infinity, a preferred foliation of a can be thought of as providing a rest frame for an isolated horizon. on the schwarzschild horizon, for example, the 2-spheres on which the eddington - finkelstein advanced time coordinate is constant which are also integral manifolds of the rotational killing fields provide such a rest frame. for the kerr metric, this foliation generalizes naturally. the question is whether a general prescription exists to select such a preferred foliation. on any non - extremal neh, the 1-form a can be used to construct preferred foliations of. let us first examine the simpler, non - rotating case in which i m 2 = 0. then equation (9) the 2-surfaces orthogonal to a must be topologically s because, on any non - extremal horizon, a 0. thus, in the non - rotating case, every isolated horizon comes equipped with a preferred family of cross - sections which defines the rest frame. note that the projection \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${{\tilde w}_a}$\end{document } of a on any leaf of this foliation vanishes identically. the rotating case is a little more complicated since a is then no longer curl - free. now the idea is to exploit the fact that the divergence of the projection \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${{\tilde w}_a}$\end{document } of a on a cross - section is sensitive to the choice of the cross - section, and to select a preferred family of cross - sections by imposing a suitable condition on this divergence. however, (in the case when the angular momentum is non - zero) this condition does not pick out the v = const. cuts of the kerr horizon where v is the (carter generalization of the) eddington - finkelstein coordinate. pawlowski has provided another condition that also selects a preferred foliation and reduces to the v = const. cuts of the kerr horizon : 10\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${\rm{div}}\tilde \omega = - \tilde \delta \ln \vert{\psi _ 2}{\vert^{1/3}},$$\end{document } where \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\tilde \delta}$\end{document } is the laplacian of \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${{\tilde q}_{ab}}$\end{document}. on isolated horizons on which |2| is nowhere zero a condition satisfied if the horizon geometry is near that of the kerr isolated horizon this selects a preferred foliation and hence a rest frame. a symmetry of an ih (, []) is a diffeomorphism of which preserves the geometry (\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${q_{ab}},{\mathcal d},[\ell ] $ \end{document }). (on a wih, the symmetry has to preserve (qab, a, []). let us denote the symmetry group by g. first note that diffeomorphisms generated by the null normals in [] are symmetries ; this is already built into the very definition of an isolated horizon. the other possible symmetries are related to the cross - sections of. since we have assumed the cross - sections to be topologically spherical and since a metric on a sphere can have either exactly three, one or zero killing vectors, it follows that g can be of only three types : type i : the pair (\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${q_{ab}},{{\mathcal d}_a}$\end{document }) is spherically symmetric ; g is four dimensional.type ii : the pair (\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${q_{ab}},{{\mathcal d}_a}$\end{document }) is axisymmetric ; g is two dimensional.type iii : diffeomorphisms generated by are the only symmetries of the pair (\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${q_{ab}},{{\mathcal d}_a}$\end{document }) ; g is one dimensional. type i : the pair (\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${q_{ab}},{{\mathcal d}_a}$\end{document }) is spherically symmetric ; g is four dimensional. type ii : the pair (\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${q_{ab}},{{\mathcal d}_a}$\end{document }) is axisymmetric ; g is two dimensional. type iii : diffeomorphisms generated by are the only symmetries of the pair (\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${q_{ab}},{{\mathcal d}_a}$\end{document }) ; g is one dimensional. in the asymptotically flat context, boundary conditions select a universal symmetry group at spatial infinity, e.g., the poincar group, because the space - time metric approaches a fixed minkowskian one. the situation is completely different in the strong field region near a black hole. because the geometry at the horizon can vary from one space - time to another however, the above result shows that the symmetry group can be one of only three universality classes. this section is divided into three parts. in the first, we discuss basic definitions, in the second we introduce an explicit example, and in the third we analyze the issue of uniqueness of dynamical horizons and their role in numerical relativity. to explain the evolution of ideas and provide points of comparison, we will introduce the notion of dynamical horizons following a chronological order. readers who are not familiar with causal structures can go directly to definition 5 of dynamical horizons (for which a more direct motivation can be found in). as discussed in section 1, while the notion of an event horizon has proved to be very convenient in mathematical relativity, it is too global and teleological to be directly useful in a number of physical contexts ranging from quantum gravity to numerical relativity to astrophysics. this limitation was recognized early on (see, e.g.,, page 319) and alternate notions were introduced to capture the intuitive idea of a black hole in a quasi - local manner. in particular, to make the concept local in time, hawking [111, 113 ] introduced the notions of a trapped region and an apparent horizon, both of which are associated to a space - like 3-surface m representing an instant of time. let us begin by recalling these ideas. hawking s outer trapped surface s is a compact, space - like 2-dimensional sub - manifold in (\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\mathcal m},{g_{ab}}$\end{document }) such that the expansion () of the outgoing null normal to s is non - positive. hawking then defined the trapped region \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\mathcal t}(m)$\end{document } in a surface m as the set of all points in m through which there passes an outer - trapped surface, lying entirely in m. finally, hawking s apparent horizon \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\partial { \mathcal t}(m)$\end{document } is the boundary of a connected component of \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\mathcal t}(m)$\end{document}. the idea then was to regard each apparent horizon as the instantaneous surface of a black hole. one can calculate the expansion () of s knowing only the intrinsic 3-metric qab and the extrinsic curvature kab of m. hence, to find outer trapped surfaces and apparent horizons on m, one does not need to evolve (qab, kab) away from m even locally. in this sense the notion is local to m. however, this locality is achieved at the price of restricting s to lie in m. if we wiggle m even slightly, new outer trapped surfaces can appear and older ones may disappear. in this sense, the notion is still very global. initially, it was hoped that the laws of black hole mechanics can be extended to these apparent horizons. however, this has not been possible because the notion is so sensitive to the choice of m. to improve on this situation, in the early nineties hayward proposed a novel modification of this framework. the main idea is to free these notions from the complicated dependence on m. he began with penrose s notion of a trapped surface. a trapped surface s a la penrose is a compact, space - like 2-dimensional sub - manifold of space - time on which ()(n) > 0, where and n are the two null normals to s. we will focus on future trapped surfaces on which both expansions are negative. a trapped region \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\mathcal t}$\end{document } a la hayward is a subset of space - time through each point of which there passes a trapped surface. finally, hayward s trapping boundary \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\partial { \mathcal t}$\end{document } is a connected component of the boundary of an inextendible trapped region. under certain assumptions (which appear to be natural intuitively but technically are quite strong), he was able to show that the trapping boundary is foliated by marginally trapped surfaces (mtss), i.e., compact, space - like 2-dimensional sub - manifolds on which the expansion of one of the null normals, say, vanishes and that of the other, say n, is everywhere non - positive. furthermore, \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${{\mathcal l}_n}{\theta _ { (\ell)}}$\end{document } is also everywhere of one sign. these general considerations led him to define a quasi - local analog of future event horizons as follows : definition 4 : a future, outer, trapping horizon (foth) is a smooth 3-dimensional sub - manifold \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\underline h$\end{document } of space - time, foliated by closed 2-manifolds \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\underline s$\end{document }, such that the expansion of one future directed null normal to the foliation, say, vanishes, () = 0;the expansion of the other future directed null normal n is negative, (n) 0,$$\end{document } since () = 0 and (n) 0, the right side is positive definite whence the gauss - bonnet invariant i is positive definite, and the topology of the cross - sections s of the dh is necessarily that of s.if = 0, then s is either spherical or toroidal. the toroidal case is exceptional : if it occurs, the matter and the gravitational energy flux across h vanishes (see section 3.3), the metric \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${{\tilde q}_{ab}}$\end{document } is flat, \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${{\mathcal l}_n}{\theta _ { (\ell) } } = 0$\end{document } (so h can not be a foth), and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${{\mathcal l}_\ell}{\theta _ { (\ell) } } = 0$\end{document}. in view of these highly restrictive conditions, toroidal dhs appear to be unrelated to the toroidal topology of cross - sections of the event horizon discussed by shapiro, teukolsky, winicour, and others [121, 167, 139 ]. in the generic spherical case, the area balance law (24) becomes 25\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${1 \over { 2g}}({r_2 } - { r_1})\int\nolimits_{\delta h } { { t_{ab}}{{\hat{\tau}}^a}\xi _ { (r)}^b{d^3}v + } { 1 \over { 16\pi g}}\int\nolimits_{\delta h } { { n_r}(\vert\sigma { \vert^2 } + 2\vert\zeta { \vert^2}){d^3}v.}$$\end{document}if 0, the right side is positive definite whence the gauss - bonnet invariant i is positive definite, and the topology of the cross - sections s of the dh is necessarily that of s. if = 0, then s is either spherical or toroidal. the toroidal case is exceptional : if it occurs, the matter and the gravitational energy flux across h vanishes (see section 3.3), the metric \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${{\tilde q}_{ab}}$\end{document } is flat, \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${{\mathcal l}_n}{\theta _ { (\ell) } } = 0$\end{document } (so h can not be a foth), and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${{\mathcal l}_\ell}{\theta _ { (\ell) } } = 0$\end{document}. in view of these highly restrictive conditions, toroidal dhs appear to be unrelated to the toroidal topology of cross - sections of the event horizon discussed by shapiro, teukolsky, winicour, and others [121, 167, 139 ]. in the generic spherical case, the area balance law (24) becomes 25\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${1 \over { 2g}}({r_2 } - { r_1})\int\nolimits_{\delta h } { { t_{ab}}{{\hat{\tau}}^a}\xi _ { (r)}^b{d^3}v + } { 1 \over { 16\pi g}}\int\nolimits_{\delta h } { { n_r}(\vert\sigma { \vert^2 } + 2\vert\zeta { \vert^2}){d^3}v.}$$\end{document } if < 0, there is no control on the sign of the right hand side of equation (24). stationary solutions with quite general topologies are known for black holes which are asymptotically locally anti - de sitter. event horizons of these solutions are the potential asymptotic states of these dhs in the distant future. for simplicity, the remainder of our discussion of dhs will be focused on the zero cosmological constant case with 2-sphere topology. let us interpret the various terms appearing in the area balance law (25). the left side of this equation provides us with the change in the horizon radius caused by the dynamical process under consideration. since the expansion () vanishes, this is also the change in the hawking mass as one moves from the cross section s1 to s2. the first integral on the right side of this equation is the flux \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\mathcal f}_{{\rm{matter}}}^{(r)}$\end{document } of matter energy associated with the vector field \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\xi _ { (r)}^a$\end{document}. the second term is purely geometrical and accompanies the term representing the matter energy flux. hence it is interpreted as the flux \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\mathcal f}_{{\rm{grav}}}^{(r)}$\end{document } of \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\xi _ { (r)}^a$\end{document}-energy carried by the gravitational radiation : 26\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${\mathcal f}_{{\rm{grav}}}^{(r) } : = { 1 \over { 16\pi g}}\int\nolimits_{\delta h } { { n_r}(\vert\sigma { \vert^2 } + 2\vert\zeta { \vert^2}){d^3}v.}$$\end{document } a priori, it is surprising that there should exist a meaningful expression for the gravitational energy flux in the strong field regime where gravitational waves can no longer be envisaged as ripples on a flat space - time. therefore, it is important to subject this interpretation to viability criteria analogous to the standard tests one uses to demonstrate the viability of the bondi flux formula at null infinity. it is known that it passes most of these tests. however, to our knowledge, the status is still partially open on one of these criteria. the situation can be summarized as follows : gauge invariance since one did not have to introduce any structure, such as coordinates or tetrads, which is auxiliary to the problem, the expression is obviously gauge invariant. this is to be contrasted with definitions involving pseudo - tensors or background fields. positivity the energy flux (26) is manifestly non - negative. in the case of the bondi flux, it was perhaps the most convincing evidence that gravitational waves are not coordinate artifacts but carry physical energy. it is quite surprising that a simple, manifestly non - negative expression can exist in the strong field regime of dhs. one can of course apply our general strategy to any space - like 3-surface \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\bar h}$\end{document }, foliated by 2-spheres. however, if \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\bar h}$\end{document } is not a dh, the sign of the geometric terms in the integral over \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\delta \bar h$\end{document } can not be controlled, not even when \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\bar h}$\end{document } lies in the black hole region and is foliated by trapped (rather than marginally trapped) surfaces \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\bar s}$\end{document}. thus, the positivity of \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\mathcal f}_{{\rm{grav}}}^{(r)}$\end{document } is a rather subtle property, not shared by 3-surfaces which are foliated by non - trapped surfaces, nor those which are foliated by trapped surfaces ; one needs a foliation precisely by marginally trapped surfaces. the property is delicately matched to the definition of dhs. locality all fields used in equation (26) are defined by the local geometrical structures on cross - sections of h. this is a non - trivial property, shared also by the bondi - flux formula., the proof of the positive energy theorem by witten provides a positive definite energy density on cauchy surfaces. but since it is obtained by solving an elliptic equation with appropriate boundary conditions at infinity, this energy density is a highly non - local function of geometry. locality of \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\mathcal f}_{{\rm{grav}}}^{(r)}$\end{document } enables one to associate it with the energy of gravitational waves instantaneously falling across any cross section s. vanishing in spherical symmetry the fourth criterion is that the flux should vanish in presence of spherical symmetry.. then one can show that each cross - section of s must be spherically symmetric. now, since the only spherically symmetric vector field and trace - free, second rank tensor field on a 2-sphere are the zero fields, ab = 0 and = 0. balance law the bondi - sachs energy flux also has the important property that there is a locally defined notion of the bondi energy e(c) associated with any 2-sphere cross - section c of future null infinity, and the difference e(c1) e(c2) equals the bondi - sachs flux through the portion of null infinity bounded by c2 and c1. the answer is in the affirmative : as noted in the beginning of this section, the integrated flux is precisely the difference between the locally defined hawking mass associated with the cross - section. in section 5 we will extend these considerations to include angular momentum. since one did not have to introduce any structure, such as coordinates or tetrads, which is auxiliary to the problem the energy flux (26) is manifestly non - negative. in the case of the bondi flux, it was perhaps the most convincing evidence that gravitational waves are not coordinate artifacts but carry physical energy. it is quite surprising that a simple, manifestly non - negative expression can exist in the strong field regime of dhs. one can of course apply our general strategy to any space - like 3-surface \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\bar h}$\end{document }, foliated by 2-spheres. however, if \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\bar h}$\end{document } is not a dh, the sign of the geometric terms in the integral over \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\delta \bar h$\end{document } can not be controlled, not even when \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\bar h}$\end{document } lies in the black hole region and is foliated by trapped (rather than marginally trapped) surfaces \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\bar s}$\end{document}. thus, the positivity of \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\mathcal f}_{{\rm{grav}}}^{(r)}$\end{document } is a rather subtle property, not shared by 3-surfaces which are foliated by non - trapped surfaces, nor those which are foliated by trapped surfaces ; one needs a foliation precisely by marginally trapped surfaces. all fields used in equation (26) are defined by the local geometrical structures on cross - sections of h. this is a non - trivial property, shared also by the bondi - flux formula., the proof of the positive energy theorem by witten provides a positive definite energy density on cauchy surfaces. but since it is obtained by solving an elliptic equation with appropriate boundary conditions at infinity, this energy density is a highly non - local function of geometry. locality of \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\mathcal f}_{{\rm{grav}}}^{(r)}$\end{document } enables one to associate it with the energy of gravitational waves instantaneously falling across any cross section s. vanishing in spherical symmetry the fourth criterion is that the flux should vanish in presence of spherical symmetry.. then one can show that each cross - section of s must be spherically symmetric. now, since the only spherically symmetric vector field and trace - free, second rank tensor field on a 2-sphere are the zero fields, ab = 0 and = 0. the bondi - sachs energy flux also has the important property that there is a locally defined notion of the bondi energy e(c) associated with any 2-sphere cross - section c of future null infinity, and the difference e(c1) e(c2) equals the bondi - sachs flux through the portion of null infinity bounded by c2 and c1. the answer is in the affirmative : as noted in the beginning of this section, the integrated flux is precisely the difference between the locally defined hawking mass associated with the cross - section. in section 5 taken together, the properties discussed above provide a strong support in favor of the interpretation of equation (26) as the (r)-energy flux carried by gravitational waves into the portion h of the dh. nonetheless, it is important to continue to think of new criteria and make sure that equation (26) passes these tests. for instance, in physically reasonable, stationary, vacuum solutions to einstein s equations, one would expect that the flux should vanish. thus, one is led to conjecture that these space - times do not admit dhs. while special cases of this conjecture have been proved, a general proof is still lacking. we will conclude this section with two remarks : the presence of the shear term || in the integrand of the flux formula (26) seems natural from one s expectations based on perturbation theory at the event horizon of the kerr family [108, 66 ]. but the term || is new and can arise only because h is space - like rather than null : on a null surface, the analogous term vanishes identically. to bring out this point, one can consider a more general case and allow the cross - sections s to lie on a horizon which is partially null and partially space - like. then, using a 2 + 2 formulation one can show that flux on the null portion is given entirely by the term ||. however, on the space - like portion, the term || does not vanish in general. indeed, on a dh, it can not vanish in presence of rotation : the angular momentum is given by the integral of a, where is the rotational symmetry.the flux refers to a specific vector field \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\xi _ { (r)}^a$\end{document } and measures the change in the hawking mass associated with the cross - sections. however, this is not a good measure of the mass in presence of angular momentum (see, e.g., for numerical simulations). the presence of the shear term || in the integrand of the flux formula (26) seems natural from one s expectations based on perturbation theory at the event horizon of the kerr family [108, 66 ]. but the term || is new and can arise only because h is space - like rather than null : on a null surface, the analogous term vanishes identically. to bring out this point, one can consider a more general case and allow the cross - sections s to lie on a horizon which is partially null and partially space - like. then, using a 2 + 2 formulation one can show that flux on the null portion is given entirely by the term ||. however, on the space - like portion, the term || does not vanish in general. indeed, on a dh, it can not vanish in presence of rotation : the angular momentum is given by the integral of a, where is the rotational symmetry. the flux refers to a specific vector field \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\xi _ { (r)}^a$\end{document } and measures the change in the hawking mass associated with the cross - sections. however, this is not a good measure of the mass in presence of angular momentum (see, e.g., for numerical simulations). as mentioned in the introduction, the discovery of the laws of black hole mechanics has led to fundamental insights in both classical and quantum gravity. in this section we discuss how the standard framework tied to stationary space - times can be extended using wihs and dhs. the isolated horizon framework has not only extended black hole mechanics, but it has also led to a deeper insight into the origin of the laws of black hole mechanics. in this section we will summarize these developments using wihs. along the way we shall also obtain formulas for the mass and angular momentum of a wih. for simplicity, in the main part of the discussion, we will restrict ourselves to type ii (i.e., axi - symmetric) wihs on which all matter fields vanish. generalizations including various types of matter field can be found in [19, 26, 15, 75, 76, 77 ]. the zeroth law of thermodynamics says that the temperature of a system in thermodynamic equilibrium is constant. its counterpart for black hole mechanics says that surface gravity of a weakly isolated horizon is constant. this result is non - trivial because the horizon geometry is not assumed to be spherically symmetric ; the result holds even when the horizon itself is highly distorted so long as it is in equilibrium. recall from section 2.1.3 that the notion of surface gravity is tied to the choice of a null normal of the isolated horizon : ():= a. now, using equation (5) in definition 2 (of wihs), we obtain : 27\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${\mathcal l_\ell}{\omega _ a } = 0.$$\end{document } next, recall from equation (9) that the curl of a is related to the imaginary part of 2 : 28\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$d\omega = 2({{\rm i m } } { \psi _ 2})\epsilon$$\end{document } where is the natural area 2-form on satisfying \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${{\mathcal l}_\ell}\epsilon = 0$\end{document } and = 0. hence we conclude = 0 which in turn implies that () is constant on the horizon : 29\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$0 = { l_\ell}\omega = d(\ell. \omega) = d{\kappa _ { (\ell)}}.$$\end{document } this completes the proof of the zeroth law. as the argument shows, given an neh, the main condition (5) in the definition of a wih is equivalent to constancy of surface gravity. note that no restriction has been imposed on 2 which determines the mass and angular momentum multipoles : as emphasized above, the zeroth law holds even if the wih is highly distorted and rapidly rotating. if electromagnetic fields are included, one can also show that the electric potential is constant on the horizon. let us restrict ourselves to space - times which admit a non expanding horizon as inner boundary. then the standard palatini action principle is not well defined because the variation produces a non - vanishing surface term at the horizon. the necessary and sufficient condition for this surface term to vanish is precisely that the gravitational (and the electromagnetic) zeroth laws hold. consequently, the standard action principle is well - defined if inner boundaries are wihs. in field theories, conserved quantities such as energy and angular momentum can be universally defined via a hamiltonian framework : they are the numerical values of hamiltonians generating canonical transformations corresponding to time translation and rotation symmetries. in absence of inner boundaries, it is this procedure that first led to the notion of the adm energy and angular momentum at spatial infinity. at null infinity, it can also be used to define fluxes of bondi energy and angular momentum across regions of \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${{\mathcal i}^ + } $ \end{document }, and values of these quantities associated with any cross - section of \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${{\mathcal i}^ + } $ \end{document } [18, 185 ]. the first ingredient required for a hamiltonian framework is, of course, a phase space. the appropriate phase space now consists of fields living in a region of space - time outside the black hole, satisfying suitable boundary conditions at infinity and horizon. let \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\mathcal m}$\end{document } be the region of space - time that we are interested in. the boundary of \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\mathcal m}$\end{document } consists of four components : the time - like cylinder at spatial infinity, two space - like surfaces m1 and m2 which are the future and past boundaries of \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\mathcal m}$\end{document }, and an inner boundary which is to be the wih (see figure 6). at infinity, all fields are assumed to satisfy the fall - off conditions needed to ensure asymptotic flatness. to ensure that is a type ii horizon, one fixes a rotational vector field on and requires that physical fields on \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\mathcal m}$\end{document } are such that the induced geometry on figure 6the region of space - time \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\mathcal m}$\end{document } under consideration has an internal boundary and is bounded by two cauchy surfaces m1 and m2 and the time - like cylinder at infinity. m is a cauchy surface in \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\mathcal m}$\end{document } whose intersection with is a spherical cross - section s and the intersection with is s, the sphere at infinity. the region of space - time \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\mathcal m}$\end{document } under consideration has an internal boundary and is bounded by two cauchy surfaces m1 and m2 and the time - like cylinder at infinity. m is a cauchy surface in \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\mathcal m}$\end{document } whose intersection with is a spherical cross - section s and the intersection with is s, the sphere at infinity. the first uses a covariant phase space which consists of the solutions to field equations which satisfy the required boundary conditions [26, 15 ]. here the calculations are simplest if one uses a first order formalism for gravity, so that the basic gravitational variables are orthonormal tetrads and lorentz connections. the second uses a canonical phase space consisting of initial data on a cauchy slice m of \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\mathcal m}$\end{document }. in the gravitational sector, this description is based on the standard adm variables. since the conceptual structure underlying the main calculation and the final results are the same, the details of the formalism are not important. for definiteness, in the main discussion, we will use the covariant phase space and indicate the technical modifications needed in the canonical picture at the end. the phase space is naturally endowed with a (pre-)symplectic structure a closed 2-form (whose degenerate directions correspond to infinitesimal gauge motions). given any two vector fields (i.e., infinitesimal variations) 1 and 2 on, the action (1, 2) of the symplectic 2-form on them provides a function on. a vector field x on is said to be a hamiltonian vector field (i.e., to generate an infinitesimal canonical transformation) if and only if \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${{\mathcal l}_x}\omega = 0$\end{document}. since the phase space is topologically trivial, it follows that this condition holds if and only if there is a function h on such that (, x) = h for all vector fields. the function h is called a hamiltonian and x its hamiltonian vector field ; alternatively, h is said to generate the infinitesimal canonical transformation x. since we are interested in energy and angular momentum, the infinitesimal canonical transformations x will correspond to time translations and rotations. as in any generally covariant theory, when the constraints are satisfied, values of hamiltonians generating such diffeomorphisms can be expressed purely as surface terms. in the present case, the relevant surfaces are the sphere at infinity and the spherical section s = m of the horizon. thus the numerical values of hamiltonians now consist of two terms : a term at infinity and a term at the horizon. consider a vector field on m which satisfies the following boundary conditions : (i) at infinity, coincides with a fixed rotational symmetry of the fiducial flat metric ; and, (ii) on, it coincides with the vector field lie derivatives of physical fields along define a vector field x() on. the question is whether this is an infinitesimal canonical transformation, i.e., a generator of the phase space symmetry. as indicated above, this is the case if and only if there exists a phase space function j satisfying : 30\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\delta { j^{(\phi) } } = \omega (\delta,{x_{(\phi)}}).$$\end{document } for all variations. if such a phase space function j exists, it can be interpreted as the hamiltonian generating rotations. now, a direct calculation shows that, in absence of gauge fields on, one has : 31\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\omega (\delta,{x_{(\phi) } }) = { 1 \over { 8\pi g}}\delta \oint\nolimits_s { [({ \varphi ^a}{\omega _ a})\;{}^2\epsilon ] } - \delta j_{{\rm{adm}}}^{(\phi) } = : \delta { j^{(\phi)}}.$$\end{document } as expected, the expression for j consists of two terms : a term at the horizon and a term at infinity. the term at infinity is the variation of the familiar adm angular momentum \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$j_{{\rm{adm}}}^{(\phi)}$\end{document } associated with. the surface integral at the horizon is interpreted as the variation of the horizon angular momentum j. since variations are arbitrary, one can recover, up to additive constants, \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$j_{{\rm{adm}}}^{(\phi)}$\end{document } and j from their variations, and these constants can be eliminated by requiring that both of these angular momenta should vanish in static axi - symmetric space - times. one then obtains : 32\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${j_\delta } : = - { 1 \over { 8\pi}}\oint\nolimits_s { ({ \omega _ a}{\varphi ^a}){}^2\epsilon } = - { 1 \over { 4\pi}}\oint\nolimits_s { f{{\rm i m } } [{ \psi _ 2}]{}^2\epsilon}$$\end{document } where the function f on is related to by af = ba. in the last step we have used equation (28) and performed an integration by parts. equation (32) is the expression of the horizon angular momentum. note that all fields that enter this expression are local to the horizon and is not required to be a killing field of the space - time metric even in a neighborhood of the horizon. therefore, j can be calculated knowing only the horizon geometry of a type ii horizon. we conclude our discussion of angular momentum with some comments : the hamiltonian j it follows from equation (31) and (32) that the total hamiltonian generating the rotation along is the difference between the adm and the horizon angular momenta (apart from a sign which is an artifact of conventions). thus, it can be interpreted as the angular momentum of physical fields in the space - time region \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\mathcal m}$\end{document } outside the black hole. relation to the komar integral if happens to be a space - time killing field in a neighborhood of, then j agrees with the komar integral of. if is a global, space - time killing field, then both \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$j_{{\rm{adm}}}^{(\phi)}$\end{document } as well as j agree with the komar integral, whence the total hamiltonian j vanishes identically. since the fields in the space - time region \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\mathcal m}$\end{document } are all axi - symmetric in this case, this is just what one would expect from the definition of j. j for general axial fields if the vector field is tangential to cross - sections of, j continues to the generator of the canonical transformation corresponding to rotations along, even if its restriction \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${{\tilde \varphi}^a}$\end{document } to does not agree with the axial symmetry of horizon geometries of our phase space fields. however, there is an infinity of such vector fields \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${{\tilde \varphi}^a}$\end{document } and there is no physical reason to identify the surface term j arising from any one of them with the horizon angular momentum. inclusion of gauge fields if non - trivial gauge fields are present at the horizon, equation (32) is incomplete. the horizon angular momentum j is still an integral over s ; however it now contains an additional term involving the maxwell field. thus j contains not only the bare angular momentum but also a contribution from its electromagnetic hair (see for details). canonical phase space the conceptual part of the above discussion does not change if one uses the canonical phase space in place of the covariant. however, now the generator of the canonical transformation corresponding to rotations has a volume term in addition to the two surface terms discussed above. however, on the constraint surface the volume term vanishes and the numerical value of the hamiltonian reduces to the two surface terms discussed above. it follows from equation (31) and (32) that the total hamiltonian generating the rotation along is the difference between the adm and the horizon angular momenta (apart from a sign which is an artifact of conventions). thus, it can be interpreted as the angular momentum of physical fields in the space - time region \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\mathcal m}$\end{document } outside the black hole. relation to the komar integral if happens to be a space - time killing field in a neighborhood of, then j agrees with the komar integral of. if is a global, space - time killing field, then both \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$j_{{\rm{adm}}}^{(\phi)}$\end{document } as well as j agree with the komar integral, whence the total hamiltonian j vanishes identically. since the fields in the space - time region \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\mathcal m}$\end{document } are all axi - symmetric in this case, this is just what one would expect from the definition of j. j for general axial fields if the vector field is tangential to cross - sections of, j continues to the generator of the canonical transformation corresponding to rotations along, even if its restriction \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${{\tilde \varphi}^a}$\end{document } to does not agree with the axial symmetry of horizon geometries of our phase space fields. however, there is an infinity of such vector fields \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${{\tilde \varphi}^a}$\end{document } and there is no physical reason to identify the surface term j arising from any one of them with the horizon angular momentum. inclusion of gauge fields if non - trivial gauge fields are present at the horizon, equation (32) is incomplete. the horizon angular momentum j is still an integral over s ; however it now contains an additional term involving the maxwell field. thus j contains not only the bare angular momentum but also a contribution from its electromagnetic hair (see for details). canonical phase space the conceptual part of the above discussion does not change if one uses the canonical phase space in place of the covariant. however, now the generator of the canonical transformation corresponding to rotations has a volume term in addition to the two surface terms discussed above. however, on the constraint surface the volume term vanishes and the numerical value of the hamiltonian reduces to the two surface terms discussed above. to obtain an expression of the horizon energy, one has to find the hamiltonian on generating diffeomorphisms along a time translation symmetry t on \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\mathcal m}$\end{document}. to qualify as a symmetry, at infinity t must approach a fixed time translation of the fiducial flat metric. at the horizon, t must be an infinitesimal symmetry of the type ii horizon geometry. thus, the restriction of t to should be a linear combination of a null normal and the axial symmetry vector, 33\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${t^a } = { b_{(\ell, t)}}{\ell ^a } - { \omega _ { (t)}}{\varphi ^a},$$\end{document } where and the angular velocity are constants on. however there is subtlety : unlike in the angular momentum calculation where is required to approach a fixed rotational vector on a, the restriction of t to can not be a fixed vector field. for physical reasons, the constants and should be allowed to vary from one space - time to another ; they are to be functions on phase space. for instance, physically one expects to vanish on the schwarzschild horizon but not on a generic kerr horizon. in the terminology of numerical relativity, unlike, the time translation t must be a live vector field. as we shall see shortly, this generality is essential also for mathematical reasons : without it, evolution along t will not be hamiltonian ! at first sight, it may seem surprising that there exist choices of evolution vector fields t for which no hamiltonian exists. but in fact this phenomenon can also happen in the derivations of the adm energy for asymptotically flat space - times in the absence of any black holes. standard treatments usually consider only those t that asymptote to the same unit time translation at infinity for all space - times included in the phase space. however, if we drop this requirement and choose a live t which approaches different asymptotic time - translations for different space - times, then in general there exists no hamiltonian which generates diffeomorphisms along such a t. thus, the requirement that the evolution be hamiltonian restricts permissible t. this restriction can be traced back to the fact that there is a fixed fiducial flat metric at infinity. at the horizon, the situation is the opposite : the geometry is not fixed and this forces one to adapt t to the space - time under consideration, i.e., to make it live. apart from this important caveat, the calculation of the hamiltonian is very similar to that for angular momentum. first, one evaluates the 1-form y(t) on whose action on any tangent vector field is given by 34\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${y_{(t)}}(\delta) : = \omega (\delta, { x_{(t)}}),$$\end{document } where x(t) is the vector field on induced by diffeomorphisms along t. once again, y(t)() will consist of a surface term at infinity and a surface term at the horizon. a direct calculation yields 35\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${y_{(t)}}(\delta) = - { { { \kappa _ { (t) } } } \over { 8\pi g}}\delta { a_\delta } - { \omega _ { (t)}}\delta { j_\delta } + \delta e_{{\rm{adm}}}^{(t)},$$\end{document } where \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\kappa_{(t) } } : = { b_{(\ell, t)}}{\ell ^a}{w_a}$\end{document } is the surface gravity associated with the restriction of t to, a is the area of, and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$e_{{\rm{adm}}}^{(t)}$\end{document } is the adm energy associated with t. the first two terms in the right hand side of this equation are associated with the horizon, while the \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$e_{{\rm{adm}}}^{(t)}$\end{document } term is associated with an integral at infinity. since the term at infinity gives the adm energy, it is natural to hope that terms at the horizon will give the horizon energy. however, at this point, we see an important difference from the angular momentum calculation. recall that the right hand side of equation (31) is an exact variation which means that j is well defined. however, the right hand side of equation (35) is not guaranteed to be an exact variation ; in other words, x(t) need not be a hamiltonian vector field in phase space. it is hamiltonian if and only if there is a phase space function the would be energy of the wih satisfying 36\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\delta e_\delta ^{(t) } = { { { \kappa _ { (t) } } } \over { 8\pi g}}\delta { a_\delta } + { \omega _ { (t)}}\delta { j_\delta}.$$\end{document } in particular, this condition implies that, of the infinite number of coordinates in phase space, \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$e_\delta ^t$\end{document }, and can depend only on two : a and j. let us analyze equation (36). clearly, a necessary condition for existence of \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$e_\delta ^t$\end{document } is just the integrability requirement 37\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${{\partial { \kappa _ { (t) } } } \over { \partial { j_\delta } } } = 8\pi g{{\partial { \omega _ { (t) } } } \over { \partial { a_\delta}}}.$$\end{document } since and are determined by t, equation (37) is a constraint on the restriction to the horizon of the time evolution vector field t. a vector field t for which \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$e_\delta ^{(t)}$\end{document } exists is called a permissible time evolution vector field. since equation (36) is precisely the first law of black hole mechanics, t is permissible if and only if the first law holds. thus the first law is the necessary and sufficient condition that the evolution generated by t is hamiltonian ! there are infinitely many permissible vector fields t. to construct them, one can start with a suitably regular function 0 of a and j, find so that (0) = 0, solve equation (37) to obtain and find a permissible t with t = b(, t) (t) on. each permissible a question naturally arises : can one select a preferred \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$t_0^a$\end{document } or, alternatively, a canonical function 0(a, j) ? now, thanks to the no - hair theorems, we know that for each choice of (a, j), there is precisely one stationary black hole in vacuum general relativity : the kerr solution. so, it is natural to set 0 (a, j) = kerr(a, j), or, more explicitly, 38\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${\kappa _ 0}({a_\delta},{j_\delta }) = { { r_\delta ^4 - 4j_\delta ^2 } \over { 2r_\delta ^3\sqrt { r_\delta ^4 + 4j_\delta ^2}}},$$\end{document } where r is the area radius of the horizon, r = (a/4). via equation (37), this choice then leads to 39\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${\omega _ { (t) } } = { \omega _ { { \rm{kerr}}}}({a_\delta},{j_\delta }) = { { \sqrt { r_\delta ^4 + 4gj_\delta ^2 } } \over { 2g{r_\delta}}}.$$\end{document } the associated horizon energy is then : 40\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$e_\delta ^{({t_0 }) } = { 1 \over { 2g{r_\delta}}}\sqrt { r_\delta ^4 + 4{g^2}j_\delta ^2}.$$\end{document } this canonical horizon energy is called the horizon mass : 41\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${m_\delta } : = e_\delta ^{({t_0})}$$\end{document } note that, its dependence on the horizon area and angular momentum is the same as that in the kerr space - time. although the final expression is so simple, it is important to keep in mind that this is not just a postulate. rather, this result is derived using a systematic hamiltonian framework, following the same overall procedure that leads to the definition of the adm 4-momentum at spatial infinity. finally, note that the quantities which enter the first law refer just to physical fields on the horizon ; one does not have to go back and forth between the horizon and infinity. we will conclude with three remarks : relation to the adm and bondi energy under certain physically reasonable assumptions on the behavior of fields near future time - like infinity i, one can argue that, if the wih extends all the way to i, then the difference madm m equals the energy radiated across future null - infinity. thus, as one would expect physically, m is the mass that is left over after all the gravitational radiation has left the system. horizon angular momentum and mass to obtain a well - defined action principle and hamiltonian framework, it is essential to work with wihs. however, the final expressions (32) and (40) of the horizon angular momentum and mass do not refer to the preferred null normals [] used in the transition from an neh to a wih. this fact is useful in the analysis of transition to equilibrium (section 4.3) and numerical relativity (section 5.1). generalizations of the first law the derivation of the first law given here can be extended to allow the presence of matter fields at the horizon [26, 15 ]. if gauge fields are present, the expression of the angular momentum has an extra term and the first law (36) also acquires the familiar extra term q, representing work done on the horizon in increasing its charge. since the uniqueness theorems for stationary black holes fail to extend beyond the einstein - maxwell theory in four space - time dimensions, it is no longer possible to assign a canonical mass to the horizon. however, as we will see in section 6, the ambiguity in the notion of the horizon mass can in fact be exploited to obtain new insights into the properties of black holes and solitons in these more general theories. relation to the adm and bondi energy under certain physically reasonable assumptions on the behavior of fields near future time - like infinity i, one can argue that, if the wih extends all the way to i, then the difference madm m equals the energy radiated across future null - infinity. thus, as one would expect physically, m is the mass that is left over after all the gravitational radiation has left the system. horizon angular momentum and mass to obtain a well - defined action principle and hamiltonian framework, it is essential to work with wihs. however, the final expressions (32) and (40) of the horizon angular momentum and mass do not refer to the preferred null normals [] used in the transition from an neh to a wih. this fact is useful in the analysis of transition to equilibrium (section 4.3) and numerical relativity (section 5.1). generalizations of the first law the derivation of the first law given here can be extended to allow the presence of matter fields at the horizon [26, 15 ]. if gauge fields are present, the expression of the angular momentum has an extra term and the first law (36) also acquires the familiar extra term q, representing work done on the horizon in increasing its charge. since the uniqueness theorems for stationary black holes fail to extend beyond the einstein - maxwell theory in four space - time dimensions, it is no longer possible to assign a canonical mass to the horizon. however, as we will see in section 6, the ambiguity in the notion of the horizon mass can in fact be exploited to obtain new insights into the properties of black holes and solitons in these more general theories. the variations in the first law (36) represent infinitesimal changes in equilibrium states of horizon geometries. in the derivation of section 4.1, these variations relate nearby but distinct space - times in each of which the horizon is in equilibrium. therefore equation (36) is interpreted as the first law in a passive form. physically, it is perhaps the active form of the first law that is of more direct interest where a physical process, such as the one depicted in the right panel of figure 1 causes a transition from one equilibrium state to a nearby one. in fact, one can consider fully non - equilibrium situations, allowing physical processes in a given space - time in which there is a finite rather than an infinitesimal change in the state of the horizon. our summary of the mechanics of dhs is divided in to three parts. in the first, we begin with some preliminaries on angular momentum. in the second, we extend the area balance law (25) by allowing more general lapse and shift functions, which leads to the integral version of the first law. in the third, as one might expect, the angular momentum balance law results from the momentum constraint (15) on the dh h. fix any vector field on h which is tangential to all the cross - sections s of h, contract both sides of equation (15) with and integrate the resulting equation over the region h to obtain4 42\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${1 \over { 8\pi g}}{\oint\nolimits_{{s_2 } } { { k_{ab}}{\varphi ^a}\hat{r } } ^b}{d^2}v - { 1 \over { 8\pi g}}{\oint\nolimits_{{s_1 } } { { k_{ab}}{\varphi ^a}\hat{r } } ^b}{d^2}v = \int\nolimits_{\delta h } { \left({{t_{ab}}{{\hat{\tau}}^a}{\varphi ^b } + { 1 \over { 16\pi g}}({k^{ab } } - k{q^{ab}}){{\mathcal l}_\varphi}{q_{ab } } } \right){d^3}v.}$$\end{document } it is natural to identify the surface integrals with the generalized angular momentum associated with cross - sections s and set 43\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$j_s^\varphi = - { 1 \over { 8\pi g}}{\oint\nolimits_s { { k_{ab}}{\varphi ^a}\hat{r } } ^b}{d^2}v \equiv { 1 \over { 8\pi g}}\oint\nolimits_s { { j^\varphi } } { d^2}v,$$\end{document } where the overall sign ensures compatibility with conventions normally used in the asymptotically flat context, and where we have introduced an angular momentum density \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${j^\varphi } : = - { k_{ab}}{\varphi ^a}{{\hat r}^b}$\end{document } for later convenience. the term generalized emphasizes the fact that the vector field need not be an axial killing field even on s ; it only has to be tangential to our cross - sections. if happens to be the restriction of a space - time killing field to s, then \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$j_s^\varphi$\end{document } agrees with the komar integral. if the pair (qab, kab) is spherically symmetric on s, as one would expect, the angular momenta associated with the rotational killing fields vanish. equation (42) is a balance law ; the right side provides expressions of fluxes of the generalized angular momentum across h. the contributions due to matter and gravitational waves are cleanly separated and given by 44\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${\mathcal j}_{{\rm{matter}}}^\varphi = - { \int\nolimits_{\delta h } { { t_{ab}}\hat{\tau } } ^a}{\varphi ^b}{d^3}v,\;\;\;{\mathcal j}_{{\rm{grav}}}^\varphi = { 1 \over { 16\pi g}}\int\nolimits_{\delta h } { { p^{ab}}{{\mathcal l}_\varphi } } { q_{ab}}{d^3}v,$$\end{document } with p = k kq, so that 45\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$j_{{{s}_{2}}}^\varphi - j_{{{s}_{1}}}^\varphi = { \mathcal j}_{{\rm{matter}}}^\varphi + { \mathcal j}_{{\rm{grav}}}^\varphi.$$\end{document } as expected, if is a killing vector of the three - metric qab, then the gravitational angular momentum flux vanishes : \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\mathcal j}_{{\rm{grav}}}^\varphi = 0$\end{document}. as with the area balance law, here we worked directly with the constraint equations rather than with a hamiltonian framework. however, we could also have used, e.g., the standard adm phase space framework based on a cauchy surface m with internal boundary s and the outer boundary at infinity. if is a vector field on m which tends to on s and to an asymptotic rotational symmetry at infinity, we can ask for the phase space function which generates the canonical transformation corresponding to the rotation generated by. when the constraints are satisfied, as usual the value of this generating function is given by just surface terms. the term at infinity provides the total angular momentum and, as in section 4.1.2, it is natural to interpret the surface term at s as the -angular momentum of s. this term can be expressed in terms of the cauchy data (\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$({{\bar q}_{ab}},{{\bar k}_{ab}})$\end{document }) on m as 46\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\overline j _ s^\varphi = - { 1 \over { 8\pi g}}\oint\nolimits_s { { { \overline k}_{ab}}{\varphi ^a}{{\overline r}^b } } { d^2}v,$$\end{document } where \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${{\bar r}^a}$\end{document } is the unit normal to s within m. however, since the right side involves the extrinsic curvature of m, in general the value of the integral is sensitive to the choice of m. hence, the notion of the -angular momentum associated with an arbitrary cross - section is ambiguous. this ambiguity disappears if is divergence - free on s. in particular, in this case, one has \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$j_s^\varphi = \bar j_s^\varphi$\end{document}. thus, although the balance law (42) holds for more general vector fields, it is robust only when is divergence - free on s. (these considerations shed some light on the interpretation of the field in the area balance law (25). for, the form of the right side of equation (43) implies that the field vanishes identically on s if and only if vanishes for every divergence - free on s. in particular then, if the horizon has non - zero angular momentum, the -contribution to the energy flux can not vanish.) finally, for to be interpreted as the angular momentum, has to be a symmetry. an obvious possibility is that it be a killing field of qab on s. a more general scenario is discussed in section 8. to obtain the area balance law, in section 3.2 we restricted ourselves to vector fields \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\xi _ { (r)}^a = { n_r}{\ell ^a}$\end{document }, i.e., to lapse functions nr = |r| and shifts \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${n^a } = { n_r}{{\hat r}^a}$\end{document}. we were then led to a conservation law for the hawking mass. in the spherically symmetric context, the hawking mass can be taken to be the physical mass of the horizon. however, as the kerr space - time already illustrates, in presence of rotation this interpretation is physically incorrect. therefore, although equation (25) continues to dictate the dynamics of the hawking mass even in presence of rotation, a more general procedure is needed to obtain physically interesting conservation laws in this case. in the case of wihs, the first law incorporating rotations required us to consider suitable linear combinations of and the rotational symmetry field on the horizon. in the same spirit, on dhs, one has to consider more general vector fields than \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\xi _ { (r)}^a$\end{document }, i.e., more general choices of lapses and shifts. as on wihs, one first restricts oneself to situations in which the metric qab on h admits a killing field so that \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$j_s^\varphi$\end{document } can be unambiguously interpreted as the angular momentum associated with each s. in the case of a wih, t was given by t = c + and the freedom was in the choice of constants c and. on a dh, one must allow the corresponding coefficients to be time - dependent. the simplest generalization is to choose, in place of \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\xi _ { (r)}^a$\end{document }, vector fields 47\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${t^a } : = { n_r}{\ell ^a } + \omega { \varphi ^a } \equiv { n_r}{\hat{r}^a } + ({ n_r}{\hat{r}^a } + \omega { \varphi ^a}),$$\end{document } where is an arbitrary function of r, and the lapse nr is given by nr = |r| for any function r of r. note that one is free to rescale nr and by functions of r so that on each cross - section (instant of time) one has the same rescaling freedom as on a wih. one can consider even more general lapse - shift pairs to allow, e.g., for differential rotation (see). using t in place of \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\xi _ { (r)}^a$\end{document }, one obtains the following generalization of the area balance equation : 48\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\begin{array}{{20}c}{{{{r_2 } - { r_1 } } \over { 2 g } } + { 1 \over { 8\pi g}}\left({\oint\nolimits_{{s_2 } } { \omega { j^\varphi}{d^2}v - } \oint\nolimits_{{s_1 } } { \omega { j^\varphi}{d^2}v - } \int\nolimits_{{\omega _ 1}}^{{\omega _ 2 } } { d\omega } \oint\nolimits_s { { j^\varphi}{d^2}v } } \right) = } \\ { \int\nolimits_{\delta h } { { t_{ab}}{{\hat{\tau}}^a}{t^b}{d^3}v + { 1 \over { 16\pi g}}\int\nolimits_{\delta h } { { n_r}(\vert\sigma { \vert^2 } + 2\vert\zeta { \vert^2}){d^3}v } } - { 1 \over { 16\pi g}}\int\nolimits_{\delta h } { \omega { p^{ab}}{{\mathcal l}_\varphi}{q_{ab } } } { d^3}v. } \end{array}$$\end{document } note that there is one balance equation for every vector field t of the form (47) ; as in section 4.1, we have an infinite number of relations, now ensured by the constraint part of einstein s equations. the right side of equation (48) can be naturally interpreted as the flux \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\mathcal f}_{\delta h}^t$\end{document } of the hence, we can rewrite the equation as 49\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${\mathcal f}_{\delta h}^t{{{r_2 } - { r_1 } } \over { 2 g } } + { 1 \over { 8\pi g}}\left({\oint\nolimits_{{s_2 } } { \omega { j^\varphi}{d^2}v - } \oint\nolimits_{{s_1 } } { \omega { j^\varphi}{d^2}v - } \int\nolimits_{{\omega _ 1}}^{{\omega _ 2 } } { d\omega } \oint\nolimits_s { { j^\varphi}{d^2}v } } \right).$$\end{document } if s1 and s2 are only infinitesimally separated, this integral equation reduces to the differential condition 50\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\begin{array}{{20}c}{\delta e_s^t = { 1 \over { 8\pi g}}{{\left. { \left({{1 \over { 2r}}{{dr } \over { dr } } } \right) } \right\vert}_s}\delta { a_s } + \omega \delta j_s^\varphi } \\{\equiv { { \overline \kappa } \over { 8\pi g}}\delta { a_s } + \omega \delta j_s^\varphi. } \\ \end{array}$$\end{document } thus, the infinitesimal form of equation (48) is a familiar first law, provided [(1/2r)(dr / dr)](s) is identified as an effective surface gravity on the cross - section s. this identification can be motivated as follows. first, on a spherically symmetric dh, it is natural to choose r = r. then the surface gravity reduces to 1/(2r), just as one would hope from one s experience with the schwarzschild metric and more generally with static but possibly distorted horizons (see appendix a of). under the change r r(r), we have \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${{\bar \kappa}_r } = (dr / dr){{\bar \kappa}_r}$\end{document }, which is the natural generalization of the transformation property \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\kappa_{c\ell } } = c{\kappa_\ell}$\end{document } of surface gravity of wihs under the change c. finally, \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${{\bar \kappa}_r}$\end{document } can also be regarded as a 2-sphere average of a geometrically defined surface gravity associated with certain vector fields on h [56, 31 ] ; hence the adjective effective. to summarize, equation (48) represents an integral generalization of the first law of mechanics of weakly isolated horizons to dynamical situations in which the horizon is permitted to make a transition from a given state to one far away, not just nearby. the left side represents the flux \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\mathcal f}_{\delta h}^t$\end{document } of the energy associated with the vector field t, analogous to the flux of bondi energy across a portion of null infinity. a natural question therefore arises : can one integrate this flux to obtain an energy \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$e_s^t$\end{document } which depends only on fields defined locally on the cross - section, as is possible at null infinity ? as discussed in the next section, the answer is in the affirmative and the procedure leads to a canonical notion of horizon mass. in general relativity, on dhs, the vector field is t. therefore, to obtain an unambiguous notion of horizon mass, we need to make a canonical choice of t = nr +, i.e., of functions nr and on h. as we saw in section 4.1.3, on wihs of 4-dimensional einstein - maxwell theory, the pair (a, j) suffices to pick a canonical time translation field \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$t_0^a$\end{document } on. the associated horizon energy \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$e_\delta ^{{t_0}}$\end{document } is then interpreted as the mass m. this suggests that the pair (\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$({a_s},j_s^\varphi)$\end{document }) be similarly used to make canonical choices \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${n_{{r^0}}}$\end{document } and on s. thanks to the black hole uniqueness theorems of the 4-dimensional einstein - maxwell theory, this strategy is again viable. recall that the horizon surface gravity and the horizon angular velocity in a kerr solution can be expressed as a function only of the horizon radius r and angular momentum j : 51\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${\kappa _ { { \rm{kerr}}}}(r, j) : = { { { r^4 } - 4{g^2}{j^2 } } \over { 2{r^3}\sqrt { { r^4 } + 4{g^2}{j^2}}}},\;\;\;\;{\omega _ { { \rm{keer}}}}(r, j) : = { { 2gj } \over { r\sqrt { { r^4 } + 4{g^2}{j^2}}}}.$$\end{document } given a cross section s of h, the idea is to consider the unique kerr solution in which the horizon area is given by as and angular momentum by js, and assign to s effective surface gravity \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${{\bar \kappa}_s}$\end{document } and angular velocity s through 52\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${\overline{\kappa}_{s } } : = { \kappa _ { { \rm{kerr}}}}({r_s }, j_s^\varphi),\;\;{\omega _ s } : = { \omega _ { { \rm{kerr}}}}({r_s},j_s^\varphi)$$\end{document } repeating this procedure on every cross - section, one obtains functions \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\bar \kappa ^0}(r)$\end{document } and (r) on h, since j is a function of r alone. the definition of the effective surface gravity then determines a function r of r and hence \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${n_{{r^0}}}$\end{document } uniquely. thus, using equation (51), one can select a canonical vector field \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$t_0^a$\end{document } and equation (49) then provides a canonical balance law : 53\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${\mathcal f}_{\delta h}^{{t_0}}{{r_2 ^ 0 - r_1 ^ 0 } \over { 2 g } } + { 1 \over { 8\pi g}}\left({\oint\nolimits_{{s_2 } } { { \omega ^0}{j^\varphi}{d^2}v - } \oint\nolimits_{{s_1 } } { { \omega ^0}{j^\varphi}{d^2}v - } \int\nolimits_{\omega _ 1 ^ 0}^{\omega _ 2 ^ 0 } { d{\omega ^0 } } \oint\nolimits_s { { j^\varphi}{d^2}v } } \right).$$\end{document } the key question is whether this equation is integrable, i.e., if 54\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${\mathcal f}_{\delta h}^{{t_0 } } = e_{{s_2}}^{{t_0 } } - e_{s1}^{{t_0}}$$\end{document } for some which depends locally on fields defined on s. the answer is in the affirmative. furthermore, the expression of \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${e^{{t_0}}}$\end{document } is remarkably simple and is identified with the horizon mass : 55\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$m(r) : = { e^{{t_0}}}(r) = { { \sqrt { { r^4 } + 4{g^2}{j^2 } } } \over { 2gr}}.$$\end{document } thus, on any cross - section s, ms is just the mass of the kerr space - time which has horizon area a and angular momentum \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$j_s^\varphi$\end{document } : as far as the mass is concerned, one can regard the dh as an evolution through a sequence of kerr horizons. the non - triviality of the result lies in the fact that, although this definition of mass is so elementary, thanks to the balance law (48) it obeys a bondi - type flux formula, 56\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${m_{{s_2 } } } - { m_{s1 } } = { \int\nolimits_{\delta h } { { t_{ab}}\hat{\tau } } ^a}t_0^b{d^3}v + { 1 \over { 16\pi g}}\int\nolimits_{\delta h } { { n_{{r^0}}}(\vert\sigma { \vert^2 } + 2\vert\zeta { \vert^2}){d^3}v}$$\end{document } for a specific vector field \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$t_0^a = { n_{{r^0}}}{\ell ^a } + { \omega ^0}{\varphi ^a}$\end{document }, where each term on the right has a well - defined physical meaning. thus, dhs admit a locally - defined notion of mass and an associated, canonical conservation law (56). the availability of a mass formula also provides a canonical integral version of the first law through equation (48) : 57\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${m_{{s_2 } } } - { m_{s1 } } = { { r_2 ^ 0 - r_1 ^ 0 } \over { 2 g } } + { 1 \over { 8\pi g}}\left({\oint\nolimits_{{s_2 } } { { \omega ^0}{j^\varphi}{d^2}v } - \oint\nolimits_{{s_1 } } { { \omega ^0}{j^\varphi}{d^2}v } - \int\nolimits_{\omega _ 1 ^ 0}^{\omega _ 2 ^ 0 } { d{\omega ^0 } } \oint\nolimits_s { { j^\varphi}{d^2}v } } \right).$$\end{document } the infinitesimal version of this equation yields the familiar first law \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\delta m = (\bar \kappa/8\pi g)\delta a + \omega \delta j$\end{document}. on weakly isolated as well as dynamical horizons, area a and angular momentum j arise as the fundamental quantities and mass is expressed in terms of them. the fact that the horizon mass is the same function of a and j in both dynamical and equilibrium situations is extremely convenient for applications to numerical relativity. conceptually, this simplicity is a direct consequence of the first law and the non - triviality lies in the existence of a balance equation (48), which makes it possible to integrate the first law. in physical situations, such as a gravitational collapse or black hole mergers,. however, the back scattering is generally quite weak and in simulations, within numerical errors, equilibrium is reached at finite times. the passage to equilibrium can be studied in detail in vaidya solutions discussed in section 2.2.2. moreover, in spherically symmetric examples such as these solutions, exact equilibrium can be reached at a finite time (see right panel of figure 4). the question then arises : in these situations, do various notions introduced on dynamical horizons go over smoothly to those introduced on wihs ? this issue has been analyzed only in a preliminary fashion [56, 31 ]. in this section first, if the dynamical horizon is a foth, as the flux of matter and shear across h tends to zero, h becomes null and furthermore a non - expanding horizon. by a suitable choice of null normals conditions under which it would also become an isolated horizon are not well - understood. fortunately, however, the final expressions of angular momentum and horizon mass refer only to that structure which is already available on non - expanding horizons (although, as we saw in section 4.1, the underlying hamiltonian framework does require the horizon to be weakly isolated [26, 15 ]). therefore, it is meaningful to ask if the angular momentum and mass defined on the dhs match with those defined on the non - expanding horizons. in the case when the approach to equilibrium is only asymptotic, it is rather straightforward to show that the answer is in the affirmative. in the case when the transition occurs at a finite time, the situation is somewhat subtle. first, we now have to deal with both regimes and the structures available in the two regimes are entirely different. second, since the intrinsic metric becomes degenerate in the transition from the dynamical to isolated regimes, limits are rather delicate. in particular, the null vector field \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\ell ^a } = { { \hat \tau}^a } + { { \hat \tau}^a}$\end{document } on h diverges, while \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${n^a } = { { \hat \tau}^a } - { { \hat \tau}^a}$\end{document } tends to zero at the boundary. a priori therefore, it is not at all clear that angular momentum and mass would join smoothly if the transition occurs at a finite time. however, a detailed analysis shows that the two sets of notions in fact agree. more precisely, one has the following results. let \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\mathcal q } = h \cup \delta$\end{document } be a c 3-manifold (with k 2), topologically \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${{\mathbb s}^2 } \times { \mathbb r}$\end{document } as in the second penrose diagram of figure 4. let the space - time metric gab in a neighborhood of \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\mathcal q}$\end{document } be c. the part h of \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\mathcal q}$\end{document } is assumed to have the structure of a dh and the part of a non - expanding horizon. finally, the pull - back qab of gab to \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\mathcal q}$\end{document } is assumed to admit an axial killing field. then we have : the angular momentum and the mass m defined in the two regimes agree on the boundary between h and.the vector field \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$t_0^a$\end{document } defined on h and used in the definition of mass matches with a preferred vector field \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$t_0^a$\end{document } used to define mass on. the angular momentum and the mass m defined in the two regimes agree on the boundary between h and. the vector field \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$t_0^a$\end{document } defined on h and used in the definition of mass matches with a preferred vector field \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$t_0^a$\end{document } used to define mass on. this agreement provides an independent support in favor of the strategy used to introduce the notion of mass in the two regimes. by their very nature, numerical simulations of space - times are invariably tied to choices of coordinates, gauge conditions, dynamical variables, etc. therefore, it is non - trivial to extract from them gauge invariant physics, especially in the strong curvature regions. traditionally, the analytical infrastructure available for this purpose has been based on properties of the kerr solution and its perturbations. however, a priori it is not clear if this intuition is reliable in the fully dynamical, strong curvature regime. on the numerical side, a number of significant advances have occurred in this area over the past few years. in particular, efficient algorithms have been introduced to find apparent horizons (see, e.g., [6, 168, 177 ]), black hole excision techniques have been successfully implemented [73, 3 ], and the stability of numerical codes has steadily improved. to take full advantage of these ongoing improvements, one must correspondingly upgrade the analytical infra - structure so that one can extract physics more reliably and with greater accuracy. these considerations provided stimulus for a significant body of research at the interface of numerical relativity and the dynamical and isolated horizon frameworks. in this section, we will review the most important of these developments. section 5.1 summarizes calculations of mass and angular momentum of black holes. specifically, we discuss the issue of constructing the quasi - equilibrium initial data and the calculation of the gravitational binding energy for a binary black hole problem. section 5.3 describes how one can calculate the source multipole moments for black holes, and section 5.4 presents a we assume that vacuum equations hold near horizons. as we saw in section 4, the mechanics of ihs and dhs provides expressions of angular momentum and mass of the horizon. these expressions involve geometric quantities defined intrinsically on the ih and dh h. numerical simulations, on the other hand, deal with the 3-metric \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${{\bar q}_{ab}}$\end{document } and extrinsic curvature \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${{\bar k}_{ab}}$\end{document } on (partial) cauchy surfaces m. therefore, the first task is to recast the formulas in terms of this cauchy data. simulations provide us with a foliation of space - time \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\mathcal m}$\end{document } by partial cauchy surfaces m, each of which has a marginally trapped 2-surface s as (a connected component of) its inner boundary. the world tube of these 2-surfaces is a candidate for a dh or an ih. if it is space - like, it is a dh h and if it is null (or, equivalently, if the shear ab of the outward null normal to s is zero) it is a wih. the situation is depicted in figure 7. it is rather simple to numerically verify if these restrictions are met. to calculate mass and angular momentum, one assumes that the intrinsic 2-metric on the cross - sections s admits a rotational killing field (see, however, section 8 for weakening of this assumption). a rather general and convenient method, based on the notion of killing transport, has been introduced and numerically implemented to explicitly find this vector field. figure 7the world tube of apparent horizons and a cauchy surface m intersect in a 2-sphere s. t is the unit time - like normal to m and r is the unit space - like normal to s within m. the world tube of apparent horizons and a cauchy surface m intersect in a 2-sphere s. t is the unit time - like normal to m and r is the unit space - like normal to s within m. let us first suppose that, in a neighborhood of the cross - section s of interest, the world tube of marginally trapped surfaces constitutes an ih. then the task is to recast equation (32) in terms of the cauchy data (\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${{\bar q}_{ab}},{{\bar k}_{ab}}$\end{document }) on m. this task is also straightforward and one arrives at5 : 58\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${j_\delta } = - { 1 \over { 8\pi g}}\oint\nolimits_s { { \varphi ^a}{r^b}{{\overline k}_{ab}}{d^2}v,}$$\end{document } where r is the unit radial normal to s in m. this formula is particularly convenient numerically since it involves the integral of a single component of the extrinsic curvature. now consider the dynamical regime, i.e., assume that, in a small neighborhood of s, the world tube of marginally trapped surfaces is a dh h. the angular momentum formula (46) on dhs involves the cauchy data on h. however, it is easy to show that it equals the expression (58) involving cauchy data on m. thus, equation (58) is in fact applicable in both the isolated and dynamical regimes. the availability of a single formula is extremely convenient in numerical simulations. from now on, we will drop the subscript and denote the angular momentum of s simply by \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$j_s^{(\varphi)}$\end{document } : 59\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$j_s^{(\varphi) } = - { 1 \over { 8\pi g}}\oint\nolimits_s { { \varphi ^a}{r^b}{{\overline k}_{ab}}{d^2}v.}$$\end{document } \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$j_s^{(\varphi)}$\end{document } is the intrinsic angular momentum (i.e., spin) of the black hole at the instant of time represented by s. note that equation (59) differs from the standard formula for the adm angular momentum jadm only in that the integral is over the apparent horizon instead of the sphere at infinity. finally, we emphasize that one only assumes that is a rotational killing field of the intrinsic 2-metric on s ; it does not have to extend to a killing field even to a neighborhood of s. in fact, the expressions (32) and (46) on ihs and dhs are meaningful if is replaced by any vector field \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${{\tilde \phi}^a}$\end{document } tangential to s ; on the expression is conserved and on h it satisfies a balance law. furthermore, they both equal equation (59) if \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\tilde \varphi ^a}$\end{document } is divergence - free ; it has to lie - drag only the area 2-form (rather than the intrinsic metric) on s. however, since s admits an infinity of divergence - free vector fields \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${{\tilde \varphi}^a}$\end{document }, there is no a priori reason to interpret \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$j_s^{(\tilde \varphi)}$\end{document } as the canonically. the most unambiguous way to achieve this is to require that it be a killing field of the intrinsic geometry of s. however, in section 8 we will introduce a candidate vector field which could be used in more general situations which are only near axi - symmetry. having calculated js, it is easy to evaluate the mass ms using equation (40) : 60\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${m_s } = { 1 \over { 2g{r_\delta}}}\sqrt { r_\delta ^4 + 4{g^2}j_\delta ^2}.$$\end{document } this method for calculating js and ms has already been used in numerical simulations, particularly in the analysis of the gravitational collapse of a neutron star to a black hole [34, 48 ]. it is found that this method is numerically more accurate than the other commonly used methods. the approach summarized above has the key advantage that it is rooted in hamiltonian methods which can be universally used to define conserved quantities. in particular, it is a natural extension of the approach used to arrive at the adm and bondi - sachs quantities at spatial and null infinity [7, 18, 185 ]. from more practical considerations, it has three important features : the procedure does not presuppose that the horizon geometry is precisely that of the kerr horizon.it is coordinate independent.it only requires data that is intrinsic to the apparent horizon. the procedure does not presuppose that the horizon geometry is precisely that of the kerr horizon. none of the commonly used alternatives share all three of these features. before the availability of the ih and dh frameworks, standard procedures of calculating angular momentum the motivation comes from the common belief, based on black - hole uniqueness theorems, that a black hole created in a violent event radiates away all its higher multipole moments and as it settles down, its near horizon geometry can be approximated by that of the kerr solution. the strategy then is to identify the geometry of s with that of a suitable member of the kerr family and read off the corresponding angular momentum and mass parameters. the very considerations that lead one to this strategy also show that it is not suitable in the dynamical regime where the horizon may be distorted and not well - approximated by any kerr horizon. for horizons which have very nearly reached equilibrium, the strategy is physically well motivated. however, even in this case, one has to find a way to match the horizon of the numerical simulation with that of a specific member in the kerr family. this is non - trivial because the coordinate system used in the given simulation will, generically, not bear any relation to any of the standard coordinate systems used to describe the kerr solution. thus, one can not just look at, say, a metric component to extract mass and angular momentum. a semi - heuristic it is based on an observation of the properties of the kerr horizon made by smarr using kerr - schild coordinates. let le be the length of the equator and lp the length of a polar meridian on the kerr horizon, where the equator is the coordinate great circle of maximum proper length and a polar meridian is a great circle of minimum proper length. define a distortion parameter as = (le lp)/le. the knowledge of, together with one other quantity such as the area, le, or lp, is sufficient to find the parameters m and a of the kerr geometry. however, difficulties arise when one wishes to use these ideas to calculate m and a for a general apparent horizon s. for, notions such as great circles, equator or polar meridian are all highly coordinate dependent. indeed, if we represent the standard two - metric on the kerr horizon in different coordinates, the great circles in one coordinate system will not agree with great circles in the other system. therefore, already for the kerr horizon, two coordinate systems will lead to different answers for m and a ! in certain specific situations where one has a good intuition about the coordinate system being used and the physical situation being modelled, this method can be useful as a quick way of estimating angular momentum. however, it has the conceptual drawback that it is not derived from a well - founded, general principle and the practical drawback that it suffers from too many ambiguities. problems associated with coordinate dependence can be satisfactorily resolved on axi - symmetric horizons, even when the coordinate system used in the numerical code is not adapted to the axial symmetry. the idea is to use the orbits of the killing vector as analogs of the lines of latitude on a metric two - sphere. the analog of the equator is then the orbit of the killing vector which has maximum proper length. the north and south poles are the points where the killing vector vanishes, and the analog of lp is the length of a geodesic joining these two points. (because of axial symmetry, all geodesics joining the poles will have the same length). hence one just needs to find the length of a curve joining the north and south poles which is everywhere perpendicular to the killing orbits. with le and lp defined in this coordinate invariant way, one can follow the same procedure as in the great circle method to calculate the mass and angular momentum. how does the generalized great circle method compare to that based on ihs and dhs ? since one must find the killing vector, the first step is the same in the two cases. in the ih and dh method, one is then left simply with an integration of a component of the extrinsic curvature on the horizon. in the generalized great circle method, by contrast, one has to determine the orbit of the killing vector with maximum length and also to calculate the length of a curve joining the poles which is everywhere orthogonal to the killing orbits. numerically, this requires more work and the numerical errors are at least as large as those in the ihdh method. thus, even if one ignores conceptual considerations involving the fundamental meaning of conserved quantities, and furthermore restricts oneself to the non - dynamical regime, the practical simplicity of the great circle method is lost when it is made coordinate invariant. to summarize, conceptually, equations (59) and (60) provide the fundamental definitions of angular momentum and mass, while the great circle method provides a quick way of estimating these quantities in suitable situations. by comparing with equations (59) and (60), one can calculate errors and sharpen intuition on the reliability of the great circle method. a completely different approach to finding the mass and angular momentum of a black hole in a numerical solution is to use the concept of a killing horizon. assume the existence of killing vectors in the neighborhood of the horizon so that mass and angular momentum are defined as the appropriate komar integrals. this method is coordinate independent and does not assume, at least for angular momentum, that the near horizon geometry is isometric with the kerr geometry. first, since the komar integral can involve derivatives of the killing field away from the horizon, one has to find the killing fields in a neighborhood of the horizon. second, existence of such a stationary killing vector is a strong assumption ; it will not be satisfied in the dynamical regime. even when the killing field exists, computationally it is much more expensive to find it in a neighborhood of the horizon rather than the horizon itself. finally, it is not a priori clear how the stationary killing vector is to be normalized if it is only known in a neighborhood of the horizon. in a precise sense, the isolated horizon framework extracts just the minimum amount of information from a killing horizon in order to carry out the hamiltonian analysis and define conserved quantities by by - passing these obstacles. in order to accurately calculate the gravitational waveforms for, say, the coalescence of binary black holes while there has been some progress on the construction of such data, the general problem is yet to be solved (see, e.g., for a recent review). however, there has been notable progress in the case of black holes which are in quasi - equilibrium. physically, this situation arises when the black holes are sufficiently far apart for their orbits to be quasi - periodic. in his section, we will first summarize this development [72, 124 ] and then review an approach to the calculation of the binding energy in the initial data. consider the problem of constructing initial data (\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\bar q_{ab}},{\bar k_{ab}}$\end{document }) on m, representing a binary black hole system. excise the region contained in each black hole (see, e.g., [3, 73 ]). however, this procedure creates inner boundaries on m and one must specify suitable boundary conditions there. the boundary conditions should be appropriate for the elliptic system of constraint equations, and they should also capture the idea that the excised region represents black holes which have certain physical parameters and which are in quasi - equilibrium at the instant represented by m. the second aspect of the problem is the choice of the free data in the bulk. to be of physical interest, not only must the free data satisfy the appropriate boundary conditions, but the values in the bulk must also have certain properties. for example, we might want the black holes to move in approximately circular orbits around each other, and require that there be only a minimal amount of spurious gravitational radiation in the initial data. the black holes would then remain approximately isolated for a sufficiently long time, and orbit around each other before finally coalescing. while a fully satisfactory method of prescribing such initial data is still lacking, there has been significant progress in recent years. when the black holes are far apart and moving on approximately circular orbits, one might expect the trajectory of the black holes to lie along an approximate helical killing vector [53, 91, 103, 104, 4 ]. using concepts from the helical killing vector approximation and working in the conformal thin - sandwich decomposition of the initial data, cook has introduced the quasi - equilibrium boundary conditions which require that each of the black holes be in instantaneous equilibrium ; see also [190, 74 ] for a similar approach. the relation between these quasi - equilibrium boundary conditions and the isolated horizon formalism has also been recently studied. in this section, physically, the quasi - equilibrium approximation ought to be valid for time intervals much smaller than other dynamical time scales in the problem, and the framework assumes only that the approximation holds infinitesimally off m. so, in this section, the type ii neh will be an infinitesimal world tube of apparent horizons. we assume that there is an axial symmetry vector on the horizon, although, as discussed in section 8, this assumption can be weakened. depending on the degree of isolation one wants to impose on the individual black holes, the inner boundary may be taken to be the cross - section of either an neh, wih, or an ih. the strategy is to first start with an neh and impose successively stronger conditions if necessary. using the local geometry of intersections s of m with, one can easily calculate the area radius r, the angular momentum j given by equation (59), the canonical surface gravity = kerr(r, j) given by equation (38), and angular velocity = kerr(r, j) given by equation (39). (note that while a wih structure is used to arrive at the expressions for j,, and, the expressions themselves are unambiguously defined also on a neh.) if, as in section 4.1.3, one requires that the restriction of the evolution vector field t to be of the form 61\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${t^a } = b\ell _ 0^a - { \omega _ { { \rm{kerr}}}}{\varphi ^a } = b{t^a } + (b{r^a } - { \omega _ { { \rm{kerr}}}}{\varphi ^a}),$$\end{document } where \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\ell _ 0^a = { t^a } + { r^a}$\end{document }, then the values of the lapse and shift fields at the horizon are given by 62\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\alpha = b,\;\;\;\;{\beta ^a } = \alpha { r^a } - { \omega _ { { \rm{kerr}}}}{\varphi ^a}.$$\end{document } we are free to choose the lapse arbitrarily on s, but this fixes the shift. the tangential part of can, if desired, be chosen differently depending on the asymptotic value of at infinity which determines the angular velocity of inertial observers at infinity. next, one imposes the condition that the infinitesimal world - tube of apparent horizons is an this requirement is equivalent to 63\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${{\mathcal l}_t}{\tilde{q}_{ab } } = 0,\;\;\;\;{{\mathcal l}_\varphi}{\tilde{q}_{ab } } = 0$$\end{document } on, which imply that the shear and expansion of vanish identically. these can be easily translated to conditions that the cauchy data (\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${{\bar q}_{ab}},{{\bar k}_{ab}}$\end{document }) must satisfy at the horizon [72, 124 ]. these boundary conditions are equivalent to the quasi - equilibrium boundary conditions developed by cook. however, the isolated horizon formalism allows greater control on the physical parameters of the black hole. in particular, we are not restricted just to the co - rotational or irrotational cases. the use of ihs streamlines the procedure : rather than adding conditions one by one as needed, one begins with a physically well - motivated notion of horizons in equilibrium and systematically derives its consequences. up to this point, the considerations are general in the sense that they are not tied to a particular method of solving the initial value problem. however, for the quasi - equilibrium problem, it is the conformal thin - sandwich method [192, 70 ] that appears to be best suited. this approach is based on the conformal method [144, 191 ] where we write the 3-metric as \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${{\bar q}_{ab } } = { \psi ^4}{{\hat q}_{ab}}$\end{document}. the free data consists of the conformal 3-metric \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${{\hat q}_{ab}}$\end{document }, its time derivative \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${{\mathcal l}_t}{{\hat q}_{ab}}$\end{document }, the lapse a, and the trace of the extrinsic curvature \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\bar k}$\end{document}. given this free data, the remaining quantities, namely the conformal factor and the shift, are determined by elliptic equations provided appropriate boundary conditions are specified for them on the horizon6. it turns out that the horizon conditions (63) are well - tailored for this purpose. while the issue of existence and uniqueness of solutions using these boundary conditions has not been proven, it is often the case that numerical calculations are convergent and the resulting solutions are well behaved. thus, these conditions might therefore be sufficient from a practical point of view. in the above discussion, the free data consisted of (\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${{\hat q}_{ab}},{{\mathcal l}_t}{{\hat q}_{ab}},\alpha,\bar k$\end{document }) and one solved elliptic equations for (,). however, it is common to consider an enlarged initial value problem by taking \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${{\mathcal l}_t}\bar k$\end{document } as part of the free data (usually set to zero) and solving an elliptic equation for. we now need to prescribe an additional boundary condition for a. it turns out that this can be done by using wihs, i.e., by bringing in surface gravity, which did not play any role so far. from the definition of surface gravity in equations (7, 8), it is clear that the expression for will involve a time derivative ; in particular, it turns out to involve the time derivative of a. it can be shown that by choosing \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${{\mathcal l}_t}\alpha$\end{document } on s (e.g., by taking \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${{\mathcal l}_t}\alpha = 0$\end{document }) and requiring surface gravity to be constant on s and equal to kerr, one obtains a suitable boundary condition for a. (the freedom to choose freely the function \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${{\mathcal l}_t}\alpha$\end{document } mirrors the fact that fixing surface gravity does not uniquely fix the rescaling freedom of the null normal.) note that is required to be constant only on s, not on. to ask it to be constant on would require \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${{\mathcal l}_t}\kappa = 0$\end{document }, which in turn would restrict the second time derivative ; this necessarily involves the evolution equations, and they are not part of the initial data scheme. one may imagine using the yet stronger notion of an ih, to completely fix the value of the lapse at the horizon. but this requires solving an elliptic equation on the horizon and the relevant elliptic operator has a large kernel [16, 72 ]. nonetheless, the class of initial data on which its inverse exists is infinite dimensional so that the method may be useful in practice. however, this condition would genuinely restrict the permissible initial data sets. in this sense, while the degree of isolation implied by the ih boundary condition is likely to be met in the asymptotic future, for quasi - equilibrium initial data it is too strong in general. it is the wih boundary conditions that appear to be well - tailored for this application. finally, using methods introduced by dain, a variation of the above procedure was recently introduced to establish the existence and uniqueness of solutions and to ensure that the conformal factor is everywhere positive. however, in place of dirichlet boundary conditions (62) on the shift, one now imposes neumann - type conditions on certain components of. this method is expected to be applicable all initial data constructions relying on the conformal method. furthermore, the result might also be of practical use in numerical constructions to ensure that the codes converge to a well behaved solution. for initial data representing a binary black hole system, the quantity eb = madm m1 m2 is called the effective binding energy, where madm is the adm mass, and m1,2 are the individual masses of the two black holes. heuristically, even in vacuum general relativity, one would expect eb to have several components. first there is the analog of the newtonian potential energy and the spin - spin interaction, both of which may be interpreted as contributing to the binding energy. but eb also contains contributions from kinetic energy due to momentum and orbital angular momentum of black holes, and energy in the gravitational radiation in the initial data. it is only when these are negligible that eb is a good measure of the physical binding energy. the first calculation of binding energy was made by brill and lindquist in such a context. they considered two non - spinning black holes initially at rest. for large separations, they found that, in a certain mathematical sense, the leading contribution to binding energy comes just from the usual newtonian gravitational potential. more recently, dain has extended this calculation to the case of black holes with spin and has shown that the spin - spin interaction energy is correctly incorporated in the same sense. in numerical relativity, the notion of binding energy has been used to locate sequences of quasi - circular orbits. the underlying heuristic idea is to minimize eb with respect to the proper separation between the holes, keeping the physical parameters of the black holes fixed. the value of the separation which minimizes eb provides an estimate of sizes of stable one finds that these orbits do not exist if the orbital angular momentum is smaller than a critical value (which depends on other parameters) and uses this fact to approximately locate the inner - most stable circular orbit (isco). in another application, the binding energy has been used to compare different initial data sets which are meant to describe the same physical system. if the initial data sets have the same values of the black hole masses, angular momenta, linear momenta, orbital angular momenta, and relative separation, then any differences in eb should be due only to the different radiation content. therefore, minimization of eb corresponds to minimization of the amount of radiation in the initial data. in all these applications, it is important that the physical parameters of the black holes are calculated accurately. to illustrate the potential problems, the topology of the spatial slice is with two points (called punctures) removed. rather, each of them represents a spatial infinity of an asymptotically flat region which is hidden behind an apparent horizon. this is a generalization of the familiar einstein - rosen bridge in the maximally extended schwarzschild solution. the black hole masses mi and m2 are taken to be the adm masses of the corresponding hidden asymptotic regions. (similarly, in, the angular momentum of each hole is defined to be the adm angular momentum at the corresponding puncture.) comparison between eb and the newtonian binding energy requires us to define the distance between the holes. this is taken to be the distance between the punctures in a fiducial flat background metric ; the physical distance between the two punctures is infinite since they represent asymptotic ends of the spatial 3-manifold. therefore, the sense in which one recovers the newtonian binding energy as the leading term is physically rather obscure. let us re - examine the procedure with an eye to extending it to a more general context. let us begin with the definition of masses of individual holes which are taken to be the adm masses in the respective asymptotic regions. how do we know that these are the physically appropriate quantities for calculating the potential energy ? furthermore, there exist initial data sets (e.g., misner data [150, 151 ]) in which each black hole does not have separate asymptotic regions ; there are only two common asymptotic regions connected by two wormholes. for these cases, a natural way to resolve these conceptual issues is to let the horizons, rather than the punctures, represent black holes. thus, in the spirit of the ih and dh frameworks, it is more appropriate to calculate the mass and angular momentum using expressions (60, 59) which involve the geometry of the two apparent horizons. (this requires the apparent horizons to be axi - symmetric, but this limitation could be overcome following the procedure suggested in section 8.) similarly, the physical distance between the black holes should be the smallest proper distance between the two apparent horizons. to test the viability of this approach, one can repeat the original brill - lindquist calculation in the limit when the black holes are far apart. one first approximately locates the apparent horizon, finds the proper distance d between them, and then calculates the horizon masses (and thereby eb) as a power series in 1/d. the leading - order term does turn out to be the usual newtonian gravitational potential energy but the higher order terms are now different from. similarly, it would be interesting to repeat this for the case of spinning black holes and recover the leading order term of within this more physical paradigm using, say, the bowen - york initial data. this result would re - enforce the physical ideas and the approach can then be used as a well defined method for calculating binding energy in more general situations. in the examples mentioned above, we focussed on mass and angular momentum ; linear momentum was not considered. a meaningful definition for the linear momentum of a black hole would be useful for several problems : it would enable one to define the orbital angular momentum, locate quasi - circular orbits and enable a comparison between different initial data sets. however, just as angular momentum is associated with rotational symmetry, linear momentum ought to be associated with a space translational symmetry. in a general curved space - time thus, whenever one speaks of linear momentum of initial data (e.g., in the bowen - york construction) the quantity is taken to be the appropriate component of the adm momentum at infinity. for reasons discussed above, so, a question naturally arises : can one define linear momentum as an integral over the apparent horizon ? if z is a vector field on m which, for some reason, can be regarded as an approximate translational symmetry, and s the apparent horizon, then one might naively define the linear momentum associated with z as 64\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$p_s^{(z) } = { 1 \over { 8\pi}}\oint\nolimits_s { ({ { \overline k}_{ab } } - \overline k { { \overline q}_{ab}}){z^a}{r^b}{d^2}s.}$$\end{document } unfortunately, this definition is not satisfactory. for example, in the boosted kerr - schild metric, if one lets z to be the translational vector field of the background minkowski space, equation (64) does not yield the physically expected linear momentum of the boosted black hole. let us begin by considering the notion of source and field multipole moments in newtonian gravity and in flat space electrodynamics. field multipoles appear in the asymptotic expansions of the fields at infinity while the source multipoles are defined in terms of the mass or charge distribution of the source. also, the well known quadrupole formula relates the rate of change of the quadrupole moment to the energy flux at infinity due to gravitational waves. the situation in exact, non - linear general relativity is not so simple. using the geometric structure of the gravitational field near spatial infinity, the field multipoles for stationary space - times were studied by geroch, hansen, beig, simon, and others [97, 107, 43, 41, 40, 42 ]. they found that, just as in electrodynamics, the gravitational field has two sets of multipoles : the mass multipoles mn and the angular momentum multipoles jn. the knowledge of these multipole moments suffices to determine the space - time geometry in a neighborhood of spatial infinity [41, 40, 42 ]. thus, at least in the context of stationary space - times, the field multipole moments are well understood. however, in problems involving equations of motion, it is the source multipoles that are of more direct interest. the answer is affirmative for black holes in equilibrium, which can be represented by isolated horizons. for simplicity, we will consider only type ii (i.e., axisymmetric), non - extremal isolated horizons in vacuum. the source multipoles are two sets mn and jn of numbers which provide a diffeomorphism invariant characterization of the horizon geometry. as before, let s be a cross - section of. we denote the intrinsic riemannian metric on it by \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${{\tilde q}_{ab}}$\end{document }, the corresponding area 2-form by \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${{\tilde \epsilon}_{ab}}$\end{document }, and the derivative operator by \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${{\tilde d}_a}$\end{document}. since the horizon is of type ii, there exists a vector field a on s such that \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${{\mathcal l}_\varphi}{{\tilde q}_{ab } } = 0$\end{document}. the two points where vanishes are called the poles of s. the integral curves of are natural candidates for the lines of latitude on s, and the lines of longitude are the curves which connect the two poles and are orthogonal to. this leads to an invariantly defined coordinate [1,1 ] the analog of the function cos in usual spherical coordinates defined by 65\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${\tilde{d}_a}\zeta = { 1 \over { { r^2}}}{\tilde{\epsilon } _ { ba}}{\varphi ^b},$$\end{document } where r is the area radius of s. the freedom of adding a constant to is removed by requiring \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\oint\nolimits_s { \varsigma \tilde \epsilon = 0}$\end{document}. with [0, 2) being an affine parameter along, the 2-metric \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${{\tilde q}_{ab}}$\end{document } takes the canonical form 66\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${\tilde{q}_{ab } } = { r^2}({f^{- 1}}{\tilde{d}_a}\zeta { \tilde{d}_b}\zeta + f{\tilde{d}_a}\phi { \tilde{d}_b}\phi),$$\end{document } where = a/r. the only remaining freedom in the choice of coordinates is a rigid shift in. thus, in any axi - symmetric geometry, there is an invariantly defined coordinate, and multipole moments are defined using the legendre polynomials in as weight functions. recall from section 2.1.3 that the invariant content in the geometry of an isolated horizon is coded in (the value of its area and) 2. the real part of 2 is proportional to the scalar curvature \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\tilde r}$\end{document } of \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${{\tilde q}_{ab}}$\end{document } and captures distortions [98, 88 ], while the imaginary part of 2 yields the curl of a and captures the angular momentum information. (the free function f in equation (66) determines and is completely determined by the scalar curvature \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\tilde r}$\end{document}.) multipoles are constructed directly from 2. the angular momentum multipoles are defined as 67\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${j_n } = - \sqrt { { { 4\pi } \over { 2n + 1 } } } { { r_\delta ^{n + 1 } } \over { 4\pi g}}\oint\nolimits_s { y_n^0(\zeta){{\rm i m } } } [{ \psi _ 2}]{d^2}v,$$\end{document } where \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$y_n^0(\varsigma)$\end{document } are the spherical harmonics. (axi - symmetry ensures that \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$y_n^m$\end{document } vanish if m 0.) the normalization factors have been chosen to ensure that the dimensions of multipoles are the physically expected ones and j1 is the angular momentum of the isolated horizon. the mass multipoles are defined similarly as the moments of \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\tilde r}$\end{document } : 68\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${m_n } = - \sqrt { { { 4\pi } \over { 2n + 1 } } } { { { m_\delta}r_\delta ^n } \over { 2\pi}}\oint\nolimits_s { y_n^0(\zeta){{\rm re } } } [{ \psi _ 2}]{d^2}v,$$\end{document } where the normalization factor has been chosen in order to ensure the correct physical dimensions and m0 = m. these multipoles have a number of physically desired properties : the angular momentum monopole vanishes, j0 = 0. when a has a wire singularity similar to the dirac monopole in electrodynamics, i.e., when the horizon has nut charge, then j0 would be non - zero.)the mass dipole vanishes, m1 = 0. this tells us that we are in the rest frame of the horizon.for a type i (i.e., spherically symmetric) isolated horizon, im[2 ] = 0 and re[2 ] is constant. this implies that m0 is the only non - zero multipole moment.if the horizon is symmetric under reflections as in the kerr solution (i.e., 2 2 when), then mn vanishes for odd n while jn vanishes for even n. the angular momentum monopole vanishes, j0 = 0. when a has a wire singularity similar to the dirac monopole in electrodynamics, i.e., when the horizon has nut charge, then j0 would be non - zero.) the mass dipole vanishes, m1 = 0. this tells us that we are in the rest frame of the horizon. for a type i (i.e., spherically symmetric) isolated horizon, im[2 ] if the horizon is symmetric under reflections as in the kerr solution (i.e., 2 2 when), then mn vanishes for odd n while jn vanishes for even n. there is a one - one correspondence between the multipole moments { jn, mn } and the geometry of the horizon : given the horizon area a and multipoles { jn, mn }, assuming the multipoles satisfy a convergence condition for large n, we can reconstruct a non - extremal isolated horizon geometry (, qab, \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${{\mathcal d}_a}$\end{document }), uniquely up to diffeomorphisms, such that the area of is a and its multipole moments are the given { jn, mn}. in vacuum, stationary space - times, the multipole moments also suffice to determine the space - time geometry in the vicinity of the horizon. thus, we see that the horizon multipole moments have the expected properties. in the extremal case, because of a surprising uniqueness result, the mn, jn are universal the same as those on the extremal kerr ih and the true multipoles which can distinguish one extremal ih from another are constructed using different fields in place of 2. finally, note that there is no a - priori reason for these source multipoles to agree with the field multipoles at infinity ; there could be matter fields or radiation outside the horizon which contribute to the field multipoles at infinity. the two sets of quantities need not agree even for stationary, vacuum space - times because of contributions from the gravitational field in the exterior region. for the kerr space - time, the source and field moments are indeed different for n 2. however, the difference is small for low n. see for further discussion and for the inclusion of electromagnetic fields, and for the numerical implementation of these results. the prospect of receiving gravitational waves offers exciting opportunities for astrophysics and for testing the dynamical, strong field regime of general relativity (see, e.g.,). black holes are among the most promising sources both for terrestrial and space - based observatories. extracting gravitational waveform resulting from, say, gravitational collapse or black hole mergers is one of the important goals of numerical relativity. the eventual aim is to provide waveforms which, in conjunction with gravitational wave detectors, can be used to study the astrophysics of these gravitational wave sources. in this section, the theory of gravitational radiation in exact general relativity is based on structures defined at future null infinity \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${{\mathcal i}^ + } $ \end{document}. in particular, associated with every cross - section of \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${{\mathcal i}^ + } $ \end{document } which represents a retarded instant of time there is a well defined notion of mass, introduced by bondi, which decreases as gravitational radiation flows across \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${{\mathcal i}^ + } $ \end{document}. on the other hand, except for those based on conformal methods, most simulations only deal with a finite portion of space - time and thus have no direct access to \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${{\mathcal i}^ + } $ \end{document}. instead, one usually uses scalars such as the weyl tensor component 4 to define the radiation waveform. however, 4 depends on the choice of a null tetrad as well as coordinates. while a natural tetrad is available for the perturbation theory on kerr back ground, we will sketch an approach to solve both these problems using the isolated horizon framework : one can construct an approximate analog of \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${{\mathcal i}^ + } $ \end{document } for a suitable, finite portion of space - time, and introduce a geometrically defined null tetrad and coordinates to extract gauge invariant, radiative information from simulations. our procedure is analogous to the one used at null infinity to construct the bondi - type coordinates starting from \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${{\mathcal i}^ + } $ \end{document}. let us assume that (, []) is an ih and consider its preferred foliation (i.e., the rest frame of the horizon) defined in section 2.1.3. using terminology from null infinity, we will refer to the leaves of the foliation as the good cuts of a. pick a null normal from the [] ; this can be done, e.g., by choosing a specific value for surface gravity. let na be the unique 1-form satisfying na = 1 and which is orthogonal to the good cuts. let (v,,) be coordinates on such that v is an affine parameter along, and the good cuts are given by surfaces of constant v. here and are coordinates on the good cut satisfying \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${{\mathcal l}_\ell}\theta = 0$\end{document } and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${{\mathcal l}_\ell}\phi = 0$\end{document}. next, consider past null geodesics emanating from the good cuts with n as their initial tangent vector. let the null geodesics be affinely parameterized and let the affine parameter be called r and set r = r0 on. lie drag the coordinates (v,,) along null geodesics. this leads to a set of coordinates (r, v,,) in a neighborhood of the horizon. the only arbitrariness in this coordinate system is in the choice of (,) on one good cut and the choice of a number r0. using vacuum einstein s equations, one can obtain a systematic expansion of the metric in inverse powers of (r r0). let m be an arbitrary complex vector tangent to the good cuts such that (\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\ell, n, m,\bar m$\end{document }) is a null tetrad on. using parallel transport along the null geodesics, we can define a null tetrad in the neighborhood. this tetrad is unique up to the spin rotations of m : m e on the fiducial good cut. the domain of validity of the coordinate system and tetrads is the space - time region in which the null geodesics emanating from good cuts do not cross. figure 8bondi - like coordinates in a neighborhood of. bondi - like coordinates in a neighborhood of. numerically, it might be possible to adapt existing event horizon finders to locate the outgoing past null cone of a cross - section of the horizon. this is because event horizon trackers also track null surfaces backward in time [82, 83 ] ; the event horizon is the ingoing past null surface starting from sufficiently close to future timelike infinity while here we are interested in the outgoing past directed null surface starting from an apparent horizon. in the schwarzschild solution, it can be shown analytically that this coordinate system covers an entire asymptotic region. in the kerr space - time, the domain of validity is not explicitly known, but in a numerical implementation, this procedure does not encounter geodesic crossing in the region of interest to the simulation. in general space - times, the extraction of wave forms requires the construction to go through only along the past light cones of good cuts which lie in the distant future, whence problems with geodesic - crossing are unlikely to prevent one from covering a sufficiently large region with these coordinates. there is only a phase factor ambiguity (which is a function independent of v and r) inherited from the ambiguity in the choice of m on the fiducial good cut. second, the past null cone of a good cut at a sufficiently late time can be used as an approximate null infinity. this should enable one to calculate dynamical quantities such as the analogs of the bondi mass and the rate of energy loss from the black hole, now on the approximate \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${{\mathcal i}^ + } $ \end{document}. however, a detailed framework to extract the approximate expressions for fluxes of energy and bondi mass, with sufficient control on the errors, is yet to be developed. this is a very interesting analytical problem since its solution would provide numerical relativists with an algorithm to extract waveforms and fluxes of energy in gravitational waves in an invariant and physically reliable manner. finally, the invariant coordinates and tetrads also enable one to compare late time results of distinct numerical simulations. for simplicity, in section 4 we restricted our review of the laws of black hole mechanics to the einstein - maxwell theory. however, there is a large body of literature on black holes in more general theories with dilatonic, yang - mills, higgs, proca, and skyrme fields. these fields are not expected to be physically significant in the macroscopic, astrophysical world. however, they are of considerable interest from a mathematical physics perspective because their inclusion brings about qualitative, structural changes in the theory. the most dramatic of these is that the uniqueness theorems that play a central role in the einstein - maxwell theory are no longer valid. consequently, the structure of these colored or hairy black - holes is much more complicated than those in the einstein - maxwell theory, and most of the work in this area has been carried out through a combination of analytic and numerical methods. by now a very large body of facts about stationary black holes with hair has accumulated. the isolated horizon framework has provided surprising insights into the properties of hairy black holes in equilibrium [19, 77, 75, 26, 21, 76 ]. while the zeroth and first laws go through in a straightforward manner, the notion of the horizon mass now becomes much more subtle and its properties have interesting consequences. the framework also suggests a new phenomenological model of colored black holes as bound states of ordinary, uncolored black holes and solitons. this model successfully explains the qualitative behavior of these black holes, including their stability and instability, and provides unexpected quantitative relations between colored black holes and their solitonic analogs. in these theories, matter fields if one allows non - minimal couplings, the first law itself is modified in a striking fashion : entropy is no longer given by the horizon area but depends also on the matter fields. for globally stationary space - times admitting bifurcate killing horizons, this result was first established by jacobson, kang, and myers, and by iyer and wald [183, 184 ] for a general class of theories. for scalar fields non - minimally coupled to gravity, it has been generalized in the setting of type ii wihs. while the procedure does involve certain technical subtleties, the overall strategy is identical to that summarized in section 4.1., we discuss the mechanics of weakly isolated horizons in presence of dilatons and yang - mills fields. in the second, this entire discussion is in the framework of isolated horizons because the effects of these fields on black hole dynamics remain largely unexplored. our primary purpose in this section is to illustrate the differences from the einstein - maxwell theory. therefore, for simplicity, we will restrict ourselves to non - rotating weakly isolated horizons. in dilaton gravity, the einstein - maxwell theory is supplemented with a scalar field called the dilaton and (in the einstein frame) the maxwell part of the action is replaced by 69\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${s_{{\rm{dil}}}}(\phi, a) = - { 1 \over { 16\pi } } \int\nolimits_{\mathcal m } { [2{{(\nabla \phi) } ^2 } + { e^ { - 2\alpha \phi } } { { \bf{f}}_{ab}}{{\bf{f}}^{ab}}],{d^4}v, } $ $ \end{document } where 0 is a free parameter which governs the strength of the coupling of to the maxwell field fab. if = 0, one recovers the standard einstein - maxwell - klein - gordon system, while = 1 occurs in a low energy limit of string theory. for our illustrative purposes, it will suffice to consider the = 1 case. at spatial infinity, one now has three charges : the adm mass madm, the usual electric charge q, and another charge \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${{\tilde q}_\infty}$\end{document } 70\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${q_\infty } = { 1 \over { 4\pi}}\oint\nolimits_{{s_\infty } } { \star{\bf{f}}\;\;\;\ ; } { \tilde{q}_\infty } = { 1 \over { 4\pi}}\oint\nolimits_{{s_\infty } } { { e^{- 2\phi}}\star{\bf{f}}}.$$\end{document } \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${{\tilde q}_\infty}$\end{document } is conserved in space - time (i.e., its value does not change if the 2-sphere of integration is deformed) while q is not. from the perspective of weakly isolated horizons, it is more useful to use a, q, and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${q_\delta}$\end{document } as the basic charges : 71\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${q_\delta } = { 1 \over { 4\pi}}\oint\nolimits_s { \star{\bf{f}},\;\;\;\ ; } { \tilde{q}_\delta } = { 1 \over { 4\pi}}\oint\nolimits_s { { e^{- 2\phi}}\star{\bf{f}}},$$\end{document } where s is any cross - section of. although the standard electric charge is not conserved in space - time, it is conserved along whence q is well - defined. it is straightforward to extend the hamiltonian framework of section 4.1 to include the dilaton. to define energy, one can again seek live time - translation vector fields t, evolution along which is hamiltonian. the necessary and sufficient condition now becomes the following : there should exist a phase space function \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$e_\delta ^t$\end{document }, constructed from horizon fields, such that 72\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\delta e_\delta ^t = { { \kappa (t) } \over { 8\pi g}}\delta { a_\delta } + { \phi _ { (t)}}\delta { \tilde{q}_\delta}.$$\end{document } thus, the only difference from the einstein - maxwell case is that q is now replaced by \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${{\tilde q}_\delta}$\end{document}. again, there exists an infinite number of such live vector fields and one can construct them systematically starting with any (suitably regular) function \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\kappa _ 0}({a_\delta},{\tilde q_\delta})$\end{document } of the horizon area and charge and requiring (t) should equal 0. the major difference arises in the next step, when one attempts to construct a preferred with the dilatonic coupling, the theory has a unique three parameter family of static solutions which can be labelled by (\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${a_\delta},{q_\delta},{{\tilde q}_\delta}$\end{document }) [101, 95, 96, 148 ]. as in the reissner nordstrm family, these solutions are spherically symmetric. in terms of these parameters, the surface gravity () of the static killing field, which is unit at infinity, is given by 73\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${\kappa _ { (\xi) } } = { 1 \over { 2{r_\delta}}}\left({1 + 2g{{{q_\delta}{{\tilde{q}}_\delta } } \over { r_\delta ^2 } } } \right){\left({1 - 2g{{{q_\delta}{{\tilde{q}}_\delta } } \over { r_\delta ^2 } } } \right)^{- { 1 \over 2}}}.$$\end{document } the problem in the construction of the preferred \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$t_0^a$\end{document } is that we need a function 0 which depends only on a and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${{\tilde q}_\delta}$\end{document } and, since () depends on all three horizon parameters, one can no longer set 0 = () on the entire phase space. thus, there is no live vector field \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$t_0^a$\end{document } which can generate a hamiltonian evolution and agree with the time - translation killing field in all static solutions. it was the availability of such live vector fields that provided a canonical notion of the horizon mass in the einstein - maxwell theory in section 4.1.3. one can weaken the requirements by working on sectors of phase space with fixed values of q. on each sector, () trivially depends only on a and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${{\tilde q}_\delta}$\end{document}. so one can set 0 = (), select a canonical t0, and obtain a mass function \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${m_\delta } = e_\delta ^{{t_0}}$\end{document}. however, now the first law (72) is satisfied only if the variation is restricted such that q = 0. for general variation, one has the modified law 74\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\delta { m_\delta } = { \kappa \over { 8\pi g}}\delta { a_\delta } + \hat{\phi } \delta { \hat{q}_\delta},$$\end{document } where \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\kappa = { \kappa _ { ({ t_0})}},{{\hat \phi}^2 } = ({ q_\delta}{{\tilde q}_\delta}/r_\delta ^2)$\end{document } and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\hat q_\delta ^2 = { q_\delta}{{\tilde q}_\delta}$\end{document}. thus, although there is still a first law in terms of \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$t_0^a$\end{document } and m, it does not have the canonical form (72) because \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$t_0^a$\end{document } does not generate hamiltonian evolution on the entire phase space. more generally, in theories with multiple scalar fields, if one focuses only on static sectors, one obtains similar non - standard forms of the first law with new work terms involving scalar fields. from the restricted perspective of static sector, this is just a fact. the isolated horizon framework provides a deeper underlying reason : in these theories, there is no evolution vector field t defined for all points of the phase space, which coincides with the properly normalized killing field on all static solutions, and evolution along which is hamiltonian on the full phase space. in the einstein - maxwell theory, with and without the dilaton, one can not construct a quantity with the dimensions of mass from the fundamental constants in the theory. the situation is different for einstein - yang - mills theory because the coupling constant g has dimensions (lm)., we will restrict ourselves to su(2) yang - mills fields, but results based on the isolated horizon framework go through for general compact groups. first, the reissner - nordstrm family constitutes a continuous 2-parameter set of static solutions of the einstein - yang - mills theory, labelled by (\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${a_\delta},q_\delta ^{{\rm{ym}}}$\end{document }). in addition, there is a 1-parameter family of embedded abelian solutions with (a fixed) magnetic charge \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$p_\delta ^0$\end{document }, labelled by (\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${a_\delta},p_\delta ^0$\end{document }). finally, there are families of genuinely non - abelian solutions. for these, the analog of the israel theorem for einstein - maxwell theory fails to hold [127, 128, 129 ] ; the theory admits static solutions which need not be spherically symmetric. in particular, an infinite family of solutions labelled by two integers (n1, n2) is known to exist. all static, spherically symmetric solutions are known and they correspond to the infinite sub - family (n1, n2 = 0), labelled by a single integer. however, the two parameter family is obtained using a specific ansatz, and other static solutions also exist. although the available information on the static sector is quite rich, in contrast to the einstein - maxwell - dilaton system, one is still rather far from having complete control. however, the existing results are already sufficient to show that, in contrast to the situation in the einstein - maxwell theory, the adm mass is not a good measure of the black hole (or horizon) mass even in the static case. let us consider the simplest case, the spherically symmetric static solutions labelled by a single integer n (see figure 9). in the zero area limit, the solution is known to converge point - wise to a regular, static, spherical solution, representing an einstein - yang - mills soliton [38, 171, 60 ]. this solution has, of course, a non - zero adm mass \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$m_{{\rm{adm}}}^{{\rm{sol}}}$\end{document }, which equals the limiting value of \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$m_{{\rm{adm}}}^{{\rm{bh}}}$\end{document}. however, in this limit, there is no black hole at all ! hence, this limiting value of the adm mass can not be meaningfully identified with any horizon mass. by continuity, then, \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$m_{{\rm{adm}}}^{{\rm{bh}}}$\end{document } can not be taken as an accurate measure of the horizon mass for any black hole along an n 0 branch. using the isolated horizon framework, it is possible to introduce a meaningful definition of the horizon mass on any given static branch. figure 9the adm mass as a function of the horizon radius r of static spherically symmetric solutions to the einstein - yang - mills system (in units provided by the yang - mills coupling constant). numerical plots for the colorless (n = 0) and families of colored black holes (n = 1, 2) are shown. (note that the y - axis begins at m = 0.7 rather than at m = 0.) the adm mass as a function of the horizon radius r of static spherically symmetric solutions to the einstein - yang - mills system (in units provided by the yang - mills coupling constant). numerical plots for the colorless (n = 0) and families of colored black holes (n = 1, 2) are shown. (note that the y - axis begins at m = 0.7 rather than at m = 0.) to establish laws of black hole mechanics, one begins with appropriate boundary conditions. in the maxwell case, the gauge freedom in the vector potential is restricted on the horizon by requiring \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${{\mathcal l}_\ell}{a_a } = 0$\end{document } on. the analogous condition ensuring that the yang - mills potential a is in an adapted gauge on is more subtle. however, it does exist and again ensures that (i) the action principle is well defined, and (ii) the yang - mills electric potential \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\phi _ { (\ell) } } : = - \vert\ell \cdot { \rm{a}\vert}$\end{document } is constant on the horizon, where the absolute sign stands for the norm in the internal space. the proof of the zeroth law and the construction of the phase space is now straightforward. there is a well - defined notion of conserved horizon charges 75\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$q_\delta ^{{\rm{ym } } } : = - { 1 \over { 4\pi}}\oint\nolimits_s { \vert\star{\bf{f}}\vert } { d^2}v,\;\;\;\;p_\delta ^{{\rm{ym } } } : = - { 1 \over { 4\pi}}\oint\nolimits_s { \vert{\bf{f}}\vert } { d^2}v,$$\end{document } where \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\vert{\bf{f}}\vert : = { [({ \epsilon ^{ab}}{\bf{f}}_{ab}^i)({\epsilon ^{ab}}{\bf{f}}_{ab}^j){k_{ij}}]^{{1 \over 2}}}$\end{document }, with being the cartan - killing metric on su(2), the alternating tensor on the cross - section s of, and where |f| is defined by replacing f with f. finally, one can again introduce live vector fields t, time evolution along which generates a hamiltonian flow on the phase space, and establish a first law for each of these t : 76\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\delta e_\delta ^t = { 1 \over { 8\pi g}}{\kappa _ { (t)}}\delta { a_\delta } + { \phi _ { (t)}}\delta q_\delta ^{{\rm{ym}}}$$\end{document } note that, even though the magnetic charge \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$p_\delta ^{{\rm{ym}}}$\end{document } is in general non - zero, it does not enter the statement of the first law. in the abelian case, a non - zero magnetic charge requires non - trivial u(1) bundles, and chern numbers characterizing these bundles are discrete. hence the magnetic charge is quantized and, if the phase space is constructed from connections, p vanishes identically for any variation s. in the non - abelian case, one can work with a trivial bundle and have non - zero \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$p_\delta ^{{\rm{ym}}}$\end{document}. therefore, \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\delta p_\delta ^{{\rm{ym}}}$\end{document } does not automatically vanish and absence of this term in the first law is somewhat surprising. a more significant difference from the abelian case is that, because the uniqueness theorem fails, one can not use the static solutions to introduce a canonical function 0 on the entire phase space, whence as in the dilatonic case, there is no longer a canonical horizon mass m function on the entire phase space. in the next section 6.2 we will see that it is nonetheless possible to introduce an extremely useful notion of the horizon mass for each static sequence. we will briefly summarize research in three areas in which the isolated horizon framework has been used to illuminate the structure of static, colored black holes and associated solitons7. let us begin with einstein - yang - mills theory considered in the last section 6.1. as we saw, the adm mass fails to be a good measure of the horizon mass for colored black holes. the failure of black hole uniqueness theorems also prevents the isolated horizon framework from providing a canonical notion of horizon mass on the full phase space. however, one can repeat the strategy used for dilatonic black holes to define horizon mass unambiguously for the static solutions [77, 26 ]. consider a connected component of the known static solutions, labelled by \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${{\vec n}_0 } \equiv (n_1 ^ 0,n_2 ^ 0)$\end{document}. using for 0 the surface gravity of the properly normalized static killing vector, in this sector one can construct a live vector field \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$t_0^a$\end{document } and obtain a first law. the energy is well - defined on the full phase space and can be naturally interpreted as the horizon mass \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$m_\delta ^{({{\vec n}_0})}$\end{document } for colored black holes with quantum number \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${{{\vec n}_0}}$\end{document}. the explicit expression is given by 77\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$m_\delta ^{({{\vec n}_0 }) } = { 1 \over { 2g}}\int\nolimits_0^{{r_\delta } } { { \beta _ { { { \vec n}_0 } } } } (r)dr,$$\end{document } where, following a convention in the literature on hairy black holes, we have used 78\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\beta { \overrightarrow { n } _ 0}(r) = 2[\kappa { \overrightarrow { n } _ 0}(r)]r$$\end{document } rather than the surface gravity \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\kappa _ { { { \vec n}_0}}}(r)$\end{document } of static solutions. now, as in the einstein - maxwell case, the hamiltonian generating evolution along \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$t_0^a$\end{document } contains only surface terms : \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${h_{{t_0 } } } = e_{{\rm{adm}}}^{{t_0 } } - e_\delta ^{{t_0}}$\end{document } and is constant on each connected, static sector if \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$t_0^a$\end{document } coincides with the static killing field on that sector. by construction, our \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$t_0^a$\end{document } has this property for the \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${{{\vec n}_0}}$\end{document}-sector under consideration. now, in the einstein - maxwell case, since there is no constant with the dimension of energy, it follows that the restriction of \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$h_{{\rm{adm}}}^{{t_0}}$\end{document } to the static sector must vanish. the situation is quite different in einstein - yang - mills theory where the yang - mills coupling constant g provides a scale. in c = 1 units, \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${(g\sqrt g)^{- 1}}$\end{document } mass. therefore, we can only conclude that 79\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$m_{{\rm{adm}}}^{(\overrightarrow { n }) } = m_\delta ^{({{\overrightarrow { n}}_{_0 } }) } + { (g\sqrt g)^{- 1}}{c^{({{\overrightarrow { n}}_{_0}})}}$$\end{document } for some \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$_{{{\vec n}_0}}$\end{document}-dependent constant \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${c^{({{\vec n}_0})}}$\end{document}. as the horizon radius shrinks to zero, the static solution [127, 181 ] under consideration tends to the solitonic solution with the same quantum numbers \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${{{\vec n}_0}}$\end{document}. hence, by taking this limit, we conclude \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${(g\sqrt g)^{- 1}}{c^{({{\vec n}_0 }) } } = m_{{\rm{adm}}}^{{\rm{sol,}}({{\vec n}_0})}$\end{document}. therefore, on any \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\vec n}$\end{document}-static sector, we have the following interesting relation between the black hole and solitonic solutions : 80\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$m_{{\rm{adm}}}^{{\rm{bh}},(\overrightarrow { n }) } = { 1 \over { 2g}}\int\nolimits_0^{{r_\delta } } { { \beta _ { \overrightarrow { n}}}(r)dr + } m_{{\rm{adm}}}^{{\rm{sol}},(\overrightarrow { n }) } = m_\delta ^{{\rm{bh}},(\overrightarrow { n }) } + m_{{\rm{adm}}}^{{\rm{sol}},(\overrightarrow { n})}$$\end{document } thus, although the main motivation behind the isolated horizon framework was to go beyond globally time - independent situations, it has led to an interesting new relation between the adm masses of black holes and their solitonic analogs already in the static sector. the relation (80) was first proposed for spherical horizons in, verified in, and extended to distorted horizons in. the relation has been confirmed in three more general and non - trivial cases : non - spherical, non - rotating black holes parameterized by two quantum numbers.non - spherical solutions to the more general einstein - yang - mills - higgs theory, where distortions are caused by magnetic dipole hair.static solutions in the born - infeld theory. non - spherical solutions to the more general einstein - yang - mills - higgs theory, where distortions are caused by magnetic dipole hair. isolated horizon considerations suggested the following simple heuristic model of colored black holes : a colored black hole with quantum numbers \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\vec n}$\end{document } should be thought of as bound states of a ordinary (colorless) black hole and a soliton with color quantum numbers \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\vec n}$\end{document }, where \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\vec n}$\end{document } can be more general than considered so far. the mass formula (80) now suggests that the total adm mass \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$m_{{\rm{adm}}}^{(\vec n)}$\end{document } has three components : the mass \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$m_\delta ^{(0)}$\end{document } of the bare horizon, the mass \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$m_{{\rm{adm}}}^{{\rm{sol,}}\vec n}$\end{document } of the colored soliton, and a binding energy given by 81\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${e_{{\rm{binding } } } } = m_\delta ^{(\overrightarrow { n})}({r_\delta }) - m_\delta ^{(0)}({r_\delta}).$$\end{document } if this picture is correct, being gravitational binding energy, ebinding would have to be negative. the model has several predictions on the qualitative behavior of the horizon mass (77), the surface gravity, and the relation between properties of black holes and solitons. we will illustrate these with just three examples (for a complete list with technical caveats, see): for any fixed value of r and of all quantum numbers except n, the horizon mass and surface gravity decrease monotonically with n.for fixed values of all quantum numbers \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\vec n}$\end{document }, the horizon mass \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$m_\delta ^{(\vec n)}({r_\delta})$\end{document } is non - negative, vanishing if and only if r vanishes, and increases monotonically with \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${r_\delta}.{\beta _ { (\vec n)}}$\end{document } is positive and bounded above by 1.for fixed \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\vec n}$\end{document }, the binding energy decreases as the horizon area increases. for any fixed value of r and of all quantum numbers except n, the horizon mass and surface gravity decrease monotonically with n. for fixed values of all quantum numbers \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\vec n}$\end{document }, the horizon mass \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$m_\delta ^{(\vec n)}({r_\delta})$\end{document } is non - negative, vanishing if and only if r vanishes, and increases monotonically with \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${r_\delta}.{\beta _ { (\vec n)}}$\end{document } is positive and bounded above by 1. for fixed \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\vec n}$\end{document }, the binding energy decreases as the horizon area increases. the predictions for fixed \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\vec n}$\end{document } have recently been verified beyond spherical symmetry : for the distorted, axially symmetric einstein - yang - mills solutions in and for the distorted dipole solutions in einstein - yang - mills - higgs solutions in. taken together, the predictions of this model can account for all the qualitative features of the plots of the horizon mass and surface gravity as functions of the horizon radius and quantum numbers. more importantly, they have interesting implications on the stability properties of colored black holes. einstein - yang - mills solitons are known to be unstable ; under small perturbations, the energy stored in the bound state represented by the soliton is radiated away to future null infinity \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${{\mathcal i}^ + } $ \end{document}. the phenomenological model suggests that colored black holes should also be unstable and they should decay into ordinary black holes, the excess energy being radiated away to infinity. in general, however, even if one component of a bound system is unstable, the total system may still be stable if the binding energy is sufficiently large. however, an examination of energetics reveals that this is not the case for colored black holes, so instability should prevail. we summarize these for the simplest case of spherically symmetric, static black holes for which there is a single quantum number n : all the colored black holes on the nth branch have the same number (namely, 2n) of unstable modes as the nth soliton. (the detailed features of these unstable modes can differ especially because they are subject to different boundary conditions in the two cases.)for a given n, colored black holes with larger horizon area are less unstable. for a given horizon area, colored black holes with higher value of n are more unstable.the available energy for the process is given by 82\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$e_{{\rm{available}}}^{(n) } = m_{{\rm{sol}}}^{(n) } - \vert e_{{\rm{binding}}}^{{\rm{initial}}}\vert.$$\end{document } part of it is absorbed by the black hole so that its horizon area increases and the rest is radiated away to infinity. note that \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$e_{{\rm{available}}}^{(n)}$\end{document } can be computed knowing just the initial configuration.in the process the horizon area necessarily increases. therefore, the energy radiated to infinity is strictly less than \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$e_{{\rm{available}}}^{(n)}$\end{document}. figure 10an initially static colored black hole with horizon in is slightly perturbed and decays to a schwarzschild - like isolated horizon fin, with radiation going out to future null infinity \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${{\mathcal i}^ + } $ \end{document}. all the colored black holes on the nth branch have the same number (namely, 2n) of unstable modes as the nth soliton. (the detailed features of these unstable modes can differ especially because they are subject to different boundary conditions in the two cases.) for a given n, colored black holes with larger horizon area are less unstable. for a given horizon area, available energy for the process is given by 82\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$e_{{\rm{available}}}^{(n) } = m_{{\rm{sol}}}^{(n) } - \vert e_{{\rm{binding}}}^{{\rm{initial}}}\vert.$$\end{document } part of it is absorbed by the black hole so that its horizon area increases and the rest is radiated away to infinity. note that \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$e_{{\rm{available}}}^{(n)}$\end{document } can be computed knowing just the initial configuration. in the process therefore, the energy radiated to infinity is strictly less than \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$e_{{\rm{available}}}^{(n)}$\end{document}. an initially static colored black hole with horizon in is slightly perturbed and decays to a schwarzschild - like isolated horizon fin, with radiation going out to future null infinity \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${{\mathcal i}^ + } $ \end{document}. expectation 1 of the model is known to be correct. prediction 2 has been shown to be correct in the n = 1, colored black holes in the sense that the frequency of all unstable modes is a decreasing function of the area, whence the characteristic decay time grows with area [181, 51 ]. to our knowledge a detailed analysis of instability, needed to test predictions 3 and 4 are yet to be made. finally, the notion of horizon mass and the associated stability analysis has also provided an consider the embedded abelian black holes which are solutions to einstein - yang - mills equations with a specific magnetic charge \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$p_\delta ^0$\end{document}. they are isometric to a family of magnetically charged reissner - nordstrm solutions and the isometry maps the maxwell field strength to the yang - mills field strength. the only difference is in the form of the connection ; while the yang - mills potential is supported on a trivial su(2) bundle, the maxwell potential requires a non - trivial u(1) bundle. therefore, it comes as an initial surprise that the solution is stable in the einstein - maxwell theory but unstable in the einstein - yang - mills theory [52, 61 ]. however, since the horizon mass arises from hamiltonian considerations, it is theory dependent : it is lower in the einstein - yang - mills theory than in the einstein - maxwell theory ! thus, from the einstein - yang - mills perspective, part of the adm mass is carried by the soliton and there is positive \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$e_{{\rm{available}}}^{ym}$\end{document } which can be radiated away to infinity. in the einstein - maxwell theory, \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$e_{{\rm{available}}}^{{\rm{em}}}$\end{document } is zero. the stability analysis sketched above therefore implies that the solution should be unstable in the einstein - yang - mills theory but stable in the einstein - maxwell theory. this is another striking example of the usefulness of the notion of the horizon mass. we will now briefly summarize the most interesting result obtained from this framework in more general theories. when one allows higgs or proca fields in addition to yang - mills, or considers einstein - skyrme theories, one acquires additional dimensionful constants which trigger new phenomena [50, 178, 181 ]. one of the most interesting is the crossing phenomena of figure 11 where curves in the phase diagram (i.e., a plot of the adm mass versus horizon radius) corresponding to the two distinct static families cross. this typically occurs in theories in which there is a length scale even in absence of gravity, i.e., even when newton s constant is set equal to zero [154, 21 ]. figure 11the adm mass as a function of the horizon radius r in theories with a built - in non - gravitational length scale. the schematic plot shows crossing of families labelled by n = 1 and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${r_\delta } = r_\delta ^{{\rm{inter}}}$\end{document}. the adm mass as a function of the horizon radius r in theories with a built - in non - gravitational length scale. the schematic plot shows crossing of families labelled by n = 1 and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${r_\delta } = r_\delta ^{{\rm{inter}}}$\end{document}. in this case, the notion of the horizon mass acquires further subtleties. if, as in the einstein - yang - mills theory considered earlier, families of static solutions carrying distinct quantum numbers do not cross, there is a well - defined notion of horizon mass for each static solution, although, as the example of embedded abelian solutions shows, in general its value is theory dependent. when families cross, one can repeat the previous strategy and use equation (77) to define a mass \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$m_\delta ^{(n)}$\end{document } along each branch. however, at the intersection point \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$r_\delta ^{{\rm{inter}}}$\end{document } of the nth and (n + 1)th branches, the mass is discontinuous. consider the closed curve c in the phase diagram, starting at the intersection point and moving along the (n + 1)th branch in the direction of decreasing area until the area becomes zero, then moving along a = 0 to the nth branch and moving up to the intersection point along the nth branch (see figure 11). discontinuity in the horizon mass implies that the integral of (r) along this closed curve is non - zero. furthermore, the relation between the horizon and the soliton mass along each branch implies that the value of this integral has a direct physical interpretation : 83\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$m_{{\rm{sol}}}^{(n + 1) } - m_{{\rm{sol}}}^{(n) } = { 1 \over { 2g}}\oint\nolimits_c { \beta (r) } \;dr.$$\end{document } this is a striking prediction because it relates differences in masses of solitons to the knowledge of horizon properties of the corresponding black holes ! because of certain continuity properties which hold as one approaches the static einstein - yang - mills sector in the space to static solutions to einstein - yang - mills - higgs equations, one can also obtain a new formula for the adm masses of einstein - yang - mills solitons. if (1 (n)(r)) for the black hole solutions of this theory is integrable over the entire positive half line, one has : 84\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$m_{{\rm{sol}}}^{(n) } = { 1 \over { 2g}}\int\nolimits_0^\infty { (1 - { \beta _ { (n)}}(r)) } \;dr.$$\end{document } both these predictions of the phenomenological model have been verified numerically in the spherically symmetric case, but the axi - symmetric case is still open. as discussed in the introduction, laws of black hole mechanics, discovered in the early seventies, provided a concrete challenge to candidate quantum theories of gravity : account for the thermodynamic, black hole entropy through a detailed, statistical mechanical counting of appropriate micro - states. indeed, this is essentially the only concrete quantitative hint we have had about the nature of quantum space - time geometry. the isolated horizon framework has been used to address this issue systematically and has led to the only available detailed calculations within a full - fledged approach to quantum gravity that encompass realistic black holes (which carry no or negligible gauge charges and may be distorted). as we will discuss in section 8, what we know about dynamical horizons does suggest that there should be interesting generalizations of these results also to non - equilibrium situations. the first and the second laws of black hole mechanics discussed in section 4, 85\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\delta e = { \kappa \over { 8\pi g}}\delta a + { \rm{work}},\;\;\;d\delta a \geq 0,$$\end{document } have a close similarity with the first and second laws of thermodynamics 86\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\delta e = t\delta s + { \rm{work}},\;\;\;\delta s \geq 0.$$\end{document } this suggest that one should assign to black holes an entropy proportional to their area. indeed, already in the early seventies, using imaginative thought experiments, bekenstein [44, 45 ] argued that such an assignment is forced upon us if, in presence of black holes, we are not going to simply abandon the second law of thermodynamics. specifically, consider an experiment in which a box containing radiation is slowly lowered into a black hole. at the end of the process, the entropy of the exterior region decreases in apparent violation of the second law. bekenstein argued that the area of the horizon increases in the process so that the total entropy would not decrease if the black hole is assigned entropy equal to an appropriate multiple of the area. hawking s celebrated calculation of black hole evaporation provided the precise numerical coefficient. for, it suggested that we should assign the black hole a temperature t = /2. by comparing the forms of the first laws of black hole mechanics and thermodynamics, one is then led to set 87\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${s_{{\rm{bh } } } } = { a \over { 4\ell _ { { \rm{pl}}}^{2}}}\;\;\;\;{\rm{with}}\;\ell _ { { \rm{pl}}}^2 = g\hbar.$$\end{document } note that the factor of is absolutely essential : in the limit 0, the black hole temperature goes to zero and its entropy tends to infinity just as one would expect classically. the relation (87) is striking and deep because it brings together the three pillars of fundamental physics general relativity, quantum theory, and statistical mechanics. however, the argument itself is a rather hodge - podge mixture of classical and semi - classical ideas, reminiscent of the bohr theory of atom. a natural question then is : what is the analog of the more fundamental, pauli - schrdinger theory of the hydrogen atom ? more precisely, what is the statistical mechanical origin of black hole entropy ? to answer this question, one has to isolate the microscopic degrees of freedom responsible for this entropy. for a classical ideal gas, these are given by the positions and momenta of all molecules ; for a magnet, by the states of each individual spin at lattice sites. how do we represent the analogous microscopic degrees of freedom for a black hole ? they can not be described in terms of quantum states of physical gravitons because we are dealing with black holes in equilibrium. in the approach based on weakly isolated horizons, they are captured in the quantum states of the horizon geometry. just as one would expect from bekenstein s thought experiments, these degrees of freedom can interact with the exterior curved geometry, and the resulting notion of black hole entropy is tied to observers in the exterior regions. a heuristic framework for the calculation of entropy it from bit. divide the black hole horizon into elementary cells, each with one planck unit of area \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\ell _ { { \rm{p}}1}^2$\end{document } and assign to each cell two microstates. then the total number of states \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\mathcal n}$\end{document } is given by \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\mathcal n } = { 2^n}$\end{document }, where \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$n = (a/\ell _ { { \rm{p}}1}^2)$\end{document } is the number of elementary cells, whence entropy is given by \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$s = \ln { \mathcal n } \sim a$\end{document}. thus, apart from a numerical coefficient, the entropy (it) is accounted for by assigning two states (bit) to each elementary cell. but it raises at least three central questions : what is the origin of the elementary cells and why is each endowed with an area \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\ell _ { { \rm{p}}1}^2$\end{document}what is the origin of the microstates carried by each elementary cell and why are there precisely two microstates?what does all this have to do with a black hole ? why does nt it apply to any 2-surface, including a 2-sphere in minkowski space - time ? what is the origin of the elementary cells and why is each endowed with an area \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\ell _ { { \rm{p}}1}^2$\end{document } what is the origin of the microstates carried by each elementary cell and why are there precisely two microstates ? what does all this have to do with a black hole ? why does nt it apply to any 2-surface, including a 2-sphere in minkowski space - time ? an understanding of geometry of quantum wihs provides a detailed framework which, in particular, answers these questions. the precise picture, as usual, is much more involved than that envisaged by wheeler. eigenvalues of area operator turn out to be discrete in quantum geometry and one can justify dividing the horizon 2-sphere into elementary cells. however, there are many permissible area eigenvalues and cells need not all carry the same area. to identify horizon surface states that are responsible for entropy, one has to crucially use the wih boundary conditions. however, the number of surface states assigned to each cell is not restricted to two. nonetheless, wheeler s picture turns out to be qualitatively correct. for simplicity of presentation, in this section we will restrict ourselves to type i wihs, i.e., the ones for which the only non - zero multipole moment is the mass monopole. the extension to include type ii horizons with rotations and distortion will be briefly summarized in the next section 7.3. details can be found in [10, 11, 20, 8, 24 ]. the point of departure is the classical hamiltonian formulation for space - times \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\mathcal m}$\end{document } with a type i wih as an internal boundary, with fixed area a0 and charges \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$q_0^\alpha$\end{document }, where runs over the number of distinct charges (maxwell, yang - mills, dilaton,) allowed in the theory. as we noted in section 4.1, the phase space can be constructed in a number of ways, which lead to equivalent hamiltonian frameworks and first laws. so far, the only known way to carry out a background independent, non - perturbative quantization is through connection variables. as in figure 6 let us begin with a partial cauchy surface m whose internal boundary in \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\mathcal m}$\end{document } is a 2-sphere cross - section s of and whose asymptotic boundary is a 2-sphere at spatial infinity. the configuration variable is an su(2) connection \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$a_a^i$\end{document } on m, where i takes values in the 3-dimensional lie - algebra su(2) of su(2). just as the standard derivative operator acts on tensor fields and enables one to parallel transport vectors, the derivative operator constructed from acts on fields with internal indices and enables one to parallel transport spinors. the conjugate momentum is represented by a vector field \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$p_i^a$\end{document } with density weight 1 which also takes values in su(2) ; it is the analog of the yang - mills electric field. (in absence of a background metric, momenta always carry a density weight 1.) \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$p_i^a$\end{document } can be regarded as a (density weighted) triad or a square - root of the intrinsic metric \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${{\tilde q}_{ab}}$\end{document } on s : \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${(8\pi g\gamma)^2}p_i^ap_j^b{\delta _ { ij } } = \tilde q{{\tilde q}^{ab}}$\end{document }, where is the cartan killing metric on su(2), \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\tilde q}$\end{document } is the determinant of and is a positive real number, called the barbero - immirzi parameter. this parameter arises because there is a freedom in adding to palatini action a multiple of the term which is dual to the standard one, which does not affect the equations of motion but changes the definition of momenta. this multiple is. the presence of represents an ambiguity in quantization of geometry, analogous to the -ambiguity in qcd. just as the classical yang - mills theory is insensitive to the value of but the quantum yang - mills theory has inequivalent -sectors, classical relativity is insensitive to the value of but the quantum geometries based on different values of are (unitarily) inequivalent (for details, see, e.g.,). thus, the gravitational part of the phase space consists of pairs \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$(a_a^i, p_i^a)$\end{document } of fields on m satisfying the boundary conditions discussed above. had there been no internal boundary, the gravitational part of the symplectic structure would have had just the expected volume term : 88\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${\omega _ v}({\delta _ 1},{\delta _ 2 }) = \int\nolimits_m { ({ \delta _ 2}{a^i } \wedge { \delta _ 1}{\sigma _ i } - { \delta _ 1}{a^i } \wedge { \delta _ 2}{\sigma _ i }), } $ $ \end{document } where \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\sigma _ { abi } } : = { \eta _ { abc}}p_i^c$\end{document } is the 2-form dual to the momentum \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$p_i^c$\end{document } and 1, and 2 denote any two tangent vectors to the phase space. however, the presence of the internal boundary changes the phase space - structure. the type i wih conditions imply that the non - trivial information in the pull - back \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\underleftarrow a _ a^i$\end{document } of \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$a_a^i$\end{document } to s is contained in a u(1) connection \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${w_a } : = \underleftarrow a _ a^i{r_i}$\end{document } on s, where r is the unit, internal, radial vector field on s. its curvature 2-form fab is related to the pull - back of the 2-form \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\underleftarrow \sigma _ { ab}^i$\end{document } to s via 89\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$f \equiv dw = - 8\pi g\gamma { { 2\pi } \over { { a_0}}}{^i}{r_i}.$$\end{document } the restriction is called the horizon boundary condition. in the schwarzschild space - time, all the 2-spheres on which it is satisfied lie on the horizon. finally, the presence of the internal boundary modifies the symplectic structure : it now acquires an additional boundary term 90\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\omega ({ \delta _ 1},{\delta _ 2 }) = { \omega _ v}({\delta _ 1},{\delta _ 2 }) + { \omega _ s}({\delta _ 1},{\delta _ 2}),$$\end{document } with 91\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${\omega _ s}({\delta _ 1},{\delta _ 2 }) = { 1 \over { 2\pi}}{{{a_0 } } \over { 4\pi g\gamma}}{\delta _ 1}w \wedge { \delta _ 2}w,$$\end{document } the new surface term is precisely the symplectic structure of a well - known topological field theory the u(1)-chern - simons theory. the symplectic structures of the maxwell, yang - mills, scalar, and dilatonic fields do not acquire surface terms. conceptually, this is an important point : this, in essence, is the reason why (for minimally coupled matter) the black hole entropy depends just on the area and not, in addition, on the matter charges. in absence of internal boundaries, the quantum theory has been well - understood since the mid - nineties (for recent reviews, see, [162, 176, 32 ]). the fundamental quantum excitations are represented by wilson lines (i.e., holonomies) defined by the connection and are thus 1-dimensional, whence the resulting quantum geometry is polymer - like. given any 2-surface \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\mathbb s}$\end{document } on m, there is a self - adjoint operator \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${{\hat a}_{\mathbb s}}$\end{document } all of whose eigenvalues are known to be discrete. the simplest eigenvectors are represented by a single flux line, carrying a half - integer j as a label, which intersects the surface s exactly once, and the corresponding eigenvalue \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${a_{\mathbb s}}$\end{document } of \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${{\hat a}_{\mathbb s}}$\end{document } is given by 92\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${a_\mathbb { s } } = 8\pi \gamma \ell _ { { \rm{pl}}}^2\sqrt { j(j + 1)}.$$\end{document } thus, while the general form of the eigenvalues is the same in all -sectors of the quantum theory, their numerical values do depend on. since the eigenvalues are distinct in different -sectors, it immediately follows that these sectors provide unitarily inequivalent representations of the algebra of geometric operators ; there is super - selection. put differently, there is a quantization ambiguity, and which -sector is actually realized in nature is an experimental question. one appropriate experiment, for example a measurement of the smallest non - zero area eigenvalue, would fix the value of and hence the quantum theory. every further experiment e.g., the measurement of higher eigenvalues or eigenvalues of other operators such as those corresponding to the volume of a region would provide tests of the theory. while such direct measurements are not feasible today we will see that, somewhat surprisingly, the hawking - bekenstein formula (87) for the entropy of large black holes provides a thought experiment to fix the value of. recall next that, because of the horizon internal boundary, the symplectic structure now has an additional surface term. in the classical theory, since all fields are smooth, values of fields on the horizon are completely determined by their values in the bulk. however, a key point about field theories is that their quantum states depend on fields which are arbitrarily discontinuous. therefore, in quantum theory, a decoupling occurs between fields in the surface and those in the bulk, and independent surface degrees of freedom emerge. these describe the geometry of the quantum horizon and are responsible for entropy. in quantum theory, then, it is natural to begin with a total hilbert space \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\mathcal h } = { { \mathcal h}_v } \otimes { { \mathcal h}_s}$\end{document } where \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${{\mathcal h}_v}$\end{document } is the well - understood bulk or volume hilbert space with polymer - like excitations, and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${{\mathcal h}_s}$\end{document } is the surface hilbert space of the u(1)-chern - simons theory. deposits on s an area equal to \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$8\pi \ell _ { { \rm{p}}1}^2\sqrt { j(j + 1)}$\end{document}. these contributions add up to endow s a total area a0. the surface chern - simons theory is therefore defined on the punctured 2-sphere s. to incorporate the fact that the internal boundary s is not arbitrary but comes from a wih, we still need to incorporate the residual boundary condition (89). thus, in the quantum theory, neither the triad nor the curvature of w are frozen at the horizon ; neither is a classical field. each is allowed to undergo quantum fluctuations, but the quantum horizon boundary condition requires that they have to fluctuate in tandem. polymer excitations in the bulk puncture the horizon, endowing it with quantized area. intrinsically, the horizon is flat except at punctures where it acquires a quantized deficit angle. polymer excitations in the bulk puncture the horizon, endowing it with quantized area. intrinsically, the horizon is flat except at punctures where it acquires a quantized deficit angle. an important subtlety arises because the operators corresponding to the two sides of equation (89) act on different hilbert spaces : while \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\hat f}$\end{document } is defined on \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${{\mathcal h}_s},\underleftarrow { \hat \sigma } \cdot r$\end{document } is defined on \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${{\mathcal h}_v}$\end{document}. therefore, the quantum horizon boundary condition introduces a precise intertwining between the bulk and the surface states : only those states \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\psi = { \sum\nolimits_i}\psi _ v^i \otimes \psi _ s^i$\end{document } in \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\mathcal h}$\end{document } which satisfy 93\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$(1 \otimes \hat{f})\psi = \left({- { { 2\pi \gamma } \over { { a_0}}}\sum\limits _ \leftarrow ^{\wedge } \cdot r \otimes 1 } \right)\psi$$\end{document } can describe quantum geometries with wih as inner boundaries. this is a stringent restriction : since the operator on the left side acts only on surface states and the one on the right side acts only on bulk states, the equation can have solutions only if the two operators have the same eigenvalues (in which case we can take to be the tensor product of the two eigenstates). thus, for solutions to equation (93) to exist, there has to be a very delicate matching between eigenvalues of the triad operators \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\underleftarrow { \hat \sigma } \cdot r$\end{document } calculated from bulk quantum geometry, and eigenvalues of \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\hat f}$\end{document }, calculated from chern - simons theory. the precise numerical coefficients in the surface calculation depend on the numerical factor in front of the surface term in the symplectic structure, which is itself determined by our type i wih boundary conditions. thus, the existence of a coherent quantum theory of wihs requires that the three cornerstones classical isolated horizon framework, quantum mechanics of bulk geometry, and quantum chern - simons theory be united harmoniously. not only should the three frameworks be mutually compatible at a conceptual level, but certain numerical coefficients, calculated independently within each framework, have to match delicately. remarkably, these delicate constraints are met, whence the quantum boundary conditions do admit a sufficient number of solutions. we will conclude by summarizing the nature of geometry of the quantum horizon that results. given any state satisfying equation (93), the curvature f of w vanishes everywhere except at the points at which the polymer excitations in the bulk puncture s. the holonomy around each puncture is non - trivial. consequently, the intrinsic geometry of the quantum horizon is flat except at the punctures. at each puncture, there is a deficit angle, whose value is determined by the holonomy of w around that puncture. each deficit angle is quantized and these angles add up to 4 as in a discretized model of a 2-sphere geometry. thus, the quantum geometry of a wih is quite different from its smooth classical geometry. let us now summarize the ideas behind counting of surface microstates that leads to the expression of entropy. to incorporate dynamics in this canonical approach, we have to first construct physical states by imposing quantum einstein equations (i.e., quantum constraints). while the procedure is technically quite involved, the result is simple to state : what matters is only the number of punctures and not their locations. to calculate entropy, fix the number n of punctures and allow only those (non - zero) spin - labels ji and charge labels \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$q_i^\alpha$\end{document } on the polymer excitations which endow the horizon with a total area in an interval (a0, a0 +) and charges in an interval \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$(q_0^\alpha - { \epsilon ^\alpha},q_0^\alpha + { \epsilon ^\alpha})$\end{document}. (here i = 1, 2,, n and and are suitably small. we denote by \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\mathcal h}_n^{{\rm{bh}}}$\end{document } the sub - space of \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\mathcal h } = { { \mathcal h}_v } \otimes { { \mathcal h}_s}$\end{document } in which the volume states v are chosen with the above restrictions on ji and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$q_i^\alpha$\end{document }, and the total state satisfies the quantum horizon boundary condition as well as quantum einstein equations. then the desired micro - canonical ensemble consists of states in \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${{\mathcal h}^{{\rm{bh } } } } = { \otimes _ n}{\mathcal h}_n^{{\rm{bh}}}$\end{document}. note that, because there is no contribution to the symplectic structure from matter terms, surface states in \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${{\mathcal h}^{{\rm{bh}}}}$\end{document } refer only to the gravitational sector. the next step is to calculate the entropy of this quantum, micro - canonical ensemble. note first that what matters are only the surface states. for, the bulk - part describes, e.g., states of gravitational radiation and matter fields far away from and are irrelevant for the entropy s of the wih. heuristically, the idea then is to trace over the bulk states, construct a density matrix bh describing a maximum - entropy mixture of surface states and calculate tr bh ln bh. as is usual in entropy calculations, this translates to the evaluation of the dimension \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\mathcal n}$\end{document } of a well - defined sub - space of the surface hilbert space, namely the linear span of those surface states which occur in. entropy s is given by ln \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\mathcal n}$\end{document}. a detailed calculation [84, 149 ] leads to the following expression of entropy : 94\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${s_\delta } = { { { \gamma _ 0 } } \over \gamma}{{{a_0 } } \over { 4\ell _ { { \rm{pl}}}^2 } } - { 1 \over 2}\ln { { { a_0 } } \over { \ell _ { { \rm{pl}}}^2 } } + { \mathcal o}\left({\ln { { { a_0 } } \over { \ell _ { { \rm{pl}}}^2 } } } \right),\;\;\;\;{\rm{with}}{\gamma _ 0 } = \approx 0.2375,$$\end{document } where \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\mathcal o}(\ln ({ a_0}/\ell _ { { \rm{p}}1}^2))$\end{document } is a term which, when divided by \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${a_0}/\ell _ { { \rm{p}}1}^2$\end{document }, tends to zero as \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${a_0}/\ell _ { \rm{p}}^2$\end{document } tends to infinity. thus, for large black holes, the leading term in the expression of entropy is proportional to area. for example if, as in the early treatments [170, 36, 163, 164, 134, 135 ] one ignores the horizon boundary conditions and the resulting chern - simons term in the symplectic structure, one would find that s is proportional \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\sqrt { { a_0}}$\end{document}. however, the theory does not have a unique prediction because the numerical coefficient depends on the value of the barbero - immirzi parameter. the appearance of can be traced back directly to the fact that, in the -sector of the theory, the area eigenvalues are proportional to. one adopts a phenomenological viewpoint to fix this ambiguity. in the infinite dimensional space of geometries admitting as their inner boundary, one can fix one space - time, say the schwarzschild space - time with mass m0 mpl, (or, the de sitter space - time with the cosmological constant \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\lambda _ 0 } \ll 1/\ell _ { { \rm{p}}1}^2$\end{document }, or,). for agreement with semi - classical considerations in these cases, the leading contribution to entropy should be given by the hawking - bekenstein formula (87). we can go ahead and calculate the entropy of any other type i wih in this theory. furthermore, in this -sector, the statistical mechanical temperature of any type i wih is given by hawking s semi - classical value /(2) [29, 135 ]. thus, we can do one thought experiment observe the temperature of one large black hole from far away to eliminate the barbero - immirzi ambiguity and fix the theory. this theory then predicts the correct entropy and temperature for all wihs with a0, irrespective of other parameters such as the values of the electric or dilatonic charges or the cosmological constant. an added bonus comes from the fact that the isolated horizon framework naturally incorporates not only black hole horizons but also the cosmological ones for which thermodynamical considerations are also known to apply. the quantum entropy calculation is able to handle both these horizons in a single stroke, again for the same value = 0 of the barbero - immirzi parameter. in this sense, recall from section 6 that in presence of a scalar field which is non - minimally coupled to gravity, the first law is modified [123, 183, 184 ]. the modification suggests that the hawking - bekenstein formula \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${s_{{\rm{bh } } } } = { a_0}/(4\ell _ { { \rm{p}}1}^2)$\end{document } is no longer valid. if the non - minimal coupling is dictated by the action 95\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\widetilde{s}[{g_{ab}},\phi ] = \int { \left({{1 \over { 16\pi g}}f(\phi)r - { 1 \over 2}{g^{ab}}{\nabla _ a}\phi { \nabla _ b}\phi - v(\phi) } \right)\sqrt { - g } \;{d^4}x,}$$\end{document } where r is the scalar curvature of the metric gab and v is a potential for the scalar field, then the entropy should be given by 96\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${s_\delta } = { 1 \over { 4g\hbar}}\oint f (\phi)\;{d^2}v.$$\end{document } an immediate question arises : can the calculation be extended to this qualitatively new situation ? at first, this seems very difficult within an approach based on quantum geometry, such as the one described above, because the relation is non - geometric. however, the answer was shown to be in the affirmative for type i horizons. it turns out that the metric \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${{\bar q}_{ab}}$\end{document } on m is no longer coded in the gravitational momentum \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$p_i^a$\end{document } alone but depends also on the scalar field. this changes the surface term s in the symplectic structure (90) as well as the horizon boundary condition (89) just in the right way for the analysis to go through. furthermore, state counting now leads to the desired expression (96) precisely for the same value = 0 of the barbero - immirzi parameter. next, one can consider type ii horizons which can be distorted and rotating. in this case, all the (gravitational, electro - magnetic, and scalar field) multipoles are required as macroscopic parameters to fix the system of interest. therefore, now the appropriate ensemble is determined by fixing all these multipoles to lie in a small range around given values. this ensemble can be constructed by first introducing multipole moment operators and then restricting the quantum states to lie in the subspace of the hilbert space spanned by their eigenvectors with eigenvalues in the given intervals. again recent work shows that the state counting yields the hawking - bekenstein formula (87) for minimally coupled matter and its modification (96) for non - minimally coupled scalar field, for the same value = 0 of the barbero - immirzi parameter [8, 25 ]. to summarize, the isolated horizon framework serves as a natural point of departure for a statistical mechanical calculation of black hole entropy based on quantum geometry. how does this detailed analysis compare with the it from bit scenario with which we began first, the quantum horizon boundary conditions play a key role in the construction of a consistent quantum theory of the horizon geometry. thus, unlike in the it from bit scenario, the calculation pertains only to those 2-spheres s which are cross - sections of a wih. one can indeed divide the horizon into elementary cells as envisaged by wheeler : each cell contains a single puncture. however, the area of these cells is not fixed but is dictated by the j - label at the puncture. furthermore, there are not just 2 but rather 2j + 1 states associated with each cell. thus, the complete theory is much more subtle than that envisaged in the it from bit scenario. in the last six sections, we summarized the isolated and dynamical horizon frameworks and their applications. these provide a quasi - local and more physical paradigm for describing black holes both in the equilibrium and dynamical regimes. one of the most pleasing aspects of the paradigm is that it provides a unified approach to a variety of problems involving black holes, ranging from entropy calculations in quantum gravity, to analytical issues related to numerical simulations, to properties of hairy black holes, to laws of black hole mechanics. more importantly, as summarized in section 1, these frameworks enable one to significantly extend the known results based on killing and event horizons, and provide brand new laws in the dynamical regime. in this section, we will discuss some of the open issues whose resolution would lead to significant progress in various areas. isolated horizons this is the best understood part of the new paradigm. nonetheless, several important issues still remain. we will illustrate these with a few examples : black hole mechanics throughout, we assumed that the space - time metric is c (with k 3) and the topology of is s r. these assumptions rule out, by fiat, the presence of a nut charge. to incorporate a non - zero nut charge in black hole mechanics, one must either allow to be topologically s, or allow for wire singularities in the rotation 1-form a on. the zeroth law goes through in these more general situations. arguments based on euclidean methods [114, 146, 147 ] show that entropy is no longer given by the horizon area but there is also contribution due to misner strings. however, to our knowledge, a systematic derivation in the lorentzian regime is not yet available. such a derivation would provide a better understanding of the physical origin of the extra terms. the covariant phase space methods used in the isolated horizon framework should be applicable in this case. application to numerical relativity we saw in section 5.4 that, in the ih framework, one can introduce an approximate analog of future null infinity \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${{\mathcal i}^ + } $ \end{document } and invariant coordinate systems and tetrads in its neighborhood. with this structure, it is feasible to extract waveforms and energy fluxes in a reliable manner within the standard 3 + 1 cauchy evolution of numerical relativity, without having to do a cauchy characteristic matching or use conformal field equations. the challenge here is to develop, on the approximate \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${{\mathcal i}^ + } $ \end{document }, the analog of the basics of the bondi framework [54, 33, 185 ] at null infinity. colored black holes as discussed in section 6, black hole uniqueness theorems of the einstein - maxwell theory fail once non - abelian fields are included. for example, there are black hole solutions to the einstein - yang - mills equations with non - trivial yang - mills fields whose only non - zero charge at infinity is the adm mass. thus, from the perspective of infinity, they are indistinguishable from the schwarzschild solution. however, this example suggests that perhaps the uniqueness theorems fail because of the insistence on evaluating charges at infinity. corichi, nucamendi, and sudarsky have conjectured that the uniqueness theorems could be restored if they are formulated in terms of all the relevant horizon charges. this is a fascinating idea and it has been pursued numerically. however, care is needed because the list of all relevant charges may not be obvious a priori. for example, in the case of static but not necessarily spherical yang - mills black holes, the conjecture seemed to fail until one realized that, in addition to the standard yang - mills charges at the horizon, one must also include a topological, winding charge in the list. once this charge is included, uniqueness is restored not only in the static sector of the einstein - yang - mills theory, but also when higgs fields and dilatons are included. the existence of these semi - numerical proofs suggests that it should be possible to establish uniqueness completely analytically. a second set of problems involves the surprising relations, e.g., between the soliton masses and horizon properties of colored black holes, obtained using isolated horizons. extensions of these results to situations with non - zero angular momentum should be possible and may well provide yet new insights. quantum black holes in the approach based on isolated horizons, the microscopic degrees of freedom responsible for the statistical mechanical entropy of black holes are directly related to the quantum geometry of horizons. therefore, their relation to the curved space - time geometry is clearer than in, say, the string theory calculations based on d - branes. therefore, one can now attempt to calculate the hawking radiation directly in the physical spacetime as a process in which quanta of area are converted to quanta of matter a direct approach requires quantum field theory (of matter fields) on a quantum geometry state which is approximated by the classical black hole space - time. the concrete open problem is the calculation of the absorption cross - section for quantum matter fields propagating on this background state. if this can be shown to equal the classical absorption cross - section to the leading order, it would follow that the spectrum of the outgoing particles would be thermal at the hawking temperature. another, perhaps more fruitful, avenue is to introduce an effective model whose hamiltonian captures the process by which quanta of horizon area are converted to quanta of matter. both these approaches are geared to large black holes which can be regarded as being in equilibrium for the process under consideration, i.e., when \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${a_\delta } \gg \ell _ { { \rm{p}}1}^2$\end{document}. however, this approximation would fail in the planck regime whence the approaches can not address issues related to information loss. using ideas first developed in the context of quantum cosmology, effects of the quantum nature of geometry on the black hole singularity have recently been analyzed. as in the earlier analysis of the big - bang singularity, it is found that the black hole singularity is resolved, but the classical space - time dissolves in the planck regime. therefore, the familiar penrose diagrams are no longer faithful representations of the physical situation. suppose that, evolving through what was singularity in the classical theory, the quantum state becomes semi - classical again on the other side. then, the indications are that information would not be lost : it would be recovered on full \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${{\mathcal i}^ + } $ \end{document }, although observers restricted to lie in the part of space - time which is completely determined by the data on \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${{\mathcal i}^ -}$\end{document } would see an approximate hawking radiation. if on the other hand the evolved state on the other side never becomes semi - classical, information would not be recovered on the available \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${{\mathcal i}^ + } $ \end{document}. an outstanding open issue is which of this possibility is actually realized. dynamical horizons the dh framework is less developed and the number of open issues is correspondingly higher. at least for the classical applications of the framework, these problems are more important. free data and multipoles since h is space - like, to find the fields (qab, kab) which constitute the dh geometry, one has to solve just the initial value equations, subject to the condition that h admits a foliation by marginally trapped surfaces. a general solution of this problem would provide the free data on dhs. in the case when the marginally trapped surfaces are round spheres, this problem has been analyzed by bartnik and isenberg. as noted in section 2.2, in this case there are no dhs in absence of matter sources. in presence of matter, one can freely specify the trace k of the extrinsic curvature and the (radial component of the) momentum density \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${t_{ab}}{{\hat \tau}^a}{{\hat r}^b}$\end{document }, and determine the geometry by solving a non - linear ordinary differential equation whose solutions always exist locally. it would be interesting to analyze the necessary and sufficient conditions on the free data which guarantee that global solutions exist and the dh approaches the schwarzschild horizon asymptotically. from the point of view of numerical relativity, a more pressing challenge is to solve the constraint equations in the vacuum case, assuming only that the marginally trapped surfaces are axi - symmetric. using the free data, as in the case of isolated horizons, one could define multipoles. since 2 is again defined unambiguously, a natural starting point is to use it as the key geometrical object as in the ih case. however, just as the bondi mass aspect acquires shear terms in presence of gravitational radiation, it is likely that, in the transition from isolated to dynamical horizons, 2 would have to be supplemented with terms involving, e.g., ab (and perhaps also). for instance, by adding a suitable combination of such terms, one may be able to relate the rate of change of the mass quadrupole moment with the flux of energy across h. geometric analysis the dynamical horizon framework provides new inputs for the proof of penrose inequalities which, when applied to time symmetric data (i.e., when the extrinsic curvature vanishes), say that the total (adm) mass of space - time must be greater than half the radius of the apparent horizon on any cauchy slice. recently, for the non - time symmetric case, ben - dov has constructed an example where the apparent horizon does not satisfy this inequality. this is not a contradiction with the original penrose inequality which referred to the area of cross - sections of the event horizon, however it does show that extending the results beyond time - symmetry would be quite non - trivial. the flows which led to the area law in section 3 and balance equations in section 4.2.2 may be potentially useful for this purpose. this approach could lead to an inequality relating the area of certain marginally trapped surfaces (the ones connected to future time like infinity via a dynamical horizon) to the future limit of the bondi mass. the framework also suggests a program which could shed much light on what john wheeler called the issue of the final state : what are the final equilibrium states of a dynamical black hole and how, in detail, is this equilibrium reached ? from a mathematical perspective an important step in addressing this issue is to analyze the non - linear stability of kerr black holes. consider, then, a neighborhood of the initial data of the kerr solution in an appropriate sobolev space. one would expect the space - time resulting from evolution of this data to admit a dynamical horizon which, in the distant future, tends to an isolated horizon with geometry that is isomorphic to that of a kerr horizon. one avenue is to first establish that the solution admits a kerr - schild type foliation, each leaf of which admits an apparent horizon, then show that the world tube of these apparent horizons is a dh, and finally study the decay rates of fields along this dh. angular momentum as we saw in section 5, most of the current work on ihs and dhs assumes the presence of an axial symmetry on the horizon. a natural question arises : can one weaken this requirement to incorporate situations in which there is only an approximate rather than an exact symmetry of the horizon geometry ? recall first that the newman - penrose component 2 is gauge invariant on ihs and dhs. let \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$f = { \vert{\psi _ 2}\vert}^2$\end{document}. (while any geometric field could be used here, f is the most natural candidate because, for ihs, 2 encodes the horizon geometry.) if the horizon geometry admits a symmetry, the orbits of the symmetry field are the level surfaces of f. more generally, let us suppose that the level surfaces of f provide a foliation of each good cut s (minus two points) of the horizon. then, using the procedure of section 2.1 of, one can introduce on s a vector field \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\phi _ 0^a$\end{document }, tangential to the foliation, which has the property that it agrees with the symmetry vector field whenever the horizon geometry admits a symmetry. the procedure fails if the metric on s is at of a round sphere but should work generically. one can then use \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\phi _ 0^a$\end{document } to define angular momentum. on ihs this angular momentum is conserved ; on dhs it satisfies the balance law of section 4.2.2 ; and in terms of the initial data, angular momentum \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${j^{({\phi _ 0})}}$\end{document } has the familiar form (59). is this proposal viable for numerical simulations ? in cases ready analyzed, it would be interesting to construct \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\phi _ 0^a$\end{document } d compare it with the symmetry vector field obtained via killing transport. a second issue associated with angular momentum is ether the kerr inequality j gm can be violated in the early stages of black hole formation or merger, particularly in a non - axisymmetric context. note that such an occurrence is not incompatible with the dh finally settling down to a kerr horizon. for, there is likely to be radiation trapped between the dh and the peak of the effective gravitational potential that could fall into the dh as time elapses, reducing its angular momentum and increasing its mass. the issue of whether a black hole can violate the kerr inequality when it is first formed is of considerable interest to astrophysics. again, numerical simulations involving, say, brill waves would shed considerable light on this possibility. black hole thermodynamics the fact that an integral version of the first law is valid even for non - equilibrium processes, during which the horizon makes a transition from a given state to one which is far removed, has interesting thermodynamic ramifications. in non - equilibrium thermodynamical processes, in general the system does not have time to come to equilibrium, whence there is no canonical notion of its temperature. therefore, while one can still interpret the difference e2 e1 (work) as the heat q absorbed by the system, in general there is no longer a clean split q = ts of this term into a temperature part and a change in entropy part. if the process is such that the system remains close to equilibrium throughout the process, i.e., can be thought of as making continuous transitions between a series of equilibrium states, then the difference can be expressed as tds, where the temperature t varies slowly during the transition. it is only when the horizon geometry is changing slowly that the effective surface gravity \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\bar \kappa}$\end{document } of section 4.2.2 would be a good measure of temperature, and the horizon area a good measure of entropy (see section 5.3 of). can the black hole entropy derivations based on counting of micro - states, such as those of, be extended to such dhs ? in the case of event horizons one would not expect such a procedure to be meaningful because, as we saw in equation 2.2.2, an event horizon can be formed and grow in a flat space region in anticipation of a future gravitational collapse. it is difficult to imagine how a quasi - local counting of micro - states can account for this phenomenon. we will illustrate these with a few examples : black hole mechanics throughout, we assumed that the space - time metric is c (with k 3) and the topology of is s r. these assumptions rule out, by fiat, the presence of a nut charge. to incorporate a non - zero nut charge in black hole mechanics, one must either allow to be topologically s, or allow for wire singularities in the rotation 1-form a on. the zeroth law goes through in these more general situations. arguments based on euclidean methods [114, 146, 147 ] show that entropy is no longer given by the horizon area but there is also contribution due to misner strings. however, to our knowledge, a systematic derivation in the lorentzian regime is not yet available. such a derivation would provide a better understanding of the physical origin of the extra terms. the covariant phase space methods used in the isolated horizon framework should be applicable in this case. application to numerical relativity we saw in section 5.4 that, in the ih framework, one can introduce an approximate analog of future null infinity \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${{\mathcal i}^ + } $ \end{document } and invariant coordinate systems and tetrads in its neighborhood. with this structure, it is feasible to extract waveforms and energy fluxes in a reliable manner within the standard 3 + 1 cauchy evolution of numerical relativity, without having to do a cauchy characteristic matching or use conformal field equations. the challenge here is to develop, on the approximate \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${{\mathcal i}^ + } $ \end{document }, the analog of the basics of the bondi framework [54, 33, 185 ] at null infinity. colored black holes as discussed in section 6, black hole uniqueness theorems of the einstein - maxwell theory fail once non - abelian fields are included. for example, there are black hole solutions to the einstein - yang - mills equations with non - trivial yang - mills fields whose only non - zero charge at infinity is the adm mass. thus, from the perspective of infinity, they are indistinguishable from the schwarzschild solution. however, their horizon properties are quite different from those of the schwarzschild horizon. this example suggests that perhaps the uniqueness theorems fail because of the insistence on evaluating charges at infinity. corichi, nucamendi, and sudarsky have conjectured that the uniqueness theorems could be restored if they are formulated in terms of all the relevant horizon charges. however, care is needed because the list of all relevant charges may not be obvious a priori. for example, in the case of static but not necessarily spherical yang - mills black holes, the conjecture seemed to fail until one realized that, in addition to the standard yang - mills charges at the horizon, one must also include a topological, winding charge in the list. once this charge is included, uniqueness is restored not only in the static sector of the einstein - yang - mills theory, but also when higgs fields and dilatons are included. the existence of these semi - numerical proofs suggests that it should be possible to establish uniqueness completely analytically. a second set of problems involves the surprising relations, e.g., between the soliton masses and horizon properties of colored black holes, obtained using isolated horizons. extensions of these results to situations with non - zero angular momentum should be possible and may well provide yet new insights. quantum black holes in the approach based on isolated horizons, the microscopic degrees of freedom responsible for the statistical mechanical entropy of black holes are directly related to the quantum geometry of horizons. therefore, their relation to the curved space - time geometry is clearer than in, say, the string theory calculations based on d - branes. therefore, one can now attempt to calculate the hawking radiation directly in the physical spacetime as a process in which quanta of area are converted to quanta of matter a direct approach requires quantum field theory (of matter fields) on a quantum geometry state which is approximated by the classical black hole space - time. the concrete open problem is the calculation of the absorption cross - section for quantum matter fields propagating on this background state. if this can be shown to equal the classical absorption cross - section to the leading order, it would follow that the spectrum of the outgoing particles would be thermal at the hawking temperature. another, perhaps more fruitful, avenue is to introduce an effective model whose hamiltonian captures the process by which quanta of horizon area are converted to quanta of matter. both these approaches are geared to large black holes which can be regarded as being in equilibrium for the process under consideration, i.e., when \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${a_\delta } \gg \ell _ { { \rm{p}}1}^2$\end{document}. however, this approximation would fail in the planck regime whence the approaches can not address issues related to information loss. using ideas first developed in the context of quantum cosmology, effects of the quantum nature of geometry on the black hole singularity have recently been analyzed. as in the earlier analysis of the big - bang singularity, it is found that the black hole singularity is resolved, but the classical space - time dissolves in the planck regime. therefore, the familiar penrose diagrams are no longer faithful representations of the physical situation. suppose that, evolving through what was singularity in the classical theory, the quantum state becomes semi - classical again on the other side. then, the indications are that information would not be lost : it would be recovered on full \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${{\mathcal i}^ + } $ \end{document }, although observers restricted to lie in the part of space - time which is completely determined by the data on \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${{\mathcal i}^ -}$\end{document } would see an approximate hawking radiation. if on the other hand the evolved state on the other side never becomes semi - classical, information would not be recovered on the available \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${{\mathcal i}^ + } $ \end{document}. an outstanding open issue is which of this possibility is actually realized. black hole mechanics throughout, we assumed that the space - time metric is c (with k 3) and the topology of is s r. these assumptions rule out, by fiat, the presence of a nut charge. to incorporate a non - zero nut charge in black hole mechanics, one must either allow to be topologically s, or allow for wire singularities in the rotation 1-form a on. the zeroth law goes through in these more general situations. arguments based on euclidean methods [114, 146, 147 ] show that entropy is no longer given by the horizon area but there is also contribution due to misner strings. however, to our knowledge, a systematic derivation in the lorentzian regime is not yet available. such a derivation would provide a better understanding of the physical origin of the extra terms. the covariant phase space methods used in the isolated horizon framework should be applicable in this case. application to numerical relativity we saw in section 5.4 that, in the ih framework, one can introduce an approximate analog of future null infinity \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${{\mathcal i}^ + } $ \end{document } and invariant coordinate systems and tetrads in its neighborhood. with this structure, it is feasible to extract waveforms and energy fluxes in a reliable manner within the standard 3 + 1 cauchy evolution of numerical relativity, without having to do a cauchy characteristic matching or use conformal field equations. the challenge here is to develop, on the approximate \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${{\mathcal i}^ + } $ \end{document }, the analog of the basics of the bondi framework [54, 33, 185 ] at null infinity. colored black holes as discussed in section 6, black hole uniqueness theorems of the einstein - maxwell theory fail once non - abelian fields are included. for example, there are black hole solutions to the einstein - yang - mills equations with non - trivial yang - mills fields whose only non - zero charge at infinity is the adm mass. thus, from the perspective of infinity, they are indistinguishable from the schwarzschild solution. however, their horizon properties are quite different from those of the schwarzschild horizon. this example suggests that perhaps the uniqueness theorems fail because of the insistence on evaluating charges at infinity. corichi, nucamendi, and sudarsky have conjectured that the uniqueness theorems could be restored if they are formulated in terms of all the relevant horizon charges. this is a fascinating idea and it has been pursued numerically. however, care is needed because the list of all relevant charges may not be obvious a priori. for example, in the case of static but not necessarily spherical yang - mills black holes, the conjecture seemed to fail until one realized that, in addition to the standard yang - mills charges at the horizon, one must also include a topological, winding charge in the list. once this charge is included, uniqueness is restored not only in the static sector of the einstein - yang - mills theory, but also when higgs fields and dilatons are included. the existence of these semi - numerical proofs suggests that it should be possible to establish uniqueness completely analytically. a second set of problems involves the surprising relations, e.g., between the soliton masses and horizon properties of colored black holes, obtained using isolated horizons. extensions of these results to situations with non - zero angular momentum should be possible and may well provide yet new insights. quantum black holes in the approach based on isolated horizons, the microscopic degrees of freedom responsible for the statistical mechanical entropy of black holes are directly related to the quantum geometry of horizons. therefore, their relation to the curved space - time geometry is clearer than in, say, the string theory calculations based on d - branes. therefore, one can now attempt to calculate the hawking radiation directly in the physical spacetime as a process in which quanta of area are converted to quanta of matter a direct approach requires quantum field theory (of matter fields) on a quantum geometry state which is approximated by the classical black hole space - time. the concrete open problem is the calculation of the absorption cross - section for quantum matter fields propagating on this background state. if this can be shown to equal the classical absorption cross - section to the leading order, it would follow that the spectrum of the outgoing particles would be thermal at the hawking temperature. another, perhaps more fruitful, avenue is to introduce an effective model whose hamiltonian captures the process by which quanta of horizon area are converted to quanta of matter. both these approaches are geared to large black holes which can be regarded as being in equilibrium for the process under consideration, i.e., when \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${a_\delta } \gg \ell _ { { \rm{p}}1}^2$\end{document}. however, this approximation would fail in the planck regime whence the approaches can not address issues related to information loss. using ideas first developed in the context of quantum cosmology, effects of the quantum nature of geometry on the black hole singularity have recently been analyzed. as in the earlier analysis of the big - bang singularity, it is found that the black hole singularity is resolved, but the classical space - time dissolves in the planck regime. therefore, the familiar penrose diagrams are no longer faithful representations of the physical situation. suppose that, evolving through what was singularity in the classical theory, the quantum state becomes semi - classical again on the other side. then, the indications are that information would not be lost : it would be recovered on full \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${{\mathcal i}^ + } $ \end{document }, although observers restricted to lie in the part of space - time which is completely determined by the data on \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${{\mathcal i}^ -}$\end{document } would see an approximate hawking radiation. if on the other hand the evolved state on the other side never becomes semi - classical, information would not be recovered on the available \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${{\mathcal i}^ + } $ \end{document}. an outstanding open issue is which of this possibility is actually realized. the dh framework is less developed and the number of open issues is correspondingly higher. at least for the classical applications of the framework, these problems are more important. free data and multipoles since h is space - like, to find the fields (qab, kab) which constitute the dh geometry, one has to solve just the initial value equations, subject to the condition that h admits a foliation by marginally trapped surfaces. free data on dhs. in the case when the marginally trapped surfaces are round spheres, this problem has been analyzed by bartnik and isenberg. as noted in section 2.2, in this case, one can freely specify the trace k of the extrinsic curvature and the (radial component of the) momentum density \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${t_{ab}}{{\hat \tau}^a}{{\hat r}^b}$\end{document }, and determine the geometry by solving a non - linear ordinary differential equation whose solutions always exist locally. it would be interesting to analyze the necessary and sufficient conditions on the free data which guarantee that global solutions exist and the dh approaches the schwarzschild horizon asymptotically. from the point of view of numerical relativity, a more pressing challenge is to solve the constraint equations in the vacuum case, assuming only that the marginally trapped surfaces are axi - symmetric. using the free data, as in the case of isolated horizons since 2 is again defined unambiguously, a natural starting point is to use it as the key geometrical object as in the ih case. however, just as the bondi mass aspect acquires shear terms in presence of gravitational radiation, it is likely that, in the transition from isolated to dynamical horizons, 2 would have to be supplemented with terms involving, e.g., ab (and perhaps also). for instance, by adding a suitable combination of such terms, one may be able to relate the rate of change of the mass quadrupole moment with the flux of energy across h. geometric analysis the dynamical horizon framework provides new inputs for the proof of penrose inequalities which, when applied to time symmetric data (i.e., when the extrinsic curvature vanishes), say that the total (adm) mass of space - time must be greater than half the radius of the apparent horizon on any cauchy slice. recently, for the non - time symmetric case, ben - dov has constructed an example where the apparent horizon does not satisfy this inequality. this is not a contradiction with the original penrose inequality which referred to the area of cross - sections of the event horizon, however it does show that extending the results beyond time - symmetry would be quite non - trivial. the flows which led to the area law in section 3 and balance equations in section 4.2.2 may be potentially useful for this purpose. this approach could lead to an inequality relating the area of certain marginally trapped surfaces (the ones connected to future time like infinity via a dynamical horizon) to the future limit of the bondi mass. the framework also suggests a program which could shed much light on what john wheeler called the issue of the final state : what are the final equilibrium states of a dynamical black hole and how, in detail, is this equilibrium reached ? from a mathematical perspective an important step in addressing this issue is to analyze the non - linear stability of kerr black holes. consider, then, a neighborhood of the initial data of the kerr solution in an appropriate sobolev space. one would expect the space - time resulting from evolution of this data to admit a dynamical horizon which, in the distant future, tends to an isolated horizon with geometry that is isomorphic to that of a kerr horizon. one avenue is to first establish that the solution admits a kerr - schild type foliation, each leaf of which admits an apparent horizon, then show that the world tube of these apparent horizons is a dh, and finally study the decay rates of fields along this dh. angular momentum as we saw in section 5, most of the current work on ihs and dhs assumes the presence of an axial symmetry on the horizon. a natural question arises : can one weaken this requirement to incorporate situations in which there is only an approximate rather than an exact symmetry of the horizon geometry ? recall first that the newman - penrose component 2 is gauge invariant on ihs and dhs. let \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$f = { \vert{\psi _ 2}\vert}^2$\end{document}. (while any geometric field could be used here, f is the most natural candidate because, for ihs, 2 encodes the horizon geometry.) if the horizon geometry admits a symmetry, the orbits of the symmetry field are the level surfaces of f. more generally, let us suppose that the level surfaces of f provide a foliation of each good cut s (minus two points) of the horizon. then, using the procedure of section 2.1 of, one can introduce on s a vector field \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\phi _ 0^a$\end{document }, tangential to the foliation, which has the property that it agrees with the symmetry vector field whenever the horizon geometry admits a symmetry. the procedure fails if the metric on s is at of a round sphere but should work generically. one can then use \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\phi _ 0^a$\end{document } to define angular momentum. on ihs this angular momentum is conserved ; on dhs it satisfies the balance law of section 4.2.2 ; and in terms of the initial data, angular momentum \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${j^{({\phi _ 0})}}$\end{document } has the familiar form (59). is this proposal viable for numerical simulations ? in cases ready analyzed, it would be interesting to construct \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\phi _ 0^a$\end{document } d compare it with the symmetry vector field obtained via killing transport. a second issue associated with angular momentum is ether the kerr inequality j gm can be violated in the early stages of black hole formation or merger, particularly in a non - axisymmetric context. note that such an occurrence is not incompatible with the dh finally settling down to a kerr horizon. for, there is likely to be radiation trapped between the dh and the peak of the effective gravitational potential that could fall into the dh as time elapses, reducing its angular momentum and increasing its mass. the issue of whether a black hole can violate the kerr inequality when it is first formed is of considerable interest to astrophysics. again, numerical simulations involving, say, brill waves would shed considerable light on this possibility. black hole thermodynamics the fact that an integral version of the first law is valid even for non - equilibrium processes, during which the horizon makes a transition from a given state to one which is far removed, has interesting thermodynamic ramifications. in non - equilibrium thermodynamical processes, in general the system does not have time to come to equilibrium, whence there is no canonical notion of its temperature. therefore, while one can still interpret the difference e2 e1 (work) as the heat q absorbed by the system, in general there is no longer a clean split q = ts of this term into a temperature part and a change in entropy part. if the process is such that the system remains close to equilibrium throughout the process, i.e., can be thought of as making continuous transitions between a series of equilibrium states, then the difference can be expressed as tds, where the temperature t varies slowly during the transition. it is only when the horizon geometry is changing slowly that the effective surface gravity \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\bar \kappa}$\end{document } of section 4.2.2 would be a good measure of temperature, and the horizon area a good measure of entropy (see section 5.3 of). can the black hole entropy derivations based on counting of micro - states, such as those of, be extended to such dhs ? in the case of event horizons one would not expect such a procedure to be meaningful because, as we saw in equation 2.2.2, an event horizon can be formed and grow in a flat space region in anticipation of a future gravitational collapse. it is difficult to imagine how a quasi - local counting of micro - states can account for this phenomenon. free data and multipoles since h is space - like, to find the fields (qab, kab) which constitute the dh geometry, one has to solve just the initial value equations, subject to the condition that h admits a foliation by marginally trapped surfaces. free data on dhs. in the case when the marginally trapped surfaces are round spheres, this problem has been analyzed by bartnik and isenberg. as noted in section 2.2, in this case, one can freely specify the trace k of the extrinsic curvature and the (radial component of the) momentum density \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${t_{ab}}{{\hat \tau}^a}{{\hat r}^b}$\end{document }, and determine the geometry by solving a non - linear ordinary differential equation whose solutions always exist locally. it would be interesting to analyze the necessary and sufficient conditions on the free data which guarantee that global solutions exist and the dh approaches the schwarzschild horizon asymptotically. from the point of view of numerical relativity, a more pressing challenge is to solve the constraint equations in the vacuum case, assuming only that the marginally trapped surfaces are axi - symmetric. using the free data, as in the case of isolated horizons since 2 is again defined unambiguously, a natural starting point is to use it as the key geometrical object as in the ih case. however, just as the bondi mass aspect acquires shear terms in presence of gravitational radiation, it is likely that, in the transition from isolated to dynamical horizons, 2 would have to be supplemented with terms involving, e.g., ab (and perhaps also). for instance, by adding a suitable combination of such terms, one may be able to relate the rate of change of the mass quadrupole moment with the flux of energy across h. the dynamical horizon framework provides new inputs for the proof of penrose inequalities which, when applied to time symmetric data (i.e., when the extrinsic curvature vanishes), say that the total (adm) mass of space - time must be greater than half the radius of the apparent horizon on any cauchy slice. recently, for the non - time symmetric case, ben - dov has constructed an example where the apparent horizon does not satisfy this inequality. this is not a contradiction with the original penrose inequality which referred to the area of cross - sections of the event horizon, however it does show that extending the results beyond time - symmetry would be quite non - trivial. the flows which led to the area law in section 3 and balance equations in section 4.2.2 may be potentially useful for this purpose. this approach could lead to an inequality relating the area of certain marginally trapped surfaces (the ones connected to future time like infinity via a dynamical horizon) to the future limit of the bondi mass. the framework also suggests a program which could shed much light on what john wheeler called the issue of the final state : what are the final equilibrium states of a dynamical black hole and how, in detail, is this equilibrium reached ? from a mathematical perspective an important step in addressing this issue is to analyze the non - linear stability of kerr black holes. consider, then, a neighborhood of the initial data of the kerr solution in an appropriate sobolev space. one would expect the space - time resulting from evolution of this data to admit a dynamical horizon which, in the distant future, tends to an isolated horizon with geometry that is isomorphic to that of a kerr horizon. one avenue is to first establish that the solution admits a kerr - schild type foliation, each leaf of which admits an apparent horizon, then show that the world tube of these apparent horizons is a dh, and finally study the decay rates of fields along this dh. as we saw in section 5, most of the current work on ihs and dhs assumes the presence of an axial symmetry on the horizon. a natural question arises : can one weaken this requirement to incorporate situations in which there is only an approximate rather than an exact symmetry of the horizon geometry ? recall first that the newman - penrose component 2 is gauge invariant on ihs and dhs. let \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$f = { \vert{\psi _ 2}\vert}^2$\end{document}. (while any geometric field could be used here, f is the most natural candidate because, for ihs, 2 encodes the horizon geometry.) if the horizon geometry admits a symmetry, the orbits of the symmetry field are the level surfaces of f. more generally, let us suppose that the level surfaces of f provide a foliation of each good cut s (minus two points) of the horizon. then, using the procedure of section 2.1 of, one can introduce on s a vector field \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\phi _ 0^a$\end{document }, tangential to the foliation, which has the property that it agrees with the symmetry vector field whenever the horizon geometry admits a symmetry. the procedure fails if the metric on s is at of a round sphere but should work generically. one can then use \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\phi _ 0^a$\end{document } to define angular momentum. on ihs this angular momentum is conserved ; on dhs it satisfies the balance law of section 4.2.2 ; and in terms of the initial data, angular momentum \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${j^{({\phi _ 0})}}$\end{document } has the familiar form (59). is this proposal viable for numerical simulations ? in cases ready analyzed, it would be interesting to construct \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\phi _ 0^a$\end{document } d compare it with the symmetry vector field obtained via killing transport. a second issue associated with angular momentum is ether the kerr inequality j gm can be violated in the early stages of black hole formation or merger, particularly in a non - axisymmetric context. note that such an occurrence is not incompatible with the dh finally settling down to a kerr horizon. for, there is likely to be radiation trapped between the dh and the peak of the effective gravitational potential that could fall into the dh as time elapses, reducing its angular momentum and increasing its mass. the issue of whether a black hole can violate the kerr inequality when it is first formed is of considerable interest to astrophysics. again, numerical simulations involving, say, brill waves would shed considerable light on this possibility. black hole thermodynamics the fact that an integral version of the first law is valid even for non - equilibrium processes, during which the horizon makes a transition from a given state to one which is far removed, has interesting thermodynamic ramifications. in non - equilibrium thermodynamical processes, in general the system does not have time to come to equilibrium, whence there is no canonical notion of its temperature. therefore, while one can still interpret the difference e2 e1 (work) as the heat q absorbed by the system, in general there is no longer a clean split q = ts of this term into a temperature part and a change in entropy part. if the process is such that the system remains close to equilibrium throughout the process, i.e., can be thought of as making continuous transitions between a series of equilibrium states, then the difference can be expressed as tds, where the temperature t varies slowly during the transition. it is only when the horizon geometry is changing slowly that the effective surface gravity \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\bar \kappa}$\end{document } of section 4.2.2 would be a good measure of temperature, and the horizon area a good measure of entropy (see section 5.3 of). can the black hole entropy derivations based on counting of micro - states, such as those of, be extended to such dhs ? in the case of event horizons one would not expect such a procedure to be meaningful because, as we saw in equation 2.2.2, an event horizon can be formed and grow in a flat space region in anticipation of a future gravitational collapse. it is difficult to imagine how a quasi - local counting of micro - states can account for this phenomenon. perhaps the most surprising aspect of the current status of the theory of black holes is that so little is known about their properties in the fully dynamical and non - linear regime of general relativity. indeed, we do not even have a fully satisfactory definition of a dynamical black hole. first, they are defined only in space - times where one can unambiguously identify infinity. even in these restricted contexts, event horizons are teleological, can form in a flat region of space - time and grow even though there is no flux of energy of any kind across them. when astronomers tell us that there is a black hole in the center of milky way, they are certainly not referring to event horizons. numerical simulations [48, 102 ] suggest that the outermost marginally trapped world - tubes become dynamical horizons soon after they are formed. as we saw, dynamical horizons have a number of attractive properties that overcome the limitations of event horizons : they are defined quasi - locally, can not be formed in flat space - time, and their growth is dictated by balance laws with direct physical interpretation. physically, then, dynamical horizons satisfying the additional physical condition \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${{\mathcal l}_n}{\theta _ { (\ell) } } < 0$\end{document } (i.e., sfoths) appear to be good candidates to represent the surface of a black hole. if a canonical dynamical horizon could be singled out by imposing physically reasonable conditions, one could use it as the physical representation of an evolving black hole. a plausibility argument for the existence of a canonical dynamical horizon note first that on physical grounds it seems natural to associate a black hole with a connected, trapped region \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\mathcal t}$\end{document } in space - time (see section 2.2.1). hayward sketched a proof that, under seemingly natural but technically strong conditions, the dynamical portion of its boundary, \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\partial { \mathcal t}$\end{document }, would be a dynamical horizon h. this h could serve as the canonical representation of the surface of an evolving black hole. however, it is not clear whether hayward s assumptions are not too strong. to illustrate the concern, then, hayward s argument implies that there are no trapped surfaces outside h. on the other hand there has been a general expectation in the community that, given any point in the interior of the event horizon, there passes a (marginally) trapped surface through it (see, e.g.,). this would imply that the boundary of the trapped region is the event horizon which, being null, can not qualify as a dynamical horizon. but it is clear that this expectation contradicts the conclusion based on hayward s arguments. the primary reason is that very little is known about trapped and marginally trapped surfaces which fail to be spherical symmetric. because of this, we do not know the boundary of the trapped region even in the vaidya solution. if it should turn out that the second expectation is correct, one would conclude that hayward s assumptions on the properties of the boundary of the trapped region are not met in physically interesting situations. however, this would not rule out the possibility of singling out a canonical dynamical horizon through some other conditions (as, e.g., in the vaidya solution). but since this dynamical horizon would not be the boundary of the trapped region, one would be led to conclude that in the dynamical and fully non - linear regime, one has to give up the idea that there is a single 3-manifold that can be interpreted as the black hole surface without further qualifications. for certain questions and in certain situations, the dynamical horizon may be the appropriate concept, while for other questions and in different situations, the boundary of the trapped region (which may be the event horizon) may be more appropriate. | over the past three decades, black holes have played an important role in quantum gravity, mathematical physics, numerical relativity and gravitational wave phenomenology. however, conceptual settings and mathematical models used to discuss them have varied considerably from one area to another. over the last five years a new, quasi - local framework was introduced to analyze diverse facets of black holes in a unified manner. in this framework, evolving black holes are modelled by dynamical horizons and black holes in equilibrium by isolated horizons. we review basic properties of these horizons and summarize applications to mathematical physics, numerical relativity, and quantum gravity. this paradigm has led to significant generalizations of several results in black hole physics. specifically, it has introduced a more physical setting for black hole thermodynamics and for black hole entropy calculations in quantum gravity, suggested a phenomenological model for hairy black holes, provided novel techniques to extract physics from numerical simulations, and led to new laws governing the dynamics of black holes in exact general relativity. |
preterm delivery (ptd) is a major public health problem world - wide, which may lead to severe complications such as cerebral palsy, chronic respiratory disease, blindness and neuro - developmental delay. it is also one of the main causes of perinatal and neonatal death and imposes a large economic burden on the health - care system. increased rate of ptd in women who had ptd, high probability of ptd in women with a previous history of ptd and studies of pregnancy outcomes in twins suggest that genetic factors may be important determinants of ptd. in addition, previous studies suggest that the inflammation caused by subclinical genital tract infection may be associated with ptd. increased concentration of proinflammatory cytokines has been found in amniotic fluid, maternal and fetal blood of a significant number of women with ptd. tumor necrosis factor- (tnf-) is one of these a proinflammatory cytokine that can trigger a cascade of events and lead to ptd. it has been reported that a genetic variant single nucleotide polymorphism (snp) in the promoter region of tnf- gene at 308 (a g / a transition resulting in tnf-amino acid (aa) or genotyping assays (ga) genotypes) may be associated with increased activation of the tnf- gene compared with the gg genotype. given the common genetic background of ptd and tnf- expression, a number of studies have been performed to investigate the role of genetic variation at the level of snps within genes that regulate the inflammatory response, to determine whether there is an association between this genetic variations and predisposition to ptd in women or not. although large - scale studies have been conducted to assess the association of the g308a polymorphism of tnf- with ptd, no strong convincing evidence of association has been found and this hypothesis remains controversial. this study was designed to search for an association between ptd and maternal or fetal snp in tnf- 308 gene. after approval of the study by the ethic committee of shahrekord university of medical sciences and obtaining informed consent, this cross - sectional study was performed on mothers who were referred to the hajar hospital for delivery and their infants. this investigation was performed in shahrekord, iran, between march 2010 and april 2012. using convenience sampling method, pregnant women aged between 18 and 35 years with a gestational age (ga) between 28 and 42 weeks exclusion criteria were any history of cervical incompetency, polyhydramnios, preeclampsia, vaginal bleeding during pregnancy, fetal abnormality, cord or placental abnormalities, psychological problems, diabetes or other chronic cardiac, kidney, pulmonary or collagen vascular diseases. according to the gestational age at the time of delivery, women who met the study criteria were allocated to term (control) and preterm (case) groups. last menstrual period date confirmed by ultrasound dating was used to determine ga genotype. mothers with preterm contractions (presence of at least 2 regular uterine contractions per 10 min combined with documented finally, a total of 135 women and their infants were recruited (71 in the term group and 64 in the preterm group). for genotyping, a maternal peripheral blood sample and a fetal cord blood sample were taken. blood samples of 5 ml were collected in 15 ml falcon tubes already containing 60 l ethylene diamine tetra acetic acid (0.5 molar). all blood samples were frozen at 20c for 24 h before polymerase chain reaction (pcr) and genotyping. the sequence of the primer was forward (5-tccagactttgagacacg-3) and reverse (5- gggcgattacagacaact-3). according to the standard protocols, deoxyribonucleic acid (dna) was extracted from blood samples by the phenol - chloroform method using lysis buffer (tris - hcl10 ml, sucrose, mgcl2, 5ml, tryptone x10, 10 ml. the pcr was carried out in 0.25 ml tubes containing 100 ng sample of dna, 4 mmol 1x - pcr buffer (500 mmolkcl, tris - hcl with ph : 8.4 and 200 mmolams), mgcl2, 3 mmol, dntps : deoxy ribo nucleotide triphosphates, (dttp = deoxy thymidine triphosphate, datp deoxy adenosine triphosphate, dgtp deoxy guanosine triphosphate, dctp deoxy cytidine triphosphate) 0.2 mmol / l, dna taq polymerase 0.5 ml and primers cycling was performed at 95c for 5 min ; then for 34 cycles of 94c, for 50 s (denaturation), 60c for 50 s (annealing) and 72c for 30 s (extension) ; and finally for 5 min at 72c (final extension). then, 2.5 l of pcr amplification products was mixed with 0.5 l of loading dye and was electrophoresed on 5% polyacrylamide gels., they were mixed with 1 l of tango buffer and 0.5 l of nco1 enzyme for 3 h at 37. at this stage, 5 l of digested products was mixed with 4 l of loading dye and the sample was assessed using the polyacrylamide gel electrophoresis and silver staining [figure 1 ]. finally, tnf- 308 genotype and alleles were determined. white arrow : mutation, red arrow : mutation, blue arrow : negative control, green arrow : digested, yellow arrow : positive control (uncut) data was analyzed by the statistical package for the social sciences (spss) 20.0 (spss inc., chicago, il, usa) using independent t - test and chi - square. after approval of the study by the ethic committee of shahrekord university of medical sciences and obtaining informed consent, this cross - sectional study was performed on mothers who were referred to the hajar hospital for delivery and their infants. this investigation was performed in shahrekord, iran, between march 2010 and april 2012. using convenience sampling method, pregnant women aged between 18 and 35 years with a gestational age (ga) between 28 and 42 weeks exclusion criteria were any history of cervical incompetency, polyhydramnios, preeclampsia, vaginal bleeding during pregnancy, fetal abnormality, cord or placental abnormalities, psychological problems, diabetes or other chronic cardiac, kidney, pulmonary or collagen vascular diseases. according to the gestational age at the time of delivery, women who met the study criteria were allocated to term (control) and preterm (case) groups. last menstrual period date confirmed by ultrasound dating was used to determine ga genotype. mothers with preterm contractions (presence of at least 2 regular uterine contractions per 10 min combined with documented finally, a total of 135 women and their infants were recruited (71 in the term group and 64 in the preterm group). for genotyping, a maternal peripheral blood sample and a fetal cord blood sample were taken. blood samples of 5 ml were collected in 15 ml falcon tubes already containing 60 l ethylene diamine tetra acetic acid (0.5 molar). all blood samples were frozen at 20c for 24 h before polymerase chain reaction (pcr) and genotyping. the sequence of the primer was forward (5-tccagactttgagacacg-3) and reverse (5- gggcgattacagacaact-3). according to the standard protocols, deoxyribonucleic acid (dna) was extracted from blood samples by the phenol - chloroform method using lysis buffer (tris - hcl10 ml, sucrose, mgcl2, 5ml, tryptone x10, 10 ml. the pcr was carried out in 0.25 ml tubes containing 100 ng sample of dna, 4 mmol 1x - pcr buffer (500 mmolkcl, tris - hcl with ph : 8.4 and 200 mmolams), mgcl2, 3 mmol, dntps : deoxy ribo nucleotide triphosphates, (dttp = deoxy thymidine triphosphate, datp deoxy adenosine triphosphate, dgtp deoxy guanosine triphosphate, dctp deoxy cytidine triphosphate) 0.2 mmol / l, dna taq polymerase 0.5 ml and primers cycling was performed at 95c for 5 min ; then for 34 cycles of 94c, for 50 s (denaturation), 60c for 50 s (annealing) and 72c for 30 s (extension) ; and finally for 5 min at 72c (final extension). then, 2.5 l of pcr amplification products was mixed with 0.5 l of loading dye and was electrophoresed on 5% polyacrylamide gels. silver staining was used to detect proteins. in order to digestion of pcr products, they were mixed with 1 l of tango buffer and 0.5 l of nco1 enzyme for 3 h at 37. at this stage, 5 l of digested products was mixed with 4 l of loading dye and the sample was assessed using the polyacrylamide gel electrophoresis and silver staining [figure 1 ]. white arrow : mutation, red arrow : mutation, blue arrow : negative control, green arrow : digested, yellow arrow : positive control (uncut) data was analyzed by the statistical package for the social sciences (spss) 20.0 (spss inc., chicago, il, usa) using independent t - test and chi - square. mean of ga and neonatal birth weight was significantly lower in the case group. however, there was no significant difference between 2 groups regarding other baseline characteristics [table 1 ]. comparison of baseline characteristics between 2 groups only 1 (1%) mother in the control group and 2 (3%) mothers in the case group had ga genotype, which was not significantly different (p = 0.46). all cord samples in both groups had gg genotype except 1 (1.5%) sample in the case group, which had ga genotype. mean of ga and neonatal birth weight was significantly lower in the case group. however, there was no significant difference between 2 groups regarding other baseline characteristics [table 1 ]. only 1 (1%) mother in the control group and 2 (3%) mothers in the case group had ga genotype, which was not significantly different (p = 0.46). all cord samples in both groups had gg genotype except 1 (1.5%) sample in the case group, which had ga genotype. in spite of much effort and extensive research over the past decades, ptd remains an important issue in perinatal care. depending on the population, ptd may occur in 5 - 10% of pregnancies and cause serious perinatal morbidity and mortality. some investigators have reported an association between elevated levels of tnf- concentration in maternal blood and/or amniotic fluid and ptd. on the other hand, the position 308 in tnf- gene may have a normal guanine (g) allele or have a variant adenine (a) allele. it has been shown that g308a transition in the promoter region of tnf- gene results in tnf- aa or ga genotypes that may induce higher levels of tnf- gene transcription and activation and cause disease susceptibility in the human subjects. based on the combination of the above evidence, some investigators have hypothesized that preterm labor may be associated with genetic mutations in tnf- gene at some positions such as 308. a number of studies suggest that this genotype variant may affect the likelihood of ptd, but some others reject this association. present study demonstrated that despite higher frequency of ga mutation in tnf- gene at 308 position in mothers and neonates with ptd, this association was not statistically significant. our findings are consistent with several previous studies, which did not find such association. menon., performed a meta - analysis of available evidence and concluded that there is no statistically significant association between a snp in the tnf- gene ptd. dizon - townson. also compared the frequencies of tnf t1 or tnf t2 allele between women or fetuses delivered preterm with the control group or previously published allele frequencies and reported that the frequency of tnf- promoter mutation is not increased in either women or fetuses delivered pretermly. in addition, in vitro assays that used 308 snp and studies that used cells derived from subjects with various tnf- 308 genotypes showed inconsistent results and raised doubts about the functional significance of 308 snp. the presence of other snps in the tnf- gene and/or variation in other genes that can modify expression, translation and post - translational modification of tnf- gene could be the probable causes of this condition. a study performed by moura. suggests that the combination of tnf-, including interferon- and interleukin-6 maternal gene polymorphisms might contribute to susceptibility to ptd, not the tnf- polymorphism alone. because immunological response is most probably the result of the interaction between several polymorphic genes, it is not be surprising to find a significant association between a single polymorphism and an event such as ptd. therefore, it would be logical to investigate the combination of various polymorphisms in different genes to make an appropriate assessment, the role ofpolymorphic genes in the pathogenesis of ptd. in summary, we conclude that there is no significant association between ptd and either maternal or fetal tnf- 308 polymorphism and frequency of ga mutation is not significantly increased in mothers and neonates delivered preterm. it means that the presence of this mutation by itself does not modify the overall risk of ptd. investigations on the combination of various polymorphisms in different genes are recommended to achieve more accurate results. | background : increased concentrations of tumor necrosis factor alpha (tnf-) in blood and amniotic fluid are observed in women with preterm delivery (ptd) and tnf- mutations at 308 position are associated with higher expression of this gene. therefore, we compared the frequency of g308a transition in the promoter region of tnf- gene of women and neonates delivered preterm with the normal subjects.methods:this cross - sectional study was performed on 135 mothers who were referred for delivery. according to the gestational age, mothers and their neonates were allocated to the case (preterm, 64 subjects) and control (term, 71 subjects) groups. using the polymerase chain reaction, restrictive fragment length polymorphism (rflp), genotyping was performed on both maternal peripheral blood and cord blood samples to determine single nucleotide polymorphism in the promoter region of tnf- gene at 308.results : two mothers in the case group, one mother in the control group and one neonate in the case group had genotyping assays (ga) mutation. all other subjects had normal gg genotype. frequency of ga mutation was not significantly different between two groups (p = 0.47).conclusions : there is no significant association between ptd and either maternal or fetal tnf- 308 polymorphism and frequency ofgamutation is not significantly increased in mothers and neonates delivered preterm. it means that the presence of this mutation by itself does not modify the overall risk of ptd. investigations on the combination of various polymorphisms indifferent genes are recommended to achieve more accurate results. |
arterial chronic hypertension (htn) is one of the established cardiovascular risk factors for development of atherosclerosis and an increased incidence of peripheral vascular disease, cerebrovascular disease, chronic renal disease, and coronary artery disease. it is also an important risk factor for heart failure, myocardial infarction, stroke, and cardiovascular death [6, 7 ]. in patients with acute myocardial infarction (ami), the prevalence of antecedent hypertension varies from 31 to 59% [8, 9 ]. according to the available evidence, it is not clear whether previously known hypertensive patients have an increased rate of adverse outcomes after ami including stroke, heart failure, and cardiovascular death. conversely, in non - st elevation acute myocardial infarction (nstemi), htn is an independent factor for major short- and long - term cardiac adverse outcome. so far, data on long - term prognostic impact of hypertension in patients with acute coronary syndrome (including st - elevation myocardial infarction (stemi), nstemi, and unstable angina) are still controversial, and scarce data are available up to now. the aim of the present paper is to focus on hypertensive patients with acs, in order to better elucidate whether these patients are at higher risk and deserve a tailored approach for management and followup. arterial hypertension is one of the main factors leading to atherogenesis and the development of vulnerable plaques whose instability or rupture (which in turn results in thrombosis and vessel occlusions) are responsible for the development of acute coronary syndromes (acs). in the general population, the prevalence of hypertension rises progressively with age in both men and women, but it is higher at all ages in blacks in whom it is a stronger risk factor for coronary artery disease in respect to whites. about 54% of the united states population aged 65 to 74 years is hypertensive while among blacks the prevalence of hypertension is 72%. the age- and sex - adjusted prevalence of hypertension (at the 140/90 mm hg threshold) is 28% in the north american countries and 44% in the european countries. in gusto-1 trial, 41021 stemi patients who presented within 6 hours of symptom onset were randomized to receive different thrombolytic regimens : in this population the prevalence of a history of previous hypertension was 38.1% (15544 of 41021). in the gissi-2 (gruppo italiano per lo studio della streptochinasi nell'infarto) which included 20491 patients with stemi randomized to a 2 2 protocol of thrombolysis, a history of htn was present in about 35% of the whole population. however, to date, the prevalence of arterial hypertension in patients with acute myocardial infarction (ami) has not been adequately investigated, since most data were obtained from studies performed in the prefibrinolytic era (when drugs such as aspirin, statins, or beta - blockers were not yet part of the routine therapeutic list and few diagnostic or interventional procedures were carried out) or from clinical trials in which the patient characteristics differed considerably from those found in routine clinical practice. more recently, other studies focused on patients with stemi submitted to primary pci [16, 17 ] in which a previous history of hypertension was present in a range of 3033%. the symphony trial showed a prevalence of htn in stemi patients of more than 50% (probably due to different criteria of selection of the study population), and a recent spanish registry (primvac) reported a 46% prevalence of hypertension in stemi patients. similarly, in a recent paper by our group, performed in 856 stemi patients all submitted to primary pci, a previous history of hypertension was detectable in 50.6% (median age 67 years). from all the registries and the data available up to now [1220 ], hypertensive patients with stemi are more likely to be older, female, of non - white ethnicity, and having a higher prevalence of comorbidities such as diabetes, hypercholesterolemia, chronic renal failure, history of cardiac heart failure, prior myocardial infarction, and prior myocardial revascularization (angioplasty and stent implantation or coronary artery bypass graft). in epidemiological studies performed in non - st elevation myocardial infarction (nstemi) patients, chronic htn is the most prevalent risk factor being detectable in almost two thirds of entire population. this higher prevalence of htn in nstemi in respect to stemi patients (about 7075% versus 3040%) could be justified by the fact that nstemi patients are usually older and affected by more co - morbidities in respect to stemi patients. the relation between htn and myocardial infarction can be mainly explained underscoring two key factors : (1) common risk factors shared by the two diseases, such as genetic risk profiles, insulin resistance, sympathetic hyperactivity, and vasoactive substances (i.e., angiotensin ii) and (2) hypertension is associated with accelerated atherosclerosis, which contributes to progression of myocardial infarction (figure 1). genetic risk factors, specifically gene polymorphisms of the angiotensinogen - converting enzyme (ace) and of the renin - angiotensin - aldosterone system (ras), could represent a common milieu responsible for both hypertension and myocardial infarction, in a specific subset of patients at high risk for cardiovascular complications [24, 25 ]. insulin resistance is another risk factor : hyperinsulinemia is known to contribute to development of both atherosclerosis and hypertension. in fact, decreased effect of insulin on vascular smooth muscle tissue may result in decreased ability to modulate vascular smooth muscle cytoplasmic calcium and enhanced contractility (the so - called insulin resistance - induced hypertension). hyperinsulinemia can also promote atherosclerosis and vascular remodeling : in insulin - resistant patients the left ventricular mass index is greater and the intimal - medial complex of the common carotid artery and the frequency of plaques in carotid artery are higher. in a recent study by our group in 253 nondiabetic stemi patients submitted to percutaneous coronary intervention, acute insulin resistance, as assessed by homa index, was quite common and helped in the early prognostic stratification, as it represented an independent predictor of in - hospital mortality : in this series 47.2% of patients had a history of htn on admission. furthermore, in hypertensive patients, an hyperactivity of sympathetic tone may promote atherosclerosis by worsening insulin resistance, through sympathetic vasoconstriction on glucose extraction in skeletal muscle, beta - adrenoreceptor - mediated insulin resistance, and vascular rarefaction because of the closure of the smaller vessels due to vascular hypertrophy. sympathetic hyperactivity itself also contributes to a higher risk of sudden death, coronary spasm, and coronary thrombosis. the role of vasoactive substances, such as angiotensin ii, endothelin, natriuretic peptides, and nitric oxide, in hypertensive patients is still under debate. recent researches underscored the role of vascular, local produced angiotensin ii, in opposition to its circulating form, in remodeling of vascular structures such as aortic wall in animal models. one of the main factors leading to the development of atherosclerosis in hypertensive patients ' vessels is mechanical stress. it consists of three - dimensional forces : shear stress, transmural pressure, and wall stress. shear stress has been shown to be responsible for the activation of angiotensin ii in htn patients since cultured endothelial cells exposed to shear stress have a higher expression of ace gene. transmural pressure produces a net effect of pressure on endothelial cells in vitro (in lack of other forces) and vascular smooth cells, thus enhancing the production and growth of new smooth cells. lastly, stretch stress or wall stress, in cultured vascular smooth muscle cells, can increase the release of factors stimulating the dna and protein synthesis ; stretched smooth cells in turn induce ace activity and cell growth, finally causing a muscle cell hypertrophy. a chronic hypertensive state causes cardiac hypertrophy which is an independent risk factor for myocardial infarction. left ventricular hypertrophy is associated with increased oxygen demand leading to the development of new arterial vessels (collaterals) to supply the myocardium. this collateral circulation, driven by pressure gradient, is more effective in the subepicardial layer than in subendocardial layer, which therefore results in being more exposed to ischemia and infarction. during an acute coronary ischemic event, systolic blood pressure and decreased wall tension of ischemic area result in patency of small vessels, giving some blood supply to the ischemic area. on the other side, large reduction of diastolic blood pressure more often leads to ischemia, despite the lack of total vessel occlusion. lastly, hypertensive state is characterized by hemorheological abnormalities, such as hyperviscosity, endothelial dysfunction, and a prothrombotic state ; moreover it has been demonstrated that vascular inflammation as well as oxidative stress is more prevalent in htn patients [33, 34 ]. the clinical course of stemi can be affected by several complications, including (1) renal failure, due both to contrast induced nephropathy and acute heart failure, (2) cardiogenic shock, (3) major and minor bleedings causing a new anemic state, and (4) acute glucose imbalance. regarding acute renal failure, in a recent paper by rembek. comparing hypertensive and nonhypertensive patients with stemi, the hypertensive group showed a higher incidence of previous renal diseases such as nephrolithiasis and chronic glomerulonephritis and higher baseline values of urea and creatinine serum level. showed that reduced creatinine clearance was a significant adverse prognostic factor for mortality, including cardiovascular deaths, in hypertensive stemi patients, while anavekar. showed that moderate renal dysfunction (predicted by glomerular filtration rate) was associated with a higher rate of mi complications, in particular heart failure. it is likely that stemi hypertensive patients are more prone to develop a acute renal failure or a contrast induced nephropathy. a recent study by thiele. confirmed that in both hypertensive and nonhypertensive stemi patients, who undergo coronary angiography and angioplasty with stenting and moderate doses of contrast medium, only optimal hydration is effective in preventing a contrast - induced nephropathy, while n - acetylcysteine only reduces oxidative stress due to reperfusion after ami in comparison with placebo. it can be supposed that the target mean arterial pressure in hypertensive patients should be achieved in order to guarantee an adequate renal perfusion and to prevent acute renal failure. cardiogenic shock remains the leading cause of death in patients hospitalized with acute mi and in particular with stemi [40, 41 ], but its incidence has been greatly reduced by the use of reperfusion therapy through mechanical revascularization. one of the wider registries including cardiogenic shock patients, the shock trial, included 1190 patients from different countries, of which about 53% had a previous history of hypertension : overall mortality was decreased in comparison with previous registries, and the main cause was ventricular septum rupture. these data agree with our experience in patients with cardiogenic shock treated with iabp (hypertension was detectable in 51.3% of the entire population). free wall and interventricular septum ruptures occur more frequently in elderly patients of female sex, in previously known hypertensive patients and in those who suffer from their first acute myocardial infarction, with a preferential involvement of left ventricle and anterior wall at the junction of the infarct and normal muscle. partial or total rupture of a papillary muscle is more rare but often fatal and can be caused by an inferior wall infarction with involvement of posteromedial papillary muscle : partial rupture (tip or head of muscle) causes a massive but not immediately fatal mitral regurgitation (mr) while total rupture can lead to death (acute massive mr). in all these conditions, especially in patients with previously known and poorly controlled hypertensive state, high blood pressure values could play a pivotal role as they highly increase intracavitary pressures and shear stress force of muscular contraction against an inert and necrotic area, leading to laceration and then to rupture. this strengthens the importance of a strict blood pressure control, in the first hours after an acute myocardial infarction. specific data on the rate of hemodynamic and bioelectric complications of an acute mi in hypertensive patients are scarce and inconsistent. have reported that a history of hypertension in ami was associated with an increased incidence of sudden death. on the other hand, abrignani. found a lower incidence of shock, ventricular fibrillation, atrioventricular conduction disturbances, intracardiac thrombus, and cardiac rupture in hypertensive patients suffering from stemi, while atrial fibrillation was more common in these patients., hypertensive stemi patients showed higher incidence of cardiogenic shock, pulmonary oedema, ventricular tachycardia and/or fibrillation and third degree atrioventricular block ; they required intraaortic balloon counterpulsation (iabp) more than nonhypertensive stemi patients. our group recently evaluated the role of prior and new anaemia and their prognostic significance in the short term in stemi patients. hypertension was present in 53% of patients, and new anemia patients were more frequently affected by hypertension (56.5%), followed by prior anemia (55.3%, p 75 years), increased glucose values were independent predictors of early death. a subanalysis of the acute myocardial infarction registry (kamir), which collected a total of 8568 korean patients with stemi, confirmed that presence of type 2 diabetes in hypertensive stemi patients is very common and is associated with worse clinical and angiographic features with a higher risk to develop heart failure and an increased risk of mace on long - term followup. several studies reported that a history of hypertension was associated with an increased rate of adverse outcomes after ami such as stroke, heart failure, and cardiovascular death [6, 10 ]. the increased incidence of ami or sudden death in hypertensive patients may be related to several factors, such as endothelial damage, atherosclerosis, insulin resistance, left ventricular hypertrophy, and ventricular arrhythmias. in the kamir study 48% of stemi patients had hypertension : at multivariate analysis a history of hypertension independently contributed to higher in - hospital mortality in patients with ami but not to one - year mortality. this was related not to antecedent hypertension but to the coexistence of other risk factors (old age, high killip class, multivessel disease). multivessel disease and complex lesions in coronary angiography are among the factors which have been proved to be associated with poor outcomes in hypertensive patients. in the gissi-2 study, in - hospital and 6-month mortality in hypertensive mi patients was significantly higher compared to normotensive patients as was the rate of left ventricular failure, recurrent angina, and recurrent mi. on the other hand, gusto-1 study showed that elevated blood pressure was not an independent prognostic factor for 30-day mortality, but in these trial patients with very high values of blood pressure were excluded due to the thrombolytic treatment. a later subanalysis of the gusto-1 trial found a higher risk of early death in patients with elevated systolic blood pressure (bp) at admission. on the other side, abrignani. stated that hypertensive subjects with first ami have a better in - hospital outcome than age- and gender - matched normotensive subjects, perhaps due to a less severe extension of the infarction area or to a different physiopathologic mechanism. other studies did not show relevant difference for in - hospital and 6-month mortality in hypertensive and normotensive patients with myocardial infarction [35, 54 ], even considering different subgroups according to bp admission values (normal, high - normal, high). in the prove - it - timi 22 trial (pravastatin or atorvastatin evaluation and infection therapy - thrombolysis in myocardial infarction) 4162 patients with acs were categorized in 10 mm hg increments of blood pressure during followup : a j- or u - shaped curve association was found between blood pressure and risk of future cardiovascular events, with the lowest event rates in the systolic pressure (sbp) range of 130 to 140 mm hg and 80 to 90 mm hg diastolic pressure (dbp), a flat curve for 110130 mm hg sbp and 7090 mm hg bdp. the latter finding strongly suggests that too low pressures (especially < 110/70 mm hg) may be dangerous ; anyway the trial considered only blood pressure values during follow - up visits, not in - hospital measurements. most of the studies regarding links between hypertension and myocardial infarction consider patients with a previous history of hypertension, but few data are available about patients who do not have a previously known history of hypertension but showed elevated blood pressure values during their hospital staying for acs. in patients with myocardial infarction admitted within 6 hours from the onset of pain, 31.7% presented with elevated blood pressure (160/100 mm hg) : only 6.3% of these patients had elevated blood pressure levels after 6 hours, though not treated with any antihypertensive drug. blood pressure should therefore be carefully monitored in patients with acs for several reasons : (a) to achieve the optimal perfusion pressure by tailoring drugs, (b) to prevent complications (ranging from drug - induced hypotension to hypertensive crisis which may promote acute heart failure syndrome), and (c) to obtain renal protection (thus preventing acute renal failure). whenever the findings of serial high bp measurements and/or previously not known left ventricular hypertrophy should lead to the suspicion that a hypertensive state was present before hospital admission for acs, a more strict followup should be recommended in these patients as well as investigations in order to rule out hypertension - related complications (i.e., fundus oculi). acs patients with hypertension represent a subset at higher risk since they are more often older and with higher comorbidities (including renal failure). in these patients blood pressure values should be carefully monitored in order to achieve an adequate perfusion pressure (usually values of mean arterial pressure higher in respect to nonhypertensive acs patients). the main goal is to prevent renal failure due to hypoperfusion. at echocardiographic evaluation, diastolic function should be carefully assessed and treated in hypertensive acs patients. in these patients, hypertensive crises comorbidities should be searched if not previously diagnosed, firstly, glucose imbalance by means of serial glucose measurements, acute insulin resistance (by means of homa index), and glycated haemoglobin (indicating the glucose control in the previous months). the occurrence of anemia on admission should induce an accurate evaluation of renal function in order to exclude the coexistence of renal failure. in presence of bp values a strict followup should be recommended in acs patients with hypertension with close monitoring of adherence to drug administration. in fact, polypharmacy is quite frequent in hypertensive patients, especially after acs, but in these patients the consequences of drug discontinuation may be severe and even lethal. | arterial chronic hypertension (htn) is a well - known cardiovascular risk factor for development of atherosclerosis. in order to explain the relation between htn and acute coronary syndromes the following factors should be considered : (1) risk factors are shared by the diseases, such as genetic risk, insulin resistance, sympathetic hyperactivity, and vasoactive substances (i.e., angiotensin ii) ; (2) hypertension is associated with the development of atherosclerosis (which in turn contributes to progression of myocardial infarction). from all the registries and the data available up to now, hypertensive patients with acs are more likely to be older, female, of nonwhite ethnicity, and having a higher prevalence of comorbidities. data on the prognostic role of a preexisting hypertensive state in acs patients are so far contrasting. the aim of the present paper is to focus on hypertensive patients with acs, in order to better elucidate whether these patients are at higher risk and deserve a tailored approach for management and followup. |
genital ulcer diseases (guds) often represent a diagnostic dilemma, especially in developing countries, like india, where few sexually transmitted infection (sti) clinics have access to reliable laboratory facility. the changing trends in stis make it imperative that a correct diagnosis be made in order to institute appropriate treatment and formulate policies for control. the annual global incidence of gud exceeds 20 million cases. in 1960s and 1970s, bacterial guds were the commonest. by 1980s, with the advent of hiv, the viral guds took over. thus, over time the trends in microorganisms causing gud have undergone considerable change across the developed world, with bacterial aetiologies giving way to viral causes. genital herpes has become the most common std among clinic attendees and the leading cause of genital ulcers worldwide. for viral guds, prevention and counseling are more important rather than early diagnosis and treatment, as is the case with bacterial guds. often, in guds, it is seen that more than one aetiological agent may be found, if careful evaluation is conducted. an accurate diagnosis of a gud is often not possible when based solely on history and physical examination, because of the lack of sensitivity and specificity of lesions, even in so - called classic cases. to add to the confusion, mixed or multiple infections, or a concurrent infection with hiv, can mask the characteristic clinical features of gud, thus affecting the institution of appropriate therapeutic regimen. thus, no clinical characteristic is predictive of aetiology of gud, which underlines the importance of performing a thorough microbiological evaluation. in india, the cultural and sociodemographic diversity is considerable, with the result that guds vary in presentation according to aetiology, length of clinical course, age, and immunity of the host, leading to diagnostic confusion and unsatisfactory therapeutic results. therefore, along with a detailed medical history and physical examination, a reliable laboratory support is essential for management of these lesions. this also implies that the syndromic management should not be indistinctly applied to all cases of gud, without aetiological diagnosis, unless there is no other recourse. fewer still in numbers are the laboratories that perform accurate diagnostic tests like culture techniques and pcr for guds. there is significant variability in morphologic presentation of guds making the clinical interpretation unreliable when used without confirmatory laboratory tests. there are not many studies from the developing countries, like india, to corroborate such findings, largely due to a lack of reliable laboratory data to back such studies. this study was conceived and carried out with the primary objective of determining the recent trends in the aetiology of guds by using newer and more accurate methods of diagnosis, to ensure authenticity of data.. it also aims at establishing the utility of existing guidelines and algorithms for the management of gud and to suggest changes in them based on the findings of the study. diagnosis of gud by real - time pcr was introduced for the first time in our institution for sti organisms, in addition to the standard culture and serological tests. an aetioepidemiological study was carried out, over a period of one year (from april 1, 2010 to march 31, 2011), as an operational research project under the aegis of national aids control organization, in safdarjang hospital, a tertiary care hospital of new delhi, india. the project was evaluated and approved by the ethics committee, of the institution. a written, informed consent was taken from each participant at the time of enrolment in the study and sociodemographic characteristics were all enquired into and recorded. for patients who were minors, the consent was obtained from the parent or a responsible adult accompanying the minor, although the history was taken and all procedures were explained to the minor in question as well. the study population comprised of 1208 male and female sti clinic attendees, of whom 90 patients presented with gud, as evidenced by disruption of genital mucous membrane or epithelium, and they constituted the study group. provision was made for enrolling patients referred from targeted intervention clinics run by the nongovernmental organizations (ngos). demographic characteristics, history of prior antibiotic use, and details of physical examination were also recorded. material was collected from the genital ulcers as swabs, from the base and edge of the ulcers, and processed for various microbiological tests, within minutes, as the clinic and laboratory are located very close to each other. swabs collected for real - time pcr were placed in specimen transport medium (stm) and frozen at 20c until further use. the various tests performed with the specimens collected included the following. for syphilis. dark field microscopy was performed, within twenty minutes of collection, on the serous exudates collected from the ulcer, to look for treponema pallidum. a positive dark field microscopy was taken to be positive for syphilis irrespective of the serological test results for syphilis. a real - time pcr for t. pallidum was also performed on ulcer swabs collected in stm. for chancroid. a swab was collected from the ulcer for smear and stained with gram 's stain, which was then observed under oil immersion objective for presence of pus cells and gram - negative coccobacilli in clusters or fish in stream appearance. a second swab was streaked on special media (gc agar base - enriched with isovitalex, haemoglobin, and foetal calf serum with antibiotics) and incubated under appropriate conditions. a third swab was collected and placed in stm for real - time pcr. for herpes simplex virus. a swab collected from the base of the ulcer was stained by giemsa 's stain to look for typical multinucleated giant cells (mngcs) indicative of hsv infections. a second swab was collected and placed in stm for real - time pcr. the serological test performed for detection of hsv-2 igm antibodies was elisa. for donovanosis. a small piece of tissue from the ulcer was crushed between two glass slides and stained by giemsa stain to look for the typical morphology of bacteria and safety pin appearance swabs from ulcers were collected for chlamydia antigen detection by dfa test. when indicated, elisa for chlamydia antigen detection in serum, was also performed. the various serological tests performed with the patient 's blood (serum) samples includedvdrl test;tpha to confirm vdrl reactive samples;fta - abs to confirm vdrl reactive samples;elisa for t. pallidum to confirm vdrl reactive samples;elisa for hsv-2 igm antibody detection;elisa / rapid test / simple tests for antibody detection of hiv antibody. this was done after appropriate pre- and post - test counseling and informed patient consent, as required by the national aids control organization (naco) guidelines. tpha to confirm vdrl reactive samples ; fta - abs to confirm vdrl reactive samples ; elisa for t. pallidum to confirm vdrl reactive samples ; elisa for hsv-2 igm antibody detection ; elisa / rapid test / simple tests for antibody detection of hiv antibody. this was done after appropriate pre- and post - test counseling and informed patient consent, as required by the national aids control organization (naco) guidelines. the special swabs in the stm kits were used to collect material from genital ulcers. these swabs were processed, dna was extracted, and real - time pcr performed for treponema pallidum, herpes simplex virus-1, herpes simplex virus-2, and haemophilus ducreyi, as per the manufacturer 's instructions. the pcr was standardized with appropriate controls provided by the firm that supplied the pcr kits. a diagnosis of gud was made when any or many of the above - mentioned tests yielded a positive result. a total of 90 cases of gud were enrolled among 1208 std clinic attendees over the study period, giving a prevalence of gud as 7.45. majority of the gud patients (62.3%) were between 15 and 34 years of age, with 66.7% males and 33.3% females. there were only two minors in the study group (aged 12 and 8 years). the study group consisted of 67.8% married persons and 85.5% literate, above secondary school level. among the female patients in the study group most of them were homemakers by occupation, while among the male patients knowledge of stis and their occurrence was poor, with over 60% being ignorant about the signs and symptoms of sti. use of condom with regular partner was 19.5%, while with nonregular partner it was 42.1%. sexual orientation of most of the patients in the study group was heterosexual (92.2%), followed by homosexual (2.2%). most of the subjects (52.2%) reported having sex with a regular partner, while 30% had sex with nonregular partners and 7.8% reported having sex with sex worker. among the participants, 31 (34.4%) gave a history of previous sti episodes, ranging from ulcers (51.6%), redness / itching to vaginal discharges. most of them consulted doctors (67.7%) and sought treatment at a government or private clinic. most of the patients completed the course of treatment advised (77.4%) and claimed to be completely relieved of symptoms (64.5%). however, the number of patients whose partners also sought treatment was low (25.8%). of the 90 cases with gud, 8 were hiv seropositive (8.9%). the commonest gud encountered in the samples from study population was herpes simplex virus78.9% clinically and 92.2% aetiologically. dark field microscopy was performed on 63 cases of gud, of which 3 were positive (4.76%). gram 's stain smear was performed in 89 samples of which 3 were positive for h. ducreyi (3.4%). giemsa stain was performed on 87 out of the 90 cases of gud. of these, 19 (21.84%) culture for h. ducreyi was put up in 75 cases of gud, all of which were negative for growth. real - time pcr was performed on 90 samples, for the three important organisms causing gud - treponema pallidum, haemophilus ducreyi and hsv 1 & 2. the comparison of clinical and laboratory diagnosis of all the cases of gud is shown in tables 2 and 3. there were 8 cases (8.89%) (2 females and 6 males) of clinically diagnosed gud that were negative for all the tests carried out, and hence no aetiological diagnosis could be made. two of these cases were treated for primary syphilitic chancre and six others for herpes genitalis, as per the clinical findings. one of the herpetic gud cases also had tinea manuum and tinea unguinum and hence received antifungal agents too. there were 10 cases where more than one gud was diagnosed clinically (11.11%). the present study revealed that guds are a common occurrence in std clinic attendees (prevalence 7.45%), especially among males (66.7%). considering the conservative nature of many indian societies, it is understandable that guds are commoner among males because they have easy access and opportunity of seeking multiple sexual partners and practising high risk behavior, in comparison to females. the age group of patients presenting to the sti clinics in our study was predominantly between 15 and 34 years. although this is in accordance with the age group seen in several other studies [1215 ], and an earlier study from this very centre, the lower limit of age seems to have fallen in the present study. this may be a significant finding indicating that stis may be occurring at an earlier age, and that initiation of sexual activity is earlier now than it was then. also, this is an economically productive age group and hence may have serious repercussions in loss of man hours at work. the literacy level in the present study was quite high (85.5%) as compared to a previous study on stis from the same centre (46.5% secondary school or higher). it was also seen that several of the clinic attendees were seeking information, or clarifications on information, gained from internet, on stis. this was a new and significant finding during this study, undoubtedly the result of higher literacy rates. sexual orientation was predominantly heterosexual in this (92.2%) and older studies (97.2%). homosexuality has made its presence in our study with 2.2%, reflecting a small, but definite change in sexual behaviour. in most young sexually active patients with gud the main causative agent of gud in this study was herpes simplex virus (whether hsv-1 or hsv-2). this is in agreement with other studies in both the developed and developing countries, like brazil and india [19, 20 ]. in an earlier report from this very centre, it was shown that syphilis was the commonest gud observed over a fifteen - year period, followed by chancroid and herpes genitalis. according to other indian studies, around the same period, hsv emerged as the commonest gud, as has also been observed by o'farrell. and hsv - gud increased from 5.7% to 22.4% in an earlier study at this centre. our study has shown this to have increased further to 78.9% clinically and 92.2% aetiologically, although the previous study reported on the percentage of hsv with reference to all stis and not just guds. this is probably why the prevalence of hsv in developing countries is increasing in recent decades. another important cause for increase in hsv - guds is the escalation of the hiv epidemic since mid to late 1990s. in a study performed at kigali, in 1985, the proportions of chancroid, syphilis, and genital herpes diagnosed among patients with gud were 18%, 28%, and 19%, respectively and 59% of these patients were identified with hiv-1 infection. most studies do not provide clear - cut data on this because they do not use the tests which differentiate between hsv-1 and 2. real - time pcr significantly increased hsv-1 and 2 detection in both early (5 days) presentations and in both first and recurrent episodes. also, the hsv detection by pcr was achieved in less than 4 hours, leading to a significant reduction in time lag with highly reproducible results. in our study hsv-1 was positive in 29 gud cases (32.2%), either alone (10), with hsv-2 (19) or with t. pallidum (1). in the study by risbud., hsv 1 and 2 prevalence was 39% and hsv-1 was the second most frequently diagnosed microorganism with 7.6% by pcr. a recent study in south africa showed 4.2% of guds to be due to hsv-1. on the other hand, hsv-1 did not seem to be the causative organism of gud in an older study in africa. thus, geographical variations exist and reliable laboratory data can establish this conclusively. in most countries around the world, syphilis ranks as the second most frequent cause of gud (225%). this was true of our study too, wherein 9 out of 90 cases yielded positive results for syphilis (10%), while the study in pune showed 10% primary syphilis and 3.3% prevalence. chancroid is encountered very rarely in most developed and much of the developing nations now. in india too the figures seem to be moving downhill, as is evident from our study where h. ducreyi was seen in only 3 out of 90 cases (real - time pcr-1 and by gram 's stain-3). in the pune study, h. ducreyi was the most common cause of gud with 26% by multiplex pcr in 1999, while no h. ducreyi has been isolated in australia since 1998. a study of guds in zambia yielded 0% prevalence of h. ducreyi, while hsv-2 (28%), hsv-1 (0.5%), t. pallidum (11.5%), and c. trachomatis (3%) were all encountered. the present study did not yield a single case of donovanosis or lgv (neither clinically nor aetiologically). this situation is true of many other nations across the world where no donovanosis has been reported in several studies. the trend in our centre may be compared with a previous data from this centre over 15 years, when the incidence of lgv decreased from 3.4% in 1990 to 0.2% in 2004 and incidence of donovanosis decreased from 3.9% in 1990 to 0.2% in 2000. since all guds are sexually transmitted, often more than one aetiological agent may be found if careful evaluation is conducted with reliable laboratory aid. in our study, there were ten cases of gud (11.11%) wherein more than one aetiological agent was diagnosed. also, our study showed that there were 8 cases of gud in which no causative agent could be established (8.9%) by any of the laboratory methods, although clinically 6 of them were diagnosed as herpetic ulcers and 2 cases diagnosed as syphilitic and were treated as such. one pune study had 34% cases with no causative agent for gud, while another more recent pune study had 38% of gud cases also, the canadian guidelines have stated that even after complete diagnostic evaluation, about 25% of patients with gud have no laboratory confirmed diagnosis. the overall sensitivity and specificity of the national syndromic management algorithm for gud were 68% and 52%, respectively, in the recent pune study, while in the present study the same figures were 92% and 83%, respectively. the positive and negative predictive values were 86% and 90%, respectively. from a public health perspective, it is more important that a diagnostic approach to gud has a high sensitivity than a high specificity. this is because a low sensitivity means more infected individuals remaining untreated, resulting in complications and the risk of secondary infections, including hiv. also, using the national algorithm 52 (42%) cases in the pune study were clinically misclassified as either herpetic (18 cases) or nonherpetic (34 cases) gud, resulting in incorrect treatment. in the present study, there were 6 cases that were clinically diagnosed as non - herpetic while aetiologically they were herpetic. there were no cases that were clinically diagnosed as herpetic but aetiologically nonherpetic, which indicates that clinical diagnosis of herpetic ulcers was accurate. there was one case each, wherein clinically a diagnosis of herpetic or nonherpetic ulcer was made, and the aetiological diagnosis yielded both herpetic and nonherpetic aetiologies. there were seven cases that were clinically diagnosed as herpetic (6) or nonherpetic (1) that were aetiologically negative for both. the hiv seropositive cases in this study were 8 (8.9%), of whom 3 were males and 5 females. all the 8 hiv seropositive cases presented with genital herpes, and pcr of these 8 cases revealed that 3 of them were positive for both hsv-1 and 2. the significance of this lies in the fact that guds are known to facilitate the transmission of hiv and hiv, in turn, can alter the presentation and response to treatment of all the organisms causing the gud. the use of acyclovir for herpetic guds is very relevant, as it has been shown to significantly decrease the population of men shedding hiv from their ulcers, as well as the mean ulcer hiv rna viral load, which suggests a possible benefit in terms of reduction in hiv transmission. a study in rwanda confirmed a very high rate of hiv infection among patients with gud (73.2%), while another study in zambia showed a similar trend (54%). the clinical picture of genital ulcer syndromes may be particularly severe and prolonged when the patient is hiv positive, thus requiring longer courses of therapy. recurrence rates after treatment may also be higher. based on surveys performed in south africa, since 2007, syphilis is generally detected now in less than 10%, and chancroid in less than 1% of guds. therefore, a new gud flowchart has been devised which gives the option of prescribing antiherpes therapy without the addition of antimicrobial agents for syphilis and chancroid in patients deemed to be at low risk of having acquired an sti. men and women with gud are at an increased risk of acquiring and transmitting hiv via a variety of biological mechanisms. this makes it imperative to strengthen measures to control guds which in turn would reduce the ability to transmit hiv by decreasing the amount and frequency of hiv shedding. gud is often caused by pathogens for which suitable therapies exist, but clinical and laboratory diagnosis may be problematic. there is dire need for rapid and accurate approach to this, as earlier studies have shown. the use of real - time pcr in routine diagnostic settings is limited by concerns over its cost and contamination. but, when validated and used, it is a highly reproducible, rapid, and labour efficient method for hsv detection in genital swabs. this study has demonstrated the potential for this technology to replace conventional culture as a sensitive and rapid means of detecting hsv, especially in a developing country. also, real - time pcr for hsv allows not only detection of hsv dna, but also differentiation between hsv-1 and hsv-2 genotypes. according to who, recurrences and subclinical shedding are much less frequent for hsv-1 infection than for genital hsv-2 infection. taken together, these findings indicate that it may be used as an important tool to detect hsv genomes in guds. another valuable contribution of this study is the validation of clinical and syndromic diagnosis of guds by making an accurate aetiological (laboratory) diagnosis. data thus obtained by using reliable laboratory diagnostic methods will aid specific treatment of gud and better define the prevalence of each microbe among the at - risk population with a view to control the common gud agents and eliminate those amenable to eradication, like chancroid and donovanosis. also, the fact that several guds that were clinically diagnosed as nonherpetic turned out to be aetiologically herpetic, while the converse was not true, points to a need to revise existing national sti treatment guidelines in india, to include the treatment for herpes simplex virus of all gud cases. the same finding has also been documented by a study in south india. the link between guds and hiv infection provides a strong case for focusing on behaviour - change education and condom promotion on patients with genital ulcers. thus, monitoring local aetiologies of guds provides valuable information that may aid policy changes for their effective treatment. also, the strengthening of an sti programme management relies heavily on evidence - based interventions which is empowered by studies on establishing aetiological and antimicrobial susceptibility studies. this is the first study validating the clinical and syndromic diagnosis of guds with accurate aetiological methods, like culture and real - time pcr.herpes simplex virus 1 & 2 were the commonest causative agents of gud, followed by syphilis and chancroid.in this study, donovanosis and lymphogranuloma venereum were conspicuous by their absence.guds with multiple aetiologies, that were missed clinically, were picked up by laboratory methods. this is the first study validating the clinical and syndromic diagnosis of guds with accurate aetiological methods, like culture and real - time pcr. herpes simplex virus 1 & 2 were the commonest causative agents of gud, followed by syphilis and chancroid. in this study, guds with multiple aetiologies, that were missed clinically, were picked up by laboratory methods. | background and objectives. genital ulcer diseases represent a diagnostic dilemma, especially in india, where few sti clinics have access to reliable laboratory facility. the changing sti trends require that a correct diagnosis be made in order to institute appropriate treatment and formulate control policies. the objective of this study was to determine recent trends in aetiology of genital ulcers, by using accurate diagnostic tools. methods. specimens from 90 ulcer patients were processed for dark field microscopy, stained smears, culture for h. ducreyi, and real - time pcr. blood samples were collected for serological tests. results. prevalence of gud was 7.45 with mean age at initial sexual experience as 19.2 years. use of condom with regular and nonregular partners was 19.5% and 42.1%, respectively. sexual orientation was heterosexual (92.2%) or homosexual (2.2%). there were 8 cases positive for hiv (8.9%). herpes simplex virus ulcers were the commonest, followed by syphilis and chancroid. there were no cases of donovanosis and lgv. conclusions. a valuable contribution of this study was in validating clinical and syndromic diagnoses of genital ulcers with an accurate aetiological diagnosis. such reliable data will aid treatment and better define control measures of common agents and help eliminate diseases amenable to elimination, like donovanosis. |
the generation of cd2/npm - alk transgenic mice was described previously as was the generation of npm1 heterozygous mice (2, 4). all mice were maintained on a c57bl/6j background (jackson laboratory via charles river, margate, uk). mice were housed under specific pathogen free (spf) conditions in a barrier facility at the university of cambridge, uk. immunohistochemical analysis was performed with the following antibody : alk (invitrogen, paisley, uk). analysis of protein expression by western blot was performed as described previously (7) using antibodies specific to npm1 (na24) (abcam, cambridge, uk) and actin (sigma). densitometric analysis of protein expression levels as a factor of the respective actin loading control were determined using aida software and a fuji las 4000 chemilluminescence analyzer (raytek, sheffield, uk) kaplan - meier survival curves were produced using medcalc software (mariakerke, belgium). the generation of cd2/npm - alk transgenic mice was described previously as was the generation of npm1 heterozygous mice (2, 4). all mice were maintained on a c57bl/6j background (jackson laboratory via charles river, margate, uk). mice were housed under specific pathogen free (spf) conditions in a barrier facility at the university of cambridge, uk. immunohistochemical analysis was performed with the following antibody : alk (invitrogen, paisley, uk). analysis of protein expression by western blot was performed as described previously (7) using antibodies specific to npm1 (na24) (abcam, cambridge, uk) and actin (sigma). densitometric analysis of protein expression levels as a factor of the respective actin loading control were determined using aida software and a fuji las 4000 chemilluminescence analyzer (raytek, sheffield, uk) kaplan - meier survival curves were produced using medcalc software (mariakerke, belgium). in order to examine the impact of npm1 genetic heterozygosity on the incidence, latency and pathological features of npm - alk - induced tumorigenesis, cd2/npm - alk mice were crossed with npm1 mice and the offspring (34 cd2/npm - alknpm1 and 25 cd2/npm - alknpm1 mice), as well as 39 genetically wild - type mice (littermates obtained from maintenance of the cd2/npm - alk mouse colony) and 12 npm1 heterozygous mice, were monitored over a two - year period (figure 1, table 1). of the 25 cd2/npm - alknpm1 and 34 cd2/npm - alknpm1 mice under observation, 11 (44%) and 13 (38%) were euthanized within the two - year period, respectively (figure 1a and table 1). lymphoma developed in both populations of npm - alk - expressing mice after a long latency that ranged from 10 to 24 months (table 1). during this time, 5 (13%) there was no evidence of tumors and the cause of ill - health remains unknown but is highly likely to be age - related. whilst cd2/npm - alknpm1 and cd2/npm - alknpm1 mice succumbed to disease significantly earlier than wild - type mice (figure 1a ; cd2/npm - alknpm1 versus wild - type mice, p = 0.01 ; cd2/npm - alknpm1 versus wild - type mice, p = 0.02), the survival distribution of the cd2/npm - alk transgenic mice was equal regardless of npm1 genetic dosage (figure 1a ; cd2/npm - alknpm1 versus cd2/npm - alknpm1 mice, p = 0.7). thus, heterozygous disruption of the npm1 locus does not significantly accelerate disease onset nor increase incidence in the context of oncogenic npm - alk signaling. table 1 details the macroscopic and microscopic pathology for each mouse that was culled on presentation of clinical signs or died unexpectedly (2 years of age). at necropsy, many of the mice that died prematurely presented with enlarged lymph nodes with or without splenomegaly. in tissue sampled from mice of either genotype, histological analysis revealed the presence of extensive infiltrates characterized by atypical lymphocytes in spleen and lymph nodes that disrupted the normal tissue architecture (figure 2a). immunohistochemical analysis with an anti - alk antibody confirmed expression of npm - alk in the tumor tissue (figure 2b). furthermore, western blot analysis revealed that loss of heterozygosity (loh) for npm1 was not a feature of these tumors as npm1 expression could be detected in tumors arising from both npm1 heterozygous and npm1 wild - type mice (figure 2c). amplification of npm1 expression was likewise not a feature of the tumors that arose in the cd2/npm - alknpm1 mice since protein lysates prepared from these tumors contained on average lower levels of npm1 than tumor lysates extracted from cd2/npm - alknpm1 mice (average densitometry level = 0.32 compared to 2.24) (figure 2c). b - cell lymphoma was the predominant tumor type presented by the cd2/npm - alknpm1 mice, occurring in 5 (20%) mice, and was the only tumor type that developed in the cd2/npm - alk npm1 mice, occurring in 6 (18%) of the mice (table 1 and figure 1b). whilst low, the lymphoma incidence was significantly different between the wt and the cd2/npm - alknpm1 cohorts (p < 0.01) but not between the cd2/npm - alknpm1 and the cd2/npm - alknpm1 groups (p = 0.8) (figure 1b). a predominance to develop b - cell lymphomas is consistent with the tumor phenotype previously described for the cd2/npm - alknpm1 transgenic mice, although an incidence of 20% is lower than published observations (4). other tumor types detected in the cd2/npm - alknpm1 mice were largely non - hematological and included two cases of hepatocellular carcinoma (table 1). however, one mouse (# 1178) in addition to developing hepatocellular carcinoma presented with histiocytic sarcoma in the liver and spleen. amongst both cd2/npm - alknpm1 and cd2/npm - alknpm1 cohorts, a small number of mice presented with an abnormal pathology in non - lymphocytic cells that was not associated with tumor formation, and therefore presumably unrelated to npm - alk expression, and possibly age - related (table 1 and figure 1a). these abnormal pathologies included inflammatory infiltrate in the heart and glomeruli of the kidney, as well as extensive red pulp expansion of the spleen. finally, 1 cd2/npm - alknpm1 and 3 cd2/npm - alk npm1 mice could not be associated with any distinct pathology following histological examination and therefore the reason for ill - health remains unknown (table 1). importantly, of those tumors for which npm - alk expression could be detected no significant difference in the incidence was observed between the two populations of mice (figure 1b), indicating that npm1 heterozygosity does not cooperate with npm - alk in lymphomagenesis or antagonize its oncogenic function in this model. whilst the cd2/npm - alk transgenic mice are prone to developing lymphoid malignancies (4), the npm1 genotype is reported to chiefly predispose to myeloid malignancies (either myeloproliferative disease - like myeloid leukemia [mpd - like ml ] or myeloid leukemia [ml ]) (2). mpd - like ml in these mice is characterized by leukocytosis together with a vast increase in the number of well - differentiated myeloid cells in the bone marrow, spleen and non - hematopoietic tissues (2). ml is characterized by severe anemia, mild leukocytosis and thrombocytopenia, as well as the presence of leukemic blasts in affected tissues (2). of the cd2/npm - alknpm1 and cd2/npm - alknpm1 mice for which blood counts were taken, all mice exhibited perturbations in at least one of the following blood parameters : white blood cell, red blood cell or platelet count or hemoglobin levels (compared to the range obtained from the peripheral blood of 10-month - old wild - type litter mate mice (n = 10)) (table s1 and figure s1). however, the blood counts were not consistent with a diagnosis of mpd - like ml or ml. extreme expansion of myeloid cells was not a feature of any of the tissues examined, which included bone marrow and analysis of blood smears (data not shown), and hence no evidence of myeloid malignancy was observed in any of the mice, regardless of npm1 genetic dosage. equally, there was no evidence of myeloid disease in the npm1 cohort (table s1). amongst all of the blood samples analyzed there was no trend to indicate a common deficiency or increase in blood cell count parameters amongst any of the cohorts and group readings did not differ significantly from one another (figure s1). this disparity with the literature may reflect generation - specific differences, subtle changes in the genetic background of the mice following outcross to the npm - alk transgenic lines or disparities in housing of the mice compared to the original mouse colonies. although transgene expression of npm - alk is sufficient to drive lymphoma development in several mouse models of npm - alk - induced tumorigenesis, none of these models faithfully recapitulate the human disease (4 - 9). for instance, disease latency is in most cases long and manifests after many months, suggesting the need for additional cooperating events to accelerate disease. in addition, in these and other models of npm - alk - driven lymphomagenesis, npm - alk - expressing b - cell lymphomas frequently develop despite supposed t - cell - restricted expression of the transgene (4, 5). in all of these models a potential role for t(2;5)-associated npm1 allelic loss in tumor development has not been considered. here, we addressed this issue by crossing mice expressing npm - alk from the cd2 promoter to npm1 heterozygous mice and examining the resultant offspring. this mouse model was chosen due to the long latency to disease development and a lower disease penetrance and hence to provide a clear window in which our hypothesized increased disease incidence and shorter latency owing to a npm1 heterozygous background could be observed (4). in the event, no difference in tumor latency, incidence or phenotype was observed, providing evidence that heterozygous expression of npm1 does not cooperate with npm - alk in tumorigenesis in this model system. these results differ to the published observations that oncogenic myc expression cooperates with npm1 heterozygosity to accelerate tumor onset in lymphoma - predisposed mice (2). we propose that whereas myc and npm1 heterozygous expression have complementary functions in oncogenesis that permit cooperation, this is not the case for npm - alk expression and npm1 heterozygosity although this remains to be determined by complementary in vitro studies. these findings are in accordance with analyzes of alk alcl patient data indicating that tumors harboring alk fusions where the partner gene is not npm1 exhibit an immunophenotypic, clinical and prognostic behaviour that is indistinguishable from that of classical t(2;5) alcl tumors (10), although the t(2;5) is the most common translocation, observed in greater than 80% of cases raising the question as to why this particular event is selected for (11). however, together, these data indicate that npm1 heterozygosity does not contribute to the genesis of t(2;5)-induced alcl. it seems likely that the significance of this translocation lies largely, if not entirely, in the creation of an oncogenic tyrosine kinase that drives tumorigenesis. however, before this statement can be firmly concluded, it is necessary to examine the consequences of npm1 heterozygosity in an npm - alk transgenic model with a more penetrant disease phenotype or even a mouse in which npm - alk expression and npm1 heterozygosity are achieved simultaneously, for example using the translocator system developed by rabbitts. yet, one must consider that the phenotype observed in mouse models of cancer is influenced by a number of tangible and intangible variables and as such a direct correlation between mouse and human pathologies is often difficult to make, although we have observed that human npm - alk forms some of the same hetero - complexes and occupies the same cellular sub - compartments in murine cells as has been observed in human cells (figure s2), suggesting that at least at the molecular level, npm - alk biology is equivalent in both mouse and human. | heterozygous expression of nucleophosmin (npm1) predisposes to hematological malignancies in the mouse and cooperates with myc in lymphomagenesis. npm1 is therefore regarded as a haploinsufficient tumor suppressor. heterozygous loss of npm1 occurs as a result of the t(2;5) which generates the oncogenic fusion tyrosine kinase, npm - anaplastic lymphoma kinase (alk), a molecule underlying the pathogenesis of anaplastic large cell lymphoma (alcl). given the aforementioned role of npm1 as a tumor suppressor we hypothesized that npm1 heterozygosity would cooperate with npm - alk in lymphomagenesis. in the event, we observed no difference in tumor latency, incidence or phenotype in npm - alk - transgenic mice heterozygous for npm1 relative to transgenic mice expressing both npm1 alleles. we propose that whilst the t(2;5) simultaneously reduces npm1 allelic dosage and creates the npm - alk fusion protein, the two events do not cooperate in the pathogenesis of alcl in our mouse model. these data indicate that a tumor - suppressive role for npm1 may be dependant on cellular and/or genetic context.alcl = anaplastic large cell lymphoma, alk = anaplastic lymphoma kinase, npm1 = nucleophosmin, mpd = myeloproliferative disorder, ml = myeloid leukemia |
therapeutic exercise and physiotherapeutics are important treatment methods in physical therapy, but recently, physiotherapeutics has become auxiliary to therapeutic exercise. however, physio -therapeutics is very often used to treat orthopedic patients and those with disability - related pain. intermittent cervical traction is a physiotherapy approach that is used to treat patients with pain or stiffness in the neck and shoulder region. many patients receive intermittent cervical traction in a daily treatment setting ; however, the objective assessment of severity of symptoms such as pain and numbness is difficult. therefore, evaluating the effectiveness of treatment must rely on self - reported symptoms. furthermore, unified settings for traction force, duration, and direction in traction therapy have yet to be determined. accordingly, a more objective index of evaluating treatment effectiveness which can determine changes in traction conditions or indicate its effectiveness is necessary. in this study, we evaluated changes to muscle thickness that occurred during intermittent cervical traction using ultrasonic diagnostic equipment that is frequently used to assess and diagnose patients with motor dysfunction. the subjects were 15 healthy males (height, 172.5 4.0 cm ; weight, 67.9 15.6 kg ; age, 21.3 1.8 years) who were free of orthopedic disease of the neck and were not receiving treatment such as cervical traction. we applied intermittent cervical traction to the subjects and measured their muscle thicknesses before and during traction using an ultrasonic diagnostic device. the research aims and methods were explained to the subjects, and the experiment was performed after receiving their consent. the study protocol was approved by the research ethics committee of kawasaki university of medical welfare (approval no. traction was performed in a seated position in a reclining wheelchair with the subject s heads on the headrest positioned at a cervical flexion angle of 10. we chose this position to prevent the muscles contracting to support the heads and to restrict movement of the head and neck to the minimum when capturing ultrasonic images. the traction forces were 5, 8, and 11 kg, and the traction cycle consisted of 20 seconds of traction and 10 seconds of rest. two cycles of each traction force were performed and ultrasonic images of muscle thickness were captured during the second cycle. we used the ultrasonic image diagnostic device prosound ssd-3500 (aloka co. ltd.). images were captured in b - mode (brightness mode) using a linear probe (10 mhz) positioned on the outside of the fifth and seventh cervical vertebra, so that the trapezius and splenius capitis muscles were visible. we drew a line marker in the middle of the ultrasonic image for measurements of muscle thickness and took images of each subject using the same frequency and contrast conditions. we used the seventh cervical spinous process as a landmark so that the same area could be imaged before and after traction, and secured the probes to the fifth and sixth cervical spinous process using an adjustable frame and a custom - made probe holder. we attached a 200 gf constant load spring to the custom probe holder so that the probe and neck region would always be secured at the same force. we measured the thickness of the trapezius and splenius capitis muscles from the images using the distance measuring functionality of the device. muscle thicknesses before and during traction were measured, and we calculated the muscle thickness ratio during traction by normalizing the contraction value to the thickness before traction. values are shown as muscle thickness ratios (%). in order to confirm that the ultrasonic images were cuptured from the same region, we calculated the interclass correlation coefficients (icc) of muscle thickness before traction for the three traction forces. for each of the three traction forces, we compared muscle thicknesses before and during traction using the t - test. we also compared the muscle thickness ratios of the three traction forces using one - way analysis of variance. we used spss statics22, and statistical significance was accepted for values of p < 5%. table 1table 1. muscle thickness and muscle thickness ratio of the three traction forcestrapeziussplenius capitis 5 kg before (mm)5.981.568.971.85during (mm)5.931.579.071.78ratio(%)100.73.62101.24.28 kg before5.981.648.911.88during5.941.578.651.87ratio99.75.8597.14.7511 kg before6.051.669.021.84during5.731.728.321.94ratio94.49.0491.85.32 indicates a significant difference between before and during, p<0.05. indicates a significant difference from 8 kg, p<0.05 shows the muscle thickness and ratios of each traction load. the iccs of muscle thickness showed high correlations for both the trapezius muscle (0.968) and the splenius capitis muscle (0.986), indicating that almost the same region was imaged before application of each of the three traction forces. indicates a significant difference between before and during, p<0.05. indicates a significant difference from 5 kg, p<0.05. indicates a significant difference from 8 kg, p<0.05 the trapezius muscle thickness at 5 kg was 5.89 1.56 mm before traction, and 5.93 1.57 mm during traction, with a muscle thickness ratio of 100.7 3.62%. similarly, at 8 kg, the values were 5.98 1.64 mm, 5.94 1.57 mm, and 99.7 5.85%, respectively. at 11 kg, the values were 6.05 1.66 mm, 5.73 1.72 mm, and 94.4 9.04%, respectively. a significant difference in muscle thickness was observed only at 11 kg between before and during traction. significant differences in the muscle thickness ratios were found between 11 kg and 5 and 8 kg. the splenius capitis muscle thickness at 5 kg was 8.97 1.85 mm before traction, and 9.07 1.78 mm during traction, with a muscle thickness ratio of 101.2 4.20%. similarly, at 8 kg, the values were 8.91 1.88 mm, 8.65 1.87 mm, and 97.1 4.75%, respectively. at 11 kg, the values were 9.02 1.84 mm, 8.32 1.94 mm, and 91.8 5.32%, respectively. significant differences in muscle thickness were found at 8 kg and 11 kg between before and during traction. significant differences in muscle thickness ratios were found between 5 kg and 8 and 11 kg, as well as between 8 kg and 11 kg. we previously investigated the effects of intermittent cervical traction by analyzing cutaneous blood flow, skin temperature, gsr (galvanic skin response), deep tissue blood flow, and electromyogram frequencies ; however, individual differences and the resulting unclear trends made it difficult to obtain a usable index for traction therapy effects1, 2. nonetheless, we believe that basic data of healthy subjects are required to support a more effective traction therapy. therefore, we investigated the changes that occur in neck muscle thicknesses during traction using an ultrasonic diagnostic device that is commonly used to diagnose and assess motor dysfunction. in studies measuring muscle thickness using ultrasound3, 4, furthermore, the effects of muscle extension are often calculated as changes in muscle / tendon transfer or muscle bundle length5,6,7. however, muscle thickness has not been used to investigate the effect of cervical vertebra traction. in this study, we found that the trapezius muscle thickness hardly changed between before and during traction at 5 kg. muscle extension may not have occurred at this traction force since it is load similar to that exerted by the head, which accounts for roughly 7% of total body weight. considering the physiology of the trapezius muscle, it may not be directly involved in extension during traction, and the effects may be difficult to observe. however, muscle thickness changes were observed at 11 kg, and at 11 kg significant differences were found from the muscle thickness ratios of 5 kg and 8 kg. the splenius capitis muscle thickness did not change at 5 kg, but significant differences were found at 8 kg and 11 kg. as noted above, a 5 kg load is similar to the load of the head, and may not induce muscle extension. physiologically, the splenius muscle can be extended directly through traction, which would explain the significant difference in the muscle thicknesses at 8 kg, and the significant difference in muscle thickness ratio at the 8 kg load from that of 5 kg. the muscle thickness ratio at the 11 kg load was also significantly different from that of 8 kg, which demonstrates that the stronger traction was effective. cervical traction force often begins at 5 kg and is gradually increased depending on the symptoms. although this load does not result in muscle extension, it is thought to be appropriate for relieving excessive muscle tension and spasms since at this load, the cervical muscles no longer support the head. differences in muscle direction between the trapezius and splenius capitis muscles may account for the contrasting results obtained at the 8 kg traction force. this finding suggests that cervical traction must be performed considering the effects on different neck regions. on the other hand, the 11 kg traction force was sufficient to affect both muscle groups. however, care is necessary since higher traction forces can lead to stress on the teeth or jaw joints and prevent cervical muscle contractions, causing the traction to become uncomfortable. the therapeutic objectives of cervical traction are as follows : disarticulation, reduction of extruded disks, extension of soft tissue, muscle relaxation, and joint mobilization. although studies of the effects of therapy have been performed, some reports have concluded that improvement effects have not been established8. furthermore, other reports have noted the low quality of research involving randomized controlled trials in this field9. thus, the lack of an established traction method and uniform assessment method has led to contrasting evaluations of traction therapy effects. accordingly, an objective index for evaluating the effectiveness of therapy is necessary to improve this situation. the accuracy and reproducibility of measuring muscle thickness using an ultrasonic diagnostic device has previously been confirmed. however, it is necessary to measure the same region, and some innovations are required for the measurement. in this study, we used a headrest to limit the movement of the neck, an adjustable frame and probe holder to stabilize the probe, and a constant load spring to standardize the contact force. as a result, we obtained high interclass correlation coefficients for muscle thickness before each traction application. we will continue to search for an objective method of assessment to determine the appropriateness of traction therapy as well as changes to the therapeutic parameters. in particular, our research will focus on the non - invasive ultrasonic diagnostic device to broaden the use of this device as an assessment tool in physiotherapy. | [purpose ] many patients receive intermittent cervical traction in a daily treatment setting. however, unified settings for traction force, duration, and direction have yet to be determined. therefore, an objective index is required to determine changes in traction conditions or to indicate its effectiveness. [subjects ] fifteen healthy males volunteers participated in this study. [methods ] the thickness of the trapezius and splenius capitis muscles before and during traction were measured using ultrasonography at three traction forces : 5, 8 and 11 kg. [results ] significant differences in muscle thickness were observed at 11 kg in the trapezius, and at 8 kg and 11 kg in the splenius capitis muscles. the muscle thickness ratio of the trapezius muscle showed a significant difference between 11 kg and 5 and 8 kg, and between 5 kg and 8 and 11 kg, as well as between 8 kg and 11 kg for the splenius capitis muscles. [conclusion ] differences in muscle direction between the trapezius and splenius capitis muscles may account for the contrasting results obtained at the 8 kg traction force. this finding suggests that cervical traction must be performed considering the effects on different neck regions. |
attention deficit hyperactivity disorder (adhd) is one of the most common psychiatric disorders in children (1 - 3). the main symptoms of adhd include hyperactivity, symptoms must last for 6 months, not match the developmental level of the child, and have appeared before the age of 7 (4, 5). this disorder is 5 folds more common in boys than in girls (6). stress in family, especially when it is chronic and exists in the early growth period of the child, has harmful effects on the health of the parents and the children as well as the parents ' relationship with the child (7). one of the factors related to the characteristics of the family that can cause stress in parents is emotional and behavioral problems in children, the most common of which being adhd (8). concerned and watchful parents, especially mothers, can be a source of stress (10). repeated calls of the parents of the adhd children with their teacher regarding their children 's misbehavior, talking to other parents about their children 's behavior, leaving job to care for their hyperactive children, and worrying about the children harming themselves are only a few of the stressors that are commonly reported by the parents of the children with adhd (11). studies suggest that maternal depression is associated with behavior problems in children (13). for instance, swanne and colleagues (2006) reported that 30.62% and 4% of the mothers of the adhd children had moderate and severe depression, respectively (14). moreover, dealing with the problems of the adhd children has a negative impact on the mental health of the parents (15). much evidence indicates that the interaction between the parents and the children and mental health of the parents, especially mothers, are improved when the adhd children are treated by various methods. danforth and colleagues showed that various methods of treatment, including medication and parent training, could be effective in improving the maternal mental health (16). among the educational programs, cognitive behavioral stress management based on four components of increased awareness, assessment, coping resources, and coping reactions, can be a more effective manner in facing the stress and reducing it. cognitive - behavioral intervention in stress management familiarizes the individuals with stress and coping with it and also neutralizes the stressors ' effects and stress responses to help a better physiological and psychological functioning. intervention can be effective by increasing the sense of control, self - efficacy, self - esteem, adaptive coping, and social support (17). according to blumenthal. employing stress management techniques improves the individuals ' confrontation with stressful situations, increases their control of the physical and emotional states, and consequently improves their health (18). despite the impact of children 's misbehavior on the mental health of the family members, the importance of the intervention programs in improvement of the mental health of the families of the children with behavioral problems has been neglected (19). therefore, the present study aimed to assess the effect of stress management program using cognitive behavior approach on mental health of the mothers of the children with adhd. the study population included all the mothers of the adhd children referring to the psychiatric clinics affiliated to shiraz university of medical sciences in 2012. cognitive behavioral stress management program consisted of eight 90-minute sessions that were held during 8 weeks. in this study, the participants in the cognitive - behavioral stress management program were placed in four groups of seven or eight mothers who received the same content. in the study, cognitive - behavioral stress management models presented by antony and colleagues (17) were used. the intervention included needs assessment of the mothers of the children with adhd, training the mothers about adhd, understanding stress and its effects, training benson 's relaxation technique, negative thoughts and cognitive distortions, replacing rational thoughts, efficient coping and implementing effective coping responses, anger management, and summary of content. in order to maintain the mothers ' attention during the intervention sessions, visual tools, such as microsoft powerpoint, graphs, and images associated with hyperactive children, were utilized. the intervention was conducted through lectures, question - and - answer, and group discussions. the first 30 minutes of each session was assigned to summary of the previous session, relaxation techniques, and reviewing homework. the session was continued with new contents, mothers ' questions about the new contents, talking about the situations which were related to the upcoming subjects during the session, and new homework. at the end of each session, stress management program utilizing cognitive behavior approach was conducted by a psychiatrist or psychiatric nurse. the inclusion criteria of the study were obtaining moderate to severe score in depression - anxiety - stress scale, having hyperactive children aging 6 to 12 years, and mothers not suffering from mental illnesses (according to a self - report form). on the other hand, the exclusion criteria of the study were unwillingness to continue participation in the study, having received similar training interventions before the study, mothers who take any medications for mental health problems, mothers who themselves suffer from any medical or mental health problems and the presence of mental or physical illness in a family member that prevents mothers from doing homework at home. in this study, besides, the mothers in the placebo group only participated in meetings. in the meetings, they talked about children 's behavior and problems at school and home such as low performance in school, repeated calls of the teachers of the adhd children regarding the children 's misbehavior, their resistance in taking medication, lack of support and cooperation of other family members. also, they expressed feelings about this behavior but did not receive any interventions. this study was approved by the ethics committee of shiraz university of medical sciences, shiraz, iran. it should be noted that the participants were informed about the purpose of the study as well as about their right to withdraw at any time. they were also assured that their personal information would remain confidential. in this study, the data were collected using strength and difficulties questionnaire (parent report), depression - anxiety - stress scale, and general health questionnaire. spss statistical software (v 18) was used for statistical analysis. at baseline, the socio - demographic characteristics of the three groups were compared using the chi - square test. in addition, inter - group comparisons of the variables were made through one - way anova and tukey 's post - hoc test. besides, within group comparisons of the variables were made using one - sample repeated measurement anova in the intervention, the placebo and the control groups. overall, 4 participants (two from the intervention group and two from the control group) dropped out before completion of the study for different reasons unrelated to the study. also, 5 participants in the placebo group dropped out because they realized that the program was useless. the study results revealed no significant difference among the three groups regarding the socio - demographic characteristics. the mean age of the children was 8.85 1.61 years in the intervention group, 8.88 1.62 years in the placebo group, and 9.03 1.60 years in the control group (p < 0.905). also, no significant difference was found among the three groups concerning the mothers ' mean age (33.51 7.00, 34.11 5.36, and 34.59 5.67 years in the intervention, placebo, and control groups, respectively ; p < 0.806). most of the study mothers had high school diploma degrees and 76.5% of the 81 children were boys. in addition, the majority of the mothers in the three groups were housewives and none of the mothers had a history of physical or mental illness. the mean stress and mental health scores in the three groups have been presented in figures 1 and 2. the mean mental health and depression - anxiety - stress scores in separate groups at baseline, immediately after, and one month after the intervention are shown in table 1. also, the mean stress and mental health variables in tukey 's post - hoc test in separate groups immediately after the intervention are shown in table 1. comparisons of the baseline scores of the study variables (stress, anxiety, depression, and mental health) revealed no significant differences among the three groups. however, immediately after, and one month after the completion of the stress management program utilizing cognitive behavioral approach, a statistically significant difference was found among the three groups regarding stress and mental health. the mean score of stress reduced in both intervention and the placebo group, but the mean mental health score improved only in the intervention group. the results of one - way anova showed that the mean score of stress immediately after the intervention was 12.29 4.32 in the intervention group and 12.33 3.75 in the placebo group (p = 0.033). also, the mean mental health score was 20.50 10.56 in the intervention group immediately after the intervention (p = 0.001). one month after intervention, improvement in the mean mental health score was detected only in the intervention group. the results of one - way anova showed that the mean mental health score was 21.73 9.43 in this group (p < 0.001). the mean anxiety and depression scores were statistically significant in the intervention and the placebo groups. within group comparisons of the variables were performed using one - sample repeated measurement anova in the intervention group. the placebo and the control groups were not changed. the changes in the scores of mental health and depression - anxiety - stress at baseline, immediately after, and one month after the intervention are presented in table 2. time 0, baseline ; time 1, immediately after the intervention ; and time 2, one month after the intervention. time 0, baseline ; time 1, immediately after the intervention ; and time 2, one month after the intervention. this study was performed using stress management program utilizing cognitive behavioral approach in three experimental, placebo, and control groups. in fact, elimination of the confounding variables and inclusion of a placebo group are important because any change in the experimental group at the end of the intervention can be attributed to the cognitive behavioral stress management intervention. this study assessed the effect of stress management program using cognitive behavior approach on mental health of the mothers of the children with adhd. the results of this study showed that cognitive behavioral stress management program in the experimental group led to improvement of mental health immediately and one month after the intervention and reduction of maternal stress immediately after the intervention. previous studies have been mostly conducted on the relationship between maternal mental health and behavioral problems in the children, stress management training for the parents, and determining the degree of stress, anxiety, and depression in mothers of the adhd children. musa and shafiee conducted a study in malaysia in order to determine depression, anxiety, and stress levels among the mothers of the adhd children and their relationships with adhd symptoms. the results of that study showed that compared to the control group, the mothers of the adhd children experienced higher levels of stress, anxiety, and depression resulting from external behaviors in the children (20). moreover, lesesne and colleagues in their study found a relationship between mothers mental health and behavioral outcomes in children (13). these results suggest that the mothers of hyperactive children need a comprehensive training to increase their information and awareness about adhd that can, in turn, create changes in attitude and behavior toward children (21). also, increase in the parents ' information and awareness can reduce the maternal concerns and anxiety and reform the parents ' false and dysfunctional beliefs. the reformation eventually paves the way to promote the mothers mental and social health (22). although perception and acceptance of the characteristics and behaviors of the children are hard by mothers, they can more easily explain their children 's behavior after participation in the training sessions. consequently, behavior problems in the children are reduced, the children will obey the maternal orders more, and the mothers will feel less under pressure and stress (21). furthermore, the findings of studies have pointed out that the parents ' training programs can be effective in improving the relationship between the parents and their adhd children. up to now, studies have used different training programs to increase the knowledge and awareness of the mothers of hyperactive children. the results of their study showed that stress management program helped the parents of hyperactive children as a valuable adjunctive strategy in the overall comprehensive management of adhd (23). regarding the effect of training on mental health of mothers of adhd children, the results of the present study are in agreement with those of the previous ones. meftagh and colleagues carried out a research on 95 mothers to determine the effectiveness of different treatment methods for children with adhd on maternal mental health. that study indicated that maternal behavioral training methods and the medications used for these children improved maternal mental health in post - test and follow - up stages (24). moreover, parand and colleagues performed a study which aimed to develop a stress management program for mothers of the children with adhd and determine its effectiveness in their mental health. the results revealed significant improvement in mental wellbeing of the mothers and diminishing their anxiety and depression (19). the findings of the current study showed a reduction in stress levels in the placebo group immediately and one month after the intervention. the observed reduction might have resulted from participation in the meetings for talking about and expression of feelings. in addition, the loss rate in the placebo group was higher compared to the other two groups since they believed that expressing their feelings would not lead to any improvement. in the control group, no significant changes were observed regarding mental health and stress, anxiety and depression. first of all, the sample size was relatively small because of inclusion of three groups and lack of facilities. thus, further studies with three groups and larger sample size are required to confirm the results of this study. also, the studies are suggested to have follow - up periods of more than 1 month and be repeated over time. the findings of the present study showed the effectiveness of cognitive behavioral stress management intervention in improvement of the mothers ' mental health and stress. nonetheless, further studies with longer follow - up periods and in the mothers who have more than one adhd child are suggested to be done in this area. first of all, the sample size was relatively small because of inclusion of three groups and lack of facilities. thus, further studies with three groups and larger sample size are required to confirm the results of this study. also, the studies are suggested to have follow - up periods of more than 1 month and be repeated over time. the findings of the present study showed the effectiveness of cognitive behavioral stress management intervention in improvement of the mothers ' mental health and stress. nonetheless, further studies with longer follow - up periods and in the mothers who have more than one adhd child are suggested to be done in this area. | background : attention deficit hyperactivity disorder is one of the most common psychiatric disorders in children.objectives:the study aimed to evaluate the effectiveness of stress management program using cognitive behavior approach on mental health of the mothers of the children with attention deficit hyperactivity disorder.patients and methods : in this interventional study, 90 mothers of the children with attention deficit hyperactivity disorder were randomly allocated into three intervention, placebo, and control groups. the general health questionnaire was used to measure mental health. besides, stress was assessed through the depression - anxiety - stress scale. the two instruments were completed at baseline, immediately after, and one month after the intervention by the mothers. afterwards, within group comparisons were made using one - sample repeated measurement anova. one - way anova was used for inter group comparisons. mothers in the placebo group only participated in meetings to talk and express feelings without receiving any interventions.results:at the baseline, no significant difference was found among the three groups regarding the means of stress, anxiety, depression, and mental health. however, a significant difference was observed in the mean score of stress immediately after the intervention (p = 0.033). the results also showed a significant difference among the three groups regarding the mean score of mental health (p < 0.001). one month after the intervention, the mean difference of mental health score remained significant only in the intervention group (p < 0.001).conclusions : the study findings confirmed the effectiveness of stress management program utilizing cognitive behavior approach in mental health of the mothers of the children with attention deficit hyperactivity disorder. |
nonspecific urethritis in children is rare and mild disease.1 2 adolescent urethritis in most cases is self - limiting.3 to our knowledge, this is the first case in which an ascending posterior urethritis extending laterally into seminal vesicles and vas deference and causing inflammation of epididymis simulating acute scrotum in whom a battery of various investigations including micturating cystourethrogram (mcug) and cystourethroscopy were a useful adjunct in establishing the diagnosis and an innovative management included intravesical instillation of triamcinolone was effective as the definitive treatment. a 12-year - old boy was first presented at a tertiary pediatric surgical hospital with a 20-hour history of left scrotal pain, dysuria, hematuria, and tenderness while he was on holidays. he was given trimethoprim for 5 days, but he continued to have dysuria and left scrotal pain postoperatively. he was then presented to urology and pediatric departments of his local general hospital with continued dysuria pain in the left scrotum with radiation to left loin and tender left epididymis 2 months later. he had at times difficulty in walking with wide - based gait due to dysuria associated with left lower abdominal, pelvic - perineal, and scrotal pain. his urine dip was negative, had normal renal functions, full blood count, and biochemical bone profile. his c - reactive protein was less than 5 and erythrocyte sedimentation rate was 2 mm / hour. he felt to be constipated and was treated with a disimpaction dose of movicol paediatric plain (norgine). he was discharged home after 7 days but continued to have dysuria and severe left scrotal pain. he was readmitted at his local hospital after 2 weeks with the same symptoms of dysuria and nonremitting pain in the left scrotum and left side of lower abdomen. urinalysis revealed 115 white blood cells (wbcs), no red cells, and no subsequent growth on culture. both epididymides were enlarged, with the right more noticeable than the left with reduced echo texture and slightly increased doppler flow suggestive of bilateral epididymitis. he was treated with 14 days of cefalexin with triple analgesia with paracetamol, ibuprofen, and codeine. he was again presented 3 weeks later to his local hospital with no improvement of symptoms. there was bilateral epididymal thickening more marked on the left side with a 4-mm cyst noted in the right epididymal head and upper body of the left epididymis. he was, therefore, referred to us for further management for dysuria associated with chronic and recurrent epididymitis with disabling pain. he was seen in clinic and an emergency admission was requested because of the severity of his symptoms that started approximately 3 months prior to this consultation. urine tests revealed no infection, and several courses of antibiotics have not helped him resolve his pain. the clinical examination ; laboratory tests ; radiological investigations ; and abdominal, renal, and testicular ultrasounds were normal. a magnetic resonance urogram was performed, which showed bilateral simplex kidneys and no abnormality of renal or collecting system. uncommon diagnosis of idiopathic urethritis and urethrovasal reflux was considered based on our previous experience. at cystourethroscopy, he was found to have severe posterior urethritis with active inflammation of verumontanum and both ejaculatory ducts were swollen and red. there was a large inflamed utricle with inflamed and swollen verumontanum with debris in the posterior urethra and bladder. the left ejaculatory duct was open and freely refluxing into the seminal vesicle and vas deference (fig. 1). triamcinolone 40 mg diluted in 10 ml of water for injection was instilled topically into the urethra and bladder. he underwent mcug that showed free reflux of the contrast into the left seminal vesicle and vas deference in the micturating film (fig. (a) bulbous urethra, (b) posterior urethra, (c) bladder neck, (d) trigone, (e) ureteric orifices, (f) bladder. micturating cystourethrogram he was seen in follow - up clinic in 2 weeks and was very much better and walking normally. the scrotal pain has gone down to 1/10 and 4/10 on deep palpation of the left epididymis. at 6-week follow - up, he was well, asymptomatic, was not on any medication, and was back to school after a period of 3 months from the onset of illness. a follow - up cystoscopy and cystogram was considered but as the patient was completely asymptomatic and well, the patient and parents were not happy to undertake it unless he gets recurrence in the long run. at 2-year follow - up, he is asymptomatic, both epididymides were normal and nontender, and the ultrasound scan showed complete reversal of thickening to normal on both epididymides. patient, parents, referring pediatricians, and adult urologists were very pleased with the outcome. idiopathic urethritis in male children is diagnosed by excluding bacterial infection and urethroscopy is helpful, but there is no specific treatment available.2 bala eradi and george ninan (2009) for the first time reported use of novel therapeutic intervention of intravesical topical instillation of steroids as an effective treatment with complete reversal of symptoms and the local inflammation.4 since then we had a large series of idiopathic urethritis patients treated with this novel therapeutic option with gratifying results. most patients need one or two instillations and only one case with extension of inflammation into the bladder and ureters needed six instillations. the series of these patients with long - term follow - up is being reported separately. ascending spread and flare is well known in balanitis xerotica obliterans5 ; we could not find any case of nonspecific urethritis having ascending spread to the genital tracts with its urethrovasal reflux and spread of inflammation in association with the literature. the suggestion that the epididymal inflammation was secondary to posterior urethritis is interesting and suggests reflux or efflux of chemical, inflammatory, or infectious agent. reflux into the seminal vesicles, vas deference, and epididymis has been reported as postoperative complication following transurethral resection of the prostate in adults.6 epididymitis is very rare in children, and occasional cases of vassal reflux as a cause of acute scrotum in children has been mentioned in english and non - english literature.7 8 9 structural urologic abnormalities are common in children and in men older than 40 years with acute epididymitis. children may have urethral abnormalities, such as a prostatic utricle, urethral duplication, posterior urethral valves, or urethrorectal fistula, or other anomalies, such as an ectopic ureter, ectopic vas deferens, detrusor sphincter dyssynergia, or vesicoureteral reflux. siegel found that 47% of prepubertal boys with epididymitis had associated urogenital abnormalities, including ectopic vas deferens or ureters, and urethral abnormalities.10 reflux of urine into the ejaculatory ducts of children may result in recurrent orchitis and sterility. urethro - ejaculatory duct reflux (uer) is an uncommonly reported condition in children. the diagnosis of this condition can be made using a mcug to demonstrate the reflux of contrast into any of the ejaculatory ducts.11 symptoms usually are mild and periodic lasting between 1 and 4 weeks at a time, but in our patient, symptoms continued unabated for several months and needed several admissions and investigations, and fortunately got cured following intravesical instillation treatment of single topical steroid. our patient needed an intervention with our novel therapeutic option to help him get out of the persistent symptoms disabling him from his school and requiring significant triple analgesia and number of antibiotics without any relief. we wonder how a single dose of topical steroid even one with the pharmacokinetic profile of triamcinolone would be effective in the long run while reflux persists. earlier in the series, we have done follow - up cystoscopy and cystogram in a patient with posterior urethritis who had inflammation extending into upper urinary tract with complete reversal of the inflammation and vesicoureteral reflux. topical steroids are helpful in early inflammations ; advanced inflammation may need oral and at times intralesional or parenteral steroids to control the severe cases.12 13 uer is more common than originally thought. all boys who present with urogenital symptoms should have an mcug as part of the investigative workup and be scrutinized for uer. management should aim at correcting any underlying anomaly and providing prolonged antibiotics, but vasectomy and the injection of a bulking agent should be considered. the indications for these newer forms of treatments are not clearly defined.11 in conclusion, our patient had a complicated history with nonspecific urethritis resulting in genital tract inflammation predominantly on the left side resulting in recurrent acute scrotum that was investigated with a variety of measures and finally diagnosed at cystourethroscopy and mcug ; it was treated with intravesical instillation of single steroid that resulted in complete resolution. | we describe a case of recurrent left - sided epididymitis secondary to severe idiopathic posterior urethritis extending to left seminal vesicle and vas deference with associated urethrovasal reflux (uvr). cystourethroscopy and micturating cystourethrogram were essential for the diagnosis. following cystourethroscopy, intravesical, and urethral instillation of topical steroid triamcinolone, patient had a full recovery. idiopathic urethritis in association with veru montentitis, utriculitis leading to left - sided uvr, inflammation of the seminal vesicle, and vas deference causing secondary epididymitis is rare. we report the first such rare case presenting as recurrent acute scrotum and response to innovative treatment we used. |
the location of egfr and its ligands in the kidney has been described in the normal kidney. constitutive egfr expression was detected in glomeruli, tubules, and interstitium of normal human kidneys and is normally expressed in several cell types in the kidney that include renal epithelial cells, podocytes, renal interstitial fibroblasts, and mesangial cells. egfr ligands, such as egf, tgf-, and hb - egf are also expressed in renal tubules. other egfr ligands such as amphiregulin, betacellulin, and epiregualtin are detected in the kidney at low levels, but their nephron location remains unclear. upregulated renal expression and de novo expression of egfr and its ligands has been observed in nearly all rodent - kidney injury models. those models include unilateral ureteral obstruction (uuo)-induced renal fibrosis, ang ii - induced renal damage, nephrotoxic nephritis, diabetic nephropathy, ischemia / reperfusion injury. cellular location of increased expression egfr and its ligands vary with the type of kidney disease. in experimental renal fibrosis, there is an upregulated expression of egfr by interstitial cells and renal epithelial cells. in the mode of nephrotoxic serum - induced nephritis, increased egfr was detected in podocytes and hb - egf was in parietal epithelial cells and podocytes. tubular expression of egfr and multiple egfr ligands such as egf, tgf-, amphiregulin, and hb - egf are also upregulated in the epithelium of a murine autosomal - recessive polycystic kidney disease model. moreover, a high level of tgf- in renal tubules was reported in ang ii - induced renal fibrosis. increased egfr although transient egfr activation is seen in acute and mild injured kidneys, its sustained expression is most observed in the kidney subjected to severe and chronic kidney damage. in a mouse model of unilateral urethral obstruction - induced renal fibrosis, egfr expression and phosphorylation increased gradually after urethral ligation and was persistent for at least 21 days. however, egfr activation after injury is not due solely to its increased expression, as egfr phosphorylation is still increased even if egfr levels are normalized. the mechanism responsible for sustained expression / activation of renal egfr after chronic injury remains incompletely understood. we have recently demonstrated that administration of ms-275, a highly selective inhibitor of class i histone deacetylases (hdacs), reduced expression and phosphorylation of egfr in the damaged kidney after uuo injury in vivo and cultured renal epithelial cells in vitro. gilbert. also indicated that application of vorinostat, a pan - hdac inhibitor, also resulted in reduced egfr protein and mrna levels, and attenuated cellular proliferation in cultured renal tubular cells. although the molecular mechanism by which the class i hdac inhibitor suppresses egfr phosphorylation (activation) remains unclear, vorinostat was reported to attenuate the egfr transcript and induce its ubiquitination and targeted it predominantly to lysosome degradation in tumor cell lines in line with this observation, inhibition of a class ii hdac member, hdac6, also accelerates segregation of egfr from early endosomes to the late endosomal and lysosomal compartments for degradation. these studies therefore suggest that both transcriptional and post - translational mechanisms are involved in the modulation of egfr. renal fibrosis is the final common pathway in end - stage renal disease, characterized by aberrant activation and growth of the renal fibroblasts and overproduction of extracellular matrix proteins. increasing evidence indicates that activation of egfr signaling is critically involved in the development and progression of renal fibrosis. demonstrated that the chronic infusion of ang ii causes renal lesions such as glomerulosclerosis, tubular atrophy and/or dilation with microcyst formation, and interstitial fibrosis accompanied by severe proteinuria. these effects of ang ii were reduced in mice overexpressing a dominant - negative form of egfr. similar to this observation, mice overexpressing a dominant - negative egfr construct exhibited significantly less tubulointerstitial injury in the kidney compared with wild - type littermates after subtotal renal ablation. in our study using the model of uuo - induced renal fibrosis, we found that pharmacological inhibition or genetic reduction of egfr activity markedly reduced the expression of -smooth muscle actin, a hallmark of activated fibroblasts, and deposition of fibronectin and collagen i, two extracelular matrix proteins, in the kidney. finally, flamant. indicated that treatment with the egfr inhibitor gefitinib prevented the development of renal vascular and glomerular fibrosis and the decline in renal function in rats with no deficiency induced hypertension. egfr ligands have also been reported to mediate renal fibrosis and deterioration in renal function. in the model of ang ii - induced renal fibrosis, increased expression of tgf- and its sheddase, metalloprotease-17, was observed in mice. specific deletion of tgf- or inhibition of tgf- cleavage with a specific metalloprotease-17 inhibitor substantially reduced ang ii - induced renal damage, suggesting that tgf- may be a key factor between ang ii signaling and egfr transactivation during ang ii - induced nephropathy. tgf- expression was also increased in mice after nephron reduction in the lesion - prone fvb / n strain of mice, and egfr inhibition reduced fibrotic lesion, indicating that tgf- acts in the genetic predisposition to chronic kidney disease (ckd) progression. in addition, sustained expression of hb - egf in myofibroblasts during remodeling of the peri - infarct region of the remnant kidney model has also been reported, suggesting a potential role for this egfr ligand in the progression of ckd. signaling pathways downstream of egfr have been studied in animal models of renal fibrosis. as stated above, egfr activation signals to several signaling pathways including stat3, erk1/2, and akt, although egfr is not the only receptor that mediates activation of these pathways. our previous studies revealed that development of renal fibrosis after uuo injury is accompanied with activation of stat3, and that blockade of stat3 with s3i-201 reduced renal fibrosis. other studies also showed that pharmacological inhibition of erk1/2 decreased levels of fibroblast myofibroblast markers in the interstitium, and that inhibition of phosphatidylinositol-3 kinase reduced the number of proliferating cells and the amount of interstitial extracellular matrix deposition. the mammalian target of rapamycin (mtor) is a major downstream component of phosphatidylinositol-3 kinase. the antifibrotic effects of mtor inhibition have been reported in several animal models of ckd, including diabetic nephropathy, chronic glomerulosclerosis, and tubulointerstitial fibrosis. in addition, mtor and egfr correlation was studied in non - renal fibrotic diseases. for example, blocking mtor with rapamycin could also attenuate tgf--induced pulmonary fibrosis in mice, which was similar to egfr inhibition in this model, suggesting that mtor is a major effector of egfr - induced pulmonary fibrosis. hence, egfr signaling may mediate renal fibrogensis and other organ fibrosis through activation of multiple signaling pathways, in particular, the phosphatidylinositol-3 kinase - mtor signaling pathway. development of renal fibrosis after injury is involved in activation of multiple signaling pathways. however, activation of tgf- signaling is the most important mechanisms. in this signaling pathway, tgf-1 binding to tgf- receptor ii, results in the recruitment, phosphorylation, and concomitant activation of tgf- receptor i. activated tgf- receptor i induces phosphorylation of smad3, which forms a complex with smad4 that is then translocated to the nucleus where it drives the expression of tgf- target genes such as collagens. studies from our and other groups have demonstrated that activation of egfr signaling is critical for increased production of tgf-1 in murine models of renal fibrosis induced by uuo injury or chronic ang ii infusion. in vitro studies indicated that erk1/2 act downstream of egfr to mediate ang ii - induced sustained expression of tgf-1. as smad3 activation is central to tgf--induced fibroblast cell activation and matrix protein deposition, we also examined the effect of egfr inhibition on smad3 phosphorylation and found that either pharmacological or genetic blockade of egfr reduces phosphorylation of smad3 in uuo - induced renal fibrosis. further, exposure of cultured renal interstitial fibroblasts to gefitinib also reduced smad3 phosphorylation and the expression of fibronectin and collagen i in response to tgf-1 stimulation. in line with this observation, chen. observed that gefitinib attenuates tgf-1-induced cell mitogenesis via the egfr - erk1/2/p38 kinase pathway in cultured renal interstitial fibroblasts. these observations suggest that egfr is coupled to tgf- signaling to regulate its activation and mediate its biological consequences. epithelial cells arrested at the g2/m phase are a profibrotic phenotype that triggers production and release of tgf-1 and connective tissue growth factor, suggesting that egfr - mediated epithelial cell g2/m phase arrest might be an important mechanism for transducing epithelial injury to renal interstitial fibroblast activation in the setting of chronic kidney injury. although the complex cascade from epithelial cell injury to fibroblast activation and extracellular matrix protein deposition is the current subject of research, we observed that inhibition of egfr by gefitinib or genetic reduction of egfr decreased the number of renal tubular cells arrested at the g2/m phase, suggesting that egfr has an essential role in this process. our recent research indicated that overexpression / activation of egfr increased the expression of p21, a molecule associated with regulation of cell cycle arrested in the g2/m phase (zhuang s, unpublished observation). multiple cytokines / chemokines including tumor necrotic factor- and monocyte chemoattractant protein-1 are upregulated and involved in this process. to understand the role of egfr in their expression in the fibrotic kidney, we examined inhibition of egfr on expression of both tumor necrotic factor- and monocyte chemoattractant protein-1 in the injured kidney by uuo. our results showed that either pharmacological or genetic inhibition of egfr reduced their expression, indicating that egfr activity is necessary for regulation of tumor necrotic factor- and monocyte chemoattractant protein-1 expression. how egfr contributes to upregulation of proinflammatory cytokines is not clear. given that tubular epithelial cells are considered to be a prominent source of cytokines / chemokines in the kidney, and egfr - mediated epithelial cell arrested at the g2/m phase is associated with overproduction of tgf-1 and connective tissue growth factor after chronic injury, it is assumed that egfr signaling may also promote production of cyokines / chemokines through a mechanism involved in epithelial cell cycle arrested at the g2/m phase. taken together, sustained activation egfr signaling regulates renal fibrogenesis through at least two mechanisms : (1) mediating activation of tgf- signaling components (i.e., smad3) and (2) facilitating production of profibrotic factors (i.e., tgf-1). development of renal fibrosis after injury is involved in activation of multiple signaling pathways. however, activation of tgf- signaling is the most important mechanisms. in this signaling pathway, tgf-1 binding to tgf- receptor ii, results in the recruitment, phosphorylation, and concomitant activation of tgf- receptor i. activated tgf- receptor i induces phosphorylation of smad3, which forms a complex with smad4 that is then translocated to the nucleus where it drives the expression of tgf- target genes such as collagens. studies from our and other groups have demonstrated that activation of egfr signaling is critical for increased production of tgf-1 in murine models of renal fibrosis induced by uuo injury or chronic ang ii infusion. in vitro studies indicated that erk1/2 act downstream of egfr to mediate ang ii - induced sustained expression of tgf-1. as smad3 activation is central to tgf--induced fibroblast cell activation and matrix protein deposition, we also examined the effect of egfr inhibition on smad3 phosphorylation and found that either pharmacological or genetic blockade of egfr reduces phosphorylation of smad3 in uuo - induced renal fibrosis. further, exposure of cultured renal interstitial fibroblasts to gefitinib also reduced smad3 phosphorylation and the expression of fibronectin and collagen i in response to tgf-1 stimulation. in line with this observation, chen. observed that gefitinib attenuates tgf-1-induced cell mitogenesis via the egfr - erk1/2/p38 kinase pathway in cultured renal interstitial fibroblasts. these observations suggest that egfr is coupled to tgf- signaling to regulate its activation and mediate its biological consequences. epithelial cells arrested at the g2/m phase are a profibrotic phenotype that triggers production and release of tgf-1 and connective tissue growth factor, suggesting that egfr - mediated epithelial cell g2/m phase arrest might be an important mechanism for transducing epithelial injury to renal interstitial fibroblast activation in the setting of chronic kidney injury. although the complex cascade from epithelial cell injury to fibroblast activation and extracellular matrix protein deposition is the current subject of research, we observed that inhibition of egfr by gefitinib or genetic reduction of egfr decreased the number of renal tubular cells arrested at the g2/m phase, suggesting that egfr has an essential role in this process. our recent research indicated that overexpression / activation of egfr increased the expression of p21, a molecule associated with regulation of cell cycle arrested in the g2/m phase (zhuang s, unpublished observation). multiple cytokines / chemokines including tumor necrotic factor- and monocyte chemoattractant protein-1 are upregulated and involved in this process. to understand the role of egfr in their expression in the fibrotic kidney, we examined inhibition of egfr on expression of both tumor necrotic factor- and monocyte chemoattractant protein-1 in the injured kidney by uuo. our results showed that either pharmacological or genetic inhibition of egfr reduced their expression, indicating that egfr activity is necessary for regulation of tumor necrotic factor- and monocyte chemoattractant protein-1 expression. how egfr contributes to upregulation of proinflammatory cytokines is not clear. given that tubular epithelial cells are considered to be a prominent source of cytokines / chemokines in the kidney, and egfr - mediated epithelial cell arrested at the g2/m phase is associated with overproduction of tgf-1 and connective tissue growth factor after chronic injury, it is assumed that egfr signaling may also promote production of cyokines / chemokines through a mechanism involved in epithelial cell cycle arrested at the g2/m phase. taken together, sustained activation egfr signaling regulates renal fibrogenesis through at least two mechanisms : (1) mediating activation of tgf- signaling components (i.e., smad3) and (2) facilitating production of profibrotic factors (i.e., tgf-1). the later mechanism is summarized in figure 1. several studies have also demonstrated increased expression of egfr and its ligands in the fibrotic kidney, and the beneficial effect of egfr inhibition in attenuating development of renal fibrosis in a variety of animal models. thus, egfr may be an attractive candidate for targeted therapeutic treatment of renal fibrosis. to date, several anti - egfr therapies have been developed (table 1), and the clinical application of egfr inhibitors for patients with high levels of egfr expression in non - small - cell lung cancers suggests that blockade of egfr with these inhibitors may also be effective in treating ckd in humans. because egfr activation and egfr - induced cellular events are mediated by downstream signaling pathways, therapies inhibiting various aspects of the egfr signaling cascade may be also beneficial in alleviation of ckds. in this regard, pharmacological inhibition of adam metallopeptidase domain 17, a protease that mediates cleavage of the mature form of egfr ligands from membrane precursors, also attenuates renal lesions induced by ang ii infusion. owing to expression of egfr in epithelia under a physiological state, side effects other side effects include nausea, vomiting, loss of appetite, diarrhea, dryness, itching, acne, and weakness. beyond its fundamental role for kidney development and physiology, the egfr signaling is implicated in many pathologic conditions including renal fibrogenesis. excessive egfr signaling induces renal fibrosis through increasing tgf-1 expression, regulating activation of tgf- signaling, promoting epithelial cell g2/m arrest, and enhancing production of inflammatory cytokines / chemockines. given that egfr is activated by ligand - dependent and -independent mechanisms and mediates fibrotic responses, egfr could serve as convergent platform for diverse insults to induce / promote renal fibrogenesis. on this basis, it is envisioned that blockade of egfr signaling pathways would have therapeutic potential for halting or slowing down progression of renal fibrosis in kidney diseases. | signaling through the epidermal growth factor receptor (egfr) is involved in regulation of multiple biological processes, including proliferation, metabolism, differentiation, and survival. owing to its aberrant expression in a variety of malignant tumors, egfr has been recognized as a target in anticancer therapy. increasingly, evidence from animal studies indicates that egfr signaling is also implicated in the development and progression of renal fibrosis. the therapeutic value of egfr inhibition has not yet been evaluated in human kidney disease. in this article, we summarize recent research into the role of egfr signaling in renal fibrogenesis, discuss the mechanism by which egfr regulates this process, and consider the potential of egfr as an antifibrotic target. |
the number of people with diabetes is increasing due to population growth, aging, urbanization, and increasing prevalence of obesity and physical inactivity. predictions show that the number of people with diabetes worldwide will double in the next generation. in the quality of diabetes management in five countries with advanced economies (australia, canada, new zealand, usa, and uk) has been analysed. yearly costs (e.g., 132 billions usd in the usa) and deaths due to diabetes (e.g., 143 308 people in the usa) are reported in that paper. in face of such a disastrous situation improving accessibility of the african population to diabetes care seems to be a big challenge in most of african countries such as the democratic republic of congo (drc), a country which is four times bigger than france and where the number of care centers is reduced. drc has 71 million inhabitants. around 7% of the population in the capital city kinshasa most of diabetic patients in the drc do not control regularly their glucose values, occasional checks are the only executed options. what are the factors which are responsible for deficiency in the successful management of diabetes in africa ? a number of obstacles make the detection, prevention, monitoring, and treatment of diabetes difficult in most african countries. the first group of difficulties is related to the health facilities which are not sufficient to cope with the scale of the problem. prevention and education are not sufficiently promoted, although they should be given high priority. there are not enough medical specialists and the few existing focus their work in big cities without addressing the needs of rural populations. therefore, patients from rural areas are forced to spend money and time travelling long and expensive journeys to access diabetes care. another point, the priority of health politics in several african countries is oriented on the management of transmissible diseases such as hiv and not to chronic diseases like diabetes and high blood pressure. it is impossible for the majority of the population to afford the cost of the diabetes medication in particular insulin. due to life condition, patients are unable to control a proper diet, especially when the family eats by hand in the same dish. limited resources push the population to focus on high calorie foods rich in fat, sugar, and salt. the third group of difficulties is related to the sociocultural context which has a considerable weight in africa. africans have difficulty accepting the concept of chronic illnesses requiring lifelong treatment, so when diabetes is well balanced, patients often stop the treatment thinking the disease is cured. moreover, patients do not find it useful to carry out regular glucose checks although this is necessary for the adaptation of the insulin therapy. the next point deals with the fact that the struggle against obesity faces the belief that being fat is a sign of wealth, good health, and beauty. according to the african tradition, women weight loss, even if it is wanted, raises suspicion of possible hiv infection in the continent. the last obstacle is caused by traditional medicine which plays a key role in many african countries. this practice is often dangerous when it advices diabetes patients to stop effective treatments and use empirical drugs which have the advantage of being cheap. information and communication technologies have great potential to address some of the challenges faced by the public health care. a number of literature have been oriented on the exploitation of mobile devices for completing traditional therapy [5, 6 ] ; other works have been focused on the architectural issues of a telemedicine intervention [7, 8 ]. a significant number of telemedical products in the area of diabetes management have been developed in the last decade. this is justified by the fact that recent studies have depicted a very critical situation ; over 10% of the united states population over the age of 20 has diabetes. as a result, diabetes is now ranked amongst the ten first leading causes of death. a web - based system for monitoring patients with type 2 diabetes is presented in. in that system weight, blood sugar, and blood pressure are recorded using an application implemented on the mobile phone. in bayer 's gluckofact deluxe software the software recognizes the bayer glucose meter connected to the personal computer and stores automatically the data in a database. the visualization of the recorded data is well organized. however, that software is not extended for online applications. this obliges the patient to regularly visit his doctor or to use another system in order to send the recorded data. the software helps in tracking daily diabetic data ; it has a simple interface for data entry and uses different devices for entering data (windows - pc, android phones, windows mobile phones, iphones, and java phones). it has been proved in [12, 13 ] that telemedicine is a suited instrument to support health care providers in the effective management and prevention of diabetes complications. solution to the four listed main difficulties in properly addressing diabetes in africa can be solved by providing a cost - effective telemedical system and a suitable educational program in order to increase the population awareness. this is the focus of this paper, in which we propose and validate an innovative mobile health application. the system manages a secure collection, processing, storage, and sharing of diabetes - related data and thus reduces the frequency of physical consultations to the treating physician. the system enables the organization of health information through a structured gathering of all relevant diabetes data in one central place enabling only access of authorized medical staff. the system helps improve the quality of service in the care process of diabetes by benefiting from rapid advances in mobile, web, and communication technologies. we strongly believe that the outcome of this work will contribute toward the establishment of a model for diabetes management in countries where access to diabetes care is limited and number of medical care personnel is reduced due to high personnel costs. the diabetes management system mobil diab, presented in this paper, can serve as a complete hospital management system which supports several hospitals, each hospital having its own medical staff and patients and every clinician coaching a group of patients assigned to him. moreover, since e - health systems store sensitive data and should have a proper security and privacy framework and mechanisms, this paper extends the state - of - the art by presenting a system which enables a secure data access, communication, and storage. investigation of the impact of using this system mobil diab in assisting the therapy and treatment of diabetes is the major focus of this paper. mobil diab system is an innovative solution for the assistance and care of diabetes patients. it enables diabetes patients to manage their self - control data around the clock using their mobile devices (android, iphone, and ipads) and/or web - based applications. medical care personnel access patients ' data through a protected web portal. the platform is responsible for the following tasks : connection of web and mobile applications, user - hierarchical model (administration, hospital, doctor, and patient), access control of different user categories, and secure interface to hospital information systems. the concept of the system aims at empowering patients to better manage their disease by allowing them to monitor their blood sugar trends over an extended period of time and draw some actionable conclusions. acquired data are then sent using http / https protocol to the central platform which ensures a secure storage and further processing of the information, figure 1. mobil diab guarantees a series of benefits for patients, health care staff, and public health system. benefits for the patients include, among others, unimpeded patient mobility, data input via smart phones and/or via web, regular self - control of diabetes - related data enables the right care to be administrated at the right time, potential to improve care process and quality of service, improvement in patient 's motivation through their involvement in the therapy process, reduced check - up frequency to doctors, and use of mobile health technologies encourages diabetes patients to change their behavior / lifestyle and improve their health. benefits for the health care staff involve, among others, the following : complete and regular data input which is helpful for individual therapy plan, minimization of errors caused by lack of information about the disease history, improvement of the care process quality, getting specialist opinions, access to patients ' data worldwide independent from time and location, and automatic alarm message in case of critical data from a patient. benefits for the health system include, among others, the following : delaying and reducing diabetes complications, minimizing hospitalization rates due to diabetes complications, reducing death rates from diabetes, speeding up the transition of patients from hospitals to their own homes which leads to a reduction in costs, and enabling the organization of health information through a structured gathering of all relevant data in one central place. the system helps to easy track patterns and trends in diabetes management process, helps in taking right treatment decisions and lead to a better glucose control, is suitable for self - care, home care, family physicians, clinics, and hospitals, presents statistics that cover everything doctors and patients need, allows risk monitoring with automated alarm message to care provider and trusted person, preserves mobility through the use of apps and web technologies, and acquires a highly secured database platform with end - to end encryption and data are worldwide available in all major languages. the mobile application mobil diab has an intuitive user interface which is self - explained. you can choose the action you wish to perform as shown on the screenshot of figure 2. data are automatically synchronized in background with the online system so that medical care providers receive these data in real time. to record diabetes - related data, these data may include, amongst others, the blood glucose measurement, insulin intake, sport done with duration, blood pressure measurements, and body weight and size. data acquisition can also take place automatically from glucose meters supporting the ieee 11073 personal health data standards. therapy plans, instructions, and recommendations sent by the doctor from the doctor portal are received directly in the mobile application. for patients without smartphones (mobil diab app), they can still get these doctors ' feedbacks in the protected patients ' portal, through their email address or as sms if this service was activated. the web structure of the system is conceived in order to serve as a complete hospital management system. four web - based portals are integrated to the system and hosted from the central platform. they are designed for four different types of users : patients, doctors, hospital administrators, and system administrator. the graphic shows the glucose measurements of one day, amount of carbohydrate taken (blue color facing up), and units of insulin injected (purple color facing down). clear graphical representation of trends and statistics enables clinicians to make appropriate decisions on therapy adjustment. the tool helps medical staff save their times while providing high quality service to diabetes patients. for emergency cases, an sms is automatically generated and sent to the treating physician, to alert him to check the critical situation in the portal and be able to give direct instructions to the patient. the patent - protected platform illustrated by figure 5 has been developed to help bridge the gap between health and mobility. diverse healthcare modules have been implemented and other are planned in order to meet a complete health care service package from diabetes, dermatology, stress, fitness, and long - term health conditions up to the assisted - living for senior. the platform helps consumers track health, wellness, and vital information using a highly secure infrastructure. moreover, interfaces to hospital information systems and practice management software are supported. in this work, the four - layer architecture of the platform enables users to securely share sensitive information. using different devices, such as smartphones and computers, users can access functionalities of applications supported by the core of the platform through the communication layer. security is a general requirement in modern computing environments, but in e - health systems security is an imperative requirement because those systems handle very sensitive data like medical and personal data. the platform acquires features enabling the following.authentication : methods and mechanisms which allow an entity to prove its identity to a remote end.authorization : access control mechanisms and the ability of an entity to access shared resources.data integrity : mechanisms which ensure that when there is an interchange of data between two peer entities, the received data and the original ones are the same, and that no intermediate alteration has occurred.data confidentiality : it assures that stored or transmitted data are well protected from possible disclosure. a mean used to achieve data confidentiality is through cryptographic mechanisms.privacy : it can be defined as an entity 's ability to control how, when, and to what extent personal information about the entity will be communicated to third parties.secure data communication and storage.data availability : data can be accessed by authorized users independently from time and location. authentication : methods and mechanisms which allow an entity to prove its identity to a remote end. authorization : access control mechanisms and the ability of an entity to access shared resources. data integrity : mechanisms which ensure that when there is an interchange of data between two peer entities, the received data and the original ones are the same, and that no intermediate alteration has occurred. data confidentiality : it assures that stored or transmitted data are well protected from possible disclosure. privacy : it can be defined as an entity 's ability to control how, when, and to what extent personal information about the entity will be communicated to third parties. data availability : data can be accessed by authorized users independently from time and location. our proposed architecture addresses authentication and authorization issues, since each user is classified to a category that defines what he / she has access to. all data are encrypted using the symmetric encryption method aes (advanced encryption standard). for the protection of user 's privacy, an implemented mechanism allows different groups of users to have access to different types of medical data. the platform is composed of several modules, each one supporting its group of tasks. this platform was also presented in [1416 ]. in order to test the effectiveness of the diabetes management system mobil diab in the context of african health care system, a trial was conducted in two cities in the eastern part of the democratic republic of congo (goma and butembo). patients diagnosed with type 2 diabetes and aged between 35 and 75 years were recruited randomly. a total of 40 patients were included in the trial phase. for classification and evaluation purpose, the cohort was divided into a control group (conventional therapy without the use of telemedicine system) and an intervention group (treatment with the use of telemedicine system mobil diab). to verify the comparability of the two groups, table 3 shows that the control and intervention groups in terms of anthropometric data and metabolic control parameter are almost the same at the beginning of the trial : the mean age for the intervention group is 53.3 years with a standard deviation of 10.7 years and for the control group is 53.35 years with a standard deviation of 9.59 years. the mean hba1c value for the whole intervention group before the trial was 8.67% and for the control group was 8.59%. the trial was conducted for a period of two months. during this trial period, medical staff of four local hospitals belonging to the health department of the baptist community in central africa (cbca) accessed the system and coached the patients based on the analysis of the sent data. at the end of the trial each participant had to fill a questionnaire evaluating the system mobil diab based on the three following metrics : usability and design, efficiency and therapy satisfaction, and acceptance and appreciations. questions for patients were as follows.q1 : how often could you successfully use the system?q2 : how easy was it for you to cope with the application?q3 : how do you evaluate the input options and design of the application?q4 : how do you evaluate the output options and visualization possibilities?q5 : how do you evaluate the design of the application?q6 : do you find mobil diab as a tool which motivates you in the control of your blood glucose levels?q7 : did feedbacks (messages and therapy) from doctors help in the diabetes management process?q11 : would you wish to continue using mobil diab for managing your diabetes?q12 : would you recommend the system to other users ? q4 : how do you evaluate the output options and visualization possibilities ? q5 : how do you evaluate the design of the application ? q6 : do you find mobil diab as a tool which motivates you in the control of your blood glucose levels ? q7 : did feedbacks (messages and therapy) from doctors help in the diabetes management process ? each medical care personnel had to evaluate the system by answering the following questions.q1 : how difficult was it for you to use the doctor web application to interact with your patients?q2 : how do you evaluate the design of the doctor portal?q3 : how often could you successfully use the system?q4 : how do you evaluate the representation of diabetes data in the portal?q5 : how do you evaluate the monthly representation of patient 's data on graph?q6 : how do you evaluate the feature for report generation?q7 : how do you appreciate the page summarizing all important patient 's data at glance?q8 : is the provided information in portal enough for improving the therapy adaptation?q12 : would you wish to continue using mobil diab for coaching your diabetes patients?q13 : would you recommend the system to other medical care personnel ? q1 : how difficult was it for you to use the doctor web application to interact with your patients ? q5 : how do you evaluate the monthly representation of patient 's data on graph ? q7 : how do you appreciate the page summarizing all important patient 's data at glance ? q12 : would you wish to continue using mobil diab for coaching your diabetes patients ? a 10-point scale from 1 to 10 was used for questionnaire, with higher scores representing a better evaluation of the system based on the given question. patients and medical care staff provided their evaluations as presented in tables 1 and 2. moreover, based on the analysis of patients ' data collected during the trial period, the improvements in clinical outcome could be evaluated. two metrics were used for this purpose : the mean amplitude of glycemic excursions (characterized by the mean blood glucose and its standard deviation) and the glycated hemoglobin (hba1c), as table 3 presents the results. before the trial could start all the involved partners (patients, medical care providers, support teams, administrators, internet and telephone providers, developers, and policy makers) worked closely together during the development and adaptation process of the system. user requirements, functionalities and design, relevant standards and rules, and methodology and market analysis were discussed with all the involved players. analysis of feedbacks from each stage constituted important input for the following stage. at the end, final modifications were made to obtain the final pilot ready for use in a larger scale. through collaboration with the local university, the ruwenzori state university in butembo, democratic republic of congo, both its faculty of medicine and faculty of applied sciences will keep on conducting research in order to contribute to the adaptation of the system to meet other local specific needs. each question was evaluated based on a 10-point - scale from 1 to 10 point with high scores reflecting positive feedback from the user. three metrics were considered for evaluation : usability and design, efficiency and therapy satisfaction, and acceptance and appreciations. score regarding usability and design was the average score for questions 1, 2, 3, 4, and 5 and resulted in a mean value of 7 points out of ten. score for efficiency and therapy satisfaction was the average score for questions 6 and 7 and resulted in a mean value of 7.43 points out of ten. at last, score related to acceptance and appreciations was averaged from questions 11 and 12 and resulted in a mean value of 8.65 points out of ten. score regarding usability and design was the average score for questions 1 and 2 and resulted in a mean value of 7.56 points out of ten. score for efficiency and therapy satisfaction was the average score for questions 3, 4, 5, 6, 7, and 8 and resulted in a mean value of 7 points out of ten. acceptance and appreciations score was averaged from questions 12 and 13 and resulted in a mean value of 8.75 points out of ten. two metrics were used to assess the improvement in the quality of metabolic control : the mean amplitude of glycemic excursions (characterized from the mean bg and its standard deviation) and the mean hba1c of each group. we should notice that the mean amplitude of glycemic excursions is the measure of diabetic instability, or a characteristic of blood glucose behavior and the hba1c is the standard measure of average glycemic control predicting diabetes complications in type 1 and type2. the approximate mapping between hba1c values given in percentage (%) and mean bg (estimated average glucose) measurements was calculated using the following equation : (1)hba1c [% ] = (mean bg[mg / dl]+86)33.3. positive results have been noticed at the end of the trial, this is particularly evident in a significant improvement in the mean hba1c values for the intervention group. the mean hba1c for that group was 8.67% before the start of the trial and could be reduced to 6.89% at the end. this value of 1.78% in the improvement of hba1c indicates that the risk of diabetes complications has been reduced or postponed, thus leading to cost savings. moreover, an important improvement could be noticed in the mean amplitude of glycemic excursions. this is shown in table 3 by an important reduction of the glucose variability (characterized by the standard deviation of the mean bg). the mean bg standard deviation was 48.6 mg / ml for the control group whereas ; this could be reduced to 33 mg / dl for the intervention group at the end of the trial. patients found the system as a helpful tool, since it helped them increase their motivation for regular glycemic control, motivated them to fix some goals or targets values for blood glucose control for the future, and helped them to control their meal since they could access easily the database containing the amount of carbohydrate for each meal and portion they were taking. medical care personnel listed such benefits as follows : the system allowed them to coach several patients at the same time and at a distance, and modernization of health care system allowed them to get rich and useful data through a secure system. the amelioration in the clinical results is due to these above listed benefits provided by the system. however, some disadvantages such as costs for internet connection and time needed for getting used to the application were the preoccupation of both categories of the users. wishes from both categories of the subjects included to the trial (medical staff and patients) for the amelioration of the system were collected and included the following : extend the drug list and meal database in the application to include all local foods, and applied drugs, since some of them were missingmore sensitizing activities and training sessions about the system to patients and medical staffinclude the support of the glucose measuring device kit in the modelcreate a framework for sport activities for patients extend the drug list and meal database in the application to include all local foods, and applied drugs, since some of them were missing more sensitizing activities and training sessions about the system to patients and medical staff include the support of the glucose measuring device kit in the model create a framework for sport activities for patients an it - enabled diabetes management with fully integrated provider - patient system has been presented in this paper. results from the trial conducted in two eastern cities of the democratic republic of congo have proved how the system mobil diab is suited not only for developed countries but also for communities traditionally underserved, those in remote or rural areas with few health services and staff. this population can access diabetes care using mobil diab, because the system overcomes distance and time barriers between health - care providers and patients. the use of mobil diab showed improvement of clinical outcomes of the patients from the intervention group involved in the conducted trial. this has been demonstrated from the amelioration of both the hba1c (from 8.67% to 6.89%) and the mean amplitude of glycemic excursions which is characterized from both the mean blood glucose and its standard deviation. the decrease of the blood glucose fluctuations is demonstrated from results of the mean blood glucose standard deviation from the intervention group compared to the control group at the end of the study (33.0 mg / ml instead of 48.6 mg / ml). this proves how the use of the system could help patients stabilize better their glucose values. positive evaluations of the system from patients and medical staff have been presented based on three metrics : usability and design, efficiency and therapy satisfaction, and acceptance and appreciations. the model developed from the conducted pilot in the democratic republic of congo can be extended and applied for other countries in order to enhance awareness on the diabetes management in the continent and to improve the quality of service in the care process. | the demand for new healthcare services is growing rapidly. improving accessibility of the african population to diabetes care seems to be a big challenge in most countries where the number of care centers and medical staff is reduced. information and communication technologies (ict) have great potential to address some of these challenges faced by several countries in providing accessible, cost - effective, and high - quality health care services. this paper presents the mobil diab system which is a telemedical approach proposed for the management of long - term diseases. the system applies modern mobile and web technologies which overcome geographical barriers, and increase access to health care services. the idea of the system is to involve patients in the therapy process and motivate them for an active participation. for validation of the system in african context, a trial was conducted in the democratic republic of congo. 40 subjects with diabetes divided randomly into control and intervention groups were included in the test. results show that mobil diab is suitable for african countries and presents a number of benefits for the population and public health care system. it improves clinical management and delivery of diabetes care services by enhancing access, quality, motivation, reassurance, efficiency, and cost - effectiveness. |
diagnostic assessment in patients with orofacial pain may be challenging due to the close proximity between teeth and other orofacial tissues, and by symptoms associated with neurological disorders (1). herpes zoster (hz) affecting the oral and maxillofacial region may pose a significant diagnostic challenge and should be considered in the differential diagnosis of those presenting with atypical odontalgia (2). other diagnoses in the early stages of symptoms may include irreversible pulpitis, acute periapical periodontitis or even acute sinusitis. prompt management is required, especially in immunocompromised individuals, to prevent complications, which may cause significant morbidity (3). we report a case of hz affecting the trigeminal nerve presenting as odontalgia in a patient and review the relevant literature. a male patient aged 50 yrs. reported to the department of oral medicine and radiology with the chief complaint of pain since 3 days. on eliciting history of presenting illness patients gave history of pain in the upper left front tooth region 3 days back which was sudden in onset, severe in intensity, continuous in periodicity, throbbing type, initiates by itself, aggravates on chewing and relieves after taking medication. the pain was thought to be arising due to root stumps which was present in the region of 23 and was subsequently removed and patient was put on amoxicillin 500 mg tds and combination of ibuprofen and paracetamol tds as analgesic. next day patient developed rash over his left mid face region which was thought to be as an allergic reaction to one of the above said drugs and subsequently these drugs were withdrawn and patient was given cetrizine and only paracetamol 650 mg was given for control of pain. on day 2, patient developed multiple blisters with erythema and swelling of left mid face region with excruciating pain. diffuse erythema and edematous area was seen on the left middle third of face extending antero posteriorly from philtrum upto the tragus and supero inferiorly from lower eyelid to upper lip. erosions with oozing watery sections were seen on the upper lip region and over the temple region. on intra oral examination, a solitary shallow ulcer measuring roughly around 0.5 cm in diameter was seen i.r.t. left buccal mucosa opposite to 26 which was surrounded by tissue tag suggestive of ruptured vesicle. on hard tissue examination, patient gave history of atypical seizures without aura for which he was on anti convulsant therapy i.e. carbamazepine 200 mg bd and phenytoin 100 mg bd since 10 years and no episode of seizures is reported in the last 10 years. on the basis of clinical examination and history given by the patient a provisional diagnosis of herpes zoster of maxillary branch of trigeminal nerve was given and a differential diagnosis of recrudescent herpes labialis and herpes simplex infection of mid face region was made. since the clinical features were characteristic i.e. unilateral and localized dermatomal presentation of the vesicles, so we made the final diagnosis of herpes zoster of maxillary branch of trigeminal nerve and patient was educated about the viral, self - limiting and contagious nature of the disease and that the disease can spread to other family members and even school children, so necessary precautions needs to be followed, and was given symptomatic treatment and was put on acyclovir 800 mg five times a day for a period of 2 weeks and topical acyclovir to be applied over the lesion. patient was referred for ophthalmic consultation for increased redness which was present in his left cornea, and ophthalmologist told that it was corneal inflammation due to local conditions, and patient was kept on follow up. after 15 days patient was relieved of the symptoms and at one month follow up, patient was totally free of any symptoms. although the lesions healed with scarring, no post therapeutic complications were reported. the majority of hz infections involve the thoracic and lumbar dermatomes ; however, approximately 13% of patients present with infections involving any of the three branches of the trigeminal nerve (4). the ophthalmic branch is most commonly affected ; however, in our case only the maxillary branch is involved ; this is rare (1.7% of cases) (5). a case similar to our case i.e. maxillary nerve involvement with prodromal tooth pain has been reported in literature (6). reactivation of varicella zoster virus(vzv) may occur spontaneously or when host defenses are compromised. increased age (7), physical trauma (8) (including dental manipulation) (9,10), psychological stress (7,8), malignancy (7), radiation therapy and immunocompromised states including transplant recipients, immunomodulatory therapy and hiv (11) infection are predisposing factors for vzv reactivation. there are 1.53 cases of herpes zoster infection (hzi) per 1,000 subjects ; this increases to 10 per 1,000 in those over 75 years (12). in our patient patient also reported increased stress at work place as he was a teacher and examinations were going in his school so that also could be traced as one of the predisposing factors. patients with hz may progress through three stages : prodromal stage, active stage (also called acute stage) and chronic stage (13). the prodromal stage presents as sensations (described as burning, tingling, itching, boring, prickly) occurring in cutaneous distribution of the dermatome and is believed to represent viral degeneration of nerve fibrils.2 during this period, if branches of the trigeminal nerve are affected, odontalgia and pulpal necrosis may occur. for the latter, it is proposed that the reactivated virus may travel the length of the nerve, infect the pulp vasculature lead to infarction and necrosis. furthermore, these symptoms may present up to one month before the acute mucocutaneous lesion, and pose significant diagnostic difficulties. the active stage is characterized by the emergence of the rash which is nearly always accompanied by systemic upset. the characteristic skin rash progresses from erythematous papules and oedema to vesicles and finally to pustules within one to seven days which dry and crust and are exfoliated over two to three weeks leaving erythematous macular lesions that may scar. diagnostic difficulties may be encountered when the vesicular rash does not occur (zoster sine herpete) (2). surprisingly, pain is reported to subside when the rash is most active ; however, it returns during the crusting and scale phase until the rash clears. eruptive phase that hz is at its most contagious and could pose a significant cross infection risk. in this particular case risk of cross infection to students the chronic stage is only seen in approximately 10% of all patients with hz, and is termed post - herpetic neuralgia (phn). it is defines as pain that lingers for 30 days or 120 days (14) after the onset of the acute rash and described as a brief recurrent shooting or shocking allodynia, with a constant, usually deep pain, lasting beyond the period of healing of the active skin lesions. phn affects 870% of patients over age 50 and upto 50% of patients over age of 50 have debilitating pain lasting more than 1 month (15). although post - herpetic neuralgia is the most common complication of hz, other complications include neurological disorders like meningo - encephalitis or aseptic meningitis ; ophthalmologic like uveitis or keratitis ; cutaneous like bacterial superinfection or cellulitis ; and visceral complications like bronchitis or pleuritis ; immunocompromised individuals with hz exhibit a significantly higher rate of complications. periapical lesions, root resorption, (16) tooth exfoliation and alveolar osteonecrosis, (17) have also been reported in association with hz infection. although hz is a self - limiting condition and resolution is usually complete, treatment is indicated in some cases to reduce the acute symptoms of pain and malaise, to limit the spread and duration of the skin lesions and to prevent complications. the pharmacological approach is based on symptomatic relief and antiviral therapy. for many years, acyclovir (acv) (800 mg five times a day) (18) has been the drug of choice for the treatment of vzv infections. recently, other antiviral agents such as valacyclovir(1000 mg three times a day) and famciclovir (500 mg three times a day) (15) have been developed to overcome the low oral bioavailability of acv and its limited and less predictable effect in preventing the development of post - herpetic neuralgia, as well as to provide a more favourable dosage regime. antiviral therapy should be initiated as early as possible, especially when patient factors that may complicate the manifestations of the condition are expected. in our case, since patient was already on anticonvulsants and also the treatment for herpes zoster was started within 72 hrs of the appearance of symptoms, there was no need for any prophylactic treatment for post herpetic neuralgia. in conclusion, a case of hz affecting the second division of trigeminal nerve is reported. this case highlights the importance of a thorough dental history and examination in patients with odontalgia as in our case. in those presenting with atypical odontalgia furthermore, special consideration must be given to patients who are immunocompromised, although in our case the patient was immunocompetent but we could consider some decrease in immunity due to his age. clinicians are urged to recognize the early features of hzi and to provide prompt antiviral therapy to prevent the complications. | herpes zoster (shingles) is caused by reactivation of the latent varicella zoster virus which is present due to an earlier varicella infection (chicken - pox). herpes zoster is a less common and endemic disease than varicella, although factors causing reactivation are still not well known, but it occurs in older and/or immunocompromised individuals. involvement of c3, t5, l1, l2 and first division of trigeminal nerve are the most frequently encountered whereas the involvement of second and third division of trigeminal nerve is rarely seen. during the prodromal stage, the only presenting symptom may be odontalgia, which may prove to be a diagnostic challenge for the dentist, since many diseases can cause orofacial pain, and the diagnosis must be properly established before final treatment. here we present a case of herpes zoster involving the second division of trigeminal nerve masquerading as odontalgia. the difficulties in diagnosis and management are discussed. |
kaposiform hemangioendothelioma (khe) is a rare distinctive vascular tumor of soft tissue that occurs almost exclusively in children. original reports mainly emphasized on skin and retroperitoneum as the most common location of the tumor ; however, zukerberg.1) later described more varied presentations of khe. histologically, it is composed of infiltrating nodules of vessel forming slit - like, crescentic capillaries with individual lumens containing red blood cells. it grows rapidly with focal extension into the adjacent skin, soft tissue and bone1,2). in the extremities there are various reports of single organ involvement of khe in different locations and also a few studies discussing multifocal khe. multifocal khe is an extremely rare entity with only occasional reports available in the literature. to the best of our knowledge, a 13-year - old boy was referred to the pediatric oncology clinic with a rapidly enlarging mass in left elbow for the past three months. physical examination was unremarkable except for the mass in left elbow with extension to the arm and upper forearm causing complete limitation of the joint motion. magnetic resonance imaging (mri) of the left elbow revealed a large 11 cm12 cm16.5 cm heterogeneously enhancing cystic / solid circumferential mass with vague border in volar aspect of distal arm, antecubital fossa and proximal forearm, infiltrating the asssociated muscles, including brachialis, brachioradialis, extensor carpi radialis and all the muscles in the proximal forearm, as well as neurovascular bundles (fig. the bone marrow signal intensity of the distal humerus, proximal radius and ulna were within normal limits with no apparent infiltration. chest computed tomography (ct) scan showed multiple variable sized pulmonary nodules predominantly in the lower zones of both lungs, ranging up to1.5 cm (fig. a whole body scan with 99mtc was normal and showed no evidence of involvement of the bones adjacent to the tumor. histologically, the tumor was composed of hyper- and hypocellular areas with a myxoid stroma. the tumor cells were predominantly composed of mildly pleomorphic cells with oval to spindle shaped vesicular nuclei and scant to moderate amount of cytoplasm. mitotic activity was infrequent in most of the tumor ; however, a few glomeruloid nests composed of pleomorphic epitheliod cells with high mitotic activity were identified (fig. the tumor was diagnosed as khe with focal atypia and increased mitotis (glomeruloid epithelioid nests). a combination of prednisone (starting dose of 2 mg / kg / day for a period of 1 month followed by gradual reduction within 3 months), propranolol and alpha - interferon (3 million units thrice weekly) was started. an obvious regression of the primary tumor resulted and interim chest ct scan after 3 months showed decrease in number of pulmonary nodules. however, he came back with high fever and severe respiratory distress 4 months later. chest ct - scan at this time revealed increase in the size and number of the pulmonary nodules. some of them had evolved into masses up to 6.2 cm with necrotic irregular centers and intense peripheral enhancement as well as bilateral pleural effusion (fig. 2b, 2c). bilateral chest tubes were inserted and lung biopsy was taken by video assisted thoracoscopic surgery. he also developed a hypodense 1 cm round lesion in the spleen in follow - up imagings that were undertaken in 3-month intervals. a repeat bone scan showed increased uptake of the radiotracer in multiple bones. due to multifocal extension of the disease and regrowth of the primary tumor of the extremity the patient received bevacizumab (avastin, roche, basel, switzerland) (15 mg / kg every 3 weeks) and thalidomide (300 mg / day). soft tissue tumor of the extremity and pulmonary involvement showed remarkable response after the first two courses of bevacizumab (fig. 2d) ; nevertheless, shortly thereafter obvious regrowth of the primary tumor ensued. likewise, progressive pulmonary involvement characterized by enlargement of bilateral huge tumoral masses and pleural effusion detected by chest x - ray and chest ct scan indicated unresponsiveness to the aforementioned therapies (fig. khe was first described in 1993 by zukerberg.1) as a distinctive lesion of childhood with common features to both hemangiomas and kaposi 's sarcoma. it is a rare vascular tumor typically seen in children as a cutaneous lesion with ill defined borders. khe is a locally aggressive spindled cell endothelial derived tumor of intermediate malignancy that may cross tissue planes from dermis into subcutis, fascia, muscle and the bone2). according to the largest recent series reported by croteau.4) the prevalence of khe is estimated to be as 0.91 case per 100,000 children. over the past decade, there has been about one new case of khe diagnosed in massachusetts per year, yielding an incidence of 0.071 case per 100,000 children. in the first description of khe by zukerberg.1), 9 cases were reported, of which only two were beyond the first decade of life. interestingly, zukerberg.1) have also reported a case with a deep soft tissue mass of the arm with involvement of adjacent soft tissues, bones and regional lymph nodes, in contrast to our case where despite having a huge soft tissue tumor of the arm, evidence of extension to the bones was absent. although, most are commonly found as visible cutaneous lesions, lesions hidden within the retroperitoneum, abdomen, mediastinum, or cervicofacial regions5) and also regional lymph node metastases have been documented6). distant metastases have not been reported in the course of khe and there is a matter of debate whether it should be considered as multifocality or a true metastasis. fernandez.7) have reviewed the english literature and finally reported 165 cases ; among these 7 were in the chest wall, 4 in the mediastinum, one in the thyroid and one in thymus. a thorough literature review has disclosed four reports of multifocal khe with unusual presentations ; a 3-year - old boy with submandibular lymphadenopathies, swelling of commisure of the lip and a lesion of right thyroid lobe8), a newborn with congenital cutanous multifocal khe9), 3 children with multiple bone involvement10) and an infant with multiple bone lesions and bilateral cervical lymph nodes11). chen.12) presented mri findings of a histologically proven case of khe of ankle in a 12-year - old boy and revealed multiple foci of the cutaneous tumor with different mri morphologies along with intact underlying bones. our case was similar to chen.12) 's with respect to the absence of overlying skin changes, lack of invasion to the underlying bone and heterogeneous enhancement of the soft tissue tumor. an interesting aspect of our case was involvement of the pulmonary parenchyma which started as few small nodules with homogeneous enhancement. during a period of 6 months, the nodules increased both in size and number and displayed irregular necrotic centers with peripheral avid enhancement despite initial response to corticosteroids and interferon. moreover, the patient developed bilateral pleural effusion with negative cytology (fig. 2c). given the patient 's clinical and radiologic deterioration we decided to treat the patient with bevacizumab ; a recombinant humanized antiangiogenic factor along with thalidomide. a search in pubmed identified a total of 6 case reports of epithelioid hemangioendothelioma with 5 cases of thoracic primaries that were treated with bevacizumab ; one had partial response for 13 months, 1 had stable disease, and 4 had progressive disease13). an open - label, multicenter study demonstrated bevacizumab had been an effective and well - tolerated treatment for metastatic or locally advanced angiosarcoma and epithelioid hemangio endotheliomas14). 2d), after a short course of about two months, massive regrowth of pulmonary masses infiltrating normal lung tissues did occur (fig. another intriguing aspect of our case was absence of any evidence of consumptive coagulopathy or kasabach - merritt syndrome, a well - known association in khe7). in conclusion, according to the published literature, this was the first case of biopsy proven khe of both lungs in a 13-year - old boy complicating soft tissue tumor of the extremity with further involvement of the spleen and multiple bones. | kaposiform hemangioendothelioma (khe) is a rare, locally aggressive vascular tumor of intermediate malignancy with resemblance to kaposi sarcoma. it occurs predominantly in pediatric age groups as a cutaneous lesion with focal infiltration into the adjacent soft tissue and bone. although visceral involvement is very uncommon, several cases with bone, retroperitoneal, or mediastinal involvement have been described. khe has been reported to occasionally occur in unusual sites such as the thymus, tonsils, larynx, paranasal sinuses, deltoid muscle, spleen, uterine cervix, thoracic spine, and even the breast. multifocal khe is an extremely rare entity with few reports available in the literature, none of which describes pulmonary involvement. herein, we report a unique case of multifocal khe in a 13-year - old boy presenting with a huge soft tissue mass in the upper extremity complicated by bilateral pulmonary nodules that developed into large, necrotic tumor masses. |
a 49-year - old man with a body mass index of 30 (height 165.4 cm, weight 81.7 kg and abdominal circumference 108.5 cm) was scheduled to have a hemorrhoidectomy under spinal anesthesia. the preoperative laboratory examinations were normal ; sinus rhythm without conduction abnormalities on 12-lead electrocardiogram (ecg). standard monitoring devices were applied ; baseline heart rate (hr) was 81 beats per minute (bpm) with normal sinus rhythm and blood pressure (bp) was 134/72 mmhg. the block level, immediately after a prone position, was l3 checked using an alcohol swab with stable vital sign (bp 102/63 mmhg, hr 92 bpm). about 2 minutes later, the patient complained of nausea and soon after lost consciousness. the ecg monitoring showed complete av block with ventricular asystole (video 1). while reaching for a stretch cart to move the patient to supine position and preparing atropine injection, a sinus rhythm returned and the patient regained consciousness. but soon another episode of av block developed and the patient became unconscious again (video 1). this time he was immediately transferred to a stretch cart for a supine position and simultaneously 0.5 mg atropine was injected. shortly after, both patient 's consciousness and sinus rhythm returned (bp 95/60 mmhg, hr 81 bpm, block level l3). 1. he did not recall syncope events and did not seem to be nervous or restless through out the anesthesia. the operation was performed under lithotomy position without any further development of av block. on questioning, he reported that he had been found unconscious at a local steam sauna with bruise on his forehead a few years ago. we believe that the patient in this case suffered syncope as a result of vagally mediated av block with prolonged ventricular asystole. sudden decrease of venous return is a well - known risk factor of a severe vagal activation. typical cases of vagally mediated av block can be discovered in any conditions which create sudden surge of parasympathetic outflow to heart, such as carotid sinus massage, tilt - induced syncope, vasovagal syncope, and emotional distress. an unopposed parasympathetic hyperactivity is the main pathophysiology responsible for both vasovagal syncope and av block. a vagal surge can cause syncope and av block simultaneously in some patients but not always. indeed, av block during tilt - induced syncope was rarely reproduced and brignole. reported that isolated depression of sinus node function was four times more common than av block during a positive tilt test. although we frequently encounter vagally mediated second- or third - degree av block as an asymptomatic in clinical practice, the resultant sustained ventricular asystole can lead to a loss of consciousness as a result of profound decrease in cerebral blood flow. in this case, the stored telemetry showed heterogeneous presentation of av block during the first episode of syncope (video 1). we suggest that the vagal overactivity in our patient is related to a reduced venous return due to prone positioning. instead of atropine, we believe the restoration of av conduction was a result of the change of the patient 's position because of the drug 's inability to reach central circulation. additionally, patient 's previous syncope episode could be attributed to a vasovagal response considering circumstances where peripheral vasodilation in hot environment could cause central blood volume depletion. we can not confirm that vasovagal av block was also present at that time because vasovagal syncope can occur without heart symptom. prone position is often required for hemorrhoidectomy, the second most common operation performed in 2013. the physiologic changes in prone position have been well described in literatures and the most consistent of which is a decrease in cardiac index. in a prone position, a hypotension in the obstruction of the inferior vena cava (ivc) is influenced by the degree of abdominal compression. since our patient was centrally obese, he is more susceptible to venous obstruction from the high abdominal pressure. the direct compression of the abdomen could have been reduced if parallel thoracic rolls were placed and the patient correctly positioned on them. peripheral venous pooling resulted from spinal anesthesia could be attributed to a sudden drop in central venous volume in this case. geffin and shapiro reported 13 cases of severe bradycardia or asystole from approximately 4000 regional anesthesia. they could not find any common risk factor from patients ' profile and concluded that a reflex, possibly associated low cardiac filling pressure could be the cause of these clinical feature. although the block level was relatively low to produce a significant effect alone in our case, when it was superimposed on ivc obstruction, it could be a significant hazard. vagally mediated av block is generally considered as benign because there is no anatomical abnormality in the av conduction system. if an asymptomatic vagally mediated av block is encountered, these patients should only be followed up in order to monitor the possible appearance of symptoms. however, it is important to understand that life - threatening syncope due to prolonged ventricular asystole may be developed in vagally mediated av block in a small percentage of patients. jang. reported a case of vasovagal cardiac arrest during spinal anesthesia for cesarean section. the symptoms of vasovagal reflex were far more serious than ours and the patient was successfully resuscitated with chest compression, mask ventilation and epinephrine injection. if a patient experiences any vasovagal symptom such as nausea, abdominal discomfort and diaphoresis, adequate venous return should be restored promptly with trendelenburg or leg elevation and relieve of ivc compression. intravenous fluid must be considered if there is hypovolemic evidence and pre - existing hypovolemic should be corrected before establishment of regional anesthesia. kinsella and tuckey recommended ephedrine as a single agent for profound bradycardia and asystole during regional anesthesia, considering the lack of vasoconstrictor effect of atropine. however, for persistent cardiac arrest, external cardiac massage with epinephrine injection must be started early to ensure circulation of resuscitation drugs and perfusion of vital organs. this case illustrates a serious consequence of vagally mediated av block in a prone positioned patient under spinal anesthesia. it seems likely that this phenomenon was chiefly induced by a decrease in venous return, secondary to the postural change from the sitting to the prone. it should be stressed again that the proper patient positioning and constant monitoring should not be overlooked only because patients are awake and under regional anesthesia. mobitz type i block preceded 2 : 1 block, which was followed by 20 s long ventricular systole. | vagally mediated atrioventricular (av) block is a condition which a paroxysmal av block occurs with the slowing of the sinus rate. owing to its unpredictability and benign nature, it often goes unrecognized in clinical practice. we present the case of a 49-year - old man who suddenly lost consciousness when he assumed a prone position for hemorrohoidectomy under spinal anesthesia ; continuous electrocardiographic recording revealed av block with ventricular asystole. he was completely recovered after returning to a supine position. this case calls our attention to fatal manifestation of vagally mediated av block leading to syncope. |
one of the main objectives of dentistry is rehabilitation of the stomatognathic system, restoring form, function, cosmesis, phonation, and self - esteem in individuals who have suffered tissue loss due to trauma, disease, atrophy, surgical intervention, or congenital defects. when a fracture occurs, an osseointegrated implant is placed, or a graft is used for bone augmentation prior to implant placement. the purpose is induction of bone regeneration, that is, the formation of new bone that, after undergoing remodeling processes, is identical to preexisting tissue.1 bone is the second most widely transplanted of all tissues ; only blood is transplanted or transfused more often. over 2.2 million bone grafts are placed worldwide every year, 450,000 in the united states alone.2 autogenous tissue is the ideal substrate for bone augmentation, but harvesting procedures increase operative time and cost and require a second surgical field, which may increase postoperative discomfort. the limited quantity of autogenous tissue that can be obtained from intraoral sites such as the maxillary tuberosity, extraction areas, edentulous alveolar ridge, symphysis menti, ramus, and retromolar space also poses a challenge.3 for these and other reasons, researchers are increasingly searching for alternative approaches or biomaterials that might circumvent these hurdles and reduce surgical morbidity. the advantages of allograft bone include greater availability, avoidance of a second surgical field, prevention of donor site morbidity, lower treatment costs, and improved patient acceptance.4 during the first hundred years (1880 - 1980) of allograft use, the main issue was availability, as there was no legislation in place to protect tissue processing and tissue donors. during the two decades that followed (1980 - 2000) more sensitive blood testing and major efforts on the part of tissue banks to develop better mechanisms for graft cleansing and removal of infectious agents have helped enable safe bone transplantation, and the key concern is now the efficacy of bone grafts.5 incorporation may be defined as the process of union between graft material and host tissue. bone graft incorporation involves a cascade of events similar to the stages of fracture consolidation.6,7 regardless of graft type, incorporation is preceded by a sequential process, which may be divided for educational purposes into the stages of inflammation, revascularization, osteoinduction, osteoconduction, and remodeling.8 allograft bone is necrotic, and incorporation to adjacent bone occurs through identification of necrotic bone spicules (lacking cells in the osteocyte lacunae) bounded by the cementing lines of new bone formation. this process, known as creeping substitution, mimics the way in which necrotic bone is resorbed and neoformed.9 incorporation of allogeneic bone is slower than that of autologous bone at the graft - host interface, as it involves a stronger initial inflammatory reaction and less revascularization and because allogeneic bone plays no role in the early stages of incorporation due to its absence of osteogenic function, allograft bone is entirely reliant on the host site to provide the vital substrate for this stage.7,10 - 12 allogeneic bone appears to act more as a mineral matrix or scaffold that supports cell migration and proliferation.13 the growing popularity of dental implants increased the possibility of treating dental loss. dental care professionals also began to seek excellence in cosmetic results, due to increasingly discerning and demanding patients. in case of major bone loss at the alveolar ridge, implant placement usually must be preceded by bone grafting to restore function, form, and cosmesis.14 - 18 restorative - driven or backwards planning, in which implant placement is determined by the ideal position of the crown for optimal function and cosmesis, is the mainstay of current dental implant treatment.18 this approach sometimes requires the use of bone grafts to correct alveolar ridge defects and thus enable optimal implant placement.4,17,19 several methods are available to improve bone repair, including the use of grafts and laser therapy.20 use of the latter began empirically in the 1960s, and has since gained credibility and adherents worldwide. over the past two decades, several studies on the theme have been published in scientific journals, enabling evidence - based clinical use of laser therapy.21 the present study thus sought to assess the effect of low - level laser therapy on deep - frozen block allograft incorporation in the rabbit calvaria, comparing irradiated and non - irradiated sites. this study was approved by the universidade do estado de santa catarina (udesc) animal experimentation ethics committee with judgment no. 1.04/08 and by the ethics of animal use committee of the pontifcia universidade catlica do rio grande do sul with registry number ceua / pucrs 09/00112. the animal model chosen for this study was the new zealand rabbit (oryctolagos cuniculus). a total of 14 adult female rabbits (age 8 - 10 months, weight 3.5 - 4.5 kg) were used in the study. twelve were randomly allocated into two groups, l (experimental) and c (control), and received allogeneic bone grafts. after measurement of weight and several clinical parameters (including respiratory rate, heart rate, and capillary refill), animals were premedicated with tiletamine / zolazepam (zoletil 100, virbac saude animal, so paulo, brazil) 20 mg / kg and xylazine (anasedan, vetbrands saude animal, brazil) 3 mg / kg i m. animals were then placed in sternal recumbency on an active warming blanket and the marginal ear vein was cannulated with a 24-gauge catheter for administration of normal saline. anesthesia was maintained with 1.0 - 1.5 mac isoflurane (isoforine, cristlia produtos qumicos e farmacuticos ltda., so paulo, brazil), provided through a non - rebreather mask, diluted in 100% oxygen (2 l / min) in a universal anesthetic vaporizer (oxygel, so paulo, brazil). the frontoparietal region was further anesthetized with local infiltration of 0.5 ml plain lidocaine (2%) (xylestesin 2%, cristlia produtos qumicos e farmacuticos ltda, so paulo, brazil). the surgical field was disinfected with povidone - iodine (povidine) and isolated with sterile drapes. a 5 cm - long, full thickness incision was made down to the periosteum overlying the sagittal suture and center of the frontal bone with a # 15 blade. soft tissues were dissected with a freer elevator (golgran, so paulo, brazil). ltda) set to 800 rpm and an 8 mm trephine (neodent, curitiba, brazil), under copious irrigation with saline solution. six bone blocks were harvested from the calvaria of each donor rabbit, for a total of 12 block allografts. block allografts were thoroughly washed with saline solution, cleaned of any soft tissues, placed into sterile containers and frozen at -70c. allografts were thawed in saline at room temperature, drilled through with a 1.6-mm bit (neodent), and fastened to the cranial vault with a 1.5 mm x 6 mm self - tapping screw (neodent) on the right, using a conventional philips screwdriver (neodent). eight - millimeter pieces of bone were trephined from a more anterior region of the skull vault and placed on the left (opposite the experimental site) to serve as an autogenous positive control for bone incorporation. the wound bed was copiously irrigated with saline solution and the incision was closed with simple running sutures (ethicon 5 - 0 nylon). postoperative analgesia consisted of meloxicam 0.1 mg / kg once daily, with rescue tramadol 2 mg / kg (tramadon 50mg / ml, cristlia produtos qumicos e farmacuticos ltda, so paulo, brazil) as needed in case of intense pain. immediately after conclusion of the procedure, animals in group l (experimental) underwent infrared laser irradiation with aluminum gallium arsenide diode laser (algaas ; wavelength 830 nm ; thera lase, dmc equipamentos ltda., brazil), which was subsequently repeated every 48 hours for a total of eight sessions. the laser was applied to four sites (anterior, posterior, left lateral and right lateral aspects of the cranial vault) at an energy density of 4 j / cm, for a total dose of 16 j / cm per session. animals in group c (control) underwent sham irradiation, with the laser unit switched off, to simulate the stress of restraint. six animals were euthanized at 35 days post - graft implantation, and the remaining six at 70 days. anesthesia was induced with tiletamine / zolazepam 20 mg / kg and xylazine 3 mg / kg i m, followed by a lethal injection of 300 mg potassium chloride (cristlia produtos qumicos e farmacuticos ltda. the surgical site was dissected and a 10-mm trephine bit was used to obtain a sample of bone containing the graft and host tissue margins. specimens were kept in 10% buffered formalin solution for 48 hours, decalcified in 5% aqueous nitric oxide, and bisected lengthwise with a microtome knife. specimens then underwent routine processing ; 5-m thick longitudinal slices were obtained and stained with hematoxylin and eosin (h&e). the other half of each sample was sent for scanning electron microscopy (sem) at the pontifcia universidade catlica do rio grande do sul (pucrs) microscopy and microanalysis center. samples were attached to specimen holders with double - stick carbon tape, sputter coated with gold, and placed in a vacuum chamber for observation under a scanning electron microscope. assessment of the effects of laser therapy was based on descriptive, semiquantitative histological analysis based on the methods proposed by weber.20, torres.22 and pinheiro.23 fisher 's exact test was used for statistical analyses. histological assessment showed significant changes, particularly regarding filling of osteocyte lacunae, replacement of graft myeloid tissue with fibrocollagenous tissue, inflammatory reaction (with a macrophage and neutrophil infiltrate), bone remodeling, and neovascularization. allograft group (35 days) : collagen fiber deposition was visible throughout the medullary spaces in both groups (moderate in the lllt group and mild to moderate in the control group). bone remodeling was also evident in both groups (mild to moderate in the lllt group and mild in the control group). in both groups, osteocyte lacunae were mostly filled at the graft - to - host interface, while the surface, center, and periphery of the trabeculae were empty in most lacunae. the inflammatory reaction was mild in the irradiated group and mild to moderate in the control group (table 2). allograft group (70 days) : collagen fibers were present in moderate to marked density in the medullary spaces of irradiated grafts and in slight to moderate density in the control group. bone remodeling was moderate in the lllt group and mild in the control group. lacunae at the graft - to - host interface and surface of irradiated grafts were mostly filled with osteocytes, whereas only moderate filling was visible in the center of the graft. in the control group, most lacunae were empty in the center and periphery, and partially filled at the graft - to - host interface. mild inflammatory reaction with a macrophage and neutrophil infiltrate was present in both groups (table 3). autograft group (35 days) : in both groups, deposition of collagen fibers ranged from mild to moderate in intensity. bone remodeling was moderate to marked in the experimental group and mild to moderate in controls. most osteocyte lacunae were filled in the irradiated group, whereas lacunae in control grafts were filled mostly at the graft - host interface and surface, with most lacunae empty at the center. mild - to - moderate inflammatory infiltrates were present in both groups (table 4). autograft group (70 days) : in the irradiated group, deposition of collagen fibers ranged from mild to marked. in the control group, fibers were seen only in some specimens, and never at a moderate or higher density. moderate to marked bone remodeling was present in the irradiated group, and moderate remodeling in the control group. most osteocyte lacunae were filled in irradiated group grafts, whereas lacunae in control grafts were mostly filled at the graft - host interface, at the surface, and, to a lesser extent, in the core area (table 5). sem at 45x (figure 1 a and b) and 500x magnification and optical microscopy at 40x magnification and 100x magnification (figures 2 a and b, 3 a and b, 4 a and b and 5 a and b) clearly showed graft incorporation at the graft - host interface in all speciments. in areas of cortical bone, grafts were in close contact with recipient areas ; filling of osteocyte lacunae was evident, as were medullary degeneration, necrosis and revascularization in the medullary spaces at the graft - host interface, with deposition of collagen fibers and bone remodeling. assessment showed the osteoconductive properties of block allografts are preserved after deep - freezing, as are their structural characteristics and bone matrix, providing a scaffold for host - to - graft migration of blood vessels and cells. histological assessment showed significant changes, particularly regarding filling of osteocyte lacunae, replacement of graft myeloid tissue with fibrocollagenous tissue, inflammatory reaction (with a macrophage and neutrophil infiltrate), bone remodeling, and neovascularization. allograft group (35 days) : collagen fiber deposition was visible throughout the medullary spaces in both groups (moderate in the lllt group and mild to moderate in the control group). bone remodeling was also evident in both groups (mild to moderate in the lllt group and mild in the control group). in both groups, osteocyte lacunae were mostly filled at the graft - to - host interface, while the surface, center, and periphery of the trabeculae were empty in most lacunae. the inflammatory reaction was mild in the irradiated group and mild to moderate in the control group (table 2). allograft group (70 days) : collagen fibers were present in moderate to marked density in the medullary spaces of irradiated grafts and in slight to moderate density in the control group. bone remodeling was moderate in the lllt group and mild in the control group. lacunae at the graft - to - host interface and surface of irradiated grafts were mostly filled with osteocytes, whereas only moderate filling was visible in the center of the graft. in the control group, most lacunae were empty in the center and periphery, and partially filled at the graft - to - host interface. mild inflammatory reaction with a macrophage and neutrophil infiltrate was present in both groups (table 3). autograft group (35 days) : in both groups, deposition of collagen fibers ranged from mild to moderate in intensity. bone remodeling was moderate to marked in the experimental group and mild to moderate in controls. most osteocyte lacunae were filled in the irradiated group, whereas lacunae in control grafts were filled mostly at the graft - host interface and surface, with most lacunae empty at the center. mild - to - moderate inflammatory infiltrates were present in both groups (table 4). autograft group (70 days) : in the irradiated group, deposition of collagen fibers ranged from mild to marked. in the control group, fibers were seen only in some specimens, and never at a moderate or higher density. moderate to marked bone remodeling was present in the irradiated group, and moderate remodeling in the control group. most osteocyte lacunae were filled in irradiated group grafts, whereas lacunae in control grafts were mostly filled at the graft - host interface, at the surface, and, to a lesser extent, in the core area (table 5). sem at 45x (figure 1 a and b) and 500x magnification and optical microscopy at 40x magnification and 100x magnification (figures 2 a and b, 3 a and b, 4 a and b and 5 a and b) clearly showed graft incorporation at the graft - host interface in all speciments. in areas of cortical bone, grafts were in close contact with recipient areas ; filling of osteocyte lacunae was evident, as were medullary degeneration, necrosis and revascularization in the medullary spaces at the graft - host interface, with deposition of collagen fibers and bone remodeling. assessment showed the osteoconductive properties of block allografts are preserved after deep - freezing, as are their structural characteristics and bone matrix, providing a scaffold for host - to - graft migration of blood vessels and cells. histological assessment showed significant changes, particularly regarding filling of osteocyte lacunae, replacement of graft myeloid tissue with fibrocollagenous tissue, inflammatory reaction (with a macrophage and neutrophil infiltrate), bone remodeling, and neovascularization. allograft group (35 days) : collagen fiber deposition was visible throughout the medullary spaces in both groups (moderate in the lllt group and mild to moderate in the control group). bone remodeling was also evident in both groups (mild to moderate in the lllt group and mild in the control group). in both groups, osteocyte lacunae were mostly filled at the graft - to - host interface, while the surface, center, and periphery of the trabeculae were empty in most lacunae. the inflammatory reaction was mild in the irradiated group and mild to moderate in the control group (table 2). allograft group (70 days) : collagen fibers were present in moderate to marked density in the medullary spaces of irradiated grafts and in slight to moderate density in the control group. bone remodeling was moderate in the lllt group and mild in the control group. lacunae at the graft - to - host interface and surface of irradiated grafts were mostly filled with osteocytes, whereas only moderate filling was visible in the center of the graft. in the control group, most lacunae were empty in the center and periphery, and partially filled at the graft - to - host interface. mild inflammatory reaction with a macrophage and neutrophil infiltrate was present in both groups (table 3). autograft group (35 days) : in both groups, deposition of collagen fibers ranged from mild to moderate in intensity. bone remodeling was moderate to marked in the experimental group and mild to moderate in controls. most osteocyte lacunae were filled in the irradiated group, whereas lacunae in control grafts were filled mostly at the graft - host interface and surface, with most lacunae empty at the center. mild - to - moderate inflammatory infiltrates were present in both groups (table 4). autograft group (70 days) : in the irradiated group, deposition of collagen fibers ranged from mild to marked. in the control group, fibers were seen only in some specimens, and never at a moderate or higher density. moderate to marked bone remodeling was present in the irradiated group, and moderate remodeling in the control group. most osteocyte lacunae were filled in irradiated group grafts, whereas lacunae in control grafts were mostly filled at the graft - host interface, at the surface, and, to a lesser extent, in the core area (table 5). sem at 45x (figure 1 a and b) and 500x magnification and optical microscopy at 40x magnification and 100x magnification (figures 2 a and b, 3 a and b, 4 a and b and 5 a and b) clearly showed graft incorporation at the graft - host interface in all speciments. in areas of cortical bone, grafts were in close contact with recipient areas ; filling of osteocyte lacunae was evident, as were medullary degeneration, necrosis and revascularization in the medullary spaces at the graft - host interface, with deposition of collagen fibers and bone remodeling. assessment showed the osteoconductive properties of block allografts are preserved after deep - freezing, as are their structural characteristics and bone matrix, providing a scaffold for host - to - graft migration of blood vessels and cells. a variety of methods have been studied as potential means of improving the speed and quality of allograft incorporation, including platelet - rich plasma (prp),3,24,25 bone morphogenetic proteins (bmps),5 bisphosphonates,26 adjuvant stem cell and gene therapy,27 and processing techniques, such as laser drilling and partial demineralization of block allografts.28 several studies using various graft materials have shown that infrared laser therapy stimulates osteoblast proliferation, collagen deposition, and new bone formation.29,30 in this study, allogeneic bone tissue was processed by deep freezing. this proved to be an adequate method for allografts, preserving the osteoconductive and structural properties of the graft tissue as a scaffold for vessel and cell proliferation, as demonstrated by partial filling of osteocyte lacunae, early - stage bone remodeling, and incorporation at the graft - host interface. the use of other methods, such as freeze - drying, demineralization, gamma irradiation, ethylene oxide, or hydrogen peroxide, can jeopardize the structural properties of the donor tissue, which are of the utmost importance in onlay grafting.9,12,13,31 - 37 in the present study, collagen deposition was greater in allogeneic and autogenous blocks exposed to laser irradiation. this was consistent with the findings of pinheiro.38, lopes.39 and lopes.40. statistical comparisons of all lllt groups versus controls showed a positive effect of lllt on bone remodeling (p = 0.0269). bone remodeling was significantly predominant (p = 0.0151) in irradiated allograft groups versus controls at 35 and 70 days. this finding is consistent with that of several prior studies that have shown greater bone remodeling after laser therapy.38 - 46 filling of osteocyte lacunae was also greater in the laser group, most markedly at 70 days in the allograft group and at 35 and 70 days in the autograft group, providing evidence of partial allograft bone vitality and preserved osteoconduction during this brief experiment. drtbudak.42 and jakse.47 both reported a similar effect on autogenous grafts. statistical analysis showed greater filling of osteocyte lacunae (p = 0.0009) and greater bone remodeling (p = 0.0267) in autogenous grafts (positive controls) than in allograft blocks. comparison of the autograft and allograft + lllt groups showed greater filling of osteocyte lacunae in the autograft group (p = 0.0375), most likely due to transport across the autogenous graft of cells that remained viable during the incorporation stage10,11,13 and to the biomodulation effects of lllt on host cells and on graft cells that remained viable in the early stages of bone regeneration, during osteoblast proliferation.48,49 inflammatory reaction in the experimental (irradiated) allograft group at 35 days was less intense than in the control group. animal experiments provide a useful means of estimating tissue reaction to bioactive materials ; however, results obtained from animal models should not necessarily be extrapolated to humans.30 optical and scanning electron microscopy showed that the combination of deep - frozen bone allografts and lllt is an adequate alternative for the treatment of bone defects. after lllt, deep - freeze - processed bone allografts showed incorporation at the graft - host interface, moderate bone remodeling, partial filling of osteocyte lacunae, less inflammatory infiltrate in the early postoperative period, and higher collagen deposition than the control group, over the course of this brief study. use of the deep - freezing method for processing of bone grafts preserves the structural and osteoconductive characteristics of bone tissue. | objective to assess the effect of low - level laser therapy (lllt) on the incorporation of deep - frozen block allografts in a rabbit model.background data studies have shown that lllt has beneficial effects on tissue repair and new bone formation.methods bone tissue was harvested from two rabbits, processed by deep - freezing and grafted into the calvaria of 12 animals, which were then randomly allocated into two groups : experimental (l) and control (c). rabbits in group l were irradiated with an aluminum gallium arsenide diode laser (algaas ; wavelength 830 nm, 4 j / cm2), applied to four sites on the calvaria, for a total dose of 16 j / cm2 per session. the total treatment dose after eight sessions was 128 j / cm2. animals were euthanized at 35 (n = 6) or 70 days (n = 6) postoperatively.results deep - freeze - processed block allografts followed by lllt showed incorporation at the graft - host interface, moderate bone remodeling, partial filling of osteocyte lacunae, less inflammatory infiltrate in the early postoperative period, and higher collagen deposition than the control group.conclusion optical microscopy and scanning electron microscopy showed that allograft bone processed by deep - freezing plus lllt is suitable as an alternative for the treatment of bone defects. use of the deep - freezing method for processing of bone grafts preserves the structural and osteoconductive characteristics of bone tissue. |
Subsets and Splits
No community queries yet
The top public SQL queries from the community will appear here once available.