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Upper urinary tract urothelial carcinoma (utuc) is uncommon, accounting for only 5% to 6% of all urothelial carcinomas . The estimated annual incidence in western countries is about 2 new cases per 100,000 individuals . The peak incidence of utuc is in the seventh and eighth decades of life, and utuc occurs three times more frequently in men than in women . Whereas just 15% to 25% of bladder tumors are invasive at diagnosis, 60% of utucs are invasive at diagnosis and utucs have a poorer prognosis . Radical nephroureterectomy (rnu) with excision of the ipsilateral bladder cuff is considered to be the gold standard treatment for utuc . However, despite radical surgical intervention, it has become increasingly recognized that a significant proportion of patients still die from their disease, possibly as a result of micrometastasis at the time of operation, with a 5-year disease - specific mortality of 15% to 30% . Several facets of endoscopic management, including the potential benefits of nephron - sparing surgery, the multifocal recurrent nature of utuc, and the evolution in endoscopic technology, have contributed to this interest . The recently published 2013 european association of urology guidelines indicate that conservative management of utuc can be considered in imperative or elective cases for low - grade, low - stage tumors . In this article advances in endoscopic technology, including the development of improved optics, progressively smaller and more durable rigid and flexible endoscopes, actively deflecting telescopes, and adjunctive instrumentation, have enabled endoscopic management to become more practical and efficacious when used for both diagnostic and therapeutic purposes in utuc cases . Traditionally, endoscopic management of utuc was recommended only for patients with imperative indications, such as renal insufficiency or a solitary functional kidney, bilateral disease, or a significant comorbidity that precluded radical surgery . Recently, however, the 2013 european association of urology guidelines suggested that endoscopic management of utuc could also be used for elective cases (when the contralateral kidney is functional) if specific criteria are met, such as a unifocal tumor, tumor size <1 cm, low - grade tumor (cytology or biopsies), no evidence of an infiltrative lesion on computed tomography (ct) urography, and an understanding of the necessity of close follow - up . Direct visualization of upper urinary tract tumors can be obtained by using tumor biopsy specimens and selective urine cytology . Tumor grading in this setting is very accurate and is 90% in agreement with the grade of the final histopathological specimen . Although ureteroscopy has not been demonstrated to be a dependable method of staging and therefore has been characterized by some authors as unreliable in determining stage, recent reports suggest a highly reliable concordance between biopsy grade and histopathological stage . In addition, ct urography can detect with high accuracy evidence of utuc extending beyond the wall of the ureter or renal pelvis . Therefore, more accurate utuc characterization can be obtained through the combination of tumor grading on biopsy and clinical staging by ct urography . Endoscopic management of utuc can be performed via either an ureteroscopic retrograde or a percutaneous antegrade approach . Advances in ureteroscopic techniques and instruments allow for retrograde access to the entire upper urinary tract . Small - diameter rigid and flexible ureteroscopes with greater deflecting abilities have been combined with endoscopic biopsy techniques and devices for tissue ablation to offer practical approaches to managing upper urinary tract tumors . However, the ureteroscopic approach is limited by the size of the instruments that can be adjusted in the ureter and the size of tumor that can be adequately managed . Some parts of the upper urinary tract, such as the lower calyces, are less accessible when using the retrograde approach . Also, the retrograde ureteroscopic approach is difficult in patients who have undergone a previous urinary diversion . Following retrograde access, a utuc of the distal ureter is cleared with a rigid ureteroscope, and the remains of the ureter and renal pelvis are observed with a flexible ureteroscope . After an initial biopsy of the lesion, available management options are performed and can include mechanical debulking (cold - cup forceps or stone basket), electrofulguration, electroresection, laser photo - coagulation, or ablation . Mechanical removal of small utucs can be very effective, and the biopsy process can remove significant volumes of tumor . An electrosurgical technique was first used for the management of ureteral tumors, but the use of this technique is mostly confined to the distal ureter owing to the rigid design of the resectoscope . In particular, the holmium: yttrium aluminum garnet (ho: yag) and neodymium: yttrium aluminum garnet (nd: yag) lasers used to cauterize and ablate utucs, which are supplied through small - diameter, flexible fibers, permit the management of relatively large utucs . The ho: yag laser is effective because it can both coagulate and ablate tissue and has minimal tissue penetration (<0.5 mm) and therefore can be used for ureteric lesions . The nd: yag laser has been used widely for the treatment of both bladder and upper tract tumors . It has a greater depth of penetration (4 - 6 mm) and provides a deeper coagulation and ablative effect on the tumor . The nd: yag laser, penetrating to a depth of several millimeters, is used to coagulate the major volume of the tumor, and then the coagulated tissue can be removed with the ho: yag laser . Treatment outcomes from the current literature of ureteroscopic management for utuc are summarized in table 1 . Upper urinary tract and bladder recurrence rates for patients managed ureteroscopically are reported to be 20% to 90% and 15% to 53%, respectively, and several studies with limited follow - up have suggested 45% to 100% overall survival (os) and 82% to 100% cancer - specific survival (css). Upper urinary recurrence occurred in 68% (50/73), and 19% of the patients (14/73) eventually proceeded to nephroureterectomy . The estimated os and css were 69.7% and 88.9%, respectively, at 5 years, and 40.3% and 77.4%, respectively, at 10 years . The estimated mean and median os times were 119 months and 107 months, respectively . Similar results were reported by cornu et al . In 35 patients undergoing ureteroscopic resection of utuc . The 3-year cancer - specific and recurrence - free survival rates were 100% and 35%, respectively . The median interval to recurrence was 10 months, and four patients ultimately underwent nephroureterectomy . The incidence of perforation is 0% to 10%, and perforations are treated by ureteral stenting or percutaneous nephrostomy drainage . Evidence suggests that a lower incidence of stricture is associated with lesions treated by laser ablation rather than by electrocoagulation . Most strictures can be treated by ureteral stenting, laser incision, or balloon dilation . Generally, ureteroscopic management has a notably lower requirement for blood transfusion and a lower overall complication rate than does percutaneous management . Painter et al . Classified 45 patients who underwent ureteroscopic treatment of utuc into elective, relative (patients who rejected radical surgery), and imperative groups . At 24 months of follow - up, the css was 100% in the elective group . In comparison, 12 of 16 patients in the relative group required nephroureterectomy, and 6 patients had stage pt2 or greater disease on the final histopathological examination . Thus, ureteroscopic management should be considered for low - volume, low - grade utuc in healthy populations who are candidates for radical treatment . Compared 121 patients who underwent nephroureterectomy, 75 patients who underwent nephroureterectomy after ureteroscopic biopsy, and 12 patients who underwent tumor ablation . No significant difference was found in css among the three groups (85%, 81%, and 83%, respectively) at a mean follow - up of 37 months . An important point is that ureteroscopic biopsy or ablation before radical surgery does not adversely affect the oncologic outcomes compared with immediate radical nephron - ureterectomy . Although the ureteroscopic retrograde approach has the benefit of conserving a closed urinary system, the percutaneous antegrade approach can be considered for low - grade or noninvasive utuc in the renal cavities . The principal advantage of the percutaneous approach is that it allows for the removal of a larger tumor volume from any site of the collecting system owing to the use of instruments with larger working channels, which allows better visualization and faster resection . The percutaneous approach also allows better access to the lower caliceal system, which is inaccessible by flexible ureteroscopy . After the establishment of a percutaneous tract, the lesion is initially biopsied and subsequently debulked . Various techniques have been used for tumor ablation, including monopolar and bipolar electrocautery, rollerball electrodes, lasers, and electrovaporization . The whole tumor is ablated, and the base of the lesion is resected or fulgurated . The established nephrostomy tract can be maintained, allowing for repeated treatment or administration of topical adjuvant therapy . Treatment outcomes in the current literature for the percutaneous management of utuc are summarized in table 2 . Upper urinary tract and bladder recurrence rates for patients managed percutaneously are reported to be 13% to 65% and 15% to 42%, respectively, and several studies with limited follow - up suggested os of 68% to 96% and css of 75% to 100% . Retrospectively reviewed 34 patients who underwent percutaneous management of utuc; 15% had grade 3 tumors with either a solitary kidney or bilateral disease . During 51 months of follow - up, upper urinary tract recurrence was found in 44% of cases, at a median time of 24 months . The rates of os and css were 74% and 94%, respectively . In a more recent study, roupret et al . Reported on the outcomes of 24 patients who underwent a percutaneous approach for utuc . During follow - up, which was a median of 62 months three recurrences were in the ipsilateral ureter, one in the contralateral ureter, and four in the bladder . Five patients with high - grade or invasive tumors subsequently required nephroureterectomy; one patient immediately and four patients during follow - up . Five patients (20.8%) died, and four of those deaths were attributed to disease progression . The authors reported 5-year disease - specific survival rates as 79.5% and tumor - free survival rates as 68% . The tumor grade and stage were predictive of disease - specific survival and recurrence - free survival . The principal disadvantage of the percutaneous antegrade approach is the increase in morbidity compared with the ureteroscopic retrograde approach . Bleeding is a latent complication of percutaneous management owing to the vascularization of the kidney . Transfusion rates are greater than 20%, while obstruction of the ureteropelvic junction from stricture, adjacent organ injury, and pleural injury are less common . There is a theoretical risk of seeding resulting from tract puncture and perforations that may occur during the procedure . For tract seeding the reported incidence of seeding of utuc within the percutaneous tract is extremely rare, being only 0.75% in the most experienced center . Data suggest the percutaneous management of utuc as a tolerable alternative to radical surgery in patients with low - grade, low - volume tumors . However, the elective indications for the percutaneous management of patients with high - grade tumors are unclear . This approach is being gradually abandoned owing to the development of enhanced materials and advances in distal - tip deflection of recent ureteroscopes . Topical immunotherapy with bacillus calmette - guerin (bcg) and chemotherapy with mitomycin c have been the mainstays of adjuvant topical therapy in bladder cancer . Adjuvant topical therapy has also been used after endoscopic management for utuc in an attempt to decrease recurrences . Instillation can be performed through a percutaneous nephrostomy tube or via a retrograde ureteral catheter . The aim of therapy is continued exposure of the urothelium to the topical agent while maintaining a low pressure system that is free of infection . Several agents have been used, including thiotepa, interferon, adriamycin, and mitomycin c, but the most commonly used agent is bcg . Adjuvant topical therapy with both mitomycin c and bcg has been the most widely studied, because these agents are considered to provide the best outcomes for reducing the recurrence rates of utuc . Treatment outcomes from the current literature for adjuvant topical therapy for utuc are summarized in table 3 . Recently analyzed data gathered over 20 years with adjuvant bcg after percutaneous management of utuc . Bcg was instilled as a 6-week course to 50 renal units, and outcomes were compared with 39 control renal units . In general, there was no statistical difference in recurrence, progression, or time to recurrence between the control and treatment groups when classified by grade and stage . The estimated five - year utuc recurrence - free survival was 53.8% for patients treated with adjuvant mitomycin c and 54.2% for those without adjuvant treatment . Even with substratification by utuc grade, adjuvant topical treatment did not show any difference in utuc recurrence - free survival for grade 1, grade 2, or grade 3 utuc . There is no proven efficiency for the administration of adjuvant topical therapy (bcg or mitomycin c) in the management of papillary utuc . Reports have typically been limited to small, retrospective cohort studies with limited follow - up (typically <36 months), mainly assessing the use of bcg and with very few assessing mitomycin c . Although adjuvant topical therapy with bcg has not shown proven efficacy for papillary utuc, several studies have reported positive outcomes for upper urinary tract carcinoma in situ (cis). These studies collectively showed an 85% positive initial response rate and a 37% upper urinary tract recurrence rate, with a follow - up of 20 to 51 months (table 4). The major limitation of these studies is that the diagnosis of upper urinary tract cis was made on the basis of the presence of positive selective urine cytology and negative radiologic imaging findings rather than biopsy - proven disease . In addition, the response rates were based on restoration of normal urine cytology rather than more specific criteria, such as biopsy and ureteroscopy . Although the initial positive response rates with adjuvant bcg therapy for upper urinary tract cis appear encouraging, the disease - specific mortality outcomes varied considerably, between 9% and 40% beyond 3 years, and have not been confirmed in longer terms . The potential side effects of adjuvant bcg therapy include fever, flu - like illness, irritative voiding symptoms, septicemia, and death . It is also important to consider the risk of mitomycin c spillage outside the urinary tract and its tendency to generate inflammation and necrosis, especially when used in a percutaneous approach . Interestingly, renal function is not impaired after administration of bcg or mitomycin c, thus preserving quality of life owing to the retained kidney . In the majority of cases, complications from the instillation of chemotherapy or immunotherapy can be precluded by sustaining low intracavitary pressures during administration . Additionally, extravasation and obstruction should be systemically controlled by antegrade or retrograde radiographic opacification before the instillation of these adjuvant topical agents . Long - term endoscopic follow - up of the upper urinary tract and bladder is obligatory . As is seen in other organ - preserving management strategies, surveillance should be stringent . All patients considered candidates for endoscopic management should be counseled and be motivated enough to adhere to a regular evaluation schedule . Most investigators agree that cystoscopy, urine cytology, and ureteroscopy should be performed in declining intensity as is done with bladder cancer surveillance protocols . This surveillance of the upper urinary tract should be tailored to the grade and stage of the utuc . According to the 2013 european association of urology guidelines, the recommended schedule would be cystoscopy, ureteroscopy, and cytology in situ at 3 and 6 months, then every 6 months for the next 2 years, and then yearly if the patient is free from tumor recurrence . Urinary cytology and ct urography are recommended at 3 and 6 months and then yearly . This surveillance after endoscopic management for utuc should be performed for at least 5 years . Generally, a 3-month interval follow - up protocol for the first year is the most conventional pattern . However, patients should be prepared to follow the schedule and undergo repeated endoscopic surveillance . The definitive purpose of endoscopic management is oncological control while preserving renal function and diminishing morbidity . The endoscopic management of utuc has traditionally been utilized only for patients with imperative indications, such as renal insufficiency or a solitary functional kidney, bilateral disease, or a significant comorbidity that precluded radical surgery . More recent studies report that endoscopic treatment can be used even for elective cases (when the contralateral kidney is functional), such as a unifocal tumor, tumor size <1 cm, low - grade tumor (cytology or biopsies), and no evidence of an infiltrative lesion on ct urography . The evaluation of the overall life expectancy of a patient is a notable consideration for elective cases . Therefore, endoscopic management for utuc has become a reasonable treatment option in highly selected patients who are expected to adhere to stringent surveillance protocols, because recurrence after endoscopic management is very common and multiple sessions may be necessary . The current literature supports the use of adjuvant bcg therapy in patients with upper urinary tract cis . However, the use of adjuvant topical therapy appears to accord little benefit compared with endoscopic management alone . Definite conclusions about the outcomes of endoscopic management for utuc cannot be made owing to the relatively low frequency of utuc and the lack of randomized controlled trials.
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The mammalian auditory system is challenged with the task of accurately encoding the pattern and source of incoming sound . Many of the initial steps involved in the manipulation of acoustic information already have been well - characterized (webster, 1992; winer, 2005). In the standard model, auditory information is first transmitted from the cochlea (ryugo, 1992) to the cochlear nucleus (ca nt, 1992), where it is distributed across multiple parallel ascending streams to the superior olivary complex, the lateral lemniscal nuclei (schwartz, 1992), and the inferior colliculus (ic) (oliver and huerta, 1992). Subsequently, information is communicated to the medial geniculate body (mgb) of the thalamus (winer, 1992), where it is then transferred to the primary auditory cortex and on to higher auditory cortical areas (kaas and hackett, 2000; lee and winer, 2008b). The tonotopic organization of frequency--specific channels established at the cochlea is one of the few organizing features that persist throughout the auditory pathway (kandler et al ., 2009). In the classical view, the principle route for -auditory information traverses through these tonotopic nuclei at each level of processing . However, numerous parallel ascending (winer, 2005) and descending (winer, 2006) pathways complicate this simple picture, and their roles in audition have yet to be adequately elucidated . For example, the ascending pathways through the non - tonotopically organized nuclei and the large number of descending projections pose unanswered questions regarding their roles in auditory information processing . Assessing the putative functions of such projections has been recently aided by anatomical and physiological findings that segregate the main glutamatergic pathways into two types (sherman and guillery, 1998) (figure 1). The first type termed drivers are similar to the potent retinogeniculate projections in the visual system, and are likely the main conduits for the transmission of auditory information (reichova and sherman, 2004; sherman and guillery, 2006; lee and sherman, 2008, 2010). In contrast, the second type termed modulators have vastly -different properties, and may instead modify the main information - bearing streams (reichova and sherman, 2004; sherman and guillery, 2006; lee and sherman, 2009b). We have used these properties to characterize several pathways in the central stages of auditory processing, and to identify the likely routes for auditory information flow from the ic through thalamus to the auditory cortex (lee and sherman, 2008, 2009b, 2010; llano and sherman, 2008). Here we review these recent findings, the open questions, and the future directions . Summary of anatomical and physiological properties of driver (red) and modulator (green) synapses onto a neuron (blue) (adapted from lee and sherman, 2009b). Assessing the information - bearing role of the central auditory pathways is an important challenge, but it is one that can be addressed partly using anatomical and physiological criteria (figure 1). As mentioned above, glutamatergic synapses can be characterized as either drivers or modulators of activity . Driver pathways are suggested to be the principal conduits for information flow, while modulator pathways modify these main information - bearing streams . Such a characterization has been derived from previous work, mostly in mice and cats, on the retinogeniculate and corticothalamic (ct) projections in the visual and somatosensory systems (li et al ., 2003; reichova and sherman, 2004), and this has been extended across multiple synapses in the auditory system (winer et al ., 1999; huang and winer, 2000; bartlett and smith, 2002; lee and sherman, 2008, 2009b, 2010; llano and sherman, 2008). The distinction between the two types of input derives in part from morphological observations of axonal arborizations . Driver input often resembles that of the retinal input to the lateral geniculate nucleus (lgn), which has been termed type 2 morphology by guillery (1966). This morphological type has thick axons, dense terminal arbors (guillery, 1966; ralston, 1971; famiglietti and peters, 1972), and large endings that contact the proximal dendrites of relay cells (winer et al ., 1999; llano and sherman, 2008), often in triadic structures in glomeruli (ralston, 1971; famiglietti and peters, 1972; hamos et al ., 1987). Like the retinal driver inputs, the driver projections produce large, all - or - none epsps by activating only ionotropic glutamate receptors (iglurs), and they exhibit synaptic depression (bartlett and smith, 2002; li et al ., 2003; in contrast, glutamatergic modulator inputs have different morphologies, called type 1 by guillery (1966) and exemplified by the corticogeniculate feedback pathway from layer 6 of visual cortex . This morphological type has thin axons, sparse arbors, and small terminals ending on distal dendrites (sherman and guillery, 2006). Their physiological properties differ as well, exhibiting synaptic facilitation and producing small, graded epsps by engaging both iglurs and metabotropic glutamate receptors (mglurs) (bartlett and smith, 2002; li et al ., 2003; reichova and sherman, 2004) (figure 1). These glutamatergic modulators should not be confused with the various neuromodulator pathways, such as those using acetylcholine (varela and sherman, 2007) and serotonin (varela and sherman, 2009) as neurotransmitters; their synaptic properties and roles may be very different . These properties likely support the roles of driver and modulator pathways in information processing . Thus, driver synapses are likely highly - reliable and efficient transmitters of information, given their close proximity to the neuronal cell body and their high probability of transmitter release (gil et al ., 1999; furthermore, the synaptic depression has been suggested to act as a dynamic gain control mechanism specific to the input, which is very useful in information flow as firing rates of the afferents change (abbott et al ., 1997). Modulator projections, with their distal dendritic locations, lower probability of transmitter release, and prolonged responses from mglurs, are less suited as conveyors of information (stratford et al ., 1996; gil et al ., 1999; sherman and guillery, 2006). However, the prolonged responses afforded by activation of mglurs not only modulates such properties as overall excitability, but also -provides control of many time- and voltage - gated ion channels in the target cell, a feature that is poorly controlled by driver inputs with their brief epsps (sherman and guillery, 2006); such prolonged mglur responses would also act like low - pass temporal filters, resulting in less information transferred across the synapse (sherman and guillery, 2006). These multiple anatomical and physiological criteria for distinguishing glutamatergic synapses have been useful for characterizing the auditory pathways from the ic, thalamus and cortex (lee and sherman, 2008, 2009b, 2010; llano and sherman, 2008), which we review below . These properties also provide the foundation for ongoing and future investigations of the information - bearing roles of the intracortical and corticocortical connections in the various auditory cortical areas . The driver and modulator framework has been particularly useful in the delineation of forebrain sensory pathways, particularly in the parcellation of thalamic relays, which can be classified as either a first order nucleus (fo) or higher order nucleus(ho) (sherman and guillery, 2002, 2006), based on the source of their driving input . Fo nuclei, such as the lgn, ventral division of the medial geniculate body (mgbv) and ventroposterior medial nucleus (vpm), receive their principal driving input from peripheral sources, while ho nuclei, such as the pulvinar (lp - pul), dorsal division of the mgb (mgbd), and posteromedial nucleus (pom), receive their driving input mainly from layer 5 of the cortex (figure 2). Note that both fo and ho thalamic nuclei receive feedback modulatory input from cortical layer 6, but only ho nuclei, in addition, receive a feedforward driver input from cortical layer 5 . Interestingly, this suggests that the ho thalamic relays transmit layer 5 driver input from one cortical area to the thalamic recipient layers of a second cortical area (sherman and guillery, 2002; reichova and sherman, 2004; lee and sherman, 2008), analogous to the fo transmission by the lgn of retinal input to the primary visual cortex (figure 2), and likewise may utilize the unique operational modes of the thalamus, including gating and the different burst versus tonic firing modes of relay cells (cox et al ., 1998; these distinctions between fo and ho pathways have particular relevance for the central auditory pathways, as we discuss below . Model of auditory information flow from the inferior colliculus (ic), medial geniculate body (mgb) and auditory cortex (ai, aii). Driver inputs (red) are the main information - bearing pathways, while modulator inputs (green) modify the information being transmitted (adapted from lee and sherman, 2010). Among the main auditory centers, the ic is particularly salient as the site of convergence from downstream sources in the cochlear nucleus, superior olivary complex and the lateral lemniscal nuclei, as well as feedback projections from the auditory cortex (oliver and huerta, 1992), and thus represents a major hub for integrating ascending and descending processing streams . The central nucleus of the inferior colliculus (icc) is the main tonotopically organized subdivision (romand and ehret, 1990; malmierca et al ., 2008), and is the principal source of information ascending to the mgbv (winer, 2005). Surrounding the icc are the lateral (icl), dorsal (icd), and caudal cortices of the ic (icca), which we collectively refer to as the shell regions (ics). These subdivisions are primarily non - tonotopically organized (romand and ehret, 1990; malmierca et al ., 2008), and project to the mgbd and medial (mgbm) divisions of the mgb (wenstrup, 2005). Interestingly, the role of these non - lemniscal projections to the mgb are not well - defined (hu, 2003; wenstrup, 2005). Previous models have suggested that the ascending tectothalamic pathways from the icc and ics to the mgbv, mgbd and mgbm, respectively, represent parallel paths for the flow of auditory information (hu et al ., 1994; hu, 2003; wenstrup, 2005). These data demonstrate that the tectothalamic synapse from the icc exhibits properties associated with driver synapses (bartlett and smith, 2002; lee and sherman, 2010), while the ics projection instead has modulator characteristics (bartlett and smith, 2002; smith et al ., 2007; lee and sherman, 2010) (table 1). Thus, in the alternative model of auditory tectothalamic transmission, the main information - bearing pathway is proposed to arise primarily from the icc, while the pathway from the ics instead modulates information flow through the higher order auditory thalamus (figure 2) (bartlett and smith, 2002; smith et al ., 2007; lee and sherman, 2010). Interestingly, the source of the driving inputs to the higher order auditory thalamic nuclei instead arises from layer 5 of the primary auditory cortex (see above) (figure 2) (bartlett and smith, 2002; llano and sherman, 2008). Thus, the tectothalamic projection from ics should act to modulate the corticothalamocortical processing stream through the higher order auditory thalamus (sherman and guillery, 2006; lee and sherman, 2010). Information ascending to the auditory cortex must first be conveyed through the mgb of the thalamus, yet the importance of the mgb in audition extends beyond a role as merely a relay (sherman and guillery, 2006; lee and sherman, 2008; lee and winer, 2008a). Of the main mgb nuclei, the mgbv is the primary conduit for tonotopic information ascending to the primary auditory cortex, whereas the mgbd and mgbm divisions are not tonotopically organized and project broadly to non - tonotopic, multimodal and limbic related areas (kaas and hackett, 2000; lee and winer, 2008a; llano and sherman, 2008). The mgb also receives major projections from layers 5 and 6 of the auditory cortex (winer et al ., 2001; llano and sherman, 2008), which either transmit or modulate information through the thalamus (see below) (figure 2). The thalamocortical projections from the non - tonotopic nuclei of the mgb have been presumed to perform alternative functions, such as regulating attention (olshausen et al ., 1993), and this view is challenged by findings that demonstrate anatomical and physiological similarity among the thalamocortical projections from the mgbv and mgbd (huang and winer, 2000; rose and metherate, 2001; llano and sherman, 2008; lee and sherman, 2009b). Morphologically, these thalamocortical projections have large, dense, bushy arborizations in layer 4 that extend into layer 3 (huang and winer, 2000; llano and sherman, 2008). Physiologically, these projections demonstrate large epsps that depress in response to paired - pulse stimulation and lack a metabotropic glutamate component (rose and metherate, 2001; lee and sherman, 2009b). Thus, projections from both the mgbv and mgbd share driver - like properties (table 1), and suggest that the projections from the mgbd have a role similar to that of the mgbv, i.e., as an -information - bearing pathway to the higher auditory cortical areas (figure 2). The main distinction between them is that mgbv is an fo relay, while mgbd is an ho relay (see above). An interesting question remaining concerns the thalamocortical projections to layer 1 from the mgbm (huang and winer, 2000; jones, 2009), part of the matrix system defined by jones (2009), whose role remains to be defined . Thus, although it is generally regarded simply a relay of ascending auditory information, the mgb has important roles ranging from the transformation of auditory information (miller et al ., 2001) to continuing the flow of intraareal processing in the cortex (see below) (sherman and guillery, 2006; lee and sherman, 2008). The auditory cortex is the ultimate target for information ascending from the periphery through the mgb (lee and winer, 2008a, b; llano and sherman, 2008). Similar to lower -stations, tonotopy is an organizing feature of the primary auditory cortex (ai), but is absent in the surrounding non - tonotopic and multimodal areas, such as the secondary auditory area (aii) (stiebler et al ., 1997; kaas and hackett, 2000; lee et al ., 2004). The size and number of these auditory cortical areas varies among species, e.g., there are two tonotopic areas in the mouse (stiebler et al ., 1997), three in the monkey (hackett et al ., 1998) and five in the cat (reale and imig, 1980). Yet, an unresolved issue is: how do these multiple areas interact to compute features in the auditory scene? In the standard hierarchical cortical model of auditory processing, information is sent progressively via direct corticocortical connections from lower auditory areas, such as ai, to higher auditory areas, such as aii (felleman and van essen, 1991; rouiller et al ., 1991). Such successive convergence of auditory inputs purportedly accounts for the increasingly complicated receptive fields of higher auditory areas, such as aii (schreiner and cynader, 1984). Interestingly, the synaptic properties of these direct corticocortical connections have not been examined until recently (covic et al ., 2009), and their salience is questionable, given recent findings that suggest an alternate route for interareal processing via a corticothalamocortical route (figure 2) (reichova and sherman, 2004; sherman and guillery, 2006; lee and sherman, 2008; llano and sherman, 2008; theyel et al ., the alternate corticothalamocortical route for interareal auditory processing is enabled by driver projections that originate from layer 5 of the primary auditory cortex and synapses in the higher order auditory thalamus, i.e., mgbd (table 1) (ojima, 1994; winer et al ., 1999; these feedforward ct projections are distinguished from the feedback layer 6 projections, which exhibit modulator characteristics and project to the originating thalamic nucleus, e.g., ai to mgbv (table 1) (ojima, 1994; bartlett et al ., 2000; thus, the ai layer 5 inputs to mgbd, in conjunction with the driver thalamocortical projections from mgbd to aii (see above), establish a transthalamic route for the interareal transfer of auditory information (figure 2). This alternate route does not negate the potential information - bearing roles of the direct corticocortical projections (rockland and pandya, 1979; lee and winer, 2008b), which are composed of an intricate pattern of laminar - specific driver and modulator projections (covic et al ., 2009). Thus, auditory forebrain computations involve multiple processes beyond those suggested by simple -cortical hierarchies (felleman and van essen, 1991; rouiller et al ., 1991). Finally, intrinsic cortical microcircuits further transform auditory information before redistribution through the cortical network (feldmeyer and sakmann, 2000; silberberg et al ., 2004; this issue is complicated by the complexity and floridness of intrinsic cortical interconnections, which account for almost half of the input to a cortical column (ahmed et al ., 1994; one potential driving circuit extends from layer 4 to layers 2/3 and then to layer 5 (hirsch and martinez, 2006b; lee and sherman, 2009a), and then outputs to the higher order thalamus (ojima, 1994; winer et al ., 1999; llano and sherman, 2008), but the bulk of intrinsic connections may be instead more likely to exhibit modulator properties, such as the layer 6 to layer 4 projections (ahmed et al ., 1994; stratford et al ., 1996; prieto and winer, 1999; tarczy - hornoch et al ., 1999; lee and sherman, 2008, 2009b). In this manner, functional connectivity within the auditory cortex may resemble that in the thalamus, where synaptic weight is inversely proportional to anatomical weight (binzegger et al . The driver and modulator framework adds a unique perspective to our ongoing understanding of the central auditory pathways . As such, these properties may fruitfully be applied to other pathways of interest throughout the auditory system, such as the thalamoamygdaloid pathway (doron and ledoux, 1999). However, for instance, how do driver and modulator pathways interact functionally to construct auditory receptive fields?, how do these properties extend across systems and species? Indeed, the utility of this framework is not constrained to the auditory modality, as its relevance in other sensory systems has already been established (reichova and sherman, 2004; petrof and sherman, 2009), but has not yet been extended to non - mammalian species . In this respect, a comparative approach that extends the investigation of these properties in other organisms may lend unique insights into the ontogeny, development and evolution of the sensory pathways in higher organisms . 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.
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The aim of vital pulp therapy (vpt) is to use biomaterials in order to safeguard as much vitality as possible for the dental pulp with carious exposure . Direct pulp capping and pulpotomy, the two main vpt procedures, are less invasive than pulpectomy / root canal therapy (rct). It is well known that the most common cause of pulp / periapical diseases is the presence of bacteria and their by - products within the involved tooth, and the most common pathway of their entrance is through carious lesions; when further bacterial recontamination is prevented with a good biological seal, the exposed dental pulp has the capability to heal and construct a new dentinal bridge and periapical tissues have the ability of regeneration . There is a body of evidence for biocompatibility and sealing ability of mineral trioxide aggregate (mta). Currently, mta is as an alternative gold standard for calcium hydroxide, a traditional gold standard, in the field of vpt . This biomaterial creates a biological seal via hydroxyapatite crystal formation which is improved the first physical seal . Despite the advantages, mta has nonpredictable antimicrobial activity, delayed setting time, poor handling characteristics, as well as high cost . The calcium - enriched mixture (cem) cement is a newly introduced endodontic biomaterial with similar clinical applications as mta, but different chemical compositions . This novel cement has an antibacterial effect comparable to calcium hydroxide and superior to mta and sealing ability similar to mta . Recent researches revealed that the biologic response of the pulpal and periapical tissues to the mta and cem cement were comparable in dogs teeth . Hydroxyapatite crystal formation was reported over the set cem even in normal saline solution . Besides the surface characteristics of the set cem cement, as well as the distribution pattern of calcium, phosphorus, and oxygen ions, is similar to dentine . Last but not least, the clinical use of cem has been approved by the iranian ministry of health and medical education . Even after carious pulp exposure of symptomatic permanent molars, the pulp can heal, as it has been shown in several studies using mta and cem cement as pulp - capping biomaterials . This case report discusses a successful treatment outcome of pulpotomy treatment in a symptomatic mature molar with established irreversible pulpitis associate with condensing apical periodontitis . Diagnostic tests were performed; the involved tooth responded with mild pain to percussion and severe lingering pain to the cold thermal test . It was not responded within normal limits to the electric pulp test (ept). A radiographic examination showed a large carious lesion associated with condensing apical periodontitis [figure 1a]. (a) preoperative radiograph shows a large carious lesion associated with a condensing apical periodontitis . (b d) six months, 1 year and 2 years follow - up radiographs, respectively; a complete resolution of condensing apical periodontitis is evident . Note the existence of a dentinal bridge beneath the cem cement; the remaining pulp canal space does not obliterated following clinical and radiographic diagnostic examination, pulpotomy of the first mandibular molar was decided as the treatment . The tooth was anesthetized with 2% lidocaine with 1:80,000 epinephrine (daroupakhsh, tehran, iran), isolated with a rubber damn and then caries was excavated; the exposed coronal pulp tissue was removed with a sterile round diamond bur with water spray . Hemorrhage was controlled with sterile cotton pellets; a ~2 mm layer of the cem cement (bioniquedent, tehran, iran) was placed without any pressure to cover the exposed pulp stump . The patient was re - examined clinically after 1 and 7 days; tooth mobility was within the normal physiological range, the percussion test was slight positive to negative, and patients were asymptomatic . Six months, one - year, and 2-years follow - ups revealed no clinical or radiographic pathological findings (i.e. Inflammation, infection, calcification, and resorption) on the first mandibular molar; radiographs showed normal periodontium apparatus, an evidence of complete periapical healing [figure 1b d]. The root canal treatment of teeth with irreversible inflamed pulps is basically a prophylactic treatment, since the radicular vital pulp is frequently free of infection and the rationale is to avoid further infection of the root canal system . Improved and more conservative vpt has the potential to reduce the need for more invasive / complicated rct . However, if this is to be achieved in permanent teeth, there is a need for suitable biomaterials for vpt, particularly pulpotomy . In this report, a case of established irreversible pulpitis associated with condensing apical periodontitis recall examinations revealed that treatment outcomes were successful in preserving pulpal vitality and complete periapical healing of the tooth . It has been suggested that the existence of a dentinal bridge may at least be an indicator of vpt success, and there are several studies that point to its clinical importance . However, researchers reported the presence of tunnel defects in dentinal bridge under calcium hydroxide in vpt. [52122] they concluded that tunnel defects can serve as pathways for bacterial leakage . Unlike ch, the calcified bridge formed by mta is continuous and has no evidence of tunnel defects . According to histological evidence from animal / human studies in mature permanent teeth, the cem cement, like mta, saves pulp vitality and also induces a hard tissue bridge formation, the both . Dentine bridge induced by the cem cement has been shown to be similar to that induced by mta in terms of thickness, absence of tunnel defects, presence of adjacent odontoblast - like cells, predentine, and dentinal tubules . An important finding was the complete healing of the condensing apical periodontitis with reconstruction of bone structures to normal appearance surrounding the involved molar . Mild inflammatory processes may at times cause the formation of condensing apical periodontitis, mostly involving the mandibular first molar . Various studies have suggested that the key factor in success of vpt is the sealing ability of the material; on the other hand, the most important cause of failure is bacterial recontamination . The cem cement favourably prevents microleakage and has sealing ability comparable to mta and superior to irm . In addition, the cem cement was an effective antibacterial agent . Therefore, when bacterial contamination is removed from the dentin - pulp complex in addition to a creation of tight coronal seal to prevent further bacterial recontamination the inflamed but vital pulp of a permanent molar has a chance to return to a healthy and functional status . All these are resulted in removing the etiologic factor of periapical lesion and complete periapical healing . Because pulp / periapical healing was achieved without any need for further intervention, it seems that the cem cement has the required and acceptable properties to be used as a pulp protective biomaterial . The exact biological mechanism by which the cem cement promotes pulp healing is currently unknown . This characteristic is likely to be the result of several properties such as sealing ability, high alkalinity, antibacterial effect, biocompatibility, i.e. Dentinogenesis, cementogenesis, low cytotoxicity, pain relief effect, anti - ierr (inflammatory external root resorption) effect, hydroxyapatite formation, and similarity to dentine . Besides, recent interesting studies have established favourable treatment outcomes for the cem cement when used in pulpotomy of human permanent teeth with irreversible pulpitis . In the future, biomaterials can play an important role in regenerative dentistry; they may completely transform our whole philosophy of endodontic treatment . Endodontics seems to be on the brink of an era when a diseased pulp can be a saved one . There is a realistic biological argument to perform pulpotomy as a promising alternative endodontic treatment in mature permanent teeth; the results of this case clearly show that cem cement can be used clinically in the treatment of a mature molar associated with irreversible pulpitis and condensing apical periodontitis.
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A healthy joint requires a fine - tuned balance between molecular signals regulating homeostasis, damage, restoration, and remodelling . This balance is determined both at the level of single cells and the whole tissue architecture, and it also involves interactions among different tissues such as cartilage, bone, synovium, ligaments, tendons, and menisci . Different factors are able to impair the maintenance of homeostasis in a joint that has been damaged or strained, and they may progressively lead to osteoarthritis (oa) [27, 29]. A wide spectrum of treatments is available, from non - pharmacological modalities to dietary supplements and pharmacological therapies, as well as minimally invasive procedures involving injections of various substances aimed at restoring joint homeostasis and providing clinical improvement and, possibly, a disease - modifying effect . When these treatments fail, more invasive surgical approaches can be attempted to avoid metal resurfacing through the restoration of the mechanical balance and the regeneration of the articular surface, although results are still controversial [21, 22]. Even though some of these approaches have been shown to offer a satisfactory clinical outcome at midterm follow - up, rehabilitation is long and results are often unpredictable, incomplete, and limited over time [10, 15, 16, 18, 37]. The search for a minimally invasive solution to improve the status of the joint surface and allow a fast return to full activity is therefore highly desirable . In this landscape, a novel promising injective treatment is platelet - rich plasma (prp), a blood derivative that has a higher platelet concentrate than whole blood . When activated, platelets release a group of biologically active proteins that bind to the transmembrane receptors of their target cells, thus leading to the expression of gene sequences that ultimately promote cellular recruitment, growth, and morphogenesis, and modulating inflammation as well . Therefore, prp represents an appealing biological approach to favour the healing of tissues otherwise doomed by a low healing potential, such as cartilage . This led to the wide use of prp, which shows promising results as a minimally invasive injective treatment of cartilage degeneration and oa, both in preclinical and clinical studies [40, 67]. However, besides the increasing interest both among physicians and the scientific community, results are sometimes contradictory with no clear treatment indications, due to low - level clinical studies and the lack of understanding on the mechanism of action of this blood derivative . The aim of this review was to analyze systematically the available evidence on the clinical application of this biological approach for the injective treatment of cartilage lesions and joint degeneration, together with preclinical studies to support the rationale for this use of platelet concentrates, to shed some light and give indications on what to treat and what to expect from intra - articular injections of prp . All in vitro, in vivo preclinical and clinical studies on prp injective treatment in the english language concerning the effect of prp on cartilage, synovial tissue, and menisci were considered . Since prp injections could be used as augmentation procedure after bone marrow stimulation techniques or other cell type transplantations, the analysis of studies dealing with the prp effect on mesenchymal stem cells (mscs) of various origins for cartilage treatment was also included . A systematic review on the pubmed database was performed using the following words: (platelet - rich plasma or prp or platelet concentrate or platelet lysate or platelet supernatant) and (cartilage or chondrocytes or synoviocytes or menisci or mesenchymal stem cells). Relevant data were then extracted and collected in three tables, separating in vitro, in vivo preclinical studies, and clinical studies (case reports were not considered) (tables 1, 2, 3). Two studies focused on in vitro and preclinical in vivo evaluations and were reported in both tables 1 and 2 . The in vitro studies were divided according to the cell population targeted . With regard to clinical trials, only comparative and randomized controlled trials (rcts) were discussed further in the present manuscript.table 1 in vitro studiespublicationsprp characteristicsprp effectschondrocytes yin platelet count: 2,604 602 10/mlactivation: no leukocytesincrease in proliferation and ecm deposition in the integration area between agarose scaffold and cartilage sampleshigher scaffold integration strength muraglia platelet count: 10 10/lno activationleukocytes: increase in cell proliferation more than fcs, also in chondrocytes from elderly patients hildner platelet count: activation: leukocytes: increase in proliferationbetter redifferentiation potential than fcs expanded cells park platelet count: 610 10/lno activationleukocytes: dose - dependent increase in chondrocytes proliferation maintained at 4 days in 5, 10, 20% prpchondrogenic phenotype maintenancetime - dependent increase in angiogenic and antiangiogenic factors expression (vegf, chm - i) lee platelet count: activation: no leukocytesincreased chondrocyte proliferation in time - dependent mannerenhanced hydrogel scaffold chondrocyte maturationimmediate increase in cb1 and cb2 mrna expression pereira platelet count: 1 10/mlactivation: freezing and thawingleukocytes: increase in cell proliferationchondrogenic phenotype maintenance but decrease over time in micromass pellet culturesinitial enhancement of inflammatory response, followed by its resolution van buul platelet count: 845.3 10/mlactivation: cacl2 leukocytes: presentnormalization of collagen ii, aggrecan, adamts4, mmp13 and ptgs2 expression altered by il-1no influence on gag contentdose - dependent down - regulation of il-1 induced nf - kb activation wu platelet count: activation: thrombinleukocytes: dose - dependent increase in chondrocyte proliferation in collagen 3d arthritic modelrestoration of collagen ii, pg, integrin 11 and cd 44 expression inhibited by il-1 and tnfinhibition of il-1, cox-2, and mmp-2 genes expression bendinelli platelet count: 1,850 320 10/mlactivation: thrombin + cacl2 leukocytes: presentantiinflammatory effect: inhibition of nf - kb transactivation activity through hgf, il4, and tnf, and inhibition of monocyte - like cells chemotaxis spreafico platelet count: 1,460 10/lactivation: ca - gluconateleukocytes: 5% prpr optimal concentration for chondrocytes proliferation increasehigher prp concentration does not further induce cell proliferationincrease in collagen ii and pg production at day 2 that decreases over time drengk platelet count: activation: cacl2 no leukocytesincrease in chondrocyte proliferation, but inhibition of chondrogenic markers expression pettersson platelet count: activation: leukocytes: no beneficial effect on chondrocyte seeded macroporous gelatin microcarriers in terms of histologic characteristics and proteoglycan deposition up to 16 weeks saito platelet count: 1,081 150 10/lactivation: thrombin + cacl2 no leukocytesincrease in gag content akeda platelet count: 1,399 174 10/mlactivation: thrombin + cacl2 leukocytes: stable cell phenotypeincrease in cell proliferation and amount of collagen ii and pg synthesis, more than ppp or fbs gaissmaier platelet count: activation: thrombin + ca gluconateno leukocytesincrease in chondrocyte proliferation in dose - dependent manner (stable above 10%) with inhibition of chondrogenic markers expression in monolayer culture as well as in 3d culture model kaps platelet count: activation: freezing and thawingno leukocytesgrowth promotion activity comparable or superior to mitogenic stimulation by fcs on articular and nasal septal chondrocytesreduction in ecm formation in chondrocyte / agarose construct yang platelet count: activation: freezing and thawingno leukocytesincrease in chondrocytes proliferation with 1% pschondrocytes mass formation with 10% psincrease in gag but inhibition of collagen ii expressionmscs + chondrocytes mifune platelet count: 230 10/mlactivation: thrombin + cacl2 leukocytes: promotion of proliferation, adhesion, and migration of mdscsincrease in cell apoptosis and number of collagen ii producing cells moreira teixeira platelet count: activation: freezing and thawingno leukocyteshigh collagen ii gene expression and synthesischemo - attractant properties in hydrogelcombination with hydrogel allowed retention of prp at the defect sitemeniscal cells gonzales platelet count: 140 20 10/lactivation: leukocytes: same positive effect as fbs for meniscal cell culturedose - dependent effect: 10 and 20% prp increased proliferation rate and influenced more type i collagen and aggrecan expression at day 7 with respect to 5% prp ishida platelet count: 104.5 10/lactivation: leukocytes: increase in meniscal cells proliferation in a dose - dependent mannerno effect on collagen i but modulation of gag synthesis, high biglycan and decorin expression, aggrecan downregulationsynoviocytes browning platelet count: activation: leukocytes: presentincrease in mmp1, 3, il-6 and decrease in pdgf-, mip-1, rantes in oa synoviocyteshigher pro - inflammatory response than ppp treatment anitua platelet count: 494 10/mlactivation: cacl2 no leukocytesincrease in ha secretion, further enhancement in the presence of il-1angiogenesis switched to a more balanced statusno effect on mmp1, 3, and vegf amounts elicited by il-1mesenchymal stem cells hildner platelet count: activation: leukocytes: increase in proliferationincrease in gag and cartilage markersbetter redifferentiation potential than fcs expanded cells kruger platelet count: 0.61.3 10/mlactivation: freezing and thawingleukocytes: <0.3 10/mlincrease csp migration with 0.1100% prp, especially with 5% prpinduction in chondrogenic markers expressioninduced formation of cartilage matrix rich in pg and collagen ii moreira teixeira platelet count: activation: freezing and thawingno leukocytesin hydrogel - pl increase in bmscs proliferation rate, adhesion, and migrationno beneficial effect on collagen ii mrna expression in mscs with chondrogenic medium and pl, but higher expression in control medium and pl murphy platelet count: 10/lactivation: cacl2 no leukocytesprp is more mitogenic than fbs on mscs derived from human and rat bm and from rat compact bonehigher increase in mscs proliferation rate and migration with ucprp with respect to aprp mishra platelet count: 10/mlno activationleukocytes: presentinduction of mscs proliferationincrease in chondrogenic markers expression (sox9, aggrecan) drengk platelet count: activation: cacl2 no leukocytesstimulation of bmscs proliferation and weak chondrogenic differentiation in a 3d environment zaky platelet count: 1 - 1.8 10/lactivation: freezing and thawingno leukocytesinduction of proliferation (more than with fbs and fgf2) during the initial culture passageinduced mscs chondrogenic differentiation in conditions without fbs kakudo platelet count: 132.26 10/lactivation: thrombin + cacl2 leukocytes: higher increase in admscs proliferation with 5% prphigher proliferation induction with activated prp versus not activated prpdecrease in a dose - dependent manner with 10 and 20% prptable 2 in vivo preclinical studiespublicationanimal modellesion typeprp characteristicsprotocolprp effectsmifune 36 ratsoaplatelet count: 230 10/mlactivation: thrombin + cacl2 leukocytes: 1 injection (30 l)promotion of collagen ii synthesis and suppression of chondrocyte apoptosis only when applied with mdscs at 4 weeksat 12 weeks, lost beneficial effecthapa 42 ratschondral lesionplatelet count: 13.8 10/lactivation: leukocytes: 1 intra - op injection (150 l)1 intra - articular injection (150 l)better cartilage healing and increase in type ii collagen expression at 6 weeksguner 20 ratsoaplatelet count: activation: thrombin + cacl2 leukocytes: 3-weekly injections (50 l)no significant effects regarding cartilage healing at short term (2 weeks after injection cycle)serra 36 rabbitsosteochondral lesionplatelet count: activation: cacl2 no leukocytes7 injections every 2 days (0.25 ml)no macroscopic, microscopic, and biomechanical additional benefits from prp injections up to 19 weekskwon 21 rabbitsoaplatelet count: 2664 970 10/lactivation: leukocytes: 1 injection (0.3 ml)better cartilage regeneration in all oa degrees at 4 weeks, in particular in moderate knee oamilano 30 sheepchondral lesionplatelet count: 868 112 10/mlno activationno leukocytes5-weekly injections (3 ml)improvement in macroscopic, histologic, and biomechanical cartilage repair after microfractures, with more durable resultsno hyaline cartilage production up to 12 monthsmilano 30 sheepchondral lesionplatelet count: 2 concno activationno leukocytes5-weekly injections (23 ml)promotion of cartilage healing until 6 months after treatment (not at 12 months)no hyaline cartilage productionlippross 15 pigsarplatelet count: 1 10/lactivation: leukocytes: 2 injections every 2 weeks (5 ml)reduction in il-6 expression and staining, and vegf stainingrecovery of chondral protein concentration levelsreduction in il-1 and igf-1 on synoviocytesmilano 15 sheepchondral lesionplatelet count: 1,415 164 10/mlliquid prp: no activationprp gel: ca gluconate + fibrin glueleukocytes: 1 injection (5 ml)improvement in macroscopic, histologic and biomechanical scores, no hyaline cartilage productionbetter results with prp gel at 6 monthssaito 33 rabbitsoaplatelet count: 1,081 150 10/lactivation: no leukocytes2 injections at 4 weeks and 7 weeks after oa induction (100 l)suppression of oa progression morphologically and histologically by prp impregnated hydrogel microspheres(not significantly by the use of prp only)carmona 4 horsesoaplatelet count: 250 71.8 10/mlactivation: cacl2 leukocytes: present3 injections at 2-week interval (1020 ml)improvement in both degree of lameness and joint effusion, with normal synovial fluid parametersmarked improvement at 2 months maintained up to 8 monthstable 3clinical studiespublicationlevel of evidencepathology n patientsprotocoldose and platelet countleukocyteactivationfollow - upresultskoh case seriesknee chondropathy or oa18 prp + mscs1 injection of prp + mscs followed by 2-weekly injections of prp3 ml prp for each injection5 basal plt count(1.28 10 plts/l)yesca - chloride24 monthsstatistical improvement in pain and functionjang case seriesknee chondropathy or oa65 prp1 injection6 ml prpplatelet count: n.a.n.a.no12 monthsincreasing age, and advanced degeneration result in a decreased potential for prp injection therapyhart case seriesknee chondromalacia50 prp6-weekly injectionsafter 3 months other 3-weekly injections6 ml prp459,000 plts/ln.a.no12 monthssignificant pain reduction and quality of live improvement in low degree of cartilage degeneration not confirmed by mripatel randomized trialknee chondropathy or oa52 single injections50 double injections46 saline injections1 injection versus 2 injections 3 weeks apart8 ml prp310 10 plts/l(238 10 plts in total)noca - chloride6 monthssignificant clinical improvement in prp group within 23 weeks until 6 months, but deteriorating after 6 monthsno differences between 1 and 2 injectionsgobbi case seriesknee chondropathy or oa50 prp2 monthly injections4 ml prp2 basal plt countyesno12 monthsstatistical improvement in pain and function . Good results also in patients with history of cartilage surgerykoh case seriesknee chondropathy or oa25 prp / mscs1 injection of prp / mscs followed by 2-weekly injections of prp3 ml prp for each injection5 basal plt count1.28 10 plts/lyesca - chloride17 monthsshort - term results revealed reduction in pain and improving functiontorrero case seriesknee chondropathy or oa30 prp1 injectionn.a.nono6 monthsone prp injection provided encouraging results in pain and function at 6 months follow - upnapolitano case seriesknee chondropathy or oa27 prp3-weekly injections of prp5 ml prp2.3 basal plt countn.a.ca-gluconate6 monthsprp proved to be an effective treatment option for oaspakova comparative trialknee chondropathy or oa60 prp versus 60 ha3-weekly injections of prp3 ml prp4.5 basal plt countyesno6 monthssuperior results in prp group at short - term evaluationsanchez randomized trialknee chondropathy or oa79 prp versus 74 ha3-weekly injections of prp8 ml prgfplatelet count: n.a.noca-chloride6 monthshigher percentage of responders in prp group but no clear superiority of the biological approachcerza randomized trialknee chondropathy or oa60 acp versus 60 ha4-weekly injections of acp5.5 ml acpplatelet count: n.a.nono6 monthssuperior clinical outcome for prp in all groups of treatmentfilardo randomized trialknee chondropathy or oa55 prp versus 54 ha3-weekly injections of prp5 ml prp5 basal plt countyesca - chloride12 monthsclinical improvement in both groups without significant inter - group difference . Better trend for prp in low - grade cartilage pathologykon comparative trialknee chondropathy or oa50 prp versus 50 lwha versus 50 hwha3-weekly injections of prp5 ml prp6 basal plt count(6 billion plts in total)yesca - chloride12 monthsbest results for prp in chondropathy group, no statistical difference among treatments for higher degree of cartilage degenerationfilardo comparative trialknee chondropathy or oa72 l - prp versus 72 l - free - prp3-weekly injections of prpprp: 5 ml949,000 plts/lprgf: 5 ml315,000 plts/lprp: yesprgf: noprp and prgf: ca - chloride12 monthscomparable clinical results with higher post - injective pain in leukocyte - rich prp groupkon [13, 36]case seriesknee chondropathy or oa100 prp3 injections of prp 2 weeks apart5 ml prp6 basal plt count(6.8 billion plts in total)yesca - chloride24 monthssignificant pain reduction and functional recoverytime - dependent effect of prp injections with a mean beneficial effect of 9 monthswang - saegusa case seriesknee chondropathy or oa261 prp3 injections of prp 2 weeks apartn.a.noca-chloride6 monthssatisfactory results at 6 months evaluation in a large cohort of patientssampson case seriesknee chondropathy or oa14 prp3 injections of prp 1 month apart6 ml prpplatelet count: n.a.n.a.thrombin in ca - chloride suspension6 monthsclinical improvement at short - term evaluationsanchez retrospective comparative trialknee chondropathy or oa30 prp versus 30 ha3-weekly injections of prp68 ml prgf2 basal plts countnoca - chloride5 weeksbetter pain control and functional outcome in prp groupbattaglia case serieship oa20 prp3-weekly injections of prp5 ml prpplatelet count: n.a.yesca-chloride12 monthsclinical improvement but gradual worsening up to 1 year of follow - upsanchez case serieship oa40 prp3-weekly injections of prp8 ml prpplatelet count: n.a.noca-chloride12 monthssignificant pain reduction and functional improvementmei - dan quasi - randomized trialosteochondral talar lesions15 prp versus 15 ha3 injections of prp 14 days apartprp: 2 ml23 basal plts countnoca - chloride7 monthsstatistically better clinical outcome in prp group plt platelet, n.a . Not assessed, ha hyaluronic acid, l - prp leukocyte - rich prp, l - free - prp leukocyte - free prp in vivo preclinical studies plt platelet, n.a . Not assessed, ha hyaluronic acid, l - prp leukocyte - rich prp, l - free - prp leukocyte - free prp according to the search strategy, 388 papers were screened, among these 59 met the inclusion criteria: 26 were in vitro, 9 were in vivo, 2 were both in vivo and in vitro, and 22 were clinical studies . The analysis of per year publication showed increasing interest in this topic with an increasing number of published studies over time, in particular with regard to reports documenting results of the clinical injective application of prp (fig . 1the analysis of per year publication shows the interest in prp application for the treatment of cartilage lesions or joint degeneration with an increasing number of published studies over time the analysis of per year publication shows the interest in prp application for the treatment of cartilage lesions or joint degeneration with an increasing number of published studies over time seventeen papers investigated the effect of prp on chondrocytes (table 1) [1, 5, 11, 19, 28, 33, 44, 48, 54, 57, 59, 61, 69, 73, 7577]. . Even showed that prp promoted cell proliferation in conditions where fetal calf serum (fcs) had no proliferation stimulating effect, as in chondrocytes from elderly patients . Four papers by drengk et al ., gaissmaner et al ., kaps et al ., and yang et al . Observed, together with the increase in cell proliferation, an inhibition of chondrogenic markers expression . Conversely, 10 papers reported an increase in chondrocyte proliferation rate without affecting chondrogenic phenotype maintenance . Even documented that proliferation and chondrogenic redifferentiation potential were higher when human articular chondrocytes were previously expanded with platelet lysate (pl) instead of fcs . Besides the overall proliferation increase with phenotype maintenance, park et al . Underlined another key point: the time - dependent regulation and the dose - dependency effect . In particular, they tested different prp concentrations (0.1, 1, 5, 10, and 20%) showing an increase in cellular viability in a dose - dependent manner . Yang et al . Reported that 1% of platelet supernatant (ps) is sufficient to stimulate chondrocyte proliferation, whereas 10% ps stimulated chondrocyte mass formation . Studied prp releasate (prpr) at 1, 5, and 10% and found that 5% was the optimal concentration to increase chondrocyte proliferation . Moreover, gaissmaner et al . Provided evidence of cell proliferation increase with 1 or 10% ps, but no further stimulation occurred using concentrations above 10% . Together with chondrogenic phenotypic maintenance, other authors also documented an increase in matrix molecule production . Documented that prp treatment led to higher amounts of collagen ii and pg synthesis than platelet poor plasma (ppp) or fetal bovine serum (fbs). Since cell matrix interactions play an important role in maintaining cartilage homoeostasis, wu et al . Designed a simple 3d chondrocyte model: in a collagen matrix, the authors mimicked an oa environment by il-1 and tnf induction . Also in this model, prp increased the membrane receptors integrin 11 and cd44 and favoured type ii collagen and pg production . In another experimental model, yin et al . Reported that prp allowed the integration of an agarose construct with cartilage samples, showing a denser extracellular matrix (ecm) deposition in the integration area . Interestingly, pereira et al . Found that the prp stimulatory effect was limited over time: after an initial positive staining for collagen type ii and pg, at 20 doublings the matrix / cells ratio decreased . Similarly, spreafico et al . Documented an increase in pg release 2 days after prpr treatment, followed by a decrease after 9 days, although at 20 days pg release remained still high . Pereira et al . Found that pl enhanced the initial inflammatory response and subsequently triggered its resolution through the regulation of nuclear factor kappa b (nf - kb) and cyclooxygenase-2 (cox-2), the principal actors of inflammatory cascade . Van buul et al . Showed a dose - dependent down - regulation of il-1 - induced nf - kb activation, whereas bendinelli et al . Showed that inhibition of nf - kb transactivation activity was mediated by hgf, a cytokine present in prp -granules . Moreover, they suggested another anti - inflammatory action by inhibiting monocyte - like cell chemotaxis . Wu et al . Also investigated the anti - inflammatory potential of prp in their 3d system: prp counteracted the inflammatory cascade elicited by il-1 and tnf, showing an inhibition of il-1, cox-2, and mmp-2 gene expression . . Showed that the addition of prp to a chondrocyte / hydrogel culture led to an immediate increase in mrna levels of cannabinoid receptor cb1 and cb2 (receptors involved in analgesic and anti - inflammatory effects). In a system of oa chondrocytes and muscle - derived mscs (mdscs), mifune et al . Observed that prp promoted proliferation, adhesion, and migration of mdscs . During chondrogenic pellet culture, prp tended not only to increase the number of type ii collagen - producing cells, but also to increase cell apoptosis, which, however, was not confirmed by the in vivo evaluation . Moreira teixeira et al . Showed high expression and synthesis of collagen ii co - culturing chondrocytes and expanded bone marrow mscs (bmscs) when pl / hydrogel was added . Moreover, they investigated the retention of pl / hydrogel construct in a cartilage fragment: the combination with hydrogel allowed the retention of prp at the defect site, filling up irregularities at the cartilage surface . Investigated the role of prgf (preparation rich in growth factors: a low - concentrate prp without leukocytes) on oa synoviocytes with or without exposition to il-1, to mimic the overproduction of proinflammatory cytokines in the joint environment during oa progression . Prgf significantly enhanced ha secretion compared to ppp both with and without il-1 and switched angiogenesis to a more balanced status, but did not modify the il-1-induced rise of matrix metallo - protease (mmp) 1, 3 and vascular endothelial growth factor (vegf) produced by synovial cells . . Even showed an increase in mmp-1 and mmp-3 in oa synoviocytes incubated with prp, thus suggesting that the application of prp to synovial joints might be associated with deleterious effects due a pro - inflammatory response that might lead to an accelerated cartilage catabolism . Ishida et al . Showed the usefulness of prp not only because of its proliferation effect, but also its induction of gag synthesis . Prp up - regulated the viability of meniscal cells in a dose - dependent manner, as well as the mrna expression of biglycan and decorin . The study results showed that prp presents the same positive effect as fbs for meniscal cell culture and showed that dosage is an important aspect of the induced effect: 10 and 20% prp increased proliferation rate and influenced more type i collagen and aggrecan expression at day 7 of culture with respect to 5% prp . Eight papers investigated the effect of prp on mscs of different origin: 1 on subchondral cortico - spongious bone (csp) cells, 1 on commercial human mscs, 4 on bmscs, and 2 on adipose - derived mscs (admscs). Kruger et al . Investigated the migration and chondrogenic differentiation of human subchondral progenitors . In particular, a chemotactic assay revealed that prp significantly stimulated the migration of csps, together with their chondrogenic differentiation and production of pg and collagen type ii . Confirmed an induced chondrogenic differentiation of bmscs, which also presented a higher proliferation rate . Mishra et al . Documented the same behaviour on mscs with a higher proliferation rate and a selective differentiation along the chondrogenic line: sox9 and aggrecan (chondrogenic markers) were increased much more than runx2 (osteogenic marker). Conversely, moreira teixeira et al . Reported that pl, besides inducing a significant increase in bmscs proliferation rate and migration, did not induce an increase in collagen type ii . Focused on admscs and showed strongly enhanced proliferation rates with retained chondrogenic differentiation potential and even a tendency toward increased chondrogenic differentiation of pl - expanded admscs compared to fcs . Studied the proliferation of admscs treated with prp with or without activation and at different concentrations (1, 5, 10, or 20%). Results showed the importance of both prp activation and correct dosage: in fact, the stronger promotion of proliferation was observed in prp activated with calcium chloride and autologous thrombin and applied at 5%, whereas at higher platelet concentrations the proliferation rate decreased in a dose - dependent manner . Finally, murphy et al . Tested two different types of prp: one derived from human adult peripheral blood and one derived from human umbilical cord blood (ucprp), showing the superiority of ucprp with regard to mscs proliferation and migration induction . Seventeen papers investigated the effect of prp on chondrocytes (table 1) [1, 5, 11, 19, 28, 33, 44, 48, 54, 57, 59, 61, 69, 73, 7577]. . Even showed that prp promoted cell proliferation in conditions where fetal calf serum (fcs) had no proliferation stimulating effect, as in chondrocytes from elderly patients . Four papers by drengk et al ., gaissmaner et al ., kaps et al ., and yang et al . Observed, together with the increase in cell proliferation, an inhibition of chondrogenic markers expression . Conversely, 10 papers reported an increase in chondrocyte proliferation rate without affecting chondrogenic phenotype maintenance . Even documented that proliferation and chondrogenic redifferentiation potential were higher when human articular chondrocytes were previously expanded with platelet lysate (pl) instead of fcs . Besides the overall proliferation increase with phenotype maintenance, park et al . Underlined another key point: the time - dependent regulation and the dose - dependency effect . In particular, they tested different prp concentrations (0.1, 1, 5, 10, and 20%) showing an increase in cellular viability in a dose - dependent manner . Yang et al . Reported that 1% of platelet supernatant (ps) is sufficient to stimulate chondrocyte proliferation, whereas 10% ps stimulated chondrocyte mass formation . Studied prp releasate (prpr) at 1, 5, and 10% and found that 5% was the optimal concentration to increase chondrocyte proliferation . Moreover, gaissmaner et al . Provided evidence of cell proliferation increase with 1 or 10% ps, but no further stimulation occurred using concentrations above 10% . Together with chondrogenic phenotypic maintenance, other authors also documented an increase in matrix molecule production . Documented that prp treatment led to higher amounts of collagen ii and pg synthesis than platelet poor plasma (ppp) or fetal bovine serum (fbs). Since cell matrix interactions play an important role in maintaining cartilage homoeostasis, wu et al . Designed a simple 3d chondrocyte model: in a collagen matrix, the authors mimicked an oa environment by il-1 and tnf induction . Also in this model, prp increased the membrane receptors integrin 11 and cd44 and favoured type ii collagen and pg production . In another experimental model, yin et al . Reported that prp allowed the integration of an agarose construct with cartilage samples, showing a denser extracellular matrix (ecm) deposition in the integration area . Interestingly, pereira et al . Found that the prp stimulatory effect was limited over time: after an initial positive staining for collagen type ii and pg, at 20 doublings the matrix / cells ratio decreased . Similarly, spreafico et al . Documented an increase in pg release 2 days after prpr treatment, followed by a decrease after 9 days, although at 20 days pg release remained still high . Pereira et al . Found that pl enhanced the initial inflammatory response and subsequently triggered its resolution through the regulation of nuclear factor kappa b (nf - kb) and cyclooxygenase-2 (cox-2), the principal actors of inflammatory cascade . Van buul et al . Showed a dose - dependent down - regulation of il-1 - induced nf - kb activation, whereas bendinelli et al . Showed that inhibition of nf - kb transactivation activity was mediated by hgf, a cytokine present in prp -granules . Moreover, they suggested another anti - inflammatory action by inhibiting monocyte - like cell chemotaxis . Wu et al . Also investigated the anti - inflammatory potential of prp in their 3d system: prp counteracted the inflammatory cascade elicited by il-1 and tnf, showing an inhibition of il-1, cox-2, and mmp-2 gene expression . . Showed that the addition of prp to a chondrocyte / hydrogel culture led to an immediate increase in mrna levels of cannabinoid receptor cb1 and cb2 (receptors involved in analgesic and anti - inflammatory effects). In a system of oa chondrocytes and muscle - derived mscs (mdscs), mifune et al . Observed that prp promoted proliferation, adhesion, and migration of mdscs . During chondrogenic pellet culture, prp tended not only to increase the number of type ii collagen - producing cells, but also to increase cell apoptosis, which, however, was not confirmed by the in vivo evaluation . Moreira teixeira et al . Showed high expression and synthesis of collagen ii co - culturing chondrocytes and expanded bone marrow mscs (bmscs) when pl / hydrogel was added . Moreover, they investigated the retention of pl / hydrogel construct in a cartilage fragment: the combination with hydrogel allowed the retention of prp at the defect site, filling up irregularities at the cartilage surface . Anitua et al . Investigated the role of prgf (preparation rich in growth factors: a low - concentrate prp without leukocytes) on oa synoviocytes with or without exposition to il-1, to mimic the overproduction of proinflammatory cytokines in the joint environment during oa progression . Prgf significantly enhanced ha secretion compared to ppp both with and without il-1 and switched angiogenesis to a more balanced status, but did not modify the il-1-induced rise of matrix metallo - protease (mmp) 1, 3 and vascular endothelial growth factor (vegf) produced by synovial cells . Indeed, browning et al . Even showed an increase in mmp-1 and mmp-3 in oa synoviocytes incubated with prp, thus suggesting that the application of prp to synovial joints might be associated with deleterious effects due a pro - inflammatory response that might lead to an accelerated cartilage catabolism . Ishida et al . Showed the usefulness of prp not only because of its proliferation effect, but also its induction of gag synthesis . Prp up - regulated the viability of meniscal cells in a dose - dependent manner, as well as the mrna expression of biglycan and decorin . The study results showed that prp presents the same positive effect as fbs for meniscal cell culture and showed that dosage is an important aspect of the induced effect: 10 and 20% prp increased proliferation rate and influenced more type i collagen and aggrecan expression at day 7 of culture with respect to 5% prp . Eight papers investigated the effect of prp on mscs of different origin: 1 on subchondral cortico - spongious bone (csp) cells, 1 on commercial human mscs, 4 on bmscs, and 2 on adipose - derived mscs (admscs). Kruger et al . Investigated the migration and chondrogenic differentiation of human subchondral progenitors . In particular, a chemotactic assay revealed that prp significantly stimulated the migration of csps, together with their chondrogenic differentiation and production of pg and collagen type ii . Confirmed an induced chondrogenic differentiation of bmscs, which also presented a higher proliferation rate . Mishra et al . Documented the same behaviour on mscs with a higher proliferation rate and a selective differentiation along the chondrogenic line: sox9 and aggrecan (chondrogenic markers) were increased much more than runx2 (osteogenic marker). Conversely, moreira teixeira et al . Reported that pl, besides inducing a significant increase in bmscs proliferation rate and migration, did not induce an increase in collagen type ii . Hildner et al . Focused on admscs and showed strongly enhanced proliferation rates with retained chondrogenic differentiation potential and even a tendency toward increased chondrogenic differentiation of pl - expanded admscs compared to fcs . Studied the proliferation of admscs treated with prp with or without activation and at different concentrations (1, 5, 10, or 20%). Results showed the importance of both prp activation and correct dosage: in fact, the stronger promotion of proliferation was observed in prp activated with calcium chloride and autologous thrombin and applied at 5%, whereas at higher platelet concentrations the proliferation rate decreased in a dose - dependent manner . Finally, murphy et al . Tested two different types of prp: one derived from human adult peripheral blood and one derived from human umbilical cord blood (ucprp), showing the superiority of ucprp with regard to mscs proliferation and migration induction . Concerning in vivo preclinical studies dealing with prp injective treatment, we found 11 papers: 3 on rat, 3 on rabbit, 3 on sheep, 1 on pig, and 1 on horse, which showed heterogeneous results for heterogeneous indications . Did not find any immediate (2 weeks after the injection cycle) benefit of prp on cartilage tissue in rat joints previously damaged with intra - articular formalin injection, mifune et al . Found in a rat oa model, induced by monosodium iodoacetate injection, that prp had no marked effect by itself, but increased the cartilage repair effect of mdscs, with a better histologic appearance, higher number of cells producing type ii collagen, and lower levels of chondrocyte apoptosis at 4 weeks, although at 12 weeks its effects were lost . Confirmed the benefit of prp in a rabbit model of collagenase - induced oa: intra - articular injections influenced positively cartilage regeneration in all oa severity degrees, with a more evident effect in moderate oa . Saito et al . Used a rabbit oa model of anterior cruciate ligament resection for the treatment with gelatin hydrogel microspheres impregnated with prp: injections markedly suppressed oa progression both morphologically and histologically (less significant results were obtained by the use of prp only). Finally, carmona et al . Used a large animal model to analyze the effect of prp injections: in a study on 4 horses with oa, 3 injections of prp led to a significant improvement in both the degree of lameness and joint effusion . The most marked improvement was observed 2 months after treatment and persisted for 8 months with no adverse events . Five studies focused on the injective treatment of chondral or osteochondral lesions . Also in this case, results were controversial . Serra et al . Performed 7 prp injections every other day in rabbit joints where a full - thickness osteochondral lesion was previously made surgically on the medial femoral condyle . Evaluated prp as augmentation in rat cartilage lesions after microfractures: at week 6, the microfracture group score was worse than that of the prp + microfracture group, which had an increased degree of type ii collagen staining . Milano et al . Used one prp injection as augmentation procedure of microfracture in a sheep model . Although no hyaline cartilage was obtained, prp offered better macroscopic, histologic, and biomechanical results . The prp administration modality proved to be important for the final outcome, with better results when prp was surgically applied as a gel over the treated lesion . [49, 50] focused on the injective approach: 5-weekly injections of prp promoted a better spontaneous repair and also a better and more durable reparative response when applied after microfractures with respect to isolated microfractures, albeit without producing hyaline cartilage . Lippross et al . Reproduced ra in pigs: the animals were systemically immunized by bovine serum albumin (bsa) injections, and arthritis was induced by intra - articular bsa injection . The injection of prp attenuated the arthritic changes on synovium and cartilage by modulating the activity of inflammation mediators . In particular, il-6 and vegf staining was reduced, but concerning gene expression, only il-6 levels were significantly lower after prp application . Focusing on protein quantification, all chondral protein concentrations returned to healthy tissue levels, and in synovial samples, besides the low levels of il-6 and vegf, the authors showed a reduction in igf-1 and il-1 in prp groups, whereas tnf was not altered . Intra - articular clinical application of prp has been tested in several clinical studies to date . The present search identified 22 clinical trials that met the inclusion criteria: among these, 13 were case series, 4 were comparative studies, and 5 were randomized trials . The first comparative evaluation was performed by sanchez et al . In 2008 who published a retrospective observational study on 60 patients, 30 treated with 3 knee intra - articular injections of prgf and 30 with 3 injections of hyaluronic acid (ha). Results at 5 weeks were encouraging, with prgf showing better efficacy in pain control . Afterwards, kon et al . In 2011 performed a prospective comparative study testing prp against low molecular weight ha (lw ha) and high molecular weight ha (hw ha) in 3 homogeneous groups of 50 patients each . The results showed a better performance for the prp group at 6 months of follow - up . Conversely, in the early oa group the difference with ha was not significant and in the severe oa group no difference in clinical outcome was observed . Another interesting finding was that patients aged up to 50 years old had a greater chance to benefit from the prp approach . The same authors were the only ones to compare two different prp preparations: high - concentrate leukocyte - rich prp versus low - concentrate leukocyte - free prp . One hundred forty - four patients were treated and evaluated up to 12 months and comparable positive results were obtained with both treatments, with the only difference being that the prp - leukocyte group suffered from more swelling and pain reaction immediately after the injections . An increase in the clinical scores was reported in both groups at 6 months, but statistically superior results were found in the prp group . Sanchez et al . Investigated the efficacy of single - spinning leukocyte - free prp compared to ha in 153 patients evaluated up 6 months of follow - up . The only aspect where a clear superiority of prp was found was the percentage of responders (patients with at least 50% of pain reduction), which was significantly higher in the prp group . Besides this finding, the study did not show that prp in moderate / severe oa was more effective than ha ., according to the preliminary results (109 patients) of their randomized double - blind trial comparing prp and ha: no statistical inter - group difference was reported and just a tendency toward better results for the prp group at 6 and 12 months of follow - up was found in patients affected by low - grade cartilage degeneration (kellgren lawrence up to 2). Conversely, cerza et al . Treated 120 patients by either autologous conditioned plasma (acp, a low - concentrate prp without leukocytes) or ha . Surprisingly, the acp group showed a significantly better performance than ha in all groups of treatment, including patients affected by grade 3 knee oa . Furthermore, the clinical gap between treatments increased over time in favour of acp . Finally, a recent randomized trial by patel et al . Was the first to test prp versus saline . Iii oa were included and treated bilaterally with one injection of prp, two injection of prp (3 weeks apart) or one injection of saline . Despite the low number of patients included, a significant difference was observed between prp and saline solution in terms of clinical outcome . Interestingly, no difference was reported among patients who received one or two prp injections . Only one paper investigated the efficacy of prp versus ha in osteochondral talar lesions on 30 patients . In the short - term 28-week evaluation a superior clinical performance was found in the prp group . This systematic review confirmed the increasing interest in prp as an injective treatment for cartilage degeneration and oa, with an increasing number of published studies over time . Prp is a fashionable treatment, offering the possibility to deliver a high concentration of autologous growth factors and bioactive molecules in physiologic proportions, with low costs and in a minimally invasive way . This explains the wide application of this blood derivative to several tissues and heterogeneous pathologies in different fields of medicine . The rationale for using platelets for the treatment of different tissues is that they constitute a reservoir of growth factors that are critical to regulate the tissue healing process, which is quite similar in all kinds of tissues . However, whereas the rationale for prp use in other tissues is clear, since platelets represent the first response to a tissue damage where they participate in stopping the vessel bleeding and trigger the healing cascade, less intuitive is the rationale for prp use in cartilage, which is a physiologically vessel - free tissue . Moreover, whereas some molecules such as tgf - might justify its use in cartilage, prp also contains other molecules such as vegf that do not take part or might even jeopardize cartilage homeostasis and regeneration [48, 72]. Thus, it is mandatory to investigate whether the overall effect of prp is also beneficial for the peculiar requirements of cartilage tissue before an indiscriminate human application . The systematic analysis of in vitro studies published up to now shows an overall positive effect of prp on cartilage tissue . Besides some controversial results, most of the findings supported the role of prp in increasing chondrocyte proliferation, without affecting chondrogenic phenotype and with an increase in the production of matrix molecules . These properties of prp have provided positive results also in the animal model: preclinical studies confirmed the usefulness of prp treatment in different pathology models, with good results in cartilage regeneration after acute focal lesions, as well as in the more complex environment of joint osteoarthritic degeneration, and even in the challenging ra setting . Nonetheless, both the rapid clinical benefit and the limited effect over time are in contrast with the timing required by a hypothetically induced cartilage regeneration process . Despite the wide majority of studies focusing on cartilage tissue, it is actually likely that the clinical benefit reported after prp injection is attributable to other action mechanisms . An intra - articular injection does not just target cartilage, instead prp might influence the entire joint environment, and some in vitro studies confirm the effects of prp on other cell sources . Synoviocytes are affected by platelet releasate, as well as meniscal cells and also mscs that seem to be induced by prp and act synergically toward tissue healing . The chemo - attractant activity of prp may contribute to the recruitment of other cells that might migrate into the damaged tissues, thus triggering the healing response [42, 53]. Prp has several potential effects by enhancing the cell signalling cascade in all joint tissues and inducing positive changes in the whole joint environment through a milieu of actions . Among these, tissue regeneration is actually not the only and maybe not the most important prp mechanism of action, and increasing evidence supports the complex role of prp in modulating inflammation . Prp showed both pro- and anti - inflammatory activities: an initial pro - inflammatory action was reported, with synoviocyte stimulation for mmp and cytokine release, followed by a limitation of the inflammatory response by decreasing inflammatory molecules and preventing chemotaxis of monocytes - like cells [44, 75]. An overall down - modulation of the joint inflammation can explain the well - documented pain reduction, which is the most prominent and disabling symptom of cartilage lesions and knee oa . However, some findings suggest another intriguing aspect of prp action mechanism, with a direct analgesic effect: lee et al . Showed the role of prp in the augmentation of cannabinoid receptors cb1 and cb2, which might be involved in the analgesic effects . Further studies need to focus on understanding and possibly optimizing the analgesic and anti - inflammatory effects of prp . Prp might not lead to hyaline cartilage regeneration and might not change the clinical history with significant disease - modifying properties, but it still might offer a clinical benefit with symptoms and function improvement and possibly a slowdown of the degenerative processes . The central feature in oa cartilage degeneration is the so - called apoptosis (programmed cell death); thus, chondrocytes apoptosis is a potential therapeutic target for oa interventions . The exact mechanism behind the prp regulation of the apoptotic pathway is unclear, but it is likely that prp might have an overall effect in slowing down the apoptosis cascade . Among the hypothesized mechanisms, recent findings identified igf-1 protein as a possible effector of apoptosis inhibition: yin et al . Found that igf-1 may down - regulate the expression of programmed cell death 5 (pdcd5), thus inhibiting the apoptosis of osteoarthritic chondrocytes . Interestingly, mifune et al . Observed an increased cell apoptosis in the in vitro setting, which, however, was not confirmed by the subsequent in vivo experiment, where lower levels of apoptosis were detected . Thus, the authors suggested that it was the complex interaction of prp with the different joint structures (synovium, fat pad, bone marrow,), which might positively influence chondrocytes apoptosis . The controversial findings reported underline the limits of preclinical studies, which do not exactly represent the peculiar human pathophysiology . Nonetheless, although such experimental settings do not replace the fundamental role of robust clinical trials, in vitro studies can suggest mechanisms of action and directions for improvement and might explain some controversial findings in the reports of prp application in humans . As for other tissues, in vitro studies have shown the importance of the dosage of the potent platelet - derived growth factors, with different platelet concentrations leading to different results . Activation might also play an important role, as well as the appropriate cell population which is also a key aspect for obtaining optimal results . With regard to this, leukocytes are a controversial prp component, since some authors attribute better results to leukocyte depletion, because of the supposed deleterious effects of proteases and reactive oxygen species released from white cells, whereas other authors consider them as a source of cytokines and enzymes that may also be important for the prevention of infections . Several other variables have to be considered, such as the preparation methods and the consequent presence of other cells, storage modalities, application protocols, and many other aspects that might not be of secondary importance for determining prp properties and clinical efficacy . The number of names and acronyms encountered searching for studies on this biological treatment approach, such as prp, prgf, acp, pl, clearly represents the complexity of this field and explains the difficulties in literature analysis, study comparison, and understanding some contradictory results . With the limits of a complex field still in its infancy, few studies and some controversial results, this systematic review still showed some important aspects . The first one is that the increasing interest in this topic is being translated into research with a growing number of papers published over time, which show promise in shedding some more light on prp use in the near future . The second one is that, besides the limits and sometimes controversial findings of in vitro and animal studies, the preclinical literature documented an overall support toward prp application for the injective treatment of cartilage lesions and oa . Moreover, some conclusions can be drawn also with regard to human application, which can be of clinical usefulness . The first one is the safety of prp injections, with no major adverse events reported in the literature and only some reports of self - limiting immediate pain and swelling reaction [17, 36, 41]. The second one is that all studies seem to agree on an overall clinical benefit of prp . Better results with respect to saline have been shown, and some studies suggest a slight superiority of prp with respect to visco supplementation [8, 14, 58, 64]. However, not all patient categories present the same results that are more significant in younger patients affected by not too advanced degeneration, and the clinical benefit is limited over time and can roughly be estimated at less than 1 year . This might suggest that this treatment could be applied in cycles to ensure longer lasting results and postpone more invasive procedures . Finally, another aspect emerges from the literature analysis: whereas among the available techniques none clearly seemed to offer superior clinical results, it appears clear that there is room for a better targeting of this prp application . Several aspects still need to be studied to understand the mechanism of action of prp and give better treatment indications and possibly to optimize the procedure and improve the potential of this biological minimally invasive approach for the treatment of cartilage degeneration and oa . One of the emerging fields of prp treatment is its injective application for cartilage degeneration and oa, as shown by an increasing number of papers published on this topic over time . Preclinical evidence supports the use of prp injections that might promote a favourable environment for joint tissues healing, targeting not only cartilage but also synovial and meniscal tissues . A few high - quality trials have been published, which showed the clinical usefulness of prp but only with an improvement limited over time and mainly in younger patients not affected by advanced degeneration . Many biological variables might influence the clinical outcome and have to be studied to optimize prp injective treatment in case of cartilage degeneration and oa.
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Teratomas are congenital tumors that are usually found in the gonads, and are only rarely detected in extragonadal areas, such as, the mediastinal, retroperitoneal and sacrococcygeal regions . To the best our knowledge, an extragonadal teratoma of the long bone has not been previously reported in the literature . We describe clinical, radiologic and pathologic findings of an exceedingly rare case of malignant teratoma of the proximal humerus . A 14-yr - old female was admitted with painful limited motion of the left shoulder joint . She was transferred to our institute for further study and proper management under impression of malignant bone tumor . On physical examination, however, a radiograph of the humerus showed ill defined moth eaten to permeative destructive lesion associated with cortical perforation of proximal humerus (fig . 1a). Magnetic resonance imaging (mri) well demonstrated the entire extent of the tumor, which showed inhomogeneous enhancement after contrast agent administration . Both the intraossoeus and extraosseous tumor components were evident on transaxial gradient - echo mr images (fig . 99 m tc - mdp scintigraphy and an f - fdg whole body positron emission tomography (pet) scan showed uneven increased uptake at the left proximal humerus, but no other abnormality was detected . Initial diagnostic considerations included; leukemic infiltration, lymphoma, osteomylelitis, and a malignant bone tumor, such as, an osteosarcoma . A subsequent surgical biopsy was performed and the pathological report revealed an immature bony teratoma . On gross inspection, the cut surface of the resected proximal humerus showed a homogenous grayish white flesh - like tissue that had replaced the whole epimetaphysis and a part of the diaphysis (fig . Malignant teratoma were graded using a method described by robboy and scully (1); degree of immaturity, presence and quantity of a neuroepithelial component . Grade 1 tumors with rare foci of immature neuroepithelial tissue occupy less than one low power field (40x), grade 2 tumors occupy 1 to 3, and grade 3 tumors occupy more than 3 low power fields . The latter looked immature in appearance, which is not found in normal adult tissue . Immunohistochemical staining demonstrated the expressions of glial fibrillarary acidic protein (gfap) and cd56 expression in neuronal tissue (fig . Based on the above - mentioned grading system, the tumor of the present case was classified as a grade 1 immature teratoma bearing immature endodermal glandular structures . There was no evidence of local recurrence or distant metastasis at 30 months postoperatively . According to the musculoskeletal tumor society scoring system (2), teratomas are germ cell neoplasms that include tissues arising from the three germ cell layers (3). They usually originate from pluripotent cells, which can differentiate to a wide variety of tissues . The majority of extragonadal teratomas develop from germinal elements, and they usually occur along the migration pathway of the germ cells (4). Resultantly, the sacrococcygeal region is the most frequent extragonadal location; other sites include the mediastinum and retroperitoneum (5). Exceptionally rarely intraosseous teratoma has reported but there has been no reported case involving a long bone in the literature . In 1992, chinoy et al . (6) reported a case of an extragonadal malignant teratoma of the foot and in 2000, vazquez et al . (7) described an intraosseous teratoma of the iliac bone . In these previously published reports, initial diagnostic considerations included fibrous dysplasia, langerhans' cell histiocytosis, aneurysmal bone cyst and a malignant bone tumor, such as, an osteosarcoma . Most skeletal germ cell tumors are metastatic (8) but the pathogenesis of teratoma in the appendicular skeleton is still enigmatic . Another possible pathogenetic theory concerns the heterotopic location of extragonadal germ cells . However, the humerus is not in the migration path of germ cells, and in fact, no teratoma has been identified previously at this site . Thus, we are uncertain about the pathogenesis of the bony teratoma in this case . Nevertheless, since the tumor was confirmed as a grade 1 immature teratoma, a wide surgical excision only was undertaken for comprehensive treatment . She is doing well without evidence of local recurrence or distant metastasis at final follow - up examination.
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Cardiovascular diseases (cvd) are becoming the major cause of mortality in developing countries.1 the 2002 world health report indicates that the most important risk factors for noncommunicable diseases include high blood pressure, high blood cholesterol, inadequate fruit and vegetable intake, overweight and obesity, physical inactivity and tobacco use.2 a recent community - based national study, involving adult saudis between the ages of 30 and 70 years, showed that the overall prevalence of coronary heart disease (chd) in the kingdom of saudi arabia is 5.5%.3 however, the expected increase in mortality as a result of ischemic heart disease in the middle east region in 2020 compared to 1990 was estimated as the highest of all the regions of the world (146% increase in women and 174% increase in men).4 this is mostly attributed to the high prevalence of major chd risk factors . The latest report involving saudis between the ages of 30 and 70 years showed the following prevalence: 23.7% for diabetes; 26% for hypertension; 12.8% for smoking; 53.9% for hypercholesterolemia, and 35.6 for obesity.3 this high prevalence of chd risk factors in the saudi population confirms many earlier findings on chd risk factors involving different segments of the saudi society.58 prevention of cvd depends on controlling the modifiable risk factors, such as, physical inactivity and obesity . In fact, physical inactivity represents an independent risk factor for a number of chronic diseases, including chd, diabetes mellitus, obesity, osteoporosis.911 however, physical activity has been shown to both prevent and treat many established atherosclerotic risk factors.101214 the world health organization (who), which has long recognized the heavy burden of noncommunicable diseases on the health services, recently launched its global strategy on diet, physical activity and health.15 the overall goal of the strategy is to improve public health through healthy eating and physical activity . This concerted effort on the part of who to improve global public health through physical activity is a continuation of previous actions taken during the last decade by numerous health and medical organizations, including the american heart association,916 the american college of sports medicine,131718 and centers for disease control and prevention.1119 the present paper, therefore, provides an overview of the importance of physical activity in health promotion and disease prevention, and discusses the public health burden of physical inactivity . It is also the aim of this paper to briefly review the status of physical inactivity in saudi arabia and to calculate the attributable risk of physical inactivity in the saudi population . In addition, some preliminary findings from the longitudinal study on physical activity and cardiovascular health of the saudi youth will be presented throughout this paper . Physical activity is defined as any bodily movement produced by the skeletal muscles that results in energy expenditure above the basal level.20 physical activity is considered a complex set of behaviors . Our ability to relate physical activity to health indicators depends on accurate, precise and dependable measures . Physical activity is commonly measured by either self - report or direct monitoring through mechanical / electronic or physiological measurements . Table 1 presents an important distinction between the definitions of physical activity and physical fitness . It also shows the meaning of metabolic equivalent (met), which is increasingly becoming a very common term for quantifying the intensity of physical activity or energy expenditure, especially for the purpose of exercise prescription or physical activity assessment . Definitions of basic terms today, it is estimated that 60 - 85% of adults around the world are simply not active enough to achieve the health benefits of physical activity.21 to deal with this epidemic of physical inactivity, who has recently established the physical activity unit as part of the department of noncommunicable disease prevention and health promotion.22 the goal of this unit is to promote higher levels of physical activity within the world population of all ages and conditions, men and women . The unit's strategic products include: (1) developing a global strategy and database on physical activity; (2) promoting the annual global move for health initiative; (3) supporting a country's actions and facilitating the development of multisectorial national policies and programmes on physical activity; and (4) developing partnership and networks for increasing population participation in physical activity with a special focus on young people . Despite the fact that most medical institutions currently do not provide their graduates with the proper training and necessary skills in physical activity assessments and exercise prescription,23 many medical organizations are now urging health care professionals to provide counseling on physical activity for their patients . The american heart association (aha) for example, stated in its recent guidelines for the primary prevention of cvd and stroke, that the assessment of the risk factors in adults should commence at 20 years of age . Physical activity is a primary chd risk factor, and should be assessed at every routine evaluation.24 the aha goes further in its recommendations and asks that: (a) schools should be encouraged to teach skills required for physically active lifestyle; (b) health care professionals should be educated about exercise as a therapeutic modality, and the importance of lifelong physical activity for patients; (c) they should routinely prescribe exercise for their patients; and (d) exercise testing should be performed before vigorous exercise in selected patients with cvd or those at high risk.24 furthermore, the importance of physical activity in health promotion and disease prevention strategy was evident in the healthy people 2010 report, published by the centers for disease control and prevention (cdc) of the united states department of health and human services.25 in that report, 10 leading health indicators (lhi) were identified . The lhi reflect the major public health concerns in the united states, and highlight the importance of health promotion and disease prevention on the societal level . Physical activity, not surprisingly, is the first on the list of lhi, followed by obesity in the healthy people 2010 report . Among the target goals for the healthy people 2010 was an increase in the proportion of adults and adolescents who engage in moderate physical activity (3 - 6 mets) for at least 30 minutes on five or more days per week . During the past three decades, the kingdom of saudi arabia has undergone tremendous changes in lifestyle, including physical activity and eating habits . These dramatic lifestyle changes have definitely had a considerable negative impact on the health of the society . Indeed, this lifestyle transformation is thought to be responsible for the epidemic of noncommunicable diseases and their complications in the country.32627 unfortunately, there exists no physical activity surveillance system in the country . However, mere observation indicates that there is a reduction in daily physical activity and energy expenditure of the saudi people relative to earlier times . Moreover, findings from a limited number of studies on the prevalence of physical inactivity in saudi population confirm that a sedentary life style is on the rise.262829 across all segments of the saudi population, physical inactivity ranged from 43% to 99%, depending on gender, age, location, and target population.30 figure 1 presents some data for physical inactivity among saudi children, youth and adults, conducted on samples from riyadh.293132 in the studies conducted on children2631 and youth,32 continuous 12-hour heart rate telemetry was used to measure physical activity . In the adult study,29 the overall prevalence rates of physical inactivity among saudi children, youth and adults were roughly 60%, 70%, and 80%, respectively . The prevalence rates for inactivity among saudis, shown in figure 1, agree with those estimates reported worldwide by who.21 prevalence of physical inactivity in saudi children, youth and adults . It is quite clear from the data presented in figure 1 that physical inactivity is prevalent among saudi children and youth . Findings from a recent study on 12 - 20-year - old schoolboys living in riyadh have also shown inactivity prevalence of about 50%.33 promoting physical activity among children and adolescents is very important in order to offset any decline in their activity level as they grow - up . Data from several countries indicated a decline in physical activity among the youth starting at age 12 and continuing up to 20 years.34,35 preliminary findings from our own longitudinal study on saudi youth showed a 30% increase in physical inactivity from childhood (7 - 12 years) to early adulthood (18 - 23 years). This was coupled with a three - fold increase in television viewing during the same period of time.36 data from our laboratory3236 also demonstrated that inactivity prevalence in youth is far more pronounced than any of the other chd risk factors, as shown in figure 2 . Therefore, a reduction in the prevalence of physical inactivity among saudi youth would have a far greater impact on risk reduction than a reduction in any of the other traditional chd risk factors . The council on cardiovascular disease in the youth of the aha has issued a statement for health professionals asking them to counsel their young patients on physical activity, including physical activity assessment and exercise prescription.37 therefore, routine counseling by local physicians on physical activity and the health of their young patients should be initiated . Prevalence of coronary heart disease (chd) risk factors among a sample of saudi youth the burden of mortality, morbidity and disability attributable to noncommunicable diseases, including sedentary life, is considerably high and continuing to grow.15 according to preliminary data from a who study on risk factors, inactivity is one of the 10 leading global causes of death and disability.21 worldwide, physical inactivity was estimated to cause 1.9 million deaths and 19 million disability - adjusted life years.2 physical inactivity was also estimated to globally cause about 22% of ischemic heart disease and about 10 - 16% of cases each of diabetes mellitus, breast, colon and rectal cancer.2 research on the epidemiology of physical activity revealed that it appears to be a far more important risk factor than previously estimated.3840 quantitative estimates from the united states indicated that sedentary life is responsible for 35% of chd deaths, 32% of deaths from colon cancer and 35% of deaths from diabetes.41 furthermore, inactivity - related disease in the united states causes over 14 times more deaths annually than acquired immune deficiency syndrome (aids).42 in saudi arabia, the prevalence of physical inactivity is extremely high, especially in women, and may be considered among the highest in the world.30 recent local data also showed a high prevalence of other chd risk factors among saudi population.3 in addition, type 2 diabetes mellitus is becoming increasingly more prevalent among saudis.343 obesity has also reached epidemic proportions, especially among saudi females.344 it is our own belief that strong associations do exist between the high prevalence of physical inactivity in the saudi population and the epidemic of modern chronic diseases and risk factors in saudi arabia . Therefore, reducing the proportion of inactive saudis would have a tremendous impact on lowering these lifestyle - related diseases and risk factors, and thus reduce future health care costs in the kingdom . Population attributable risk (par) is one of the more useful methods of estimating the proportion of a public health burden resulting from a particular risk factor.41 par is the risk in total population minus the risk in the unexposed group . Thus, par provides an estimate of how much of a particular disease could be prevented if exposure to the risk factor was eliminated.45 it can be calculated from the estimate of relative risk (rr) and the population prevalence of the risk factor . Rr is used to assess the magnitude of risk of exposed individuals to a particular disease relative to unexposed individuals . However, the societal impact of exposure depends not only on the magnitude of the relative risk but also on the prevalence of the risk factor in the population.45 the concept behind par is to help provide a balanced view between a relatively strong risk factor that affects fewer people and a relatively weaker risk factor that is more prevalent in a population.39 figure 3 shows par for major chd risk factors in the united states (usa)40 and the united kingdom (uk).46 par for physical inactivity in the us and the uk were 35% and 37%, respectively . This means that approximately one third of chd mortality could be attributed to physical inactivity in the us and the uk . In both countries, physical inactivity as a contributing risk factor to chd deaths was next in magnitude to hypercholesterolemia . Estimated population attributable risk (par) for major chd risk factors in the usa and uk par of sedentary living for mortality from chd and diabetes to the data from saudi population were applied . Physical inactivity prevalence data were taken from a recent study on saudi adult males living in riyadh, where there were three levels of exposure and prevalence estimates.29 for relative risk, estimates that were previously reported elsewhere were used.4041 of course, the relative risk means the number of deaths among active population divided by the number of deaths among the inactive population . As indicated in table 2, par of sedentary living for mortality from chd and diabetes in saudi arabia was considerably high and much greater than what is reported in figure 3 for the uk and the usa since physical inactivity is much higher in saudi arabia than in the usa or in the uk . Estimated population attributable risk (par) of sedentary living for mortality from coronary heart disease (chd) and diabetes mellitus in saudi arabia table 3 presents the relative risk, prevalence and par of some major chd risk factors applied to the saudi population . The relative risk data were taken from powell, et al.47 prevalence data for physical inactivity were taken from an earlier study on adult males in riyadh.29 the prevalence data of other chd risk factors were from a newly published paper on saudi adults between the ages of 30 and 70 years.3 as clearly shown in table 3, physical inactivity represents a far more important risk factor than was previously thought . Therefore, reducing the proportion of inactive saudi adults to 40% from the current figure of 80% would definitely reduce the burden of physical inactivity on public health . In the usa, healthy people 2010 calls for reducing to no more than 20%, the proportion of people 18 years and older who are inactive.25 relative risk (rr), prevalence (%) and population attributable risk (par) of major coronary heart disease risk factors applied to saudi population it was clear from the available evidence that physical inactivity is becoming more prevalent in the saudi population of different ages and both sexes . This high prevalence of inactivity in saudi arabia represents a major public health burden, as evidenced by the high par of physical inactivity, compared with those of the usa and uk . Moreover, due to the high prevalence of other chd risk factors among saudis, the rate of lifestyle - related diseases (chd, diabetes, obesity, etc) in the society may keep escalating to epidemic proportions in the near future . Unless concrete steps are taken to reduce physical inactivity in the saudi population, the future public health cost will be heavily burdened . Physical activity is associated with numerous health benefits and plays a major role in modifying many other chd risk factors . The following recommendations for reducing physical inactivity and promoting active living are made: (1)national policy and legislative initiatives are urgently needed to encourage active lifestyle and discourage sedentary living habits . This recommendation was clearly stated as an important objective in the global strategy on diet and physical activity, launched recently by who. (2)there is a need to establish a surveillance system to monitor physical activity in the saudi population . Monitoring physical activity of the saudi population at regular intervals would definitely provide important database . Such a database would represent a cornerstone for any programs that would aim at combating physical inactivity and promoting active living. (3)medical communities and health care providers must play a leading role in promoting physical activity, by providing routine assessments and counseling on physical activity and exercise prescription for their patients . This is consistent with many recent appeals from leading medical and public health organizations, such as the american heart association, the american academy of pediatrics, the american college of sports medicine, the centers for diseases control and prevention, and the who. (4)implementation of daily physical education for students from kindergarten to grade 12 is necessary to promote life - long physical activity among saudis . Emphasis should be on quality curricula and instructions that help students develop the knowledge, attitudes, motor skills and confidence needed to adopt and maintain physically active lifestyles. (5)opportunities for physical activity should be available for a wide range of people, including the elderly, children and women . Given that walking is acceptable across sociodemographic subgroups of the saudi population, efforts must be made to increase outdoor as well as indoor walking trails . Promoting brisk walks as a means of physical activity could markedly increase the proportion of physically active saudis . National policy and legislative initiatives are urgently needed to encourage active lifestyle and discourage sedentary living habits . This recommendation was clearly stated as an important objective in the global strategy on diet and physical activity, launched recently by who . There is a need to establish a surveillance system to monitor physical activity in the saudi population . Monitoring physical activity of the saudi population at regular intervals would definitely provide important database . Such a database would represent a cornerstone for any programs that would aim at combating physical inactivity and promoting active living . Medical communities and health care providers must play a leading role in promoting physical activity, by providing routine assessments and counseling on physical activity and exercise prescription for their patients . This is consistent with many recent appeals from leading medical and public health organizations, such as the american heart association, the american academy of pediatrics, the american college of sports medicine, the centers for diseases control and prevention, and the who . Implementation of daily physical education for students from kindergarten to grade 12 is necessary to promote life - long physical activity among saudis . Emphasis should be on quality curricula and instructions that help students develop the knowledge, attitudes, motor skills and confidence needed to adopt and maintain physically active lifestyles . Opportunities for physical activity should be available for a wide range of people, including the elderly, children and women . Given that walking is acceptable across sociodemographic subgroups of the saudi population promoting brisk walks as a means of physical activity could markedly increase the proportion of physically active saudis.
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The facial nerve trunk exits through the stylomastoid foramen, and enters the parotid gland where it divides into the cervicofacial and temporofacial divisions . Then divides into five peripheral branches to supply the muscles of facial expression (1). The branches arising from the rami form to parotid plexus in parotid gland . The superior buccal nerve arising from temporofacial ramus and the inferior buccal nerve arising from the cervicofacial ramus form to the buccal plexus without parotid gland (1). The zygomatic (2), the marginal mandibular (3 - 5), the buccal (6), and the temporal branches (7) of the facial nerve were investigated by many researchers . Different surgical approaches and landmarks to the trunk of the facial nerve have been reported (8 - 12). Many landmarks such as the mastoid process (8 - 10), the transverse process of the atlas (8) and axis (11), the temporomandibular joint, the angle of the mandible (9, 11), insertion of the sternocleidomastoid muscle, pointer cartilage of the ear (10), tragal pointer (11) and the marginal mandibular branch of the facial nerve (12) can be used to identify the trunk of the facial nerve . But identification of this trunk may be difficult because it is encompassed by dense connective tissue (13). In this paper the double facial nerve trunk emerged from the stylomastoid foramen and petrotympanic fissure was found in a 65-yr - old caucasian male cadaver during a routine dissection course . Firstly, the skin and superficial fascia between the mastoid process and ramus of the mandible were reflected in all subjects . The anterior border of the parotid gland was carefully elevated and the rami of the nerves followed proximally up to the stylomastoid foramen under a stereomicroscope (stemi 2000; carl zeiss, jena, germany). Finally, magnetic resonance (mr) imaging was performed by using 1.5 t scanner to this cadaver . To be able to demonstrate the stylomastoid foramen and the petrotympanic fissure in same section, we obtained t1 and t2 weighted oblique sagittal scans . In the present cadaver, difference with the exit point of the facial nerve was observed . In this specimen, in this side, although cervicofacial ramus of the facial nerve exited from the stylomastoid foramen, temporofacial ramus of the facial nerve exited from petrotympanic fissure (fig . The buccal branch dividing from temporal branch of temporofacial ramus and first buccal branch dividing from cervicofacial ramus formed to first buccal plexus . The buccal branch dividing from zygomatic branch of temporofacial ramus and second buccal branch dividing from cervicofacial ramus formed to second buccal plexus (fig . 2). These two buccal plexuses and other branches formed to structures like to polygon (fig . The temporofacial ramus exiting from petrotympanic fissure was seen on magnetic resonance image (fig . Several studies relating to the trunk of the facial nerve have been reported in the literature (14 - 19). Katz and catalano (14) reported three cases (3%) presenting two main trunks, known as the major and minor trunks, with the latter joining the larger temporofacial division, the origin of the main buccal branch . The minor trunk of the facial nerve was noted in eight of 30 cases (26.7%) and, in all of them, the minor trunk entered the lower division of the facial nerve (15). Botman and jongkees (16) reported that the facial nerve within the mastoid segment of the temporal bone can split into two or three branches, and each branch exits through a separate osseous foramen . In this study, a trunk of the facial nerve exiting from the petrotympanic fissure is present one case . In addition, baker and conley (17) reported the possibilities of trifurcation, quadrifurcation, or even a plexiform branching pattern of the trunk of the facial nerve . Salame et al . (18) identified one case of trifurcation out of 46 cases . But we did not identify any case of trifurcation, quadrifurcation or a plexiform branching pattern of the trunk . The facial nerve can easily be injured by sharp or penetrating trauma to the cheek . There are number of studies concerning protection extracranial branches of the facial nerve during plastic surgery procedures and operations intended for the parotid gland, but relatively little care has been given exit point of the facial nerve . Knowledge of the trunk of the facial nerve is essential for preserving the nerve during surgical procedures of the mastoid process, parotid gland, the cranial base and the facial nerve (13, 17). In this study, we exposed exit point of the trunk of the facial nerve through the cranium . Surgeons should be aware of the possible anatomical variations of the trunk of the facial nerve, because a trunk of the facial nerve exiting from the petrotympanic fissure may also be present.
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Medical literature defines recklinghausen's disease, also known as neurofibromatosis type 1 (nf1), as an autosomal dominant disease characterised by abnormalities affecting tissues derived from the neural crest . Researchers indicate that the disease is directly caused by mutation of the neurofibrin 1 gene located on chromosome 17q11.2 . Studies carried out by doctors from france and finland show that benign neurofibromatosis type 1 is connected with homozygosis of constitutional mutations in one of the genes responsible for dna repair by way of deleting unpaired nucleotides such as mlh1, msh2, msh6 and pms2 [2, 3]. Recklinghausen's disease is diagnosed when at least 2 out of the 7 following criteria are met: first degree relative afflicted with neurofibromatosis type 1, min . 6 caf - au - lait spots with a diameter exceeding 5 mm (children) and 15 mm (adults), min . 2 neurofibromas of any type or one plexiform neurofibroma, freckles and/or discolouration found on covered parts of the body, optic nerve glioma, min . Two lisch nodules as well as characteristic bone lesion such as sphenoid wing dysplasia or thinning of the long bone cortex . According to a study carried out in 2007, the differential diagnosis of nf1 should focus on excluding such disorders as rare mosaic or segmental neurofibromatosis type 1, neurofibromatosis type 2, schwannomatosis, noonan syndrome, watson phenotype, caf - au - lait spots inherited as an autosomal dominant condition, autosomal dominant mccune - albright syndrome, leopard syndrome, klippel - trnaunay syndrome, proteus syndrome, multiple lipomas, bannayan - riley - ruvalcaba syndrome, fibromatosis, multiple endocrine neoplasia syndrome type 2b as well as homozygosity for one of the genes responsible for causing hereditary non - polyposis cancer of the colon . The study aimed at identifying changes in the masticatory organ and specifying possibilities of orthodontic treatment of a patient suffering from recklinghausen's disease . The present study describes a case of a 9-year - old girl suffering from recklinghausen's disease with neurofibromatosis of the face, salivary gland and external ear in the form of an extensive tumour . In consequence, the extraoral examination of facial features revealed that the central part of the chin had moved towards the unaffected side of the face, while the angle of the mouth lowered significantly towards the affected side of the face (fig . The clinical intraoral examination of the patient revealed right buccal occlusion and lingual occlusion on the opposite side (fig . The computer - assisted tomography examination identified acute atrophy of the maxilla, zygomatic bone, mandible alveolar ridge and right base of the skull (fig . 3a, b). Despite a number of surgical procedures aimed at removing the tumour (the first procedure was when the patient was 6 years old), the patient received orthodontic treatment of the co - existing malocclusion which was to prevent further mandible movement towards the right - hand side of the face a metzelder appliance (removable orthodontic appliance for the upper and lower jaw) was used . Once placed in the mouth, this functional appliance forced proper placement of the mandible versus the maxilla . The proper spatial relation between the mandible and maxilla was specified and recorded with a construction bite . The treatment lasted 3 years, yet although the appliance was used conscientiously by the patient, no significant clinical improvement was identified . Extraoral photographs of the patient intraoral photograph of the patient computer - assisted tomography image of patient's skull according to researchers, neoplasia is an inherent characteristic of a phenotype seen in patients suffering from recklinghausen's disease; therefore, it is the main factor influencing the selection of a therapy . Although there are certain single attempts of pharmacological treatment of ganglioneurofibromas at their early development stage, it is agreed that, in fact, no preventive actions are possible . Hence, in the case of neurofibromatosis type 1, reconstruction and aesthetic procedures are widely applied in treating the disease . The aim of the orthodontic appliance used in the present case was, among others, to improve facial features by making a functional change in the position of the mandible . Recklinghausen's disease in the oral cavity often manifests with gingival overgrowth, erosions, increased proneness to caries and to attachment loss due to problems with maintaining proper hygiene . Another phenomenon observed in the disease is a lack of teeth buds, teeth migration or intrusion . It is often underlined that until the skeletal development is complete, prosthetic restorations which could impact bone growth should not be used . The best solution seems to be the one offered by temporary restorations, the use of which should be consulted with an orthodontist . It was also suggested that in many cases orthodontic treatment suffices, as restoration may be combined with the treatment of existing malocclusion using orthodontic appliances . The appliances should be constructed with consideration to the missing teeth and they should restore them, prevent teeth migration, maintain space for erupting permanent teeth, restore correct articulation and occlusal conditions and stimulate maxilla and mandible bone development . In the case of the described patient, orthodontic treatment with the removable appliance prevented further intensification of the malocclusion and it was not necessary to introduce any other elements to the metzelder appliance . Surgical treatment of a patient with a hemimandibular hyperplasia was conducted in the maxillo - facial surgery department in pozna . The 18-year - old patient with right hemimandibular hyperplasia underwent (based on pre - operative and operative measurements) a correction procedure sagittal mandibular osteotomy at the side of the hyperplasia combined with dissection of the inferior alveolar nerve and genioplasty . The correction of the occlusion involved le fort i osteotomy . In the analysed case, the surgical action consisted in its enucleation, but despite the procedure relapses occurred requiring repeated surgical procedures . Studies conducted in 2007 suggested that in the case of children suffering from recklinghausen's disease deciduous teeth erupt earlier . Researchers explained this phenomenon by the activity of osteoclasts, which are more prone to migrate and proliferate in comparison with the cells of healthy individuals . This leads to a faster alveolar process of bone resorption around deciduous teeth buds and thus earlier eruption of the teeth . The applied orthodontic appliance, owing to its construction, could be used by the patient during the entire process of the functional treatment, regardless of the presence of particular teeth during the teeth replacement stage and did not inhibit the functioning of the masticatory organ . During visits to the orthodontic surgery, special surfaces in the acrylic mass of the appliance finnish scientists made a groundbreaking discovery of a sex - dependent characteristic in the case of patients suffering from neurofibromatosis type 1 . Based on an analysis of pantomographic images of 55 patients (29 women and 26 men), the scientists proved that cement dysplasia around lower teeth root apexes affects only women . Additionally, they also pointed to the fact that 85% of cases showed enlargement of the mandibular foramen and canal . Although the changes often accompany plexiform tumours located in this area, it is a characteristic of recklinghausen's disease and may be taken into consideration in differential diagnostics also in cases in which neoplastic changes are not present in those anatomical areas . Radiological testing of the patient showed no dysplastic cement changes around teeth roots, but it did show slightly enlarged mandibular foramina and canals . Considering the malocclusion they are suffering from, children diagnosed with recklinghausen's syndrome should undergo orthodontic consultation and, if necessary, should be treated using removable appliances, so that the malformation of the masticatory organ does not intensify.
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Recurrent pharyngotonsillitis is a common health problem causing medical appointments, antimicrobial treatments, and missed workdays or schooldays . Previously, we conducted two randomised controlled trials in adults with recurrent pharyngotonsillitis on the efficacy of tonsillectomy [2, 3]. While we noticed that tonsillectomy reduced the number of acute pharyngotonsillitis episodes and increased the quality of life, we also found out that the patients graded most of their episodes as nonsevere [3, 4]. Thus, it seems like something other than the severity of throat pain may affect whether or not the patient consults the health care system during a pharyngitis episode . Here, we sought patient- and episode - related factors that would explain whether or not the patient made a medical visit for acute sore throat both before and after tonsillectomy . Study design was a secondary analysis of two randomised controlled trials exploring the effect of tonsillectomy in reducing further episodes of sore throat in adult patients with recurrent pharyngotonsillitis . These trials have been described previously [2, 3]. In brief, the first trial (streptococcal material) included 70 adults who had had at least 3 episodes of pharyngotonsillitis in 6 months or 4 episodes in 12 months, with one of them having to be of streptococcal origin . The second trial (pharyngotonsillitis material) involved 86 adults who had had at least 3 episodes of pharyngotonsillitis of any origin in 12 months . Half of the participants were randomly assigned to immediate tonsillectomy and the other half to a waiting list . Thus, we followed up both operated and unoperated patients for approximately 5 to 6 months . Grades 3 and 4 tonsils according to the grading scale by brodsky were regarded as large . Tonsils were regarded as chronically infected if they were inflamed and cryptic and these crypts contained debris . The patients were given a symptom diary where they recorded the presence of throat pain, fever, cough, and rhinitis . In the pharyngotonsillitis material, also the severity (mild, moderate, or severe) of the throat pain was recorded . The patients were told to seek medical advice for their acute symptoms during the trial in exactly the same way they had done before . Physicians recorded the date, location, diagnosis, and treatment of acute episodes as guided by trial instructions . Acute pharyngitis is a sudden inflammation or infection of the pharynx, usually causing a sore throat . Acute tonsillitis, on the other hand, is a sudden infection or inflammation of one or both of the palatine tonsils . In this paper, we use the term pharyngotonsillitis for simplification for all the patients, since we have studied patients with sore throat episodes both before and after tonsillectomy . For descriptive data, we calculated means with standard deviations or ranges . To explore patient - related variables and medical consultations, we constructed kaplan - meier curves for each of the 156 patients, starting from the date of the randomisation . We calculated the cumulative incidence of a medical consultation up to 5 months of follow - up using life - table analysis . We used a cox proportional - hazards regression model to explore associations between various patient - related factors and medical consultations for sore throat . The cumulative risk of visiting a physician was calculated as an adjusted hazard ratio (hr) with 95% confidence intervals (cis). The model included the following variables: age (20 or> 30 years versus 2130 years); gender; tobacco use; prior streptococcal pharyngotonsillitis; number of episodes of pharyngotonsillitis in the prior 6 months (3 versus> 3); and chronically infected tonsils . The associations between patient - related factors and medical consultations were similar in the episodes before and after tonsillectomy, so these two datasets were combined . To study episode - related factors and medical consultations, we similarly constructed kaplan - meier curves of each of the 208 episodes of acute pharyngotonsillitis, starting from symptom onset . The primary end point was the first visit to a physician because of pharyngotonsillitis during the first 7 days . We calculated the cumulative incidence of a medical consultation up to the first week of symptoms using life - table analysis . We used a cox regression model to explore the associations between various episode - related factors and medical consultations . The cumulative risk of visiting a physician was calculated as an adjusted hr with 95% cis . The model included the following variables: maximum throat pain during the episode (mild versus moderate versus severe); fever; other respiratory symptoms; and pre- versus postoperative episode . The mean length of the follow - up was 5.9 (sd 1.2) months . Female gender (hr 3.3, 95% ci 1.48.0) and chronically infected tonsils at clinical examination (hr 2.7, 95% ci 1.26.1) were factors that associated with medical consultation during a pharyngotonsillitis episode (table 2) (figure 1(a)). Age, tobacco use, prior streptococcal pharyngotonsillitis, and frequent pharyngotonsillitis episodes did not affect the consultation rate . Seventy - eight patients had 208 acute pharyngotonsillitis episodes (mean duration of 6.2 days, sd 4.5). Thirty - six episodes (17%) led to a medical consultation during the first 7 days of symptoms and the mean duration of symptoms before consultation was 4.1 (sd 3.6) days . The risk for medical visit increased after the third day of symptom onset (figure 1(b)). Only the fact that the patient had had severe throat pain during the first 7 days correlated significantly with the medical consultation (hr 4.3, 95% ci 1.018.5) (table 2). In contrast, having had moderate throat pain, fever, the presence of other respiratory symptoms, and whether the episode was pre- or postoperative were not associated with the medical consultation . We found that acute pharyngotonsillitis episodes both before and after tonsillectomy lasted less than a week; on average, majority of cases involved at most only nonsevere throat pain and less than one - third involved fever . Thus, it was not surprising that only one of every six episodes led to medical consultation . We found that the medical appointment rate for acute pharyngotonsillitis was higher in women than in men . This higher consultation rate may lead to an increased amount of recurrent pharyngotonsillitis diagnoses and may further increase referrals to specialist care for tonsillectomy among women . In fact earlier literature reports that women have higher consultation rates with specialists for consideration of paranasal surgery due to recurrent acute rhinosinusitis, as well [79]. Severe throat pain during the acute pharyngotonsillitis episode increased the probability of a medical visit after the third day of symptom onset . This finding indicates that patients are mainly looking for relief from severe pain and also suggests that patients may be aware that milder episodes usually heal by themselves without medical care [10, 11]. In contrast, such factors as age, tobacco use, streptococcal pharyngotonsillitis in history, fever, and whether the episode occurred before or after tonsillectomy did not correlate with a medical visit . To minimize the effect of participating in a trial, we emphasized that it was important that the patients seek medical advice for their symptoms during the trial in exactly the same way they had done before . This study was done in a country where national guidelines suggest that tests should be done for -haemolytic streptococcus a when treating acute pharyngotonsillitis, which must be considered when the generalizability of the results is evaluated . To conclude, even among adult patients who suffered from recurrent pharyngotonsillitis episodes, the overwhelming majority of the acute pharyngotonsillitis episodes were mild and only few resulted in a medical consultation, with female gender, chronically infected tonsils, and having severe throat pain increasing the consultation rate.
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Breast cancer is one of the major causes of mortality in middle - aged women, especially in developed countries . At present, there are no effective ways to prevent breast cancer since its cause remains unknown . Therefore, early detection becomes the key to improving the breast cancer prognosis and reducing the mortality rates . Mammography has been widely recognized as being one of the most effective imaging modalities for early detection of breast cancer . However, it is a hard work for radiologists to provide both accurate and uniform evaluation for the enormous number of mammograms generated in widespread screening . A computer - aided detection or diagnosis (cad) system, which uses computer technologies to detect the typical signs of breast cancer, has been developed to provide a second opinion for radiologists and to improve the accuracy and stability of diagnosis . In general, there are three signs of breast cancer in a mammogram: microcalcification clusters (mcs), architectural distortions, and masses . In this paper, we particularly focus on the detection of mcs since they appear in 3050% of mammographic diagnosed cases and show a high correlation with breast cancer . According to the breast image reporting and data system (bi - rads) lexicon, mcs are tiny calcium deposits that appear as small bright spots in mammograms . As an example, figure 1 shows an mc in a mediolateral - oblique (mlo) mammogram . It is often hard for radiologists to find individual mcs in mammograms because they are very small (typically, 0.051 mm) in the size and the contrast between the mcs and the surrounding breast tissue is not high enough . Over the past two decades, there has been extensive research focused on developing the cad tools for automatic detection of mcs in mammograms . Several review papers have also been published on this topic [2, 48]. As described in, mc detection can be classified into four categories: (1) image enhancement methods [913]; (2) multiscale decomposition methods [1417]; (3) stochastic modeling methods [18, 19]; and (4) machine learning methods [2028]. In, a performance evaluation was summarized using free - response receiver operating characteristic (froc) in which a support vector machine (svm) approach is found to be superior to conventional methods and is capable of achieving a true positive rate of approximately 90% when the false - positive (fp) rate is on average of 1.1 fp clusters per image . However, such fp is not low enough for clinical application . In this paper, we present a new method based on a hybrid method which combines a morphological technique and wavelet decomposition processing for detecting the mcs in mammograms . Compared with previous methods, the proposed method can achieve not only a high sensitivity but also a lower fp . In the proposed method, we first use a multistructuring - elements (ses-) based top - hat transform to enhance the intensity of microcalcification . Subsequently, we employ a wavelet decomposition to refine the enhanced results for removing the false enhanced microcalcifications . Then, based on the feature of malignant mcs, threshold processing is used to segment the mcs from mammograms . The rest of paper is organized as follows: section 2 presents a morphological processing technique to enhance the mcs in mammogram . In section 3 section 4 presents the detection of the mcs and gives the experimental results by using the proposed method . As mentioned in section 1, the mcs appear as small bright spots in mammograms . In this sense, mcs can be directly segmented by using a threshold process . However, since most mammograms have a low dynamical rang and the intensity contrast between mcs and surrounding tissue is quite low, selection of a threshold for the whole image is not an easy task . As a solution, the difference of gaussian (dog-) based method which approximates the individual microcalcification as a two - dimensional (2d) gaussian kernel has been reported in, but this method only is suitable for detecting the microcalcifications with approximate circle shape . In this section, we propose a new method which is based on a morphological filtering technique to enhance the individual microcalcifications for mc detection . The basic idea of the method is to use a set of top - hat transforms based on multi - structuring elements (ses) of which sizes and shapes are fitted to the individual microcalcifications to enhance them . The top - hat transform of a gray - scale image f is defined as f minus its opening by structuring element b: (1)t = f(f b), where denotes the opening operation; the difference operation yields an image in which only the components fitting to the se remain . Figure 2 illustrates the concept of the top - hat transform in one dimension . In the top - hat transform, selection of an appropriate se fitting to the target objects is the key . Since individual microcalcifications in mammograms frequently vary both in size and shape, it is imposable to use a single se to remove all of them . To solve this problem, we use a multi - ses - based method which uses eight different flat ses, denoted as bi (i = 1,2,, 8) to remove the individual microcalcifications in the opening processing . In this paper, the ses are a set of lines revolving around its center in a 15 15 array . For a mammogram image with resolution 0.05 mm / pixel, the 15 15 array indicates that the objects whose size are larger than 0.75 0.75 mm will be removed in the opening operation . Figure 3 shows the flat ses used in the top - hat transform where the dots denote the centers of the ses . For each se bi, i = 1,2,, 8, the opening operation yields an image, denoted by (f bi), in which the individual microcalcifications fitting to the se are removed . Then, in order to enhance these individual microcalcifications removed by the opening operation, we use a subtraction between the original image and the maximum of the opening results to obtain an image, given by (2)e = fargmax(f bi). Figure 4 shows an original mammogram and an enhanced image obtained from (2). Comparing figure 4(b) with figure 4(a), we can see that individual microcalcifications appearing on a complex background were enhanced successfully . These results indicate that the microcalcifications can be easily segmented by using a threshold process . However, the top - hat transform also enhances some undesired objects in the mammograms, such as mammary glands, vessels, and so on . Therefore, a refinement processing is required to remove these undesirable objects from the enhanced mammogram images . As mentioned above, although the top - hat transform is capable of enhancing microcalcifications varying in size and shape, a side effect is that the undesirable objects are also enhanced . Based on an investigation, we found that these unwanted objects are mainly caused by the soft tissues, such as mammary glands and vessels . The typical feature of them is that they have a relatively higher intensity compared with their surrounding area as well as an inner nonhomogeneous intensity . Figure 5 shows a comparison between the microcalcification and the mammary gland after the top - hat transform . Comparing figures 5(c) and 5(g), we find that the intensity of microcalcification in the enhanced image approximately equals that of the mammary glands . However, since the individual microcalcification has almost homogeneous intensity, the size of the enhanced microcalcification is generally larger than that of the mammary gland . Figures 5(d) and 5(e) show the expanded view of the dashed rectangles in figures 5(c) and 5(g), respectively . We can find that the size of the microcalcification is almost twice as large as the size of the mammary gland . Therefore, the enhanced mammary glands can be treated as noise that can be removed according to their size . In this paper, we employ a wavelet denoising method to remove the mammary glands because the wavelet decomposition can easily separate them according to their particular size . The wavelet - based procedure for denoising the image consists of the following four steps . A four - scale wavelet transform is used to decompose the image obtained from the top - hat transform . Symlet-2, as the decomposition filters since they have the least asymmetry and highest number of vanishing moments . Figures 6(a) and 6(b) show the scaling function and wavelet function used in the decomposition filtering . Figure 7 shows the decomposition results in which w(4, m, n), w(4, m, n), and w(4, m, n) denote the detail coefficients at level 4 size of half the original image . A four - scale wavelet transform is used to decompose the image obtained from the top - hat transform . Symlet-2, as the decomposition filters since they have the least asymmetry and highest number of vanishing moments . Figures 6(a) and 6(b) show the scaling function and wavelet function used in the decomposition filtering . Figure 7 shows the decomposition results in which w(4, m, n), w(4, m, n), and w(4, m, n) denote the detail coefficients at level 4 size of half the original image . Since the size of the microcalcification is almost twice as large as the size of the mammary gland, the noise caused by the mammary gland can be decomposed in level 4 . Therefore, we set the detail coefficients at level 4 as well as the approximation coefficients at level 1 to zeros . Figure 7 shows the result of the four - scale wavelet transform in which the shaded coefficients marked are set to zero . Thresholding the approximation and detail coefficients . Since the size of the microcalcification is almost twice as large as the size of the mammary gland, the noise caused by the mammary gland can be decomposed in level 4 . Therefore, we set the detail coefficients at level 4 as well as the approximation coefficients at level 1 to zeros . Figure 7 shows the result of the four - scale wavelet transform in which the shaded coefficients marked are set to zero . Figures 8(a) and 8(b) show the results of the inverse wavelet transform of the microcalcification and mammary gland in figures 5(d) and 5(h). We see that the intensity of the mammary gland is clearly reduced so that the microcalcifications and mammary glands can be easily separated using a threshold . Figures 8(a) and 8(b) show the results of the inverse wavelet transform of the microcalcification and mammary gland in figures 5(d) and 5(h). We see that the intensity of the mammary gland is clearly reduced so that the microcalcifications and mammary glands can be easily separated using a threshold ., we use a thresholding processing to obtain a binary image in which each microcalcification is segmented as connected components . We use a thresholding processing to obtain a binary image in which each microcalcification is segmented as connected components . In this section, we introduce a procedure that segments the mcs from the mammogram image obtained from the above sections and give some experimental results by using the proposed method . As mentioned in section 1, the mcs are tiny calcium deposits clustering together in the mammogram images . Based on an investigation on the bi - rads, we found that a typical mc generally has the following two features:(1)an mc usually consists of four or more individual microcalcifications;(2)an mc generally appears within a limited area of size 10 10 mm in mammogram images . An mc usually consists of four or more individual microcalcifications; an mc generally appears within a limited area of size 10 10 mm in mammogram images . According to these two features, we use the following two steps to detect the mcs . First, the binary image is subdivided into blocks that overlap their neighbors both horizontally and vertically . The blocks are of size 200 200 pixels; for each pair of overlapping blocks, the overlapping boundary region is of size 100 100 pixels . Second, for each block, if the total number of the connected components is larger than three, this block will be labeled as an mc . We developed and tested the proposed method using a database collected by the tohoku university school of medicine . These mammograms are of size 4740 3540 pixels, with a spatial resolution of 0.05 mm / pixel and 16-bit grayscale . The performance of the proposed method is summarized by the true positive rate and false positive cluster per image . Table 1 summarizes the experimental results of the proposed method comparing with several previous methods [8, 9, 13, 14, 32, 33]. The proposed method is capable of detecting 92.9% of true microcalcification clusters with an average of 0.08 false microcalcification clusters detected per image . To our best knowledge, this performance is better than most state - of - the - art methods in mc detection [2, 34]. In the experiments, we found that the fps are mainly caused by the linear - structure tissues interlacing with each other or benign calcifications with nonhomogenous intensity . Since the characteristics of linear - structure tissues are quite different with that of the mcs, we consider that fps can still be reduced by using a statistic classifier, such as a support vector machine (svm). In this paper, we presented a high - accuracy method for the detection of mcs in mammograms . The proposed method combined a multi - ses top - hat transform and a wavelet - based denoising approach to enhance the individual microcalcifications in mammograms . Experimental results demonstrated that the proposed method is capable of detecting the mcs in mammograms with high accuracy . In further work, we will conduct more experiments on a wider database and improve its performance both in accuracy and computational cost.
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Rheumatoid arthritis (ra) is a multisystem autoimmune disease of unknown etiology . The characteristic pathology of affected joints is the inflammation of hyperplastic synovial membrane that can result in destruction of adjacent cartilage and bone . Though the exact molecular pathogenesis of ra remains elucidated, evidence suggests that interleukin- (il-) 17-producing t cell, th17, is a central player . Il-17 is known to act synergistically with tumor necrosis factor- (tnf-) and il-1, which are abundant proinflammatory cytokines found in arthritic joints enhancing the activation of fibroblasts, chondrocytes, and osteoclasts . In contrast, regulatory t (treg) cell has been shown to have an anti - inflammatory role where its regulatory function is known to be deranged in ra patients . Th17 and treg have been reported to have plasticity and can be converted to each other according to the cytokine milieu when the cells encounter . Therefore, a therapeutic approach that can increase treg cell while diminishing th17 cell simultaneously may be very promising in ra treatment . Earlier studies have suggested that the majority of pharmacologic effect of metformin is dependent on its ability to activate amp - activated protein kinase (ampk), a major cellular regulator of lipid and glucose metabolism . Metformin inhibits mitochondrial respiratory chain resulting in energy deficiency noted as an increase in amp, activating ampk and protein kinase a (pka). Ampk activation also promotes glucose uptake and fatty acid oxidation in muscle, enhancing the lowering effect of glucose . In addition to the glucose lowering effect, metformin has been reported to exert an anti - inflammatory effect, which is also mediated by metformin - activated ampk . Metformin - activated ampk suppresses mammalian target of rapamycin (mtor), which regulates t cell effector differentiation in vitro and in vivo . Ampk has been shown to be associated with th17 cell suppression by inhibiting mtor and signal transducer and activator of transcription 3 (stat3), suggesting the therapeutic potential of ampk agonist . While th17 cells are responsible for perpetuating chronic inflammation in ra, osteoclasts resorb bone resulting in subsequent joint destruction . The role of mtor in osteoclastogenesis has been reported where a downstream molecule of the mtor pathway (s6k) conveys cell survival signal in osteoclasts . Recently, indo et al . Reported that ampk - mediated inhibition of mtor and hypoxia - induced factor (hif)-1 negatively regulated osteoclastogenesis . These regulatory effects of ampk on th17 cells and osteoclasts have prompted the investigation on the effect of the ampk agonist, metformin, on autoimmune arthritis . In the present study, the suppression of metformin in a collagen - induced arthritis (cia) mouse model was demonstrated by focusing on the suppression of th17 differentiation and enhancing treg differentiation . Phosphorylation of stat3 was diminished by metformin while phosphorylation of stat5 increased, which seems to contribute to the alteration of th17/treg population . In addition, osteoclastogenesis was suppressed via inhibition of mtor and ampk activation by metformin . 12-week - old male c57bl/6 mice (orient bio, korea) were maintained under specific pathogen - free conditions and fed standard laboratory mouse chow (ralston purina, st . All experimental procedures were examined and approved by the animal research ethics committee of the catholic university of korea, which conforms to all national institutes of health of the usa guidelines . All surgeries were performed under isoflurane anesthesia and all efforts were made to minimize suffering . Mice were immunized into the base of the tail with 100 g of chicken cii (chondrex inc ., redmond, wa, usa) in complete freund's adjuvant (chondrex inc . ). 100 g of chicken cii in incomplete freund's adjuvant (chondrex inc .) Was injected at tail and one foot on day 14 . Cia mice were orally fed 3 times a week for 9 weeks with 5 mg / mouse metformin (sigma - aldrich) or saline as a control beginning on day 7 after first immunization (permit number: cumc-2013 - 0128 - 01). Arthritis in these mice was examined visually two times per week for the appearance of arthritis in the peripheral joints . The severity of arthritis was recorded using the mean arthritis index on scale of 04, as previously reported, as follows: (0), no evidence of erythema and swelling; (1), erythema and mild swelling confined to the midfoot (tarsals) or ankle joint; (2), erythema and mild swelling extending from the ankle to the midfoot; (3), erythema and moderate swelling extending from the ankle to the metatarsal joints; (4), erythema and severe swelling encompassing the ankle, foot, and digits . The severity of arthritis was analyzed by the sum of scores from all legs, assessed by two independent observers with no knowledge of the experimental groups . Joints of each mouse were fixed in 10% formalin, decalcified in 10% edta, and embedded in paraffin wax . The sections were stained with hematoxylin - eosin (h&e), safranin o, and toluidine blue to detect proteoglycans . For immunohistochemistry, the sections were performed using the vectastain abc kit (vector laboratories, burlingame, ca, usa). Tissues were stained anti - receptor activator of nf-b (rank), anti - receptor activator of nf-b ligand (rankl), anti - tnf-, anti - tnf receptor - associated factor 6 (traf6), anti - il-17, and anti - il-6 (all from santa cruz biotechnology inc .) And anti - phosphorylated(p) stat3 (y705), anti - pstat3 (s727), and anti - pampk anti - pmtor (all from cell signaling, danvers, ma). The tissue was stained using pe - conjugated anti - cd4, fitc - conjugated anti - forkhead box p3 (foxp3), apc - conjugated anti - cd25, fitc - conjugated anti - il-17, fitc - conjugated anti - pstat3 (y705), and fitc - conjugated anti - pstat3 (s727) (all from ebiosciences, san diego, ca, usa). Stained sections were analyzed using a zeiss microscope (lsm 510 meta; carl zeiss, oberkochen, germany). Anti - igg, igg1, and igg2a were measured by mouse igg, igg1, and igg2a elisa quantitation kits (bethyl laboratories, montgomery, tx). To analyze intracellular cytokines, splenocytes were stained with percp - conjugated anti - cd4, apc - conjugated anti - cd25, fitc - conjugated anti - il-17, and pe - conjugated anti - foxp3 (ebiosciences), followed by fixation and permeabilization with a foxp3 staining buffer kit (bd bioscience) according to the manufacturer's instructions . Four hours before the staining, the cells were stimulated with phorbol myristate acetate (25 ng / ml) and ionomycin (250 ng / ml) (all from sigma - aldrich) and golgistop (bd bioscience). All data was analyzed using flowjo software (tree star, ashland, or, usa). The mrna expression levels were estimated using a lightcycler 2.0 instrument (roche diagnostic, mannheim, germany) with the version 4.0 software . All reactions were performed with the lightcycler faststart dna master sybr green i (takara, shiga, japan), following the manufacturer's instructions . The primers sequences are shown in table 1 . To purify splenic cd4 + t cells, the splenocytes were incubated with cd4-coated magnetic beads and isolated using magnetic activated cell sorting (macs) separation columns (miltenyi biotec). Isolated cd4 + t cells were stimulated with th17 cell - polarizing conditions for 3 days: plate - bound anti - cd3 (0.5 g / ml), anti - cd28 (1 g / ml) (both from bd pharmingen, ca, usa); anti - interferon (ifn)- (2 g / ml), anti - il-4 (2 g / ml), transforming growth factor (tgf)- (2 ng / ml), and il-6 (20 ng / ml) (all from r&d systems, minneapolis, mn, usa). Osteoclasts were stimulated in the presence of macrophage colony - stimulating factor (m - csf) (10 ng / ml) (r&d systems) and receptor activator of nuclear factor kappa - b ligand (rankl) (50 ng / ml) (peprotech, london, uk) and absence or presence of metformin 1 mm . The protein was separated by sds - page and transferred on nitrocellulose membranes (amersham pharmacia biotech, piscataway, nj, usa). Western blot was performed by snap i.d . The hybridized bands were detected by enhanced chemiluminescence (ecl) detection kit (thermo scientific brand of thermo fisher scientific, inc . ). The antibodies were as follows: anti - ampk, anti - pampk, anti - mtor, anti - pmtor, anti - pstat3 y705, anti - pstat3 s727, and anti - stat3 (all from cell signaling); traf6 (santa cruz); and -actin (sigma). Comparing numerical data between groups was performed with 2-way anova and nonparametric mann - whitney tests . Differences in the mean values of various groups were analyzed by using anova with a post hoc test . To investigate the antiarthritic effect of metformin, mice with cia were orally fed with either metformin or saline three times a week from day 7 after first immunization . Metformin - treated cia mice showed significantly reduced severity (figure 1(a), left) and incidence of clinical arthritis (figure 1(a), right). Histological and cartilage scores measured based on inflammatory cell infiltration and cartilage damage were also significantly lower in metformin - treated mice (figure 1(b)). Il-17, il-6, and tnf positive cells decreased in metformin - treated cia mice (figure 1(c)). Total igg, igg1, and igg2a antibodies significantly decreased in metformin - treated cia mice when compared to saline - treated controls (figure 1(d)). The numbers of cd4+il-17 + t cells and cd4 + pstat3 + (both at s727 and y705) t cells were reduced in metformin - treated cia mice more than in the controls . In contrast, the numbers of cd4 + cd25 + foxp3 + treg cells and cd4 + pstat5 + t cells were higher in the metformin - treated group (figure 2). Next, spleen cells isolated from each group were analyzed for the expression of il-17 and foxp3 using flow cytometry . The results showed that cd4+il-17+th17 cells were lower in the metformin - treated cia, whereas cd4+cd25 + foxp3+treg cells were increased in metformin group (figure 3(a)). As well, mrna expressions of the th17-related cytokines and transcription factors were significantly reduced in the splenocytes from metformin - treated cia mice and there was greater expression of foxp3 (figure 3(b)). As the proportion of th17 cell was decreased in metformin - treated cia mice in vivo, the effect of metformin on th17 cell and treg cell differentiation in vitro was examined . Cd4 + t cells isolated from healthy c57bl/6 mice were cultured in th17 cell - polarizing condition in the presence or absence of metformin . The differentiation of th17 cells was inhibited with metformin as there was a simultaneous increase in treg differentiation (figure 3(d)). In addition, metformin inhibited the expression levels of the th17-associated genes, il-17, ahr, runx1, and rort, as it increased mrna levels of foxp3 (figure 3(e)). Treatment with metformin increased the expression of pampk in il-6 10 ng / ml stimulated cd4 t cell (figure 3(f))). These results indicate that metformin suppresses th17 cell differentiation while enhancing treg differentiation and ampk activation in a proinflammatory condition (figure 6). Metformin - treated cia mice showed reduced bone erosion and cartilage destruction in the arthritic joints (figure 1(b)). To determine effect of metformin on osteoclastogenesis, we were immunochemical staining for rank and rankl in joint tissues . As expected, the expression of rank and rankl decreased in the inflamed joint tissue of metformin - treated cia (figure 4(a)). The preosteoclasts derived from the control cia mice and metformin - treated cia mice were stimulated with m - csf and rankl . The results showed that the osteoclasts from metformin - treated cia mice were less capable of differentiating into osteoclasts (figure 4(b)). The expression levels of various osteoclastogenic markers, such as trap, mmp-9, integrin 3, calcitonin receptor, and cathepsin k, were decreased, also . The reduction in osteoclastic activity was observed following metformin - treated mice (figure 4(c)). To verify the observation that metformin reduced the expression of osteoclastogenic markers ex vivo, we investigated whether metformin could inhibit osteoclast differentiation in vitro . The results showed that metformin treatment inhibited osteoclast differentiation as determined by the trap staining assay (figure 5(a)). We also measured osteoclastogenic markers mrna expression levels in control osteoclasts and metformin - treated ones . The mrna expression of the osteoclast - related markers, trap, cathepsin k, mmp-9, calcitonin receptor, carbonic ii, and integrin 3, decreased by meformin treatment . We also found that metformin suppressed the expression of hif-1 mrna whereas it induced expression of ampk mrna (figure 5(b)). As the pharmacologic effect of metformin is largely dependent on ampk, the activation of ampk, mtor, and stat3 during osteoclastogenesis was addressed . Western blot analysis demonstrated that expressions of traf6, pmtor, and pstat3 (y705 and s727) were reduced with metformin treatment . Consistently, immunohistochemical staining of arthritic joints revealed that expressions of these molecules were significantly lower in metformin - treated cia mice (figure 5). Collectively, these results suggest that metformin suppresses osteoclastogenesis via inhibition of mtor and stat3, which is mediated by metformin - activated ampk . In the present study, we demonstrated that metformin suppressed cia via reciprocal regulation of th17 cell and treg cell . Metformin - activated ampk seems to be involved in this regulation through the inhibition of mtor and its downstream molecules, hif-1 and stat3 . As th17 receives the spotlight in the pathogenesis of ra, a number of medications that target th17 have been developed . However, tocilizumab, an il-6 receptor blocking antibody, is currently being used, whereas tofacitinib, a jak inhibitor, is under clinical trials . The molecules shown to inhibit stat3 or rort have demonstrated promising results in animal models . Recently, metabolism has been raised as an important check point in regulating th17/treg balance . Therefore, the role of ampk, the master metabolic sensor, in t cell fate was investigated . Kang et al . Reported that metformin - activated ampk inhibited mtor - stat3 pathway contributing to a reduction in the population of th17 cells . Furthermore, our results revealed that metformin not only suppressed th17 cells but also simultaneously enhanced treg cells . This might be explained as the metabolic check point determines the differentiation of th17 cell or treg cell . For instance, th17 cells are highly glycolytic while treg cells have a high lipid oxidation rate . The high glycolytic activity of th17 cell is dependent on mtor, which is inhibited by ampk . Mtor enhances the glycolytic pathway promoting th17 cell differentiation whereas ampk inhibits mtor and promotes mitochondrial oxidative metabolism to enhance treg cell . Furthermore, the high glycolytic activity of th17 cells is attributed to hif-1, a downstream molecule of mtor pathway . In addition, hif-1 promotes the nuclear translocation of rort and degrades foxp3, resulting in a decrease in treg cell . It is widely known that pstat3 competes for the same binding locus as pstat5 in il-17 promoter to enhance il-17 . Therefore, activating ampk and inhibiting mtor are an efficient way to regulate th17 and treg concurrently . Our results revealed that metformin suppressed th17 cell while increasing treg cell in cia mice . Though confocal microscopy showed an increase in stat5 phosphorylation, it seems to result from reduced pstat3 and consequently increase the number of treg rather than the direct effect of metformin on stat5 phosphorylation . Undeniably, metformin did not affect il-2 induced stat5 phosphorylation levels in vitro (data not shown). It was reported that metformin ameliorated experimental autoimmune encephalitis and collagen antibody induced arthritis, both involving th17 cells . Our results confirmed that metformin - activated ampk could suppress cia via reciprocal regulation of th17 cell and treg cell . Therefore, future investigation of whether metformin can be used as a therapeutic in th17-driven chronic inflammatory diseases is promising . The number of trap+ multinucleated cells was lower in the joints of metformin - treated mice . This may partially result from decreased expression of inflammatory cytokines that promote osteoclastogenesis via the upregulation of rankl in the arthritic joint . Metformin stimulates osteoprotegerin and reduces rankl expression in osteoblasts, and ampk inhibits rank signaling, which might also contribute to reduced osteoclastogenesis . To investigate the direct effect on osteoclastogenesis, preosteoclasts were cultured with m - csf and rankl in the presence or absence of metformin, decreasing osteoclastogenesis in the metformin - treated cells . Our results also revealed that the suppression was associated with an increase in ampk phosphorylation and subsequent decrease in mtor and stat3 phosphorylation . The decreased expression of hif-1 in metformin - treated osteoclasts verifies the recent observation that mtor - hif-1 pathway was critical in osteoclastogenesis . To date, there is no prevention in the development of rheumatoid arthritis . However, proper early treatment can prevent progressive damage of the joints . In this study, the effect of metformin on joint destruction and regulation of immune cells in occurrence of rheumatoid arthritis was evaluated . Collectively, these results suggest a new role of metformin . Metformin has been shown to ameliorate autoimmune arthritis by regulating the th17/treg balance and inhibiting osteoclastogenesis through ampk . In cases where secondary diabetes mellitus develops due to excessive corticosteroid use in ra patients, metformin can be the optimal therapeutic option due to its glucose lowering and anti - inflammatory properties . The present study demonstrated that metformin had an anti - inflammatory effect on cia due to the inhibition of th17 cell differentiation and the upregulation of treg cell differentiation along with the suppression of osteoclast differentiation . Our data suggest that metformin might be a potential candidate for therapeutic modulation of experimental animal model with rheumatoid arthritis.
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Human activities, in particular the burning of fossil fuels, have been identified as key drivers of the climatic changes observed since the mid - twentieth century (hegerl et al . The effects of these activities are altering the environment on which we depend, and threaten our collective future through very serious effects on human health (hegerl et al . A range of health impacts from climate change have been identified, such as increased cardiopulmonary, infectious, allergic, and mental illnesses as well as a host of weather - related injuries (see table 1) (mcmichael et al . Key health impacts relate to increases in air pollution, water- and food - borne contamination, the spread of disease carrying vectors and rodents and extreme weather events . Some population groups such as seniors, the socially disadvantaged, aboriginal people, the chronically ill and disabled people, are more vulnerable to the health impacts (seguin 2008). Particular challenges, such as food insecurity, socio - economic dislocation and political instability and conflict, may be faced by developing countries as a result of these changes (costello et al . 2009).table 1health impacts of climate change (haines and patz 2004; seguin 2008; friel et al . 2011)climate change effectsexamples of related health riskstemperature changescardiovascular, cerebrovascular, and respiratory diseaseextreme weather, changes in precipitationinjuries, diarrheal diseases, malnutrition, respiratory infections, depression, anxietyair pollutionrespiratory, cardiovascular diseasepollen productionallergic diseasesmicrobial contamination and transmissionmalaria, dengue fever, schistosomiasis, lyme disease, hantavirus pulmonary syndrome, leptospirosisreduced crop yieldmalnutritiondisplaced populationspoverty, depression, anxiety, malnutrition health impacts of climate change (haines and patz 2004; seguin 2008; friel et al . 2011) many adaptation strategies to address health risks from climate change have been identified (confalonieri et al . 2007; seguin 2008; cash 2011). We summarized a non - exhaustive list of common adaptation strategies to five key health outcomes identified by public health authorities and researchers those related to increased air pollution, extreme heat events, water- and food - borne diseases, vector- and rodent - borne diseases, and extreme weather events . See table 2 for examples of adaptations.table 2examples of adaptation strategies (confalonieri et al . 2007; seguin 2008; keim 2011; cash 2011)health risksadaptation strategiesheat - relatedurban planning and increase in shadingestablish hot - weather response plans and early warning systemsbuddy system to check on neighbors during heat wavesextreme weather relatedimprove land - use planning and environmental management (i.e., limit development in high - risk areas such as floodplains or coasts, defensive structures to minimize flash floods)enhance quantitative data on short - term and long - term health impacts of extreme weather eventsmaintain and improve disaster management programs for local public health facilities to provide rapid health needsair pollution relatedprovide information to the public about actions to reduce exposure to air pollution, air quality index, emission standardsidentify vulnerable populationsincrease capacity for hospital care and physician clinicsfood and water - borne diseasemaintain and upgrade water treatment, sewage and sanitation facilitiessurveillance of water- and food - borne diseasesmake available new drugs and treatmentsvector and rodent - borne diseasetravel, importation and quarantine lawssurveillance of vector populationsdevelop and make available new vaccines examples of adaptation strategies (confalonieri et al . 2007; seguin 2008; keim 2011; cash 2011) the world health organization (who 2009) has called for the adoption of both greenhouse gas (ghg) mitigation measures and adaptation strategies to address climate change . Many specific mitigation and adaptation strategies which reduce the effects of climate change have been identified by international organizations such as the intergovernmental panel on climate change (ipcc) (confalonieri et al . 2007) and who (mcmichael et al . 2003), and by national and sub - national health agencies (seguin 2008; cash 2011). Potentially large improvements to health maybe attained through well - designed adaptation strategies that either directly reduce health risks or indirectly improve health outcomes through the achievement of health co - benefits (ebi et al . 2012). Health co - benefits occur when adaptation strategies produce health advantages outside of the scope of the original health outcomes targeted to be improved; conversely, adaptation actions can pose risks to health if not designed and implemented with human health considerations in mind (ebi et al . Proper design and implementation of health adaptation strategies can maximize co - benefits and reduce negative consequences . At the time of writing, there has yet to be a comprehensive review of the research into the health co - benefits and risks of climate change with respect to health adaptation strategies . Existing literature on adaptation costs and benefits is scattered in terms of sectoral and regional coverage, and does not directly address health co - benefits (adger et al . The focus of much of the research is on the economic costs of implementing a wide range of adaptation strategies and the expected benefits from addressing specific concerns such as sea - level rise and impacts on coastal areas (nicholls and tol 2006; nrtee 2011). Limited information is available on the costs of public health adaptations (oecd 2008). Methods for estimating the economic costs of implementation and direct benefits to aid in the assessment of adaptation strategies do exist in the literature . However, they do not routinely address the health co - benefits and risks of these actions (callaway 2004; nrtee 2011; ebi et al . 2006). With a focus on the developed world, this report reviews current literature on health co - benefits and risks to gain a better understanding of how adaptation strategies undertaken by public health professionals may affect the health of the populations they serve . This information can be used in their decision - making processes to prioritize the development and implementation of specific measures in order to protect people from the impacts of climate change . While measures to mitigate climate change through the reduction of ghgs may also result in significant health co - benefits or risks (who 2009; haines et al . 2009; ebi et al . A literature review was conducted in april 2012 using electronic databases including medline, web of science, and geobase . In addition, relevant websites and materials from the ipcc and who were searched for further data . Peer - reviewed literature from january 2000 to march 2012 was retrieved using a standard search strategy based on the key words climate, climate change, adaptation, health status, public policy, health co - benefits, health risk, and public health . A total of 812 articles were identified and titles and abstracts were scanned for relevance . Articles that did not directly address the health co - benefits or risks of adaptation strategies were excluded, with 22 peer - reviewed articles remaining . The types of article were not used as inclusion and exclusion criteria, due to the limited number of articles that addressed this topic . There are limited numbers of published articles discussing the health co - benefits and risks of public health adaptation strategies . In most articles, health co - benefits and risks of adaptation the co - benefits and risks identified in the literature most commonly related to impacts on health associated with actions that increase social capital and improve urban design . We have also discussed more general, transferrable public health measures that help to address the impact of climate change . See table 3 for a summary of the results.table 3health co - benefits and risks of adaptation strategies available in the climate change literatureadaptation strategies that increase social capitalsourceshealth co - benefitsdecreased heat - related illnessesnaughton et al . (2002)improved overall health status due to increase capital, independent of other predictors of healthdhombres et al . (2010), fujiwara and kawachi (2008), giordano and lindstrom (2010), lochner et al . (2008)health risksmisinformation may be spread with increased social capital leading to worse heat adaptabilitywolf et al . (2010)adaptation strategies that influence urban designsourceshealth co - benefitsincreased physical activity, decreased cardiovascular and respiratory diseases, thermal comfort, improved mental healthfriel et al . (2011), woodcock et al . (2008), harlan and ruddell 2011, frumkin and mcmichael (2008), bambrick et al . (2011)health risksincreased allergic diseases, pests, animals, obscured views and increased fear of crimecarinanos and casares - porcel (2011), escobedo et al . (2011)air conditioning leading to increased ghg emissions and worsening all health effects of climate changeakbari et al . (2001), calm (2002)indirect effectsimproved public health including better food handling practices, electricity outage preparednesskjellstrom and weaver (2009), keim (2011), ebi et al . (2012) health co - benefits and risks of adaptation strategies available in the climate change literature a common source of health co - benefits identified in the literature was adaptation strategies to improve social capital as part of efforts to address climate change health risks stemming from an increase in public health emergencies or national disasters (ebi and semenza 2008; kjellstrom and weaver 2009; bambrick et al . Social capital refers to the ability of actors to secure benefits by virtue of membership in social networks or other social structures (portes 1998). It can have a positive influence on health outcomes by increasing access to network - based resources such as counseling during and after a weather - related emergency and by enhancing collective action and the enforcement of social norms that support healthy behaviors (eicher and kawachi 2011). Evidence suggests that belonging to a social network can have a protective effect against heat - related illness (naughton et al . 2002). A low level of social capital, or social isolation, is regarded as contributing to the vulnerability of population groups who are excluded from access to resources needed to protect health (cutter et al . Examples of adaptation strategies that increase social capital include the development of buddy systems and community outreach initiatives that target vulnerable populations (e.g., seniors, people with chronic illnesses, socially disadvantaged people) to reduce heat - health risks (seguin 2008). In terms of health co - benefits, social capital is thought to positively influence health, including mental health, independently of other determinants (dhombres et al . 2010; fujiwara and kawachi 2008; giordano and lindstrom 2010; lochner et al . Potential risks stemming from an inadequate understanding of how social capital works were also identified (pelling and high 2005). In an article by wolf et al . (2010), the authors argued that strong bonding networks could actually have the effect of exacerbating vulnerability to the health impacts of climate change . For example, social networks may reinforce misperceptions common among older adults about possible health effects of heat waves and this could act as a barrier to adaptation . A survey of older adults in the united kingdom in 2007 revealed that many respondents did not feel at significant risk from heat waves personally . Ultimately, the authors offer a cautionary note about social capital suggesting that it is more complex than simply positive for climate change vulnerability greater understanding is needed about the most effective ways to employ social capital to reduce climate change health risks . Adaptation strategies designed to decrease extreme heat events and air pollution in cities both of which are expected to increase with climate change commonly involve adjustments to urban design and planning . Strategies such as increased or improved shade and green spaces, smarter road design, the development of walkable neighborhoods, the creation and maintenance of bike paths, and improvement to public transportation are suggested in the literature (akbari et al . 2001; addy et al . 2004; frank et al . 2004; besser and dannenberg 2005; younger et al . 2008; woodcock et al . 2009; stone et al . In addition to directly reducing heat stress and cardiorespiratory disease, these adaptations can lead to increased physical activity, cooling spaces (e.g., shaded areas), and social connectivity which result in a range of health co - benefits such as reduced obesity, cardiovascular disease, and improved mental health (friel et al . 2011; woodcock et al . 2009; younger et al . 2008; harlan and ruddell 2011; frumkin and mcmichael 2008; bambrick et al . Green spaces have also been shown to contribute to flood risk mitigation and so can enhance community safety (eigenbrod et al . The resulting co - benefits from these adaptation strategies can have both immediate and long - term positive influences on public health and may have significant economic payoff (grabow et al . In addition, strategies to reduce heat stress by addressing the urban heat island effect (e.g., increasing green spaces, reducing heat absorbing concrete or paved surfaces) and to reduce air pollution by redesigning transportation infrastructure (e.g., building more bike paths and smarter road systems) and through better urban planning (e.g., densification, walkable neighborhoods) may also significantly reduce ghg emissions if designed appropriately (who 2009; susca et al . 2011; haines et al . 2009; woodcock et al . 2009; ebi et al . If broadly implemented, these adaptation actions may also therefore convey long - term health co - benefits by lessening the rate of climate change and its associated health impacts . Communities which do not reduce the urban heat island effect through proper urban design and planning may be forced to rely heavily on air conditioning to reduce heat stress . The use of air conditioning to reduce heat - related morbidity and mortality is identified as an important adaptation measure in many heat adaptation plans (health canada 2012). However, it can be a significant source of energy consumption in communities during the summer months (akbari et al . Air conditioning produces combustion products, including carbon dioxide and nitrous oxide (calm 2002), when energy systems rely largely upon the burning of fossil fuels . Both of these gases can lead to increased ghg emissions, increased air pollution, and can exacerbate the urban heat island effect (calm 2002). For this reason air conditioning should be supplemented with other measures to provide citizens with relief from the heat (e.g., extended pool hours, shaded areas) or to reduce temperatures in urban environments (e.g., uhi mitigation measures) (health canada 2012). As noted above, increasing green spaces is one of the commonly suggested adaptation strategies for reducing the urban heat island . However, in a recent article by carinanos and casares - porcel (2011), a lack of planning in the design of green spaces in urban areas was noted as a potential trigger for pollen allergies . (2011) argues that in the specific case of urban forests, there are costs to the economy and society that must be weighed against the benefits . These might include allergens, pests, insects, leaf litter, debris, falling branches, wild animal droppings and bites, obscured views and increased fear of crime (escobedo et al . Indirect benefits to health may also be achieved through adaptations that support or strengthen the resources, infrastructures, information systems and processes that underpin existing public health functions . (2012) suggest that because of the current adaptation deficit and associated impacts on health in most countries, climate change - related investments to prepare the health sector can help address health outcomes not directly attributed to a warming world . This involves the transferability of adaptation strategies for climate change to other public health threats (kjellstrom and weaver 2009; keim 2011). In an examination of international climate change and health adaptation frameworks clarke and berry (2012) reported that existing public health risk management standards in the province of ontario, canada, were able to address climate change health risks, provided data about impacts to inform interventions . Therefore, the benefits of climate change and health adaptation strategy development are often directly transferable to existing community and regional activities and processes to improve population health . These public health functions constitute the core interests for public health agencies at different levels population health assessment, health surveillance, health promotion, health protection, disease and injury prevention (frank et al ., communities will improve their public health systems and public health infrastructure, which will have knock - on positive effects on public health capacity and on the delivery of services and programs to the public . Their development is not only beneficial to the health of the public, but also essential . A common source of health co - benefits identified in the literature was adaptation strategies to improve social capital as part of efforts to address climate change health risks stemming from an increase in public health emergencies or national disasters (ebi and semenza 2008; kjellstrom and weaver 2009; bambrick et al . Social capital refers to the ability of actors to secure benefits by virtue of membership in social networks or other social structures (portes 1998). It can have a positive influence on health outcomes by increasing access to network - based resources such as counseling during and after a weather - related emergency and by enhancing collective action and the enforcement of social norms that support healthy behaviors (eicher and kawachi 2011). Evidence suggests that belonging to a social network can have a protective effect against heat - related illness (naughton et al . 2002). A low level of social capital, or social isolation, is regarded as contributing to the vulnerability of population groups who are excluded from access to resources needed to protect health (cutter et al . Examples of adaptation strategies that increase social capital include the development of buddy systems and community outreach initiatives that target vulnerable populations (e.g., seniors, people with chronic illnesses, socially disadvantaged people) to reduce heat - health risks (seguin 2008). In terms of health co - benefits, social capital is thought to positively influence health, including mental health, independently of other determinants (dhombres et al . 2010; fujiwara and kawachi 2008; giordano and lindstrom 2010; lochner et al . Potential risks stemming from an inadequate understanding of how social capital works were also identified (pelling and high 2005). In an article by wolf et al . (2010), the authors argued that strong bonding networks could actually have the effect of exacerbating vulnerability to the health impacts of climate change . For example, social networks may reinforce misperceptions common among older adults about possible health effects of heat waves and this could act as a barrier to adaptation . A survey of older adults in the united kingdom in 2007 revealed that many respondents did not feel at significant risk from heat waves personally . Ultimately, the authors offer a cautionary note about social capital suggesting that it is more complex than simply positive for climate change vulnerability greater understanding is needed about the most effective ways to employ social capital to reduce climate change health risks . Adaptation strategies designed to decrease extreme heat events and air pollution in cities both of which are expected to increase with climate change commonly involve adjustments to urban design and planning . Strategies such as increased or improved shade and green spaces, smarter road design, the development of walkable neighborhoods, the creation and maintenance of bike paths, and improvement to public transportation are suggested in the literature (akbari et al . 2001; addy et al . 2004; frank et al . 2004; besser and dannenberg 2005; younger et al . 2008; woodcock et al . 2009; stone et al . In addition to directly reducing heat stress and cardiorespiratory disease, these adaptations can lead to increased physical activity, cooling spaces (e.g., shaded areas), and social connectivity which result in a range of health co - benefits such as reduced obesity, cardiovascular disease, and improved mental health (friel et al . Green spaces have also been shown to contribute to flood risk mitigation and so can enhance community safety (eigenbrod et al . The resulting co - benefits from these adaptation strategies can have both immediate and long - term positive influences on public health and may have significant economic payoff (grabow et al . In addition, strategies to reduce heat stress by addressing the urban heat island effect (e.g., increasing green spaces, reducing heat absorbing concrete or paved surfaces) and to reduce air pollution by redesigning transportation infrastructure (e.g., building more bike paths and smarter road systems) and through better urban planning (e.g., densification, walkable neighborhoods) may also significantly reduce ghg emissions if designed appropriately (who 2009; susca et al . 2011; haines et al . 2009; woodcock et al . If broadly implemented, these adaptation actions may also therefore convey long - term health co - benefits by lessening the rate of climate change and its associated health impacts . Communities which do not reduce the urban heat island effect through proper urban design and planning may be forced to rely heavily on air conditioning to reduce heat stress . The use of air conditioning to reduce heat - related morbidity and mortality is identified as an important adaptation measure in many heat adaptation plans (health canada 2012). However, it can be a significant source of energy consumption in communities during the summer months (akbari et al . Air conditioning produces combustion products, including carbon dioxide and nitrous oxide (calm 2002), when energy systems rely largely upon the burning of fossil fuels . Both of these gases can lead to increased ghg emissions, increased air pollution, and can exacerbate the urban heat island effect (calm 2002). For this reason air conditioning should be supplemented with other measures to provide citizens with relief from the heat (e.g., extended pool hours, shaded areas) or to reduce temperatures in urban environments (e.g., uhi mitigation measures) (health canada 2012). As noted above, increasing green spaces is one of the commonly suggested adaptation strategies for reducing the urban heat island . However, in a recent article by carinanos and casares - porcel (2011), a lack of planning in the design of green spaces in urban areas was noted as a potential trigger for pollen allergies . (2011) argues that in the specific case of urban forests, there are costs to the economy and society that must be weighed against the benefits . These might include allergens, pests, insects, leaf litter, debris, falling branches, wild animal droppings and bites, obscured views and increased fear of crime (escobedo et al . Indirect benefits to health may also be achieved through adaptations that support or strengthen the resources, infrastructures, information systems and processes that underpin existing public health functions . (2012) suggest that because of the current adaptation deficit and associated impacts on health in most countries, climate change - related investments to prepare the health sector can help address health outcomes not directly attributed to a warming world . This involves the transferability of adaptation strategies for climate change to other public health threats (kjellstrom and weaver 2009; keim 2011). In an examination of international climate change and health adaptation frameworks clarke and berry (2012) reported that existing public health risk management standards in the province of ontario, canada, were able to address climate change health risks, provided data about impacts to inform interventions . Therefore, the benefits of climate change and health adaptation strategy development are often directly transferable to existing community and regional activities and processes to improve population health . These public health functions constitute the core interests for public health agencies at different levels population health assessment, health surveillance, health promotion, health protection, disease and injury prevention (frank et al ., communities will improve their public health systems and public health infrastructure, which will have knock - on positive effects on public health capacity and on the delivery of services and programs to the public . Their development is not only beneficial to the health of the public, but also essential . There are few published articles examining the health co - benefits and risks of public health adaptation strategies . No comprehensive list of adaptations currently exists that ranks or quantifies potential health co - benefits or risks; consequently, there is little information that is easily accessible for decision - makers to use when identifying priority measures to protect health from climate change impacts . The two examples of specific adaptations discussed here increasing social capital to reduce health risks from natural hazards and improving urban design to address air pollution and to reduce heat stress suggest that improvements to health can be achieved through the realization of co - benefits . Increasing social capital to reduce the health impacts of one type of natural hazard (e.g., heat stress) may convey significant health co - benefits associated with protection from other types of hazards . Adaptation strategies designed to decrease extreme heat events and air pollution in cities can additionally lead to increased physical activity, cooling spaces (e.g., shaded areas), and social connectivity, and in turn, contribute to reductions in obesity, cardiovascular disease, and improve mental health . Examination of public health adaptations that could result in sizeable co - benefits also revealed that care needs to be taken when developing and implementing actions to not harm health . Some measures may inadvertently contribute to increasing the rate of climate change (e.g., wide spread use of air conditioning) or exacerbate the impacts of climate change (e.g., poor design of urban green spaces). They should be examined closely for potential benefits and risks so that the measures adopted will maximize positive health outcomes . The health co - benefits and possible risks of both adaptation and ghgmitigation measures need to be taken into consideration by decision - makers when addressing climate change . Climate change ghg mitigation strategies prompt a rethinking of lifestyles, energy choices, and consumption patterns which can have substantial health co - benefits associated with urban land transport (woodcock et al . 2009), and low - carbon electricity generation (markandya et al . 2009). Similar to adaptation, ghg mitigation can present a valuable opportunity to achieve substantial health co - benefits (cifuentes et al . Kjellstrom and weaver 2009; frumkin and mcmichael 2008). The who reports that many policies and individual choices have the potential both to reduce greenhouse gas emissions and produce major health co - benefits these local and immediate benefits can offset a large part of the costs of climate change mitigation, and provide a strong political and personal motivation for action . (who 2009, p. 3). For example, the adoption of active transport and rapid transit / public transit which is based upon improved land use results in greater immediate health gains than improving fuel efficiency in vehicles (who 2011). Therefore, the awareness of such opportunities and information to guide decision - making is crucial for the public health community to optimize their effects (frumkin and mcmichael 2008; huang et al . 2011). Lastly, the international community has recognized that the effects of climate change will be differentially experienced by countries around the world . For this paper nonetheless, some adaptation strategies particularly those that build public health and health system capacity will be transferrable to low - middle income countries (lmics). The international community has noted the importance of adaptation strategies for lmics (ciscar et al . Establishes that by 2020, higher income countries (hics) will provide $100 (us dollars) billion annually to address the needs of developing countries . This will include funding for adaptation, and efforts to achieve ghg mitigation and temperature targets (copenhagen accord 2009). Since adaptation co - benefits often accrue to local populations, adaptation strategies that are common to hics and lmics may have differential co - benefits and risks (callaway 2004). We theorize that the potential co - benefits and risks associated with public health adaptations to climate change may be magnified in lmics . There are a limited number of articles addressing the health co - benefits and risks of climate change adaptation strategies . Through this review, we have found health co - benefits and related risks from increases in social capital, and changes in urban design strategies . We have also discussed more general, transferrable public health measures that help to address the impact of climate change; these methods are often core functions of public health . Future research should focus on both short- and long - term, region - specific, positive and negative consequences of adaptation . . Only then can public health practitioners at every level be informed of the full range of health impacts when planning adaptation measures . There are a limited number of articles addressing the health co - benefits and risks of climate change adaptation strategies . Through this review, we have found health co - benefits and related risks from increases in social capital, and changes in urban design strategies . We have also discussed more general, transferrable public health measures that help to address the impact of climate change; these methods are often core functions of public health . Future research should focus on both short- and long - term, region - specific, positive and negative consequences of adaptation . . Only then can public health practitioners at every level be informed of the full range of health impacts when planning adaptation measures.
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Rapid advances in the crystallographic determination of transmembrane (tm) protein structures, and especially of g - protein coupled receptors (gpcrs), have established the lipidic cubic phase (lcp) as a suitable medium for growing high quality crystals of these proteins (see ref (1,2) and citations therein). The lcp consists of a highly curved lipid bilayer structure (see figure 1b c) that is continuous in three dimensions (3d) and separates in space two nonintersecting intertwined aqueous channels . Because of the continuous nature of both the lipid and the water compartments, lcp has been described as a bicontinuous mesophase with its bilayer midplane tracing a triply periodic minimal surface (tpms, characterized by zero mean curvature) (see refs (68) and citations therein). The particular kind of lcp utilized during in meso crystallization trials is of pn3 m symmetry type, where each aqueous network of the lcp assumes a tetrahedral geometry (see figure 2d e). (a) ball - and - stick representation of the monoolein (9.9 mag) lipid . Double bonded carbon atoms (c9 and c10) in the hydrocarbon chain of 9.9 mag are highlighted . The grouping of atoms in martini coarse - grained beads (eth, gl1, c1a, c2a, d3a, c4a, and c5a) is highlighted by ellipsoids . (b, c) comparison of the triply periodic minimal surface (tpms) of pn3 m symmetry calculated from the improved nodal approximation given in eqs 13 (b), to a snapshot after 6 s - long coarse - grained md simulations of spontaneous self - assembly of 9.9 mag / water complex at 40% (w / w) water concentration and at 20 c temperature (c). The figure in c (see also figures 2 and 3) shows the location of the c5a coarse - grained beads from the 9.9 mag lipid (see a); note the cubic phase structure . The similarity evident in b and c underscores the close relation between the organization of the c5a beads (representing the density of terminal methylene and methyl groups in 9.9 mag lipids) and the tpms of the pn3 m cubic phase . The rapid pace of new structural data acquisition using the features of the in meso method underscores the usefulness and success of the technology . Thus, structural information obtained in this manner for several rhodopsin - like gpcrs (reviewed in ref (6)) and a gpcr - g protein complex, provided breakthrough insights about the structural basis for signal transduction through these 7-tm helical proteins . However, the present lack of a mechanistic understanding, at the molecular level, of the events that lead to in meso crystallization of membrane proteins, makes the success of the method somewhat enigmatic and results in the need for extensive trials to identify specific conditions, that is, the host and additive lipid, precipitant etc . Such in meso trials begin with the target protein being reconstituted into the lcp, and a hypothetical descriptive mechanism based on the membrane curvature and hydrophobic mismatch has been proposed to address the crucial step during in meso crystallization the preferential partitioning of proteins from the lcp to the lamellar phase . In the original trials, the lipid used to form the lcp was monoolein (9.9 mag, see figure 1a), however recent experiments have utilized shorter chain analogs of monoolein as well as mixtures of monoolein with cholesterol (see refs (12, 18,21)and citations therein). The reconstituted proteins appear to be well - accommodated, functional, and mobile inside the lcp where they are believed to reside largely in a monomeric state . The addition of a precipitant of suitable composition, which is the next step in the in meso trial, is proposed to result in local structural and compositional changes in the mesophase and likely in protein conformation as well, which alter the interactions between the proteins and the cubic phase lipid bilayer; ultimately this drives the preferential partitioning of the proteins into the lamellar phase that presumably appears within the lcp . The critical aspect of the nucleation and subsequent crystal growth process that is triggered in this manner, and ultimately determines the quality of the grown crystals, is that once they have diffused from the lcp to the lamellar phase the proteins form tight 3d oligomeric arrays . The oligomerization of tm proteins in lipid bilayers has generally been suggested to involve the hydrophobic mismatch effects originating from the difference in the lengths of the hydrophobic core of the protein and the surrounding lipid hydrocarbon region . Such mismatch effects have been shown from experiments to play a role not only in the spatial organization but also in the function of tm proteins, such as the gpcr rhodopsin . The hydrophobic mismatch is considered to drive the membrane to deform in order to alleviate the energetically unfavorable hydrophilic / hydrophobic exposure this mismatch entails . When such membrane deformation cannot achieve a complete hydrophobic adaptation, the residual exposure (or residual mismatch) to unfavorable interactions gives rise to an unfavorable contribution to the free energy of protein / membrane interaction . The mitigation of this energy term through oligomerization has been suggested as an important mechanistic contribution to membrane - driven oligomerization of multihelical tm proteins, such as gpcrs, in lipid bilayers . Therefore, we investigated the potential role of differential hydrophobic exposure of such proteins in lipid bilayers of the cubic and lamellar phases as a possible determinant of the favorable outcome of in meso crystallization trials . Here we present the results of a molecular dynamics - based study of a gpcr reconstituted into an lcp environment that reveal differences in the hydrophobic / hydrophilic exposure of the gpcr to lipid the quantitative results reflect the drive for differential oligomerization behavior of these proteins in the two lipid environments that essentially define the end points along the in meso crystallization pathway . With the ability to connect quantitatively the difference in gpcr oligomerization behavior in the two different media, to the curvature and mismatch alleviation capabilities of the two environments, we are able to predict lipid types that would enhance / diminish the difference in the residual exposure for a gpcr in the cubic and lamellar phases . Given the recognized importance of protein membrane interaction for the functional properties of gpcrs, molecular dynamics simulation studies using all - atom and coarse - grained representations have been carried out for the prototypical gpcr, rhodopsin, in lamellar membranes . To our knowledge the present work is the first molecular dynamics - based study of a gpcr reconstituted into the lcp . The results enable quantitative inferences about the energetically unfavorable hydrophobic hydrophilic interactions that differentiate the behaviors of gpcrs in the two different lipid environments, lcp and lamellar membranes . We attain these insights from comparative microsecond time - scale coarse - grained simulations with the martini force - field of rhodopsin in monoolein - based cubic and lamellar lipid bilayers . Using our recently reported computational framework for quantifying residual mismatch energies from md trajectories, we evaluated the protein - dependent membrane deformations and their attendant energy cost, and identified specific tm regions and residues of rhodopsin that exhibit differential hydrophobic exposure in the lcp and lamellar bilayers . We show that the bilayer of the lcp more efficiently shields the protein from unfavorable interactions, and that the reduced level of hydrophobic mismatch in the lcp is attributable to the specific, highly curved geometry of the cubic phase . Better protection from unfavorable hydrophobic exposure of rhodopsin in the lcp phase is especially evident for residues in tm helix 1 (tmh1) and tmh5 . Lcp environment, the planar bilayer of the lamellar phase provides a more favorable setting for gpcr oligomerization as a prelude to nucleation and crystal growth . Thus, our findings provide novel energy - based insights into driving elements of in meso crystallization mechanisms and lay the foundation for future quantitative exploration of rational approaches for the generation of structure - quality crystals of membrane proteins . Coarse - grained (cg) md simulations of the lipid cubic phases (lcp) with or without rhodopsin were done with the martini force field, grouping atoms into coarse - grained beads (see figure 1a), and the gromacs 3.3.1 package as listed for the various molecular constructs in table 1 . As a host lipid for the lcp, we used monoolein (9.9 mag) which consists of an 18 carbon fatty acid with a cis double bond between carbons 9 and 10 in ester linkage to the primary hydroxyl of glycerol (figure 1a). Simulation times reported are effective times, taking into consideration typical for martini force - field based coarse - grained simulations factor of 4 . Simulations with protein were initiated from self - assembled lipid diamond cubic phase of 500 lipids by replicating the monoolein / water system 27 times (3 times in positive and negative x, y, z directions) and inserting rhodopsin randomly into the expanded lcp phase . In the first phase of the study, we conducted extensive self - assembly simulations of randomly mixed cg 9.9 mag lipids and water molecules into a lipidic diamond cubic phase . As listed in table 1 and detailed in the results, the diamond lipid phase was successfully achieved in multiple simulations conducted at 20 c temperature and 40% (w / w) water weight percent . All the self - assembly simulations (see table 1) were initiated from a random placement of 500 cg 9.9 mags (a number that is within the experimentally determined range of 9.9 mag molecules in the unit cell of the diamond cubic phase), and a corresponding number (determined by the desired percentage) of cg water beads in a cubic box . Each system was first equilibrated for a short period of time with the isotropic pressure coupling scheme and the lennard - jones (lj) parameters of all the atoms set to those of the beads representing water molecules in the martini force - field . This setup, implemented as well in earlier cg self - assembly simulations of the lcp, allowed efficient adaptation of the volume of the simulation box to the number of cg molecules in the system, and ensured complete mixing of lipids and waters prior to self - assembly simulation . After this initial equilibration phase, the lj parameters for 9.9 mag lipids were reset to their proper values (see details of force - field parametrization, below), and 6 s - long self - assembly md simulations were performed (the simulation times reported throughout are effective times, taking into consideration the factor of 4 typical for martini force - field - based coarse - grained simulations), and using isotropic pressure coupling (with 3.0 ps and 3e bar time constant and compressibility, respectively), and a 30 fs time step . To simulate rhodopsin in the diamond phase, we constructed the system by replicating 27 times (along positive and negative x, y, z directions) one of the cubic phase structures obtained from the self - assembly simulations conducted at 20 c and at 40% (w / w) water . The diamond cubic phase in this enlarged construct remained stable in a microsecond - long cg md simulation (data not shown). A rhodopsin molecule was inserted at a random position in this large cubic phase (at 122:1 9.9mag / rhodopsin molar ratio, corresponding to 4 mg / ml concentration, see figure s1 in the supporting information). After removing overlapping waters and 9.9 mag molecules and adding counterions for electroneutrality, a 4.8 s - long cg md simulation was carried out at 20 c . The total number of cg beads in this large construct was 139 979, which corresponds to 1 400 000 actual atoms . The simulation utilized the isotropic pressure coupling scheme as above, and a 30 fs time step . Rhodopsin was simulated in 9.9 mag lipid lamellar bilayers with the cg martini force - field . The cg model of rhodopsin (see below) was inserted into a pre - equilibrated cg 9.9 mag lipid membrane containing 1772 lipids, and after solvation and ionization steps, a 3.2 s - long md simulation was carried out at 20 c temperature using a 40 fs integration time step and the semi - isotropic pressure coupling scheme . The details of the martini cg force fields and the parametrization scheme employed for 9.9 mag lipid and rhodopsin can be found in the supporting methods section of supporting information . The residual exposure energy is the energy cost of the hydrophobic mismatch that persists in the equilibrium state of the membrane - protein interaction, even after membrane remodeling around multi - tm proteins has taken place to reduce the hydrophobic mismatch . This result of the anisotropy of the protein membrane boundary when multi - tm proteins are inserted in the bilayer has been discussed in detail . For rhodopsin, the residual exposure of residues in the tm segments was quantified with the computational protocol described in ref (30) in both the lipid cubic and lamellar phases, using solvent accessible surface area (sasa) calculations with the naccess software and a probe radius of 1.4 . As described previously, sasa values were obtained either with the solute comprising the protein only (saprot), or with the solute containing the protein and the hydrophobic core of the lipid bilayer (samem)the latter being defined as the 9.9 mag bilayer region consisting of the gl1-c1a - c2a - d3a - c4a - c5a cg beads (see above for the cg bead definition, depicted in figure 1a). The residual exposure sares was then calculated as sares = saprot samem for polar residues and as sares = samem for hydrophobic residues (see ref (30) for details). These accessible surface values were used to obtain the residual mismatch energy penalty gres associated with a particular residue as gres = ressares, where res = 0.028 kcal/(mol) is a constant of proportionality related to the free energy of transfer of amino acids between aqueous and lipid environments . According to a well - accepted protocol, interfacial trp, arg, and lys residues were not penalized for residual mismatch due to their ability to adapt favorably to hydrophobic / hydrophilic interfaces . For a quantitative assessment of the spatial organization of the bicontinuous lipid / water structures assembled computationally, we utilized an analytical fit of the data obtained from the simulations . To obtain this fit we represented the location of the lipid bilayer midplane of the bicontinuous cubic phase by the triply periodic minimal surface (tpms, surface with zero mean curvature):1where f(x, y, z,) is the improved nodal approximation given by:2with3 and x = x, y = y, z = z, and p, q, r = c, s (c and s stand for cosine and sine, respectively). The {h, k, l} in the above equations are the crystallographic miller indices, and is a fitting parameter such that 1/ represents the linear size of the primitive unit cell . This surface was shown to provide an accurate representation of the surface in the pn3 m symmetry group (see, for example, ref (3) and citations therein). For each 9.9mag / water trajectory listed in table 1, a fit to the analytical solution was performed on 20 separate frames spaced 350 ns apart . To this end we extracted the coordinates {xi} of n c5a coarse - grained beads representing the location of the terminal methylene and methyl groups in the hydrocarbon tail of 9.9 mag lipids for each frame, and the fit was done by optimizing f(xi,) = ci through a search for the translational and rotational transformations that minimized = (1/n)i=1ci . The quality of the fit was assessed from the distribution of {ci} values obtained from the minimization procedure; this distribution is centered around the theoretical mean, zero (i.e., the analytical solution of eq 1). Such a distribution was collected for each md trajectory by merging the {ci} coefficients obtained from fitting all 20 separate frames, and the quality was assessed as described above (see distribution plots and captions in figure 4 and in figure s3 in the supporting information). To compare quantitatively the structural arrangement of the lcp bilayer near the protein, to that in the bulk cubic phase, we employed two complementary approaches . In the first, we obtained an analytical solution for the lipid bilayer midplane around the protein by fitting the simulation data (coordinates of c5a beads) to f(x, y, z,) = ci, as described above in the tpms representation, except that for this purpose the {ci} coefficients were also considered as variable parameters that must be self - consistently minimized (see above). In this manner, the extent of the deviation of the mean value of the {ci} distribution obtained from the solution for the pn3 m phase (ci 0, see above), measures the distortion of the diamond cubic phase due to the protein . The alternative approach we developed for quantifying the perturbation of lipid bilayer shape induced by the gpcr departs from the symmetry group definition and focuses on the lipid water boundary . In this approach, different trajectory frames from the simulation of rhodopsin in lcp are aligned onto snapshots from the simulations of the large protein - depleted 9.9 mag / water system . Using a locally defined scoring function (see below), we assess how well the lipid bilayers of the protein - containing and protein - free systems align (the large protein - free system was constructed by replicating 27 times the equilibrated system composed of 500 9.9 mag lipids/6800 water molecules, and subjecting it to microsecond long cg md simulation during which the pn3 m phase was maintained; data not shown). To align the protein - free and protein - containing structures, we used water densities to first align 10 frames from the last 100 ns of the protein - free simulation, which served as a reference on which we aligned 10 frames from the last 100 ns of the protein - containing trajectory . Only water beads at least 50 away from the protein were considered, in order to avoid degradation of the alignment quality due to deformations of the lipid cubic phase near the protein . The alignments utilized the electron density (y) at any point y of a set of beads with coordinates ri; the density function was constructed as a superposition of gaussians, so that for a particular trajectory frame4where c and k are the amplitude and width of the gaussians, respectively, chosen to produce a 10 resolution density map . The quality of the superposition of two frames was estimated by measuring the overlap of the water - containing and water - free regions using:5where x = {xj} (j = 1,2,...,m1, m1 + 1,...,n) are coordinates of beads in the fitted frame which contains m1 water beads and n m1 lipid beads; w is the density of water in the reference frame obtained using eq 5; sj = 1 for j m1, and sj = 1 for j> m1 . The best alignment was calculated by maximizing s with respect to the rotational and translational transformations applied to x. the local quality of the alignment was assessed by a normalized scoring function defined as:6where (xj) represents the electron density of the beads in the reference frame . The self - assembly of the 9.9 mag / water complex with 40% (w / w) water composition was probed at 20 c, which are conditions similar to those used in the original in meso crystallization trials of gpcr proteins (see ref (14) and citations therein). Figures 2 and 3 show several views of 9.9 mag / water complexes obtained under these conditions produced by the 6 s - long self - assembly simulation of 500 9.9 mag lipids . In figure 2, panels a c offer views of the unit simulation cell, whereas figure 3 depicts various snapshots of the same system replicated 27 times (3 times in each orthogonal direction). Typically, in our simulations the initially randomly mixed 9.9 mag / water system became organized as illustrated in figures 23 within the first 350 ns of simulation, and remained stable for the remainder of the 6 s trajectory . (a, b) views of the unit simulation cell containing 9.9 mag / water complex at 40% (w / w) water concentration and at 20 c temperature, after 6 s of self - assembly simulation . (a) entire system where different components are colored as following: 9.9 mag lipid headgroup beads, blue; 9.9 mag lipid hydrocarbon tail beads, red; and water beads, cyan . (b) separately 9.9 mag hydrocarbon chain beads from the same perspective as in a. (c) view of the tetrahedral arrangement of the water channel from a. the four arms of the tetrahedral geometry are visible . (d) snapshot of two nonintersecting continuous intertwined water channels (in gold and cyan, respectively). The representations are for the expanded system obtained by replicating the simulated system 9 times (in positive x, y, and z directions). Views of the 9.9 mag / water complex from figure 2 replicated 27 times (3 times in positive and negative x, y, and z directions) reveal bicontinuous diamond cubic phase nature of the self - assembled structure . Different panels show: the entire system (a), 9.9 mag headgroup and water cg beads (b), 9.9 mag hydrocarbon chain beads (c), only water beads (d). The bicontinuous cubic phase nature of the self - assembled structure is apparent from inspection of the spatial organization of both lipid and water components . Indeed, the hydrophobic core of the 9.9 mag lipids (shown in red in figures 23) traces a single highly curved continuous surface in 3d, surrounded by 9.9 mag headgroups (dark blue shades in figures 23) adjacent to aqueous compartments (cyan colors in figures 2a, c and 3). The aqueous part of the system consists of two nonintersecting three - dimensional continuous networks of water channels (figure 2d), which makes the assembled structure a bicontinuous cubic phase . Figure 2c shows that in each of the water compartments four aqueous channels meet in a tetrahedral arrangement . A similar arrangement can be observed as well for the surface traced by the hydrocarbon chains of 9.9 mag lipids (see figures 2b and 3c). Thus, the assembled structure resembles closely the doubly bicontinuous lipidic diamond cubic phase . One general feature of such mesophases is that the surface traced by the midplane of the lipid bilayer, which separates the two aqueous compartments, can be approximated by the triply periodic minimal surface (tpms) that has a special property: it is characterized at every point by zero mean curvature . Consequently, the tpms is saddle - shaped (figure s2, supporting information). This saddle - shaped geometry of the 9.9 mag / water assembly resulting from our calculations can be appreciated, for example, by tracing the surface formed by the 9.9 mag hydrophobic core (see figures 23). For a quantitative assessment of the adherence of the bicontinuous structure from our simulations to the organization of the pn3 m diamond cubic phase, we performed a fit of the simulation data to the analytical solution, as described in methods . The quality of the fit was assessed quantitatively from the distribution of the fitting coefficients {ci} (see methods) shown in figure 4 . Clearly, the distribution is centered around zero mean, and is relatively narrow (with a standard deviation of 0.4), suggesting that the surface traced by the c5a beads is consistent with the tpms of the pn3 m cubic phase (see eqs 13). Distribution of {ci} coefficients from the fitting of the c5a bead positions from the simulations to the analytical tpms approximation (see methods). The test established the significance of the fit with probability p <0.05 . As indicated in table 1, the self - assembly of 9.9 mag / water mixture was investigated in two additional simulations performed at t = 20 c and 40% (w / w) water content . In all cases 9.9 mag and water molecules aggregated in doubly bicontinuous diamond lipid cubic phases, similar to that described in figures 23 (see figure s3 in the supporting information). To explore the organization of a gpcr inside the cubic mesophase we simulated the prototypical class - a gpcr, rhodopsin, in 9.9 mag - containing lipidic cubic phase in a large system constructed as described in methods . The simulation results were used as described below to compare quantitatively the interactions of rhodopsin with bilayers of the cubic and lamellar phases, as a basis for interpreting the mechanistic role of the hydrophobic mismatch in triggering the nucleation process during the in - meso crystallization . Residues in the rhodopsin tm bundle that participate in unfavorable interfacial hydrophobic / hydrophilic interactions with the lipid bilayers of the cubic and lamellar phase were identified, as described in methods, with a sasa - based approach . The last 1.5 s intervals from the respective cg md trajectories for rhodopsin in the 9.9 mag lcp (4.8 s), and in the 9.9 mag lipid lamellar phase (for 3.2 s) were used for the residual exposure analysis (see methods for definitions), and the corresponding residual exposure energies were evaluated as described . Table 2 lists rhodopsin tm residues that were found to exhibit different hydrophobic exposures in the lcp and lamellar phase simulations, alongside their respective residual exposure energy penalties (gres). All other residues in the tm bundle experienced a residual penalty of <1 kbt (kb denoting the boltzmann constant) in both lpc and lamellar phases . Analysis was carried out on the last 1.5 s time interval of the respective trajectories where sasa values and corresponding residual exposure energies remained, within fluctuations, unchanged . Numbering shown in parentheses corresponds to the generic residue numbering scheme for gpcrs defined by ballesteros and weinstein . Different residual exposure in the two lipid environments became evident for residues in the extracellular (ec) end of tmh1, and the intracellular side (ic) of tmh5 (table 2). Specifically, the pro34/gln36 pair on tmh1 (superscript numbers identify the residues by the ballesteros and weinstein generic residue numbering scheme for gpcrs), and phe228 in tmh5 were found to generate a lower residual exposure energy penalty in the lcp than in the lamellar phase . Especially remarkable is the difference in gres for the hydrophobic pro34 residue, that is, 4.7 kbt in the lamellar bilayers, but <1 kbt in the cubic phase . This difference results from pro34 being largely exposed to the lipid polar headgroups and/or water environment in the planar membrane, but shielded from such unfavorable interactions in the lcp . The large residual exposure predicted for pro34 in the lamellar 9.9 mag bilayers is consistent with findings from our earlier all - atom md simulations of rhodopsin in planar membranes composed of dicn:1pc (n = 14, 16, 18, 20) lipids; the largest residual exposure for pro34 was found in the bilayers with the thinnest hydrophobic core, that is, dic14:1pc and dic16:1pc membranes . Due to the substantial difference between the hydrophobic length of tmh1 in rhodopsin (37, measured along the membrane normal) and the bulk hydrophobic thickness of the thinner bilayers (27.2 and 30.5, respectively for dic14:1pc and dic16:1pc membranes), the mismatch was not alleviated by membrane deformations around the protein (see ref (30)). Here, the hydrophobic thickness of the 9.9 mag lamellar bilayers, calculated from the average distance between the lipid gl1 backbone beads on the two leaflets, is 31.5, so that the hydrophobic mismatch between tmh1 and the bilayer is again substantial, and can be alleviated only partially by membrane deformation, as shown below . The average thickness of the 9.9 mag lamellar bilayer around rhodopsin, calculated from the last 1.5 s of the md trajectory and the membrane thinning around tmh2the helix adjacent to tmh1is evident in figure 5 . Interestingly, we find that the bilayer deforms near the polar ser98 residue (see figure 5d), which is apparently shielded from the unfavorable exposure to the hydrophobic lipid core by the local thinning of the lipid membrane . Usually, the membrane - facing oh of ser residues in a tm bundle can avoid hydrophobic contact by forming hydrogen bonds with the helix backbone . When this is not feasible energetically, the bilayer thins around this residue . In our simulations we find that in order to shield ser98 from unfavorable hydrophobic exposure, the thinning at tmh2 also constrains the lipids at the adjacent tmh1 (see ref (30) for a discussion of this type of constraint), so that the membrane cannot deform significantly and thus leaves the pro34 residue highly exposed (see figure 5d and table 2). (a c) views of membrane deformation patterns around the rhodopsin immersed into 9.9 mag lamellar bilayers . The average positioning of membrane leaflets is identified by the two surfaces colored according to the local thickness of the bilayer (see the bar for color definition). The gpcr is shown in van der waals representation with the tm helices colored as follows: tm1 in gray, tm2 in orange, tm3 in white, tm4 in pink, tm5 in purple, tm6 in black, and tm7 in lime the rest of the protein (loops, etc .) (d) magnified view of the region containing residues pro34 and ser98 (both rendered in green) showing the membrane deformations in their vicinity . Figure 5 shows that the 9.9 mag planar bilayer deforms around other tm helices as well . Specifically, the membrane thins around tmh4, alleviating the residual mismatch for this helix, and around tmh7 where the deformation efficiently accommodates the juxtaposed amphipathic helix 8 at the hydrophobic / hydrophilic interface . Overall, the heterogeneous pattern of membrane deformations we observe around rhodopsin in the current simulations agrees with earlier findings from all - atom md simulations showing the pattern of membrane remodeling around rhodopsin and the homologous serotonin 5-ht2a gpcr . Consistent with those studies, we find here that the deformations of the lamellar 9.9 mag bilayer do not completely alleviate the hydrophobic mismatch with the receptor (table 2). The different organization of the two lipid phases around the gpcr, as presented below, explains why the residual exposure was larger for rhodopsin in the lamellar 9.9 mag bilayer than in the 9.9 mag lcp . Figure 6 shows the organization of the lipid cubic phase around rhodopsin in the initial configuration where the protein was randomly inserted in the lcp (see also figure s1 in the supporting information), compared to the organization after 4.8 s of cg md simulations . C with d f in figure 6 shows the substantial rearrangement of the lipids and solvent around rhodopsin during the long md trajectory . Specifically, it appears that the lcp tends to minimize hydrophobic / hydrophilic contacts with the gpcr as the water and lipid headgroup beads organize around polar regions of the protein (yellow colors in figure 6), whereas the lipid hydrocarbon chain beads surround the hydrophobic core of the protein (see caption of figure 6 for tm definitions). Snapshots illustrating initial (a c), and final (after 4.8 s simulations) positioning (d f) of rhodopsin (in yellow / white) inside the cubic phase . A and d show the organization of water beads (in cyan), b and e illustrate the organization of 9.9 mag lipid headgroup beads (in pink), and c and f show the organization of 9.9 mag lipid hydrophobic core beads (in purple). In all panels rhodopsin transmembrane (tm) helices are colored in white and the rest of the protein in yellow . Shown are only water and 9.9 mag lipids that are within 20 of rhodopsin; rhodopsin is oriented so that its intracellular and extracellular ends (as defined in the lipid bilayer) point toward the bottom and top of the panels, respectively . The snapshots in figure 6 also show that rhodopsin equilibrates, as expected, around the saddle - point inside the lcp so that the tm bundle perpendicularly traverses a narrow but relatively flattened region of the lcp bilayer, much like the gpcr protein does in the lamellar bilayers . However, due to continuous and intrinsically saddle - shaped geometry of the diamond cubic phase, the lcp bilayer is dramatically curved near tmh1 and tmh5, which effectively confines the protein to a small region of the lcp . This special arrangement of the cubic mesophase around rhodopsin shields the gpcr in the lcp from the kind of unfavorable hydrophobic / hydrophilic interactions with the lipid bilayer seen in the lamellar membranes . Thus, as illustrated in figure s4 (in the supporting information) and reflected in the residual energy penalties (in table 2), residues pro34 and phe228 are in contact with the hydrophobic core of the lcp bilayer and therefore do not incur a residual mismatch penalty, although gln36 experiences somewhat higher residual exposure in the lcp compared to the lamellar phase (see figure s4 and table 2). Glamellar to be 2.7 kbt for the pro34/gln36 pair, and 1.8 kbt for phe228 (table 2). Notably, the shielding of ser98 that faced the 9.9 mag lipid bilayer in the lamellar phase simulations, occurs in the lcp simulations through interaction with the protein backbone . To relate the calculated residual mismatch energies to the organization of the lipidic cubic mesophase around the gpcr, we quantitatively compared the structural arrangement of the lcp bilayer near the protein, to that in the bulk cubic phase . As described in methods, two different approaches were used: in the first, we obtained an analytical fit of the lipid bilayer midplane shape around the protein by fitting the coordinates of 9.9 mag lipid c5a methyl terminus beads (figure 1a) from the simulations to f(x, y, z,) = ci, considering {ci} coefficients as variable parameters that must be minimized during the fitting (see methods). The application of this procedure to spherical lipid shells situated at different distances from rhodopsin (see figure 7a) revealed that the organization of the lcp close to the gpcr is different from that expected for the pn3 m lipid bilayer (ci 0 in figure 7a). However, already at distances 50 from the center of rhodopsin (compare to 3540 linear dimension of a rhodopsin - like gpcr in bilayer x y plane directions), the simulated structure relaxes to the organization of the diamond cubic phase (ci = 0, figure 7a). The second approach confirms that the distorted lcp bilayer near the protein tends to relax toward the pn3 m phase arrangement at larger distances (figure 7b). In this alternative approach we locally quantify the shape of the lipid bilayer around the gpcr by aligning trajectory frames from the simulation of rhodopsin in lcp onto snapshots from the simulations of the pure lcp system, so as to optimize the overlap of the water - containing and water - free regions (see methods). A scoring function defined locally (see eq 5) is used to assess the quality of the alignment . As seen from figure 7b, this procedure revealed that the fit between the densities of c5a lipid beads in protein - containing and protein - free systems progressively improves with the radial distance from the gpcr (increasing s in figure 7b). Indeed, consistent with the result in figure 7a, the scoring function reaches its plateau 50 away from the protein, indicating that at these distances the lcp arrangement becomes similar to that expected for the pn3 m phase . Distance dependence of the relaxation of membrane deformation near rhodopsin in the cubic phase . (a) analytical fit of the density of c5a 9.9 mag lipid beads from cg md simulation of rhodopsin in the lcp phase to the surface defined by the f(x, y, z,) = ci equation (see methods). The mean values of the {ci} fitting coefficient distribution as a function of r, are shown for 10 spherical lipid shells located at r = 20, 30, 40, and 50 away the protein center - of - mass . (b) reasults from the same measurements performed with an alternative approach based on the alignment of the lipid bilayer midplane in protein - containing and protein - free cubic phases (see methods). The quality of the fit upon alignment was determined for 10 spherical lipid shells located at distances r from the protein center - of - mass, and the panel shows the dependence of the scoring function s on r. note that the deformed cubic phase near rhodopsin relaxes to the arrangement that is characteristic of the pn3 m phase 50 away from the protein . Taken together, our results establish a quantitative link between the residual mismatch penalty and the lipid bilayer deformations around the gpcr in the cubic phase . Thus, the areas where the largest perturbations from the diamond cubic phase were observed included residues pro34 and phe228 (see figure s5 in the supporting material), which are also the residues with the largest value of glcp glamellar (table 2). The main findings from this study offer unprecedented insight into molecular level processes related to in meso crystallization, which are likely leading to protein nucleation and eventually to crystal growth . Thus, we found that the difference in the hydrophobic / hydrophilic exposure of the protein to the lipid bilayers of 9.9 mag lcp and lamellar phases is responsible for a lower residual mismatch (and its corresponding energy penalty) in the cubic mesophase compared to the lamellar membranes . This indicates why gpcrs reconstituted into the lcp in the initial stages of the in meso trial are well accommodated inside the cubic phase, where they are believed to reside mostly in the monomeric form . Indeed, from our findings there is insignificant drive toward rhodopsin oligomerization in the lcp . We showed, however, why this drive is increased in the planar bilayer environment of the lamellar phase where there are several critical mechanistic factors that determine the probability for proteins to oligomerize . One important energy component that has been established from both experiments and computations, involves the hydrophobic mismatch . In particular, a plausible mechanism to relieve the energy penalty due to residual exposure is protein oligomerization, whereby the tm segments incurring the highest energy penalty from the residual exposure come together in the lipid bilayer . For such hydrophobic - mismatch driven association to occur, the residual exposure penalty alleviated by the protein association must counteract other critical factors that could favor proteins in the monomeric state . One such important consideration is the mobility of proteins, because constrained diffusion reduces the chance for protein protein encounter . As shown in figure 8, the spatial restriction encountered by the gpcr inside the lcp (see figure 6) significantly affects protein mobility, with the diffusion coefficient of the gpcr in the cubic phase being smaller (dlcp = 1.1 10 m / sec) than that in the lamellar bilayers (dlamellar = 5.5 10 m / sec). The restricted diffusion mode for rhodopsin in the lcp environment is consistent with the suggested high energy cost in the lcp for gpcr - sized proteins to cross between different regions of the mesophase . This energy penalty was quantified from phenomenological principles to be as high as 1220 kbt in the pn3 m phase with lattice parameter values in the 75110 range . This estimated energy cost of protein diffusion in the lcp is significantly higher than the residual mismatch penalty we calculated for rhodopsin in the cubic phase (table 2). When considered together, the relatively low residual exposure energy for the gpcr in the lcp is not likely to be sufficient to compensate for the high energy barrier for protein mobility in the lcp and drive protein association . The prediction is, therefore, that in the cubic mesophase gpcr proteins will remain largely in the monomeric state . Mean - square - displacement (msd) as a function of time calculated for rhodopsin in the 9.9 mag lipidic cubic phase (lcp) and in the 9.9 mag lamellar membrane (lamellar). Corresponding diffusion coefficient values, calculated from the linear fit performed in [0 s;1 s] time interval, are dlcp = 1.1 10 m / sec and dlamellar = 5.5 10 m / sec . In contrast to the lcp, the lamellar membrane environment is more favorable for gpcr oligomerization: the residual energy penalty for the rhodopsin tm bundle in the lamellar bilayers is 8 kbt (table 2) and is significantly higher compared to that in the lcp, indicating a stronger drive for gpcr oligomerization in the lamellar membranes . A higher propensity for association in the planar bilayers is also supported by our calculations of the protein mobility in the two lipid environments, whereby we find a 5-fold larger diffusion coefficient for the gpcr in the lamellar bilayers (figure 8). Taken together, results from our simulations suggest that planar 9.9 mag lipid membranes provide a more suitable setting for oligomerization of the gpcr proteins . Notably, this prediction is based on results for oligomerization in a plane of the lipid bilayer . This 2d nucleation process together with the formation of protein - enriched lamellar stacks in 3d should ultimately drive the emergence of a bulk crystal . The question remains regarding the mechanism that drives the proteins from the lcp to the lamellar membranes and the potential role of the differential residual interactions between the protein and the bilayers of the two lipidic phases in the nucleation process . We note that the nucleation is triggered by the addition of the precipitant which is known to cause transient water depletion from the lcp interior, resulting in structural changes in the cubic phase bilayer . Our current study has not attempted to quantify the effects of the precipitant, but it is reasonable to speculate that dehydration of the cubic phase will only increase the unfavorable residual interactions between the gpcr and the lcp bilayer . This is likely to increase the drive for protein oligomerization inside the lcp upon precipitant addition, which could lead to the formation of locally flattened lamellar bilayers as a prelude to protein crystallization . In order to address quantitatively the structural perturbations due to precipitant, such as changes in curvature of the lcp bilayer, it is critical to calculate the corresponding deformation energies in the presence of the protein . However, the representation of the complex geometry of the lcp around the insertion (figures 67) in the numerical approach developed in the current work for quantifying the lcp shape (i.e., fitting the md data to the analytical solution) is not sufficiently refined to serve in the calculation of reliable energies we note, however, that recent experimental studies showed that, compared to 9.9 mag, in meso trials conducted on the gpcr - gs protein complex with a shorter chain mag analog, 7.7 mag (acyl chain 14 carbon atoms long with the cis double bond between carbon atoms 7 and 8) resulted in better quality crystals . Thus, results from our current studies as well as from recently reported all - atom simulations of gpcrs in membranes of different thicknesses, explain why 7.7 mag would provide a better platform in which gpcrs can aggregate more extensively: based on our findings we can predict that, due to the expected 78 difference in the hydrophobic thickness of planar 9.9 mag and 7.7 mag membranes, the residual exposure of the gpcr will be substantially higher in the thinner 7.7 mag lamellar bilayers . As an example, we recently reported that the rhodopsin tm bundle will exhibit a 12 kbt higher residual energy penalty in dic14:1pc than in dic18:1pc bilayers . A more complete quantitative elucidation of the gpcr nucleation process in relation to the residual mismatch energy component requires further studies that would take into consideration the effect of the different precipitants on various lipids . The structurally specific predictions of the regions where the residual mismatch with lipid bilayers of cubic and lamellar phases differed for the rhodopsin tm bundle made it possible to probe the resulting predictions and general character of the findings by examining crystallographic data . In particular, we showed here that the intracellular (ic) end of tmh1 (pro34), and the extracellular (ec) part of the tmh5 including phe228 have higher residual exposure in lamellar membranes, which leads to the prediction (e.g., see shan et al .) That in the planar bilayers rhodopsin will exhibit strong hydrophobic - mismatch driven tendency for oligomerization through tmh1 and tmh5 . To assess this hypothesis in the context of structural information available for gpcrs, we examined crystallographic contact interfaces for 12 different structures of rhodopsin - like gpcrs obtained by means of the in meso technology (2 receptor pbd codes: 2rh1, 3pds, 3sn6; a2a receptor pdb codes: 3eml, 3qak; chemokine cxcr4 receptor pdb codes: 3odu, 3oe0, 3oe6, 3oe8, 3oe9; dopamine d3 receptor pdb code: 3pbl, and histamine h1 receptor pdb code: 3rze). For all these gpcr structures, we analyzed the content of the unit crystallographic cell and quantified for each tm helix (1) the frequency of its occurrence at the crystallographic contact interface and (2) the number of residue interactions each tm helix forms at the contact interface . Canonical interfaces were considered, where the crystallographic contacts were formed exclusively through tm tm interactions between monomers in parallel orientations . Remarkably, we found that tmh1 and tmh5, the two helices implicated in the largest residual mismatch in our simulations of rhodopsin with 9.9 mag lamellar bilayers, contribute to the most common contact interfaces in the crystallographic structures of the homologous gpcrs . More specifically, as illustrated in figure s6a (see supporting information), all but 4 of the 14 distinct intermolecular tm tm interfaces identified in this analysis, involve tmh1 and/or tmh5 . Furthermore, examining the residues involved in the interactions at the contact interfaces revealed that the tmh1 stretch of residues in positions 1.291.34 (that would include pro34 in the homologous rhodopsin gpcr) contribute significantly to the formation of the crystallographic interface . During the final preparation of this manuscript, two new crystal structures of rhodopsin - like gpcrs, the -opioid receptor (kor) and the -opioid receptor (mor) were reported to be obtained by means of in meso crystallization . Consistent with the data presented above, the crystallographic interface of kor consists of tm1, tm2 and h8, with the n - terminal end of tm1 (region harboring the residue homologous to pro34 in rhodopsin) forming extensive dimeric contacts . For mor, two crystallographic interfaces were reported: the more prominent interface consisted of tm5/tm6 helices with the i256 residue (aligning with the residue next to phe228 in rhodopsin) residing at the dimer interface, and more limited contacts formed by tm1, tm2 and h8 . Taken together, our findings support the mechanistic inferences that (1) the reduced level of hydrophobic mismatch in the lcp is attributable to the specific highly curved geometry of the cubic phase that provides for more coverage from unfavorable hydrophobic exposure; (2) compared to the lcp, lamellar structures provide a more favorable setting in which gpcrs can oligomerize as a prelude to nucleation and crystal growth; and (3) the extent of the residual mismatch penalty is likely one of the critical mechanistic factors that determines not only the drive for gpcr proteins to oligomerize during in meso crystallization, but also the structural elements that are likely to participate in contact interfaces . These findings provide novel energy - based insights into in meso crystallization mechanisms and lay a foundation for future computational explorations involving other class - a gpcrs toward designing rational approaches for generation of structure - quality crystals of membrane proteins.
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Prostate cancer (pca) has mostly affected the male population of the united states, and in 2012 us cases constituted approximately 29% (240,890) of the incidence of pca . In south korea, the incidence of pca has significantly increased while the mortality rate has steadily decreased . From 2007 to 2013, the incidence pca in korean men doubled (5,516 to 10,855 per year), while the prevalence tripled (18,830 to 51,411) during the same period . The mortality rate increased slightly, from 4.2 in 2000 to 5.9 in 2007 and 6.0 in 2013 (per 100,000, age - adjusted). Pca incidence increased significantly faster in men <70 years of age than in the older age group . The emergence of nanotechnology has drawn much attention in the 21st century for applications in medicine such as " nanomedicine . " Nanomedicine combines engineering, physics, biology, chemistry, mathematics, and medicine and strives to improve disease detection, imaging, and drug delivery through the use of nanodevices . Nanotechnology encompasses the materials, devices, and delivery systems for disease diagnostics, prevention, and treatment . Both researchers and the pharmaceutical industry have shown particular interest in nanotechnology for medical applications with potential benefits for patients . Nanotechnology - based medicine can hurdle both physiological and biological barriers, such as the blood - brain barrier, endothelial barriers, cell membranes, and even nuclear envelopes, achieving both passive and active disease targeting . Nanosized particles are normally composed of thousands of atoms and exhibit unique physical and biochemical properties with high surface area for therapeutic loads such as cancer drugs . However, challenges to nanomedicine exist, such as safety issues, bulk manufacturing issues, and compatibility with the human body of nano - sized drug delivery systems . Meeting these challenges is essential for successful application of drug - loaded nanoparticles (nps) in the field of pharmaceutics . Different approaches have been taken to conjugate nps with targeting molecules that specifically bind to the surface markers expressed in various tumors, such as prostatespecific membrane antigen (psma) in pca . Prepared nps are also used in real - time monitoring of treatment by attaching various imaging moieties that help in visualizing the distribution of nps and the drugs . Nps continue to be investigated for their potential to improve existing therapies and to develop novel therapies . At the length scale of less than 200 nm, nps with encapsulated therapeutics can be injected intravascularly without concern for embolization . At this scale, nps have the potential to permeate and traffic through different tissues, bind to cell surface receptors, enter target cells for intracellular delivery of their cargos, and influence the intracellular tracking pathways . Owing to these unique characteristics, a multitude of applications have been identified for np - mediated drug delivery . Nps can be used as probes with many advantages such as rapid clearance from the bloodstream by macrophages and depending on the size of the nps renal clearance that helps them to remain in the circulation for a long time . Indeed, it is important that the nps have a multivalency effect with more than one type of targeting ligand . Studies have shown that combined therapy and diagnosis (nanotheranostics), with both imaging and therapeutic capacity, can be achieved . Np composition plays a critical role in drug delivery systems and determines the physicochemical nature of the particle surface for effective delivery . However, some factors of nano - formulated drugs create challenges for batch - to - batch manufacturing, reproducibility, and safety . The following sections discuss the nanomedicines that have been explored in pca in vitro and in vivo, respectively . Animal imaging technology and advances in nanotechnology are providing new research opportunities in the preclinical and clinical development of nanosized drug carriers in cancer therapy . Nanoparticulate - based medicine has been expanding and has attracted significant interest in recent years, with a primary focus on efficient delivery systems for various chemotherapy drugs . The unique properties of nanomedicines are frequently used to improve the therapeutic value of various water - soluble and insoluble medicinal drugs and bioactive molecules by improving pharmacokinetics behavior, bioavailability, solubility, and retention time . Nanoencapsulation of chemotherapy drugs (nanomedicines) increases drug efficacy, specificity, tolerability, and the therapeutic index of corresponding drugs . Several disease - related drugs and bioactive molecules have been successfully encapsulated to improve bioavailability, bioactivity, and controlled delivery . Nanomedicines are being developed not only for cancer, but also in the area of acquired immunodeficiency syndrome, diabetes, malaria, prion disease, and tuberculosis . Hyaluronic acid has attracted interest for targeted delivery in cancer therapy mainly because of its specificity to bind cancer cells . Currently, cisplatin is used in various solid tumor treatments . However, cisplatin has dose - limiting side effects such as neurotoxicity, gastrointestinal disturbance, and nephrotoxicity that limit its clinical use . Np formulations have been developed for efficient delivery of cisplatin and targeting of pca stem cells by use of cd44 surface marker tagging with hyaluronic acid . With the use of a drug - induced ionic gelation method, cisplatin glyconanoparticles have been investigated as an efficient drug delivery system targeting pca stem cells . A proven concept for drug delivery applications in nanomedicine is the use of carriers such as nanovectors including liposomes, micelles, and dendrimers as well as metallic, ceramic, protein, and polymeric nps and carbon - based nanostructures . In particular, carbon nanotubes possess unique features for biomedical applications, including the possibility of chemical functionalization with drugs and their internalization by various cell types through endocytosis and passive diffusion across cellular membranes . Carbon nanotubes have been shown to efficiently transport and release various anticancer drugs . With the use of a carbon nanotube - based approach, the natural compound catechin was recently explored as a potential therapeutic nanomedicine for pca treatment . Catechin is in the family of polyphenolic flavonoids, and catechin and its derivatives (2)-epigallocatechin-3-gallate, (2)-epigallocatechin, (2)-epicatechin-3-gallate, and (2)-epicatechin inhibit cancer cell proliferation, suppress tumor growth and metastasis, and sensitize tumors to various chemotherapy drugs and radiotherapy . Recently, a study showed that biocompatible catechin - loaded and gelatin - conjugated carbon nanotubes have good anticancer properties with the potential for targeting prostate pca stem cells in combination with x - ray irradiation ., the semisynthetic chemotherapy drug known as docetaxel, which is used alone and in combination for various tumors, has limitations including poor aqueous solubility and low oral bioavailability . Moreover, docetaxel has intolerable side effects such as acute hypersensitivity reactions, fluid retention, neurotoxicity, and febrile neutropenia . To overcome these disadvantages of docetaxel, nanomedicine has been explored for passive and active targeting by enhancing permeability and retention (epr) effect, avoidance of the reticuloendothelial system, prolonged blood circulation time, controlled release of drug, and providing an opportunity for surface modification for active drug targeting . To enhance the permeability and bioavailability of docetaxel, one researcher used d--tocopheryl polyethylene glycol succinate (tpgs, or vit e tpgs), a food and drug administration - approved adjuvant for drug delivery, and showed that cyclic peptide (crgdfk) can be used to conjugate succinoyl tpgs nanomicelles for docetaxel delivery in a successful pca - targeted drug delivery system . Another interesting nanoparticulate system for cancer treatment is magnetic iron oxide np clusters, which have been shown as promising drug delivery vehicles for both diagnostic and therapeutic applications . Magnetic np clusters have been shown to be stable in a biological circulation system, to readily interact with cells or other biological units of interest, and to be capable of releasing the drug once the selected targeting is realized . In addition, the magnetic np clusters are biocompatible, have been proven to have no toxicity in vivo, and have shown efficient contrast in magnetic resonance imaging (mri). Magnetic np clusters are excellent near - infrared photothermal mediators in the combination of photothermal therapy and chemotherapy . Recently, doxorubicin (dox) when loaded into magnetic np clusters achieved greater pca cell cytotoxicity owing to both cluster - mediated photothermal ablation and cytotoxicity of light - triggered dox release . Compared with photothermal therapy or chemotherapy alone, the chemophotothermal therapy approach with dox in magnetic np clusters showed synergistically higher therapeutic efficacy both in vitro and in vivo for treatment of the pca cell line pc3 . Natural gum arabic glycol protein has recently been explored for homogeneous dose distribution and efficient emission . Researchers have reported that nps coated with gum arabic are stable for in vivo applications . One study showed that intralesional injection of gum arabic - coated radioactive gold nps resulted in minimal short - term systemic toxicity in a naturally occurring large animal model of pca . To achieve better therapeutic efficacy, the use of liposomal micelles to encapsulate dexamethasone was recently reported for sustained, antitumor effects versus free dexamethasone in metastatic bone disease from human pca . These studies showed that intravenously administered liposomes localized efficiently to bone metastases in vivo and that treatment of established bone metastases with (liposomal) dexamethasone resulted in a significant inhibition of tumor growth up to 26 days after the initiation of treatment . Furthermore, 1.0-mg / kg liposomal dexamethasone significantly outperformed 1.0-mg / kg free dexamethasone and was found to be well tolerated at clinically relevant dosages . Delivery of liposomal - loaded glucocorticoid dexamethasone was thus proposed for future clinical evaluation as a promising new treatment option for advanced, metastatic pca . In contrast to the overexpression of a membrane lectin, expression of the cation - independent mannose 6-phosphate receptor (m6pr) was specifically demonstrated in pca cell lines and tissues . M6pr was proposed as a new, efficient target and alternative biomarker to psma for nanomedicine applications . M6pr is a ubiquitous receptor involved in several biological functions, mainly in addressing enzymes to lysosomes . In one study, a mannose-6-phosphate analogue was synthesized and grafted onto the surface of functionalized mesoporous silica nps as a theranostic therapy for pca . Therefore, m6pr appears as a promising new target for nanomedicine applications in pca, particularly for noninvasive and personalized therapy of small - sized prostate tumors . In the area of novel drug inhibitors, histone deacetylase inhibitors (hdacis) moreover, the efficacy of the first - generation hdacis was limited in solid tumor indications owing to their suboptimal potency for specific hdac enzymes and transient induction of histone acetylation in tumor tissue . Considering the several drawbacks of hdacis, nanomedicine applications have been designed with novel np formulations of hdacis, vorinostat and quisinostat, which release the drug in a slow and controlled fashion . The nps vorinostat and quisinostat demonstrated higher therapeutic efficacy than small - molecule hdacis in chemoradiotherapy in murine models of pca . The chemotherapy drug docetaxel, a second - generation taxoid, has antimitotic chemotherapeutic properties and has been used for a variety of cancers, especially pca . In response to drawbacks such as docetaxel solubility, toxicity issues, and inaccurate tumor targeting, researchers have successfully fabricated docetaxel as a nanomedicine for sustained drug release by use of a copolymer - based poly(styrene)-b - poly(dl - lactide) (ps - pdlla) for intravenous injection . Moreover, improved inhibition of cancer cell growth was shown in the group treated with ps - pdlla / docetaxel compared with the control group . Decreased in vivo drug clearance, elevated systemic exposure, and prolonged circulation of the drug were verified in a pharmacokinetic study in rats, which further supports the potential use of nanomedicines in pca therapy . In a similar study, researchers developed a novel nanoplatform based on n-(2-hydroxypropyl) methacrylamide (hpma) copolymers that allows covalent bonding of two chemotherapeutics acting via different anticancer mechanisms and that can enter target cells by receptor - mediated endocytosis . In that study, dox and 5-fluorouracil (5-fu) were covalently conjugated to a nanosized hpma copolymer via a ph - sensitive hydrazone bond and an enzymatically degradable oligopeptide gly - phe - leu - gly sequence . Interestingly, these two drugs showed similar release profiles in vitro, suggesting synergistic effects in the co - delivery system . Studies in the cellular system showed that overexpression of galectin-3 in human pc3 prostate carcinoma cells treated with a combination of the drug p-(g3-c12)-dox - fu surprisingly exhibited comparable cytotoxicity to free dox at high concentrations by increasing cell internalization and exerting synergistic genotoxic effects of cell cycle arrest, caspase-3 activation, and dna damage . In mice bearing pc3 tumor xenografts, the use of tumor - targeting ligand substantially enhanced the intracellular delivery of p-(g3-c12)-dox - fu with no adverse effects . Thus, potential exists for synergistic combination therapy using targeted nanomedicines for efficient treatment of pca . Like other imaging modalities such as computed tomography, transurethral ultrasound, and nuclear imaging, mri cannot adequately detect small tumors . Improvements in tumor imaging technologies are urgently required for early detection of disease, staging, and realtime assessment of response to therapy in pca patients . Similar to other metal nanocarriers for nanomedicine, iron oxide magnetic nps have emerged as powerful contrast agents for mri . One research group developed iron oxide magnetic nps to enhance mri of pca with the psmatargeting antibody, j591, via a 1,2-distearoyl - sn - glycero-3-phosphoethanolamine - n-[amino(polyethylene glycol) (pegdspe) linkage . In this preclinical model, psma - targeted magnetic nps effectively enhanced mri of pca . Another interesting area of research on phototherapy is the quantum dot (qd) technique, and numerous studies have been done with qd - based fluorophores with high photostability for diagnostics and biomedical assays . Recently, researchers designed a new multiplexed diagnostic system based on qd - encoded beads for simultaneous detection of free and total psa for ultrasensitive, accurate, early pca diagnosis . In contrast to the nano - micelle area, other research groups have developed a carrier system for castrate - resistant pca by using raloxifene - loaded poly(styrene - co - maleic acid) (sma) micelles (sma - raloxifene) to improve the biodistribution of raloxifene and increase its anticancer efficacy . The effect of sma - raloxifene on apoptosis, reduction of cell proliferation, migration, and invasion of castration - resistant pca (crpc) cells was superior compared with free drug in vitro . In addition, sma - raloxifene induced changes in expression and localization of the estrogen receptors, as well as downstream effectors associated with cell proliferation and survival . This study examined the cellular internalization and efflux of sma - raloxifene compared with free raloxifene . The biodistribution and anticancer efficacy of sma - raloxifene were then compared with free raloxifene in a crpc mouse xenograft model . In the area of radiation therapy for prostate tumors, researchers have developed gold nps that can be targeted by using goserelin - conjugated gold nanorods . In this approach, megavoltage radiosensitization in vivo using tumor goserelin - targeted gold nps showed independent mechanisms of tumor accumulation in vivo (epr effect as a consequence of leaky tumor vasculature and active uptake by target cells). In combination with megavoltage radiation therapy, this led to delays in tumor growth in mice with heterotopic pca . More importantly, significant radiosensitization to megavoltage radiation was not observed with unconjugated gold nps despite their intratumoral concentration being only 3 folds less than that of conjugated gold nps . This suggests that radiosensitization is improved by active targeting that leads to cellular internalization of nps and the consequent increase in ionization density within the cytoplasm, rather than merely passive accumulation of nps in the perivascular space by epr . This report concluded that conjugation of nps to tumor - specific antigens, which promotes internalization within cancer cells, causes radiosensitization and that goserelin - conjugated gold nps can be effective pca radiosensitizers when used with megavoltage radiation . Another strategy being developed is to combine a chemotherapy drug and a natural drug as a hybrid nanomedicine for metastatic crpc . One such study used lipid - polymer hybrid nps to co - encapsulate dtx and curcumin (cur). In mice bearing pc3 tumor xenografts, the dtx - cur lipid - polymer nps synergistically inhibited tumor growth to a greater extent with no adverse effects . Nanomedicine offers great promise for dual drug delivery to pca cells, with the potential for synergistic combination therapy in summary, interesting nanomedicines have been developed for pca in recent years . These namomedicines need to be further evaluated in higher animals in terms of their safety and toxicity profiles . Gene therapy offers a solution to controlled and specific delivery of dna, rna, and protein to cells . Chitosan - based carriers are nonviral vectors that have gained popularity as a safe delivery system for gene materials including plasmid dna, oligonucleotides, and small inhibitory rna (sirna). Chitosan has beneficial qualities such as low toxicity, low immunogenicity, and excellent biocompatibility . Our group reported a preliminary investigation with sirna targeted to agr2 conjugated with chitosan to form nps . The results of scanning electron microscopy and gel electrophoresis showed efficient condensation of sirna for agr2 . To improve the transfection efficiency of the agr2 sirna, the complexes were tested in the human pc3 prostate cell line, which overexpresses agr2 protein . In pc3 cells, the chitosan and sirna agr2 complex was found to be efficient and nontoxic to overall cells by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay . Thus, chitosan nps have been shown to be a safe vector system for efficient gene delivery in cancer . Our study proposed further development of chitosan - based formulations for the delivery of agr2 sirna for clinical applications, which may represent a promising approach in pca treatment . Interestingly, another delivery strategy for sirna in a vector involved coupling stearic acid to the n - terminus of hhhpkpkrkv . Str - hk formed stable complexes with sirna via noncovalent interactions and mediated efficient delivery of sirna to pc3 cells with minimal cytotoxicity compared with a gene - silencing chemical transfection agent like lipofectamine . These results proved that str - hk is a promising vector for sirna delivery . In a similar fashion, research also demonstrated that intratumoral injections of sirnas loaded on biodegradable chitosan nps led to a downregulation of rxfp1 receptor expression and a dramatic reduction in tumor growth . In the xenograft models treated with sirna against rxfp1, the smaller tumor size was associated with decreased cell proliferation and increased apoptosis, which suggests that the suppression of rln / rxfp1 might have potential therapeutic benefits in pca . Another interesting report showed that dicer complex of sirna (dsirna), when delivered using a structurally flexible triethanolamine - core poly(amidoamine) dendrimer as the nanocarrier, gives rise to a much greater rnai response than that with conventional sirna . Dendrimer / dsirna complexes with a dual targeting peptide simultaneously promoted cancer cell targeting by interacting with integrins and cell penetration via the interaction with neuropilin-1 receptors, which led to improved gene silencing and anticancer activity . The role of nanomedicines and nanogene delivery in theranostics approaches for pca are summarized in fig . This review has highlighted nanomedicine and nanogene therapy research in association with nanotechnology concepts for modern medicine, especially for improving the therapeutic outcomes of cancer . As the technology is perfected, nanomedicines can be used in the future in benign prostatic hyperplasia at the level of expression and location of proteins . As image - guiding techniques such as magnetic iron oxide nps (fe3o4 nps) are perfected, image - guided radiation therapy can be used for pca . Moreover, application of nanotechnology to extraperitoneal laparoscopic radical prostatectomy can enable patients to have a better quality of life and improve the survival rate in the near future.
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The replacement of tissue lost through trauma, disease, or congenital anomalies is a continuing clinical challenge . Current reconstructive options, including autologous tissue transfer, allograft, xenograft, and alloplastic implantation, are limited by donor site morbidity, tissue scarcity, disease transmission or antigenic incompatibility, hardware infection, and implant extrusion . Ideally, tissue reconstruction should avoid sacrificing healthy tissue or using alloplastic materials by instead engineering autologous replacement tissue de novo . Tissue engineers have successfully cultured the cellular constituents necessary to build a variety of tissue types in vitro . However, traditional two - dimensional (2d) cell - culture techniques (e.g., petri dishes and culture flasks) are inadequate for three - dimensional (3d) tissue engineering . In 2d culture, a monolayer of cells is in continuous contact with culture medium, and simple diffusion is sufficient to maintain cell viability . As scaffolds gain 3d volume, however, the central core becomes increasingly separated from the penumbra of fresh medium; simple diffusion provides inadequate oxygen delivery and waste removal from cells in the core . As a result, only cells in a thin construct (with a large surface area - to - volume ratio) survive, and typically only on the peripheral crust of the scaffold (up to 2 mm deep). Nature has addressed this problem in native bone by establishing a complex lacunocanalicular network within which a nutrient - rich fluid circulates [4, 5]. Thus, successful engineering of thick 3d osseous tissue constructs large enough to solve actual clinical problems will require novel tissue - engineering strategies that address chemotransportative requirements in their design and implementation . For example, cell - seeded porous scaffolds have been set upon orbital shakers, hung in spinner flasks [6, 7], continuously perfused through glass columns, or tumbled in rotational bioreactors [6, 9, 10]. These methods increase medium fluid flow across the external surface of the scaffold, offering an incremental improvement over traditional static culture techniques . While these technologies satisfy the external requirement for medium flow, convection of medium at the external surface does not guarantee chemotransportation within the porous confines of the scaffold interior . In fact, the majority of medium in the systems described above follow the path of least resistance and circumnavigate the scaffold (figure 1(a)). Consequently, convection of medium alone does not result in penetrating flow that perfuses the porous construct to provide effective chemotransportation . In contrast to technologies like the spinner flask, we designed a flow - perfusion bioreactor to address the internal requirement for flow within the porous network of the scaffold (figures 1(b) and 1(c)). In this system, porous scaffolds are press fit into an experimental chamber, and medium flows by gravity head or by generated hydrostatic pressure through the scaffold . Because the fluid path is confined to pass through the scaffold the flow - perfusion bioreactor promises improved chemotransportation to all regions of a 3d scaffold . Furthermore, the flow - perfusion bioreactor is, in theory, a scalable technology that should support porous scaffolds of any thickness . Nevertheless, to date, most exogenous tissue - engineering research has been constrained to using scaffolds 2 mm in thickness or less, which are readily sustainable by medium convection or static culture methods . Therefore, we evaluated the efficacy of a novel flow - perfusion bioreactor in sustaining thick specifically, as proof of principle, we tested cylindrical scaffolds measuring 24 mm in diameter and 6 mm in thickness . Since these scaffolds are thicker than the critical depth of 2 mm (from core to surface), without effective fluid flow, they should suffer central core necrosis . We hypothesized that dynamic cell culture with a flow - perfusion bioreactor will provide adequate chemotransportation to the core of a thick scaffold, thereby, maintaining cell viability and activity . The flow - perfusion bioreactor was machined from solid teflon (sabic polymershape; jacksonville, fl). It contains 8 independent experimental chambers each measuring 24 mm in diameter and able to accommodate scaffolds up to 10 mm in thickness (figures 1(c) and 1(d)). The floor of each experimental chamber is tapered to ensure flow from the outer edges of the scaffold as well as the center to the exit port of the chamber (figure 1(b)). Screw caps are fitted with viton-75 o - rings (mcmaster - carr; aurora, oh) to ensure a tight seal and prevent leakage . The bioreactor rests upon an 8-chamber medium reservoir, with each experimental chamber directly overlying its respective medium chamber . The junction between the bioreactor and reservoir is sealed by a gas - permeable membrane (tegaderm; 3 m; st . Paul, mn). An 8-channel peristaltic roller pump (manostat - carter; barnant co.; barrington, il) draws medium from the reservoir and administers it to each experimental chamber via 0.89 mm i d platinum - cured silicone tubing (cole - parmer; vernon - hills, il). Equipment was sterilized by plasma - phase hydrogen peroxide (sterrad) processing (bioreactor, screw caps, and reservoir) and steam autoclave (tubing). The entire bioreactor was placed in a standard cell - culture incubator (37c, 95% humidified air, 5% co2). 24 6 mm cylindrical polyurethane scaffolds (biomerix; somerset, nj) with an average pore size of 200 m and 100% pore interconnectivity were used in all experiments . Scaffolds were sterilized by ethylene - oxide gas sterilization by the manufacturer and sealed in single - use packets . Mc3t3-e1 murine preosteoblastic cells (riken cell bank; ibaraki, japan) were expanded by traditional 2d static culture at 37c and 5% co2 . Louis, mo) supplemented with 10% fbs (gibco invitrogen, carlsbad, ca) was used for this and all subsequent experiments . At confluence, cells were lifted using trypsin, resuspended with the same medium, and used to seed the scaffolds . Each scaffold was placed in a separate well of a 6-well tissue - culture plate for seeding . Due their hydrophobic nature, the scaffolds were compressed to allow sponge - like absorption of the cellular suspension upon release of the compressing force . The scaffolds were seeded at a standard concentration of 4 10 cells / cm (scaffolds processed for histology 12 hours after seeding confirmed even distribution of cells throughout the scaffold). After seeding, the scaffolds were surrounded by medium (to prevent desiccation) and placed in the cell - culture incubator overnight to allow cellular adherence . After 24 hours in static culture, cell - seeded scaffolds were either place in flow - perfusion culture or continued in static culture . For static culture controls, seeded scaffolds were maintained in 6-well tissue culture plates with enough media to cover the scaffold in its entirety (10 ml). Medium was changed every other day to remove waste products of cell metabolism and provide fresh growth supplements (this protocol was selected following optimization experiments in which static culture of scaffolds in larger volumes of media, such as that used in flow - perfusion, without media changes led to accumulation of waste products and lack of growth supplements in the vicinity of the scaffold), but otherwise the plates were left undisturbed in the cell - culture incubator . Scaffolds were harvested at days 0, 2, 4, 6, and 8 (n = 3 per time point). For dynamic culture, scaffolds were loaded into the bioreactor experimental chambers . Each reservoir chamber was loaded with 80 ml of fresh medium, which was recycled for the duration of the experiment (maximum 8 days) at a rate of 1.0 ml / min as it entered the experimental chamber . This rate was sufficient to provide perfusion and permit chemotransportation, but only generated a fluid shear stress of approximately 0.02 dynes / cm . The fluid flow rate was based upon optimization studies in which three - dimensional finite element fluid mechanics and mass transport models were developed (data not shown). Scaffolds were harvested at days 0, 2, 4, 6, and 8 (n = 3 per time point). Sections were viewed on an olympus bx51 microscope (olympus; center valley, pa). The depth below the scaffold edge was measured using an objective micrometer (olympus). Sections representing the periphery (top third and bottom third) and core (middle third) of the scaffold were reviewed . The number of cells per 4 low - power field (lpf) was counted (3 nonconsecutive sections for each region at each time point) by two blinded investigators . An mtt (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay was used (sigma) to measure cellular activity in the scaffolds . In this assay, the conversion of yellow mtt to a purple formazan crystal by viable, metabolically active cells is measured using spectrophotometry . Scaffolds were harvested from static and flow - perfusion culture at 1, 2, and 6 days, washed in phosphate - buffered saline (pbs), and homogenized . Each homogenate was subjected to 6 ml of 0.5 mg / ml mtt (in pbs) solution and allowed to incubate for 4 hours at 37c . Excess mtt solution was then decanted and 5 ml of extraction solution (5 ml isopranolol containing 0.01 n hcl) was added to the homogenate and allowed to incubate at 20 minutes at 37c . 100 l aliquots of the resulting supernatant were then added to a 96-well plate and the absorbance at 570 nm was determined using spectrophotometry . Cell activity was then expressed as the absorbance at 570 nm per gram of scaffold . Data were analyzed with a two - tailed student's t - test assuming unequal variance using sigmastat (spss science; chicago, il). Scaffolds harvested 12 hours after seeding demonstrated a homogenous distribution of cells in pores throughout the scaffold (figure 2). At this early time point, cell density in the periphery (109.3 5.5 cells / lpf) and core (106.8 3.9 cells / lpf) was not significantly different (p = .67). Scaffolds in static culture had a slow, but significant decline in the peripheral cell density over the course of the experiment (figure 3). Compared to the initial seeding density (109.3 5.5 cells / lpf), there were 106.7 2.0 cells / lpf (97.6% of the initially seeded cells) at day 2 (p = .4), 82.3 3.8 cells / lpf (75.2%) at day 4 (p = .003), 76.3 5.2 cells / lpf (69.8%) at day 6 (p = .004), and 72.3 1.2 cells / lpf (66.1%) at day 8 (p = .001). Cell density in the periphery was similar to a depth of approximately 2 mm from the superior and inferior surface of the scaffold . There was no statistical difference in cell density between superior and inferior portions of the periphery at any time point (data not shown). In marked contrast to the peripheral crust (outer 2 mm) of the scaffold, the core (central 2 mm) of these same scaffolds exhibited a rapid decline in cell density (figure 4(a)). Within the core, cell density was 70.3 5.5 cells / lpf (64.3% remaining of the initially seeded cells) at day 2 (p = .003), 39.0 4.4 (35.7%) at day 4 (p = .001), 4.67 1.8 (4.2%) at day 6 (p <.001), and 0.25 0.25 (0.2%) at day 8 (p <.001). The cusp of viability for cells in the core was day 4; between days 4 and 6, there was a dramatic 88% reduction (p = .002) in cell density (figure 4(b)). The metabolic activity of cells in static culture also declined over the course of the experiment (figure 5). Mtt cell assay demonstrated that cellular activity in scaffolds in static culture declined to 74% of initial activity (0.39 0.04 od / g) at day 2 and 70% of initial activity (0.37 0.04 od / g) at day 6 . The change in metabolic activity was significant by day 6 (p = .025). In static culture, metabolically active cells were limited to the periphery of the scaffold . Similar to static culture, scaffolds placed in flow - perfusion had a peripheral cell density of 108.0 5.9 cells / lpf (98.9% of the initially seeded cells) at day 2, 99.3 6.1 (90.9%) at day 4, 92.7 1.8 (84.9%) at day 6, and 100.0 1.5 (91.5%) at day 8 (figure 3). Pairwise comparisons demonstrated a significantly greater number of cells in the periphery of scaffolds placed in flow - perfusion on days 6 (p = .04) and 8 (p = .0001). Similar to static culture samples, there was no difference in cellular density between the inferior and superior thirds of the scaffold (data not shown). In marked contrast to static culture, scaffolds placed in flow - perfusion maintained significantly higher cell density within the core of the scaffold (figures 6(a) and 6(b)). Core cell density was 103.7 2.3 cells / lpf (94.9% of the initially seeded cells) at day 2, 80 5.5 cells / lpf (73.2%) at day 4, 83.5 1.5 cells / lpf (76.4%) at day 6, and 81.7 6.4 cells / lpf (74.7%) at day 8 (figure 4(a)). The differences in core cell density between static and flow - perfusion culture were statistically significant at days 2 (p = .01), 4 (p <.001), 6 (p = .006), and 8 (p = .002). The metabolic activity of cells in flow - perfusion culture significantly increased over the course of the experiment (figure 5). Mtt cell assay demonstrated that cellular activity in scaffolds in dynamic culture increased 125% (0.66 0.03 od / g) at day 2 and 285% (0.37 0.04 od / g) at day 6 compared to the initial activity . The increase in metabolic activity in flow - perfusion was not only significantly greater than the initial activity (day 2, p = .035; day 6, p = .018), but it was also significantly greater than the activity in static culture at similar time (day 2, p = .002; day 6, p = .006). To account for the possibility of confounding shear stress (despite subthreshold mechanotransductive flow) or differences in cell distribution in static versus dynamic culture, the core: periphery ratio was calculated (figure 6(c)). In static culture, the ratio was 0.66 0.05 at day 2, 0.48 0.07 at day 4, 0.06 0.02 at day 6, and 0.004 0.004 at day 8 . In contrast, flow perfusion maintained the core: periphery ratio to 0.97 0.07 at day 2, 0.82 0.10 at day 4, 0.61 0.30 at day 6, and 0.81 0.07 at day 8 . The differences in core: periphery ratio of static versus dynamic were statistically significant: day 2 (p = .004), day 4 (p = .005), day 6 (p = .01), and day 8 (p = .004). The ability to maintain cell viability in vitro in thick 3d scaffolds has important implications for tissue engineering . For example, attempts at 3d in vitro bone culture without adequate chemotransportation have invariably shown an inverse relationship between construct thickness and cellular survival [2, 12]. This inverse relationship is due to a decline in nutrients and accumulation of waste products in the core of the construct during bone matrix deposition and mineralization . Traditional cell - culture methods are not well suited to the maintenance of 3d tissue - engineered constructs due to the inherent limitation in chemotransportation . In our study, we demonstrated that, under static culture conditions, cells in the peripheral crust up to 2 mm deep from the external surface were able to survive by static diffusion, a finding that is in agreement with other published accounts describing survival to a depth of 1 - 2 mm [3, 13]. However, we noted a linear decline in core cell density over time, with less than 5% of cells remaining after 6 days of static culture . These observed temporospatial differences in cell density with static culture may be explained by two phenomena associated with poor nutrient diffusion: (1) death of cells in the interior core of the scaffold, and (2) chemotaxis of cells from the core toward the periphery . In an effort to support 3d tissue - engineered constructs, various bioreactor systems have been designed, including the spinner flask and the rotational bioreactor . However, because these designs simply move fluid across the exterior of the scaffold, chemotransportation to the interior is not guaranteed . The flow - perfusion bioreactor differs in that it ensures nutrient transport by perfusing medium through the interconnected pores of the scaffold . Moreover, the design allows the investigator to control mechanical forces; the pump speed may be set from 3.5 to 200 revolutions per minute for perfusion ranging from 0.26 to 14.8 mls / min, replicating shear stress from 0.0110 dynes / cm - physiologic in vivo range for osteocytes (830 dynes / cm). We found that dynamic culture using a flow perfusion bioreactor significantly improved core cell activity and density compared to static culture . After a slight initial decrease in cell density between days 2 and 4, core cell density in flow perfusion was maintained throughout the course of the experiment at approximately 80% of the initial seeding density . Likewise, the core: periphery ratio, which compensates for differences in cell distribution and potential effects of fluid shear stress between culture systems, was steady (approximately 0.60.8) from day 2 to day 8 . While originally designed to improve chemotransportation, the flow - perfusion bioreactor has the ability to generate fluid - shear forces at the cellular level at higher rates of medium flow . Although fluid shear forces were intentionally kept subphysiologic (i.e., less than 8 dynes / cm), to reduce / eliminate the effects mechanical stimulation during this study (to concentrate on the effect of chemotransportation, and not mechanical stimulation), other studies have suggested that fluid shear forces may be useful for bone or vascular tissue engineering [11, 16, 17]. Ultimately, in order to solve clinical problems, engineered bony tissue must be fashioned into 3d patient - specific sizes and shapes . Custom - printed scaffolds (e.g., those designed from actual patient computed tomography data) are too thick to survive by nonpenetrating chemotransportation . However, the flow - perfusion bioreactor is in theory a scalable technology that should support porous scaffolds of any thickness . This study demonstrates that thick (> 6 mm) 3d constructs are sustainable using a flow - perfusion bioreactor . Future work will concentrate on computational modeling of the fluid dynamics and mass transport using specific scaffold designs, and on analysis of cellular proliferation, differentiation, and organization within the construct.
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One of the earliest controlled ethological studies of risk - taking began in the experimental ponds at cornell university, where david sloan wilson and his colleagues began searching for different personality types in the pumpkinseed sunfish (lepomis gibbosus). Based on work in humans, wilson and his team hypothesized that risk - taking sunfish would be especially likely to explore novel objects and novel environments.3, 4 they collected fish from a pond with a series of funnel - shaped traps designed so that if a fish swam into one, it was very hard to swim back out . Presumably, risk - taking fish would be more likely to explore such a novel trap, and the researchers would catch more risk - taking fish than risk - averse fish . The researchers also dragged a large net through the pond and scooped up all the fish they could, both bold and shy . If wilson and his team were correct, the fish from the funnel traps would be bolder on average than those from the netted samples . For example, stomach - content analysis indicated that fish caught in the funnel traps had been eating more often and in areas of the pond that were the least safe the sorts of behaviors that we might expect from risk - taking individuals . Funnel - trapped fish also had many more parasites than did netted fish, suggesting that they explored many areas of their pond and thus exposed themselves to myriad parasites . When funnel - trapped and netted fish were marked individually with colored beads and released back into their pond, funnel - trapped fish were much more likely to be found leaving their group and foraging on their own than were net - trapped fish . And, finally, when pumpkinseed fish were brought into the laboratory and exposed to a novel object there, funnel - trapped individuals were much more likely to investigate the object . These results suggest that, just as in humans, a continuum of risk - taking propensities exists in nonhumans . Since i was in graduate school, i have been fascinatedobsessed might be a better word with what is called predator inspection behavior in the guppy (poecilia reticulata). One or a few fish break away from the safety of their group and slowly approach a potential predator to gather information about its possible danger . At first, my interest in predator inspection focused on the cooperative nature of the ways that pairs of fish inspected ways that matched predictions from economic models of cooperation . But then i realized that predator inspection would also allow me to measure the costs and benefits of risk - taking . After all, i was finding that some fish were consistently willing to take the risk of inspecting predators, while other guppies consistently avoided such risky behaviors . What were the underlying costs and benefits that drove the evolution of risk - taking in this system? The cost of predation inspection was fairly easy to measure and arguably intuitive . If you take risks in the presence of a predator, you get eaten more often and indeed, controlled experimental work, in which groups with different numbers of risk - takers were monitored, confirmed that risk - takers are more likely to end up in the guts of predators.5 measuring the benefits of predator inspection behavior proved to be more conceptually challenging . What were the guppies getting out of these risky endeavors? For natural selection to favor any amount of predator inspection behavior, there must be compensating benefits for undertaking this risky behavior, but what are they? Much research had already demonstrated that female guppies generally preferr more colorful males as mates . What we were seeing was that more colorful males also tended to be risk - takers.6 we ran a series of experiments designed to see if it was a male s color, his risk - taking tendencies, or both that made him attractive as a potential mate to females . When we experimentally decoupled boldness and color (using a nifty experimental device that a former engineer at nasa built for us), female preference was most directly linked to risk - taking behavior in males . Why females prefer bolder males is still not understood, but it may be that boldness is a signal of genetic quality . What does seem clear is that natural selection has favored males that temper their risk - taking behavior as a function of the benefits available: colorful males inspect predators far less often when females are not watching them than when females are observing what they do in the presence of a predator . Attraction to the opposite sex may not be the only good thing to come from risky bouts of predator inspection . My colleague michael alfieri and i also found that risk - takers were better at learning associative - memory tasks than were risk - averse fish . We measured a guppy s tendency to inspect predators and, in a separate test, its ability to pair food with an arbitrary cue . Bolder guppies learned this associative - learning task more quickly than did their shyer group mates.7 but there is an interesting catch here: bold fish were better learners only after they had recently expressed their boldness . Over the last two decades, peter drent and his colleagues have been studying risk - taking in the great tit bird (parus major). Fast birds explore new environments with rapid - fire speed and are quite aggressive . Slow birds show the opposite behavior pattern . In a clever experiment involving tutors, the researchers also found that fast birds would switch habitats when a so - called tutor passed on information that a new habitat was profitable, but slow birds, which spent more time focused on their environment and less on the tutors, paid less attention to tutors.8 drent and his team followed birds in natural populations with an eye toward studying the mating patterns of fast and slow birds . They found that fast birds that mate with fast birds and slow birds that mate with slow birds are the pairings that produce the most offspring.9 these researchers have even gone so far as to create experimental lines of great tit birds, by mating the fastest birds with the fastest and the slowest with the slowest . They then examine how genetics and development interact to shape personality.10 this work, in conjunction with other studies, shows a clear genetic basis to risk - taking personality traits in this species . Including the orangutans at the opening of this piece, we have looked at personality in mammals, fish, and birds . But some researchers have argued, and rightly so, that this is a very vertebrate - centered view of personality, and that invertebrates may also have much to teach us about the evolution of risk - taking and personality . Work in this area is scant, but studies on red octopuses (octopus rubescens) and dumpling squid (euprymna tasmanica) have uncovered evidence that they, too, show a bold shy continuum.11, 12 one especially appealing aspect of future work in invertebrates is that the nervous system of cephalopods, like octopuses and squids, is both well understood and easy to study (cephalopods are famous in neurobiology for their giant neurons), thus allowing potential insight into the underlying neurophysiology of risk - taking . The study of risk - taking behavior has practical, as well as conceptual, value . For example, conservation biologists have had some success at reintroducing large carnivores, like wolves, into their native habitats . Though these programs should be applauded, they sometimes have unexpected negative consequences, such as the rekindling of old rivalries between the conservation biologists implementing these programs and ranchers who keep domesticated animals on which the reintroduced large carnivores feed . There is mounting evidence that much of the hunting of domesticated animals by wild carnivores is done by a small number of problem individualsthat is, the same individuals repeatedly attack and kill ranchers livestock . Studies of hunting behavior in wolves, cougars, leopards, seals, lions, tigers, bears, and other species all hint at the presence of such repeat offenders . Attacking ranchers livestock carries high risk: a predator must first circumvent any fencing or other defensive measures put into place and then risk being shot by ranchers . Predators that consistently attempt to attack such livestock display many of the personality traits associated with boldness.13 suppose we stop thinking in terms of repeat offenders and instead focus on measurable characteristics like boldness . We might examine whether bold predators use particular paths to reach prey, hunt at particular times, or are more or less attracted to certain stimuli than risk - averse predators are . Problem individuals, perhaps wildlife workers could build traps tailored to the hunting strategy of bold predators . Understanding the science of risk - taking might help spare the lives of these carnivores, while at the same time helping ranchers protect their stock . Another practical application of work on animal personalities centers on the use of animals to aid disabled people . To become a guide for blind people, for example, dogs must pass through a series of tests, and the factor that eliminates the most dogs from the pool of possible guides is fear . Some dogs are just more frightened by novel things in their environment, and they are less willing to take risks to help the person they are guiding . Based on this phenomenon, researchers have developed a scoring system for fear and fearlessness and implemented it across dog breeds to find the breed most likely to aid blind people . We ve made some progress, but much work remains to be done on the evolution of boldness in animals . We need a better understanding of the molecular genetics and the underlying neurobiology and endocrinology of this trait . We need mathematical models of risk - taking; these models are only just emerging in the growing animal literature on behavioral syndromes . We need a deeper understanding of the phylogenetic that is, evolutionary history of boldness . Almost nothing is known about this, though alexander weiss has some fascinating ideas about the relationship between the evolution of personality traits in humans and that of our closest living relatives.14 i m optimistic on all fronts . The more species we investigate, the more costs and benefits we measure, the more theory we develop, and the more new tools we develop to study the genetics, neurobiology, and hormonal underpinnings of boldness, the better we will understand boldness in nonhumans and, eventually, humans.
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It is known since the work of bardeen, bekenstein, carter and hawking [42, 32, 162] that black holes are thermodynamical systems equipped with a temperature and an entropy . In analogy to bolzmann s statistical theory of gases, one expects that the entropy of black holes counts microscopic degrees of freedom . Understanding what these degrees of freedom actually are is one of the main challenges that a theory of quantum gravity should address . Since the advent of string theory, many black holes enjoying supersymmetry have been understood microscopically . In many cases, supersymmetry and its non - renormalization theorems allow one to map the black - hole states to dual states in a weakly - coupled description, which also provides a method to microscopically reproduce hawking radiation; see [253, 60] and subsequent work . For all supersymmetric black holes that contain in their near - horizon limit a factor of three - dimensional anti - de sitter spacetime ads3 or a quotient thereof, a simpler microscopic model is available . Since quantum gravity in asymptotically ads3 geometries is described by a two - dimensional conformal field theory (2d cft) [58, 251], one can account for the entropy and the hawking radiation of these supersymmetric or nearly supersymmetric black holes using only the universal properties of a dual cft description defined in the near - horizon region [209, 104] (for reviews, see [155, 113]). Ultraviolet completions of these ads / cft correspondences can be constructed using string theory [205, 265]. These results can be contrasted with the challenge of describing astrophysical black holes that are non - supersymmetric and non - extremal, for which these methods cannot be directly applied . Therefore, the main physical focus should be to understand the microstates of the kerr black hole and to a smaller extent the microstates of the schwarzschild, the kerr - newman and the reissner - nordstrm black hole . Recently, considerable progress has been made in reproducing the entropy of the kerr black hole as well as reproducing part of its gravitational dynamics using dual field theories that share many properties with two - dimensional cfts [156, 53, 68] (see also).1 the kerr / cft correspondence will be the main focus of this review . Indeed, it turns out that the ideas underlying the correspondence apply as well to a large class of black holes in supergravity (in four and higher dimensions) independently of the asymptotic region (asymptotically - flat, anti - de sitter) far from the black hole . These extensions of the kerr / cft correspondence only essentially require the presence of a u(1) axial symmetry associated with angular momentum . It is important to state that at present the kerr / cft correspondence and its extensions are most understood for extremal and near - extremal black holes . Only sparse but non - trivial clues point to a cft description of black holes away from extremality [104, 68, 107]. Before jumping into the theory of black holes, it is important to note at the outset that rotating extremal black holes might be of astrophysical relevance . Assuming exactly zero electromagnetic charge, the bound on the kerr angular momentum derived from the cosmic - censorship hypothesis is j gm . No physical process exists that would turn a non - extremal black hole into an extremal one . Using details of the accretion disk around the kerr black hole, thorne derived the bound j 0.998 gm assuming that only reasonable matter can fall into the black hole . Quite surprisingly, it has been claimed that several known astrophysical black holes, such as the black holes in the x - ray binary grs 1905 + 105 and cygnus x-1, are more than 95% close to the extremality bound . Also, the spin - to - mass - square ratio of the supermassive black holes in the active galactic nuclei mcg-6 - 30 - 15 and 1h 0707 - 495 has been claimed to be around 98% . However, these measurements are subject to controversy since independent data analyses based on different assumptions led to opposite results as reviewed in: the spin - to - mass - square ratio of the very same black hole in the x - ray binary grs 1905 + 105 has been evaluated as j/(gm) = 0.15, while the spin of the black hole in cygnus x-1 has been evaluated as j/(gm) = 0.05 . If the measurements of high angular momenta are confirmed or if precise measurements of other nearby highly - spinning black holes can be performed, it would promote extremal black holes as nearly physical objects of nature . In this review, we will present a derivation of the arguments underlying the kerr / cft correspondence and its extensions starting from first - principles . For that purpose, it will be sufficient to follow an effective field theory approach based solely on gravity and quantum field theory . In particular, we will not need any detail of the ultraviolet completions of quantum gravity except for one assumption (see section 1.1 for a description of the precise classes of gravitational theories under study). We will assume that the u(1) electromagnetic field can be promoted to be a kaluza - klein vector of a higher - dimensional spacetime (see section 1.2 for some elementary justifications and elaborations on this assumption). If this assumption is correct, it turns out that the kerr / cft correspondence can be further generalized using the u(1) electric charge as a key quantity instead of the u(1) angular momentum . We will use this assumption as a guiding principle to draw parallels between the physics of static charged black holes and rotating black holes . Our point of view is that a proper understanding of the concepts behind the kerr / cft correspondence is facilitated by studying in parallel static - charged black holes and rotating black holes . Since extremal black holes are the key objects of study, we will spend a large amount of time describing their properties in section 2 . We will contrast the properties of static extremal black holes and of rotating extremal black holes . We will discuss how one can decouple the near - horizon region from the exterior region . We will then show that one can associate thermodynamical properties with any extremal black hole and we will argue that near - horizon geometries contain no local bulk dynamics . Since we aim at drawing parallels between black holes and two - dimensional cfts, we will quickly review some of their most relevant properties for our concerns in section 3 . After this introductory material, we will discuss the core of the kerr / cft correspondence starting from the microscopic counting of the entropy of extremal black holes in section 4 . There, we will show how the near - horizon region admits a set of symmetries at its boundary, which form a virasoro algebra . Several choices of boundary conditions exist, where the algebra extends a different compact u(1) symmetry of the black hole . Following semi - classical quantization rules, the operators, which define quantum gravity in the near - horizon region, form a representation of the virasoro algebra . We will then argue that near - horizon quantum states can be identified with those of a chiral half of a two - dimensional cft . This thesis will turn out to be consistent with the description of non - extremal black holes . The thermodynamical potential associated with the u(1) symmetry will then be interpreted as the temperature of the density matrix dual to the black hole . The entropy of the black hole will finally be reproduced from the asymptotic growth of states in each chiral half of these cfts via cardy s formula . In section 5 we will move to the description of non - extremal black holes, and we will concentrate our analysis on asymptotically - flat black holes for simplicity . We will describe how part of the dynamics of probe fields in the near - extremal kerr - newman black hole can be reproduced by correlators in a family of dual cfts with both a left and a right - moving sector . The left - moving sector of the cfts will match with the corresponding chiral limit of the cfts derived at extremality . In section 6 we will review the hidden local conformal symmetry that is present in some probes around the generic kerr - newman black hole . We will also infer from the breaking of this conformal symmetry that the kerr - newman black hole entropy can be mapped to states of these cfts at specific left and right - moving temperatures . Finally, we will summarize the key results of the kerr / cft correspondence in section 7 and provide a list of open problems . This review complements the lectures on the kerr black hole presented in by providing an overview of the kerr / cft correspondence and its extensions for general rotating or charged black holes in gravity coupled to matter fields in a larger context . Since we follow an effective field - theory approach, we will cover string - theory models of black holes only marginally . We refer the interested reader to the complementary string theory - oriented review of extremal black holes .2 the kerr / cft correspondence is an effective description of rotating black holes with an infrared cft . Embedding this correspondence in string theory has the potential to give important clues on the nature of the dual field theory . Efforts in that direction include [225, 22, 157, 98, 23, 116, 31, 243, 246, 115, 130]. However, the details of particular cfts are irrelevant for the description of astrophysical black holes, as long as we do nt have a reasonable control of all realistic embeddings of the standard model of particle physics and cosmology in string theory . Despite active research in this area, see, e.g., [153, 117, 217], a precise description of how our universe fits in to the landscape of string theory is currently out - of - reach . In an effective field theory approach, one concentrates on long - range interactions, which are described by the physical einstein - maxwell theory . However, it is instructive in testing ideas about quantum gravity models of black holes to embed our familiar einstein - maxwell theory into the larger framework of supergravity and study the generic properties of rotating black holes as toy models for a physical string embedding of the kerr - newman black hole . Another independent motivation comes from the ads / cft correspondence [205, 265]. Black holes in anti - de sitter (ads) spacetime in d + 1 dimensions can be mapped to thermal states in a dual cft or cft in d dimensions . Studying ads black holes then amounts to describing the dynamics of the dual strongly - coupled cft in the thermal regime . Since this is an important topic, we will discuss in this review the ads generalizations of the kerr / cft correspondence as well . How the kerr / cft correspondence fits precisely in the ads / cft correspondence in an important open question that will be discussed briefly in section 7.2 . In this review we will consider the following class of four - dimensional theories, 1\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} {s = {1 \over {16\pi g}}\int {{d^d}} x\sqrt {- g} \left({r - {1 \over 2}{f_{ab}}(\chi){\partial _ \mu}{\chi ^a}{\partial ^\mu}{\chi ^b} - v(\chi) - {k_{ij}}(\chi)f_{\mu \nu}^i{f^{j\mu \nu}}} \right .} \\ we focus on the case where fab() and kij() are positive definite and the scalar potential v() is non - positive in (1). This ensures that matter obeys the usual energy conditions and it covers the case of zero and negative cosmological constant . Some theories of interest contained in the general class (1) are the einstein - maxwell gravity with negative or zero cosmological constant and the bosonic sector of \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}${\mathcal n} = 8$\end{document} supergravity . Note that the phenomenology described by the action (1) is limited by the absence of charged scalars, massive vectors, non - abelian gauge fields and fermions . The explicit form of the most general single - center spinning - black - hole solution of the theory (1) is not known;however, see [270, 221] for general anstze . For einstein and einstein - maxwell theory, the solutions are, of course, the kerr and kerr - newman geometries that were derived about 45 years after the birth of general relativity . For many theories of theoretical interest, e.g., \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}${\mathcal n} = 8$\end{document} supergravity, the explicit form of the spinning - black - hole solution is not known, even in a specific u - duality frame (see, e.g., and references therein). However, as we will discuss in section 2.3, the solution at extremality greatly simplifies in the near - horizon limit due to additional symmetries and takes a universal form for any theory in the class (1). It is for this reason mainly that we find convenient to discuss theory (1) in one swoop . Since the work of kaluza and klein, one can conceive that our u(1) electromagnetic gauge field could originate from a kaluza - klein vector of a higher - dimensional spacetime of the form \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}${{\mathcal m}_4} \times x$\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 m}_4}$\end{document} is our spacetime, x is compact and contains at least a u(1) cycle (the total manifold might not necessarily be a direct product). Experimental constraints on such scenarios can be set from bounds on the deviation of newton s law at small scales [197, 2]. If our u(1) electromagnetic gauge field can be understood as a kaluza - klein vector, it turns out that it is possible to account for the entropy of the reissner - nordstrm black hole in essentially the same way as for the kerr black hole . This mainly follows from the fact that the electric charge becomes an angular momentum j2 = q in the higher - dimensional spacetime, which is on the same footing as the four - dimensional angular momentum j1 = j lifted in the higher - dimensional spacetime . Assumption we will assume throughout this review that the u(1) electromagnetic gauge field can be promoted as a kaluza - klein vector . As far as the logic goes, this assumption will not be required for any reasoning in section 2, even though it will help to understand striking similarities between the effects of rotation and electric charge . The assumption will be a crucial input in order to formulate the reissner - nordstrm / cft correspondence and its generalizations in section 4 and further on . This assumption is not required for the kerr / cft correspondence and its (extremal or non - extremal) extensions, which are exclusively based on the u(1) axial symmetry of spinning black holes . In order to make this idea more precise, it is important to study simple embeddings of the u(1) gauge field in higher - dimensional spacetimes as toy models for a realistic embedding . In asymptotically - flat spacetimes, let us introduce a fifth compact dimension + 2r, where 2r is the length of the u(1) kaluza - klein circle and let us define 2\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$d{s^2} = ds_{(4)}^2 + {(d\chi + a)^2}.$$\end{document} the metric (2) does not obey five - dimensional einstein s equations unless the metric is complemented by matter fields . One simple choice consists of adding a u(1) gauge field a(5), whose field strength is defined as 3\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$d{a_{(5)}} = {{\sqrt 3} \over 2}{\star _ {(4)}}f,$$\end{document} where (4) is the four - dimensional hodge dual . The five - dimensional metric and gauge field are then solutions to the five - dimensional einstein - maxwell - chern - simons theory, as reviewed, e.g., in . These considerations can also be applied to black holes in anti - de sitter spacetimes . However, the situation is more intricate because no consistent kaluza - klein reduction from five dimensions can give rise to the four - dimensional einstein - maxwell theory with cosmological constant . As a consequence, the four - dimensional kerr - newman - ads black hole cannot be lifted to a solution of any five - dimensional theory . Rather, embeddings in eleven - dimensional supergravity exist, which are obtained by adding a compact seven - sphere [69, 109]. Therefore, in order to review the arguments for the reissner - nordstrm / cft correspondence and its generalizations, it is necessary to discuss five - dimensional gravity coupled to matter fields . We will limit our arguments to the action (1) possibly supplemented by the chern - simons terms 4\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${s_{{\rm{cs}}}} = {1 \over {16\pi {g_5}}}\int {{d^5}} x{c_{ijk}}{a^i} \wedge {f^j} \wedge {f^k},$$\end{document} where cijk = c(ijk) are constants . This theory will suffice to discuss in detail the embedding (2)(3) since the five - dimensional einstein - maxwell - chern - simons theory falls into that class of theories . We will not discuss the supergravities required to embed ads - einstein - maxwell theory . Let us finally emphasize that even though the scale r of the kaluza - klein direction is arbitrary as far as it allows one to perform the uplift (2), it is constrained by matter field couplings . For example, let us consider the toy model of a probe charged massive scalar field (x) of charge qe in four dimensions, which is minimally coupled to the gauge field . The wave equation reads as 5\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${1 \over {\sqrt {- g}}}{d_\alpha}\left({\sqrt {- g} {g^{\alpha \beta}}{d_\beta}\phi (x)} \right) + {\mu ^2}\phi (x) = 0,$$\end{document} where the derivative is defined as d = iqea. This wave equation is reproduced from a five - dimensional scalar field (5d) (x,) probing the five - dimensional metric (2), if one takes 6\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${\phi _ {(5d)}}(x,\chi) = \phi (x){e^{i{q_e}\chi}},$$\end{document} and if the five - dimensional mass is equal to \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$\mu _ {(5d)}^2 = {\mu ^2} + q_e^2$\end{document}. However, the five - dimensional scalar is multivalued on the circle unless 7\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${q_e}{r_\chi} \in \mathbb{n}.$$\end{document} this toy model illustrates that the scale r can be constrained from consistent couplings with matter . In this section, we review some key properties of extremal black holes in the context of four - dimensional theories of gravity coupled to matter . In one glance, we show that the near - horizon regions of extremal black holes are isolated geometries, isolated thermodynamical systems and, more generally, isolated dynamical systems . We first contrast how to decouple from the asymptotic region the near - horizon region of static and rotating black holes . We finally discuss uniqueness of near - horizon geometries and their lack of local bulk dynamics . For simplicity since the kerr / cft correspondence and its extensions are only concerned with the region close to the horizon, one could only require that the near - horizon region is stationary, while radiation would be allowed far enough from the horizon . Such a situation could be treated in the framework of isolated horizons [11, 10] (see for a review). However, for our purposes, it will be sufficient and much simpler to assume stationarity everywhere . We expect that all results derived in this review could be generalized for isolated horizons (see for results along these lines). Many theorems have been derived that characterize the generic properties of four - dimensional stationary black holes that admit an asymptotically - timelike killing vector . First, they have one additional axial killing vector they are axisymmetric3 and their event horizon is a killing horizon4 . In asymptotically - flat spacetimes, extremal black holes are defined as stationary black holes with vanishing hawking temperature, 8\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${t_h} = 0.$$\end{document} equivalently, extremal black holes are defined as stationary black holes whose inner and outer horizons coincide . No physical process is known that would make an extremal black hole out of a non - extremal black hole.5 if one attempts to send finely - tuned particles or waves into a near - extremal black hole in order to further approach extremality, one realizes that there is a smaller and smaller window of parameters that allows one to do so when approaching extremality . In effect, a near - extremal black hole has a potential barrier close to the horizon, which prevents it from reaching extremality . Also, if one artificially continues the parameters of the black holes beyond the extremality bound in a given solution, one typically obtains a naked singularity instead of a black hole . Such naked singularities are thought not to be reachable, which is known as the cosmic censorship hypothesis . Extremal black holes can then be thought of as asymptotic or limiting black holes of physical black holes . The other way around, if one starts with an extremal black hole, one can simply throw in a massive particle to make the black hole non - extremal . Nevertheless, as we will discuss, studying the extremal limit is very interesting because many simplifications occur and powerful specialized methods can be used . Extremal spinning or charged rotating black holes enjoy several interesting properties that we will summarize below . In order to be self - contained, we will also first provide some properties of generic (extremal or non - extremal) black holes . We refer the reader to the excellent lecture notes for the derivation of most of these properties . Spinning black holes are characterized by a chemical potential the angular velocity j conjugate to the angular momentum . The angular velocity can be defined in geometrical terms as the coefficient of the black - hole - horizon generator proportional to the axial killing vector 9\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\xi = {\partial _ t} + {\omega _ j}{\partial _ \phi}.$$\end{document} the net effect of the angular velocity is a frame - dragging effect around the black hole . Part of the intuition behind the extremal spinning black hole / cft correspondence is that the degrees of freedom responsible for the black hole entropy are rotating at the speed of light at the horizon.electrostatic potential . Electrically - charged black holes are characterized by a chemical potential the electrostatic potential e conjugated to the electric charge . It is defined on the horizon r = r+ as 10\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\phi _ e^i = - {\xi ^\mu}a_\mu ^i{\vert _ {r = {r _ +}}}, $$\end{document} where is the horizon generator defined in (9). Similarly, one can associate a magnetic potential \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$\phi _ m^i$\end{document} to the magnetic monopole charge . The form of the magnetic potential can be obtained by electromagnetic duality, or reads as the explicit formula derived in (see also for a covariant expression). Part of the intuition behind the extremal charged black hole / cft correspondence is that this kinematics is the sign of microscopic degrees of freedom moving along the gauge direction . Although the killing generator associated with the mass of the black hole, t, is timelike at infinity, it does not need to be timelike everywhere outside the horizon . The region where t is spacelike is called the ergoregion and the boundary of that region where t is lightlike is the ergosphere . If there is no ergoregion, t is a global timelike killing vector outside the horizon . However, it should be noted that the presence of an ergoregion does not preclude the existence of a global timelike killing vector . When the horizon radius is smaller than the ads length, the horizon generator becomes spacelike at large enough distances and there is no global timelike killing vector, as for the kerr black hole . On the contrary, when the horizon radius is larger than the ads length, the horizon generator is timelike everywhere outside the horizon and is therefore a global timelike killing vector.superradiance . One of the most fascinating properties of some rotating black holes is that neutral particles or waves sent towards the black hole with a frequency and angular momentum m inside a specific band 11\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$0 <\omega <m{\omega _ j}$$\end{document} come back to the exterior region with a higher amplitude . This amplification effect or penrose effect allows the extraction of energy very efficiently from the black hole . Superradiance occurs for the kerr and kerr - newman black hole and is related to the presence of the ergoregion and the lack of a global timelike killing vector . Because of the presence of a global timelike killing vector, there is no superradiance for large kerr - ads black holes (when reflective boundary conditions for incident massless waves are imposed) [165, 264].electromagnetic analogue to superradiance . Charged black holes contain electrostatic energy that can also be extracted by sending charged particles or waves with frequency and charge qe inside a specific band (see for a review) 12\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$0 <\omega <{q_e}{\phi _ e}.$$\end{document} there is no ergoregion in the four - dimensional spacetime . However, for asymptotically - flat black holes, there is a five - dimensional ergoregion when considering the uplift (2). For the reissner - nordstrm black hole, <r <2 m, where m is the mass and r the standard boyer - lindquist radius.the combined effect of rotation and charge allows one to extract energy in the range 13\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$0 <\omega <m{\omega _ j} + {q_e}{\phi _ e}.$$\end{document} when considering a wave scattering off a black hole, one can define the absorption probability abs or macroscopic greybody factor as the ratio between the absorbed flux of energy at the horizon and the incoming flux of energy from infinity, 14\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${\sigma _ {{\rm{abs}}}} = {{d{e_{{\rm{abs}}}}/dt} \over {d{e_{{\rm{in}}}}/dt}}.$$\end{document} in the superradiant range (13), the absorption probability is negative because the outgoing flux of energy is higher than the incoming flux.no thermal radiation but spontaneous emission . Taking quantum mechanical effects into account, non - extremal black holes radiate with a perfect black - body spectrum at the horizon at the hawking temperature th . The decay rate of a black hole as observed from the asymptotic region is the product of the black - body spectrum decay rate with the greybody factor abs, 15\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\gamma = {1 \over {{e^{{{\omega - m{\omega _ j} - {q_e}{\phi _ e}} \over {{t_h}}}}} - 1}}{\sigma _ {{\rm{abs}}}}.$$\end{document} the greybody factor accounts for the fact that waves (of frequency, angular momentum m and electric charge qe) need to travel from the horizon to the asymptotic region in the curved geometry . In the extremal limit, the thermal factor becomes a step function . The decay rate then becomes 16\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${\gamma _ {{\rm{ext}}}} = - \theta (- \omega + m{\omega _ j} + {q_e}{\phi _ e}){\sigma _ {{\rm{abs}}}}.$$\end{document} as a consequence, ordinary hawking emission with abs> 0 and> mj + qee vanishes while quantum superradiant emission persists . Therefore, extremal black holes that exhibit superradiance, spontaneously decay to non - extremal black holes by emitting superradiant waves.innermost stable orbit approaching the horizon in the extremal limit . Near - extremal black holes have an innermost stable circular orbit (isco) very close to the horizon . (in boyer - lindquist coordinates, the radius of such an orbit coincides with the radius of the horizon . However, since the horizon is a null surface, while the isco is timelike, the orbit necessarily lies outside the horizon, which can be seen explicitly in more appropriate coordinates . See figure 2 of 6). As a consequence, the region of the black hole close to the horizon can support accretion disks of matter and, therefore, measurements of electromagnetic waves originating from the accretion disk of near - extremal rotating black holes contain (at least some marginal) information from the near - horizon region . For a careful analysis of the physical processes around rotating black holes, see also for a recent discussion.classical singularities approaching the horizon in the extremal limit . Stationary axisymmetric non - extremal black holes admit a smooth inner and outer horizon, where curvatures are small . However, numerical results [52, 50, 51, 112] and the identification of unstable linear modes using perturbation theory [220, 125, 124] showed that the inner horizon is unstable and develops a curvature singularity when the black hole is slightly perturbed . The instability is triggered by tiny bits of gravitational radiation that are blueshifted at the inner cauchy horizon and which create a null singularity . In the near - extremality limit, the inner horizon approaches the outer horizon and it can be argued that test particles encounter a curvature singularity immediately after they enter the horizon of a near - extremal black hole . Spinning black holes are characterized by a chemical potential the angular velocity j conjugate to the angular momentum . The angular velocity can be defined in geometrical terms as the coefficient of the black - hole - horizon generator proportional to the axial killing vector 9\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\xi = {\partial _ t} + {\omega _ j}{\partial _ \phi}.$$\end{document} the net effect of the angular velocity is a frame - dragging effect around the black hole . Part of the intuition behind the extremal spinning black hole / cft correspondence is that the degrees of freedom responsible for the black hole entropy are rotating at the speed of light at the horizon . Electrostatic potential . Electrically - charged black holes are characterized by a chemical potential the electrostatic potential e conjugated to the electric charge . It is defined on the horizon r = r+ as 10\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\phi _ e^i = - {\xi ^\mu}a_\mu ^i{\vert _ {r = {r _ +}}}, $$\end{document} where is the horizon generator defined in (9). Similarly, one can associate a magnetic potential \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$\phi _ m^i$\end{document} to the magnetic monopole charge . The form of the magnetic potential can be obtained by electromagnetic duality, or reads as the explicit formula derived in (see also for a covariant expression). Part of the intuition behind the extremal charged black hole / cft correspondence is that this kinematics is the sign of microscopic degrees of freedom moving along the gauge direction . Although the killing generator associated with the mass of the black hole, t, is timelike at infinity, it does not need to be timelike everywhere outside the horizon . The region where t is spacelike is called the ergoregion and the boundary of that region where t is lightlike is the ergosphere . If there is no ergoregion, t is a global timelike killing vector outside the horizon . However, it should be noted that the presence of an ergoregion does not preclude the existence of a global timelike killing vector . When the horizon radius is smaller than the ads length, the horizon generator becomes spacelike at large enough distances and there is no global timelike killing vector, as for the kerr black hole . On the contrary, when the horizon radius is larger than the ads length, the horizon generator is timelike everywhere outside the horizon and is therefore a global timelike killing vector . One of the most fascinating properties of some rotating black holes is that neutral particles or waves sent towards the black hole with a frequency and angular momentum m inside a specific band 11\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$0 <\omega <this amplification effect or penrose effect allows the extraction of energy very efficiently from the black hole . Superradiance occurs for the kerr and kerr - newman black hole and is related to the presence of the ergoregion and the lack of a global timelike killing vector . Because of the presence of a global timelike killing vector, there is no superradiance for large kerr - ads black holes (when reflective boundary conditions for incident massless waves are imposed) [165, 264]. Electromagnetic analogue to superradiance . Charged black holes contain electrostatic energy that can also be extracted by sending charged particles or waves with frequency and charge qe inside a specific band (see for a review) 12\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$0 <\omega <{q_e}{\phi _ e}.$$\end{document} there is no ergoregion in the four - dimensional spacetime . However, for asymptotically - flat black holes, there is a five - dimensional ergoregion when considering the uplift (2). For the reissner - nordstrm black hole, <r <2 m, where m is the mass and r the standard boyer - lindquist radius . The combined effect of rotation and charge allows one to extract energy in the range 13\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$0 <\omega <m{\omega _ j} + {q_e}{\phi _ e}.$$\end{document} when considering a wave scattering off a black hole, one can define the absorption probability abs or macroscopic greybody factor as the ratio between the absorbed flux of energy at the horizon and the incoming flux of energy from infinity, 14\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${\sigma _ {{\rm{abs}}}} = {{d{e_{{\rm{abs}}}}/dt} \over {d{e_{{\rm{in}}}}/dt}}.$$\end{document} in the superradiant range (13), the absorption probability is negative because the outgoing flux of energy is higher than the incoming flux . Non - extremal black holes radiate with a perfect black - body spectrum at the horizon at the hawking temperature th . The decay rate of a black hole as observed from the asymptotic region is the product of the black - body spectrum decay rate with the greybody factor abs, 15\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\gamma = {1 \over {{e^{{{\omega - m{\omega _ j} - {q_e}{\phi _ e}} \over {{t_h}}}}} - 1}}{\sigma _ {{\rm{abs}}}}.$$\end{document} the greybody factor accounts for the fact that waves (of frequency, angular momentum m and electric charge qe) need to travel from the horizon to the asymptotic region in the curved geometry . In the extremal limit, the thermal factor becomes a step function . The decay rate then becomes 16\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${\gamma _ {{\rm{ext}}}} = - \theta (- \omega + m{\omega _ j} + {q_e}{\phi _ e}){\sigma _ {{\rm{abs}}}}.$$\end{document} as a consequence, ordinary hawking emission with abs> 0 and> mj + qee vanishes while quantum superradiant emission persists . Therefore, extremal black holes that exhibit superradiance, spontaneously decay to non - extremal black holes by emitting superradiant waves . Innermost stable orbit approaching the horizon in the extremal limit . Near - extremal black holes have an innermost stable circular orbit (isco) very close to the horizon . (in boyer - lindquist coordinates, the radius of such an orbit coincides with the radius of the horizon . However, since the horizon is a null surface, while the isco is timelike, the orbit necessarily lies outside the horizon, which can be seen explicitly in more appropriate coordinates . See figure 2 of 6). As a consequence, the region of the black hole close to the horizon can support accretion disks of matter and, therefore, measurements of electromagnetic waves originating from the accretion disk of near - extremal rotating black holes contain (at least some marginal) information from the near - horizon region . For a careful analysis of the physical processes around rotating black holes, see . See also for a recent discussion . Stationary axisymmetric non - extremal black holes admit a smooth inner and outer horizon, where curvatures are small . However, numerical results [52, 50, 51, 112] and the identification of unstable linear modes using perturbation theory [220, 125, 124] showed that the inner horizon is unstable and develops a curvature singularity when the black hole is slightly perturbed . The instability is triggered by tiny bits of gravitational radiation that are blueshifted at the inner cauchy horizon and which create a null singularity . In the near - extremality limit, the inner horizon approaches the outer horizon and it can be argued that test particles encounter a curvature singularity immediately after they enter the horizon of a near - extremal black hole . As a warm - up, let us first review the near - horizon limit of static extremal black holes . In that case, the generator of the horizon (located at r = r+) is the generator of time translations t and the geometry has so(3) rotational symmetry . Since the horizon generator is null at the horizon, the coordinate t diverges there . The near - horizon limit is then defined as 17\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\begin{array}{*{20}c} {t \rightarrow {{{r_0}t} \over \lambda},} \\ {r \rightarrow {r _ +} + \lambda {r_0}r,} \\ the scale r0 is introduced for convenience in order to factor out the overall scale of the near - horizon geometry . In the presence of electrostatic potentials, a change of gauge is required when taking the near - horizon limit (17). Indeed, in the near - horizon coordinates (17) the gauge fields take the following form, 18\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${a^i} = - {{\phi _ e^i} \over \lambda}{r_0}dt + a_r^idr + a_\theta ^id\theta + a_\phi ^id\phi, $$\end{document} where \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$\phi _ e^i$\end{document} is the static electric potential of the gauge field a. upon taking the near - horizon limit one should, therefore, perform a gauge transformation a a + d of parameter 19\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${\lambda ^i} = {{\phi _ e^{i,{\rm{ext}}}} \over \lambda}{r_0}t,$$\end{document} where \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$\phi _ e^{i,{\rm{ext}}}$\end{document} is the static electric potential at extremality . It is important to note that one is free to redefine the near - horizon limit parameter as for any> 0 . This transformation scales r inversely proportionally to t. therefore, the near - horizon geometry admits the enhanced symmetry generator 20\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${\zeta _ 0} = r{\partial _ r} - t{\partial _ t}$$\end{document} in addition to 1 = t and the so(3) symmetry generators . Using the properties of static horizons, one can further derive an additional symmetry generator at the horizon 1, which together with 1 and 0 forms a sl(2,) algebra . This argument is purely kinematical and does not involve the field equations; see, e.g., for a detailed derivation . The general near - horizon solution compatible with an sl(2,) so(3) symmetry is then given by 21\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\begin{array}{*{20}c} {d{s^2} = {v_1}(- {r^2}d{t^2} + {{d{r^2}} \over {{r^2}}}) + {v_2}(d{\theta ^2} + {{\sin}^2}\theta d{\phi ^2}),} \\ {{\chi ^a} = \chi _ \star^a,\quad \quad {a^i} = {e_i}rdt - {{{p^i}} \over {4\pi}}\cos \theta d\phi,} \\ \end{array}$$\end{document} where v1, v2, \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$\chi _ \ast^a$\end{document}, ei, p are parameters, which are constrained by the equations of motion . The geometry consists of the direct product ads2 s. for some supersymmetric theories, the values v1, v2, \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$\chi _ \ast^a$\end{document}, ei are generically completely fixed by the electric (q) and magnetic (p) charges of the black hole and do not depend continuously on the asymptotic value of the scalar fields in the asymptotic region the scalar moduli . It was then realized that it still applies in the presence of certain higher - derivative corrections [199, 200, 198]. The attractor mechanism was also extended to non - supersymmetric extremal static black holes [139, 240, 150, 179]. As a consequence of this mechanism, the entropy of these extremal black hole does not depend continuously on any moduli of the theory.7 the index that captures the entropy can still have discrete jumps when crossing walls of marginal stability in the scalar moduli space [227, 118]. This allows one to account for their black - hole entropy by varying the moduli to a weakly - coupled description of the system without gravity, where states with fixed conserved charges can be counted . Therefore, the attractor mechanism led to an explanation [18, 111] of the success of previous string theory calculations of the entropy of certain nonsupersymmetric extremal black holes [181, 172, 110, 260, 131, 132]. As will turn out to be useful in the development of the reissner - nordstrm correspondence, let us discuss the near - horizon geometry (21) under the assumption that one gauge field a can be lifted as a kaluza - klein vector to a higher - dimensional spacetime, as discussed in section 1.2 . In the simple model (2), the change of gauge a a + d is implemented as the change of coordinates + . Using the definition of the electrostatic potential \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$\phi _ e^{{\rm{ext}}}$\end{document} (10) at extremality, it is straightforward to obtain that in the geometry (2) the horizon is generated by 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 _ {{\rm{tot}}}} = {\partial _ t} + \phi _ e^{{\rm{ext}}}{\partial _ \chi}$\end{document}. The change of coordinates (17) combined with + with defined in (19) then maps this vector to 22\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${\xi _ {{\rm{tot}}}} \rightarrow {\lambda \over {{r_0}}}{\partial _ t}.$$\end{document} let us now consider extremal spinning black holes . Let us denote the axis of rotation to be, where + 2 and let r = r+ be the black - hole horizon . The generator of the horizon is \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$\xi \equiv {\partial _ t} + \omega _ j^{{\rm{ext}}}{\partial _ \phi}$\end{document} where \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$\omega _ j^{{\rm{ext}}}$\end{document} is the extremal angular velocity . We choose a coordinate system such that the coordinate t diverges at the horizon, which is equivalent to the fact that g diverges at the horizon . As in the static case, one needs to perform a gauge transformation of parameter (19), when electrostatic fields are present . One can again interpret this change of gauge parameter as a change of coordinates in a higher - dimensional auxiliary spacetime (2). The near - horizon limit is then defined as 23\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\begin{array}{*{20}c} {t \rightarrow {r_0}{t \over \lambda},} \\ {r \rightarrow {r _ +} + \lambda {r_0}r,} \\ {\phi \rightarrow \phi + \omega _ j^{{\rm{ext}}}{{{r_0}t} \over \lambda},} \\ {a \rightarrow a + {{\phi _ e^{{\rm{ext}}}} \over \lambda}{r_0}dt,} \\ \end{array}$$\end{document} with 0 . The scale r0 is again introduced in order to factor out the overall scale of the near - horizon geometry . The additional effect with respect to the static near - horizon limit is the shift in the angle in order to reach the frame co - moving with the horizon . The horizon generator becomes = /r0t in the new coordinates . Including the gauge field, one has precisely the relation (22). As in the static case, any finite energy excitation of the near - horizon geometry is confined and amounts to no net charges in the original (asymptotically flat of ads) geometry . One is free to redefine as for any> 0 and, therefore, the near - horizon geometry admits the enhanced symmetry generator 24\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${\zeta _ 0} = r{\partial _ r} - t{\partial _ t},$$\end{document} in addition to 1 = t and l0 = . Together 0 and 1 form a non - commutative algebra under the lie bracket . Now, contrary to the static case, the existence of a third killing vector is not guaranteed by geometric considerations . Nevertheless, it turns out that einstein s equations derived from the action (1) imply that there is an additional killing vector 1 in the near - horizon geometry [194, 19] (see also for a geometrical derivation). The vectors 1, 0, 1 turn out to obey the sl(2,) so(2, 1) algebra . This dynamical enhancement is at the origin of many simplifications in the near - horizon limit . More precisely, one can prove that any stationary and axisymmetric asymptotically - flat or anti - de sitter extremal black - hole solution of the theory described by the lagrangian (1) admits a near - horizon geometry with sl(2,) u(1) isometry . The result also holds in the presence of higher - derivative corrections in the lagrangian provided that the black hole is big, in the technical sense that the curvature at the horizon remains finite in the limit where the higher - derivative corrections vanish . The general near - horizon geometry of extremal spinning black holes consistent with these symmetries is given by 25\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\begin{array}{*{20}c} {d{s^2} = \gamma (\theta)\left [{- {r^2}d{t^2} + {{d{r^2}} \over {{r^2}}} + \alpha {{(\theta)}^2}d{\theta ^2} + \gamma {{(\theta)}^2}{{(d\phi + krdt)}^2}} \right],} \\ {{\chi ^a} = {\chi ^a}(\theta),\quad \quad {a^i} = {f^i}(\theta)(d\phi + krdt) - {{{e_i}} \over k}d\phi,} \\ \end{array}$$\end{document} where ()> 0, () 0, (), f() and k, ei are fixed by the equations of motion . By inverting t and redefining a a, we can always set k 0, ei 0 . The function () 0 can be removed by redefining but it is left for convenience because some near - horizon geometries are then more easily described.8 the term \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$- {{{e_i}} \over k}d\phi$\end{document} in (25) is physical since it cannot be gauged away by an allowed gauge transformation . For example, one can check that the near - horizon energy \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}${{\mathcal q}_{{\partial _ t}}}$\end{document} would be infinite in the kerr - newman near - horizon geometry if this term would be omitted . One can alternatively redefine f() = b() + ei / k and the gauge field takes the form 27\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${a^i} = {b^i}(\theta)(d\phi + k\;rdt) + {e_i}rdt.$$\end{document} the static near - horizon geometry (21) is recovered upon choosing only so(3) covariant quantities with a well - defined static limit . This requires k 0 and it requires the form 28\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${b^i}(\theta) = {{{p^i}} \over {4\pi}}\cos \theta, $$\end{document} where p are some pure numbers, which are the magnetic charges . Going back to the spinning case, the sl(2,) u(1) symmetry is generated by 29\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\begin{array}{*{20}c} {{\zeta _ {- 1}} = {\partial _ t},\quad \quad {\zeta _ 0} = t{\partial _ t} - r{\partial _ r},} \\ {{\zeta _ 1} = \left({{1 \over {2{r^2}}} + {{{t^2}} \over 2}} \right){\partial _ t} - tr{\partial _ r} - {k \over r}{\partial _ \end{array}$$\end{document} in addition, the generator 1 should be accompanied by the gauge transformation of parameter = ei / r so that \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}${{\mathcal l}_{{\zeta _ \mu}{\lambda ^i} = 0$\end{document}. Note that all of these symmetries act within a three - dimensional slice of fixed polar angle . The metric is also invariant under discrete symmetry, which maps 30\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$(t,\phi) \rightarrow (- t, - \phi).$$\end{document} this is often called the t- reflection symmetry in black - hole literature . The parity / time reversal transformation (30) reverses the electromagnetic charges of the solution . The geometry (25) is a warped and twisted product of ads2 s. the (r, t) coordinates are analogous to poincar coordinates on ads2 with an horizon at r = 0 . One can find global coordinates in the same way that the global coordinates of ads2 are related to the poincar coordinates . Let 31\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$r = {(1 + {y^2})^{1/2}}\cos \tau + y,\quad \quad tr = {(1 + {y^2})^{1/2}}\sin \tau .$$\end{document} the new axial angle coordinate is chosen so that d + krdt = d + kyd, with the result 32\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\phi = \varphi + k\log \left\vert {{{\cos \tau + y\sin \tau} \over {1 + {{(1 + {y^2})}^{1/2}}\sin \tau}}} \right\vert .$$\end{document} in these new coordinates, the near - horizon geometry becomes 33\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\begin{array}{*{20}c} {d{s^2} = \gamma (\theta)\left [{- {{(1 + y)}^2}d{\tau ^2} + {{d{y^2}} \over {1 + {y^2}}} + \alpha {{(\theta)}^2}d{\theta ^2} + \gamma {{(\theta)}^2}{{(d\varphi + kyd\tau)}^2}} \right]}, \\ {\quad \quad \;{\chi ^a} = {\chi ^a}(\theta),\quad \quad {a^i} = {f^i}(\theta)(d\varphi + kyd\tau) - {{{e_i}} \over k}d\varphi}, \\ \end{array}$$\end{document} after performing an allowed gauge transformation (as the change of gauge falls into the boundary conditions (115) derived in section 4.1). Note that the = 0 hypersurface coincides with the t=0 hypersurface, and that = on this hypersurface . The geometry has two boundaries at y = and y = + . Geodesic completeness of these geometries has not been shown in general, even though it is expected that they are geodesically complete . For the case of the near - horizon geometry of kerr, geodesic completeness has been proven explicitly in after working out the geodesic equations . At fixed polar angle, the geometry can be described in terms of 3d warped anti - de sitter geometries; see for a relevant description and [226, 158, 238, 127, 223, 175, 174, 6, 119, 43, 26, 93, 222] for earlier work on these three - dimensional geometries . Warped anti - de sitter spacetimes are deformations of ads3, where the s fiber is twisted around the ads2 base . Because of the identification + 2, the geometries at fixed are quotients of the warped ads geometries, which are characterized by the presence of a killing vector of constant norm (namely). These quotients are often called self - dual orbifolds by analogy to similar quotients in ads3 .9 the geometries enjoy a global timelike killing vector (which can be identified as) if and only if 34\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$k\gamma (\theta) <1,\quad \quad \forall \theta \in [0,\pi].$$\end{document} if there is no global timelike killing vector, there is at least one special value of the polar angle, where k() = 1 . At that special value, the slice = is locally an ordinary ads3 spacetime and acquires a local sl(2,) sl(2,) isometry . At all other values of, one sl(2,) note that there is still a global time function for each near - horizon geometry . Constant global time in the global coordinates (33) are spacelike surfaces because their normal is timelike, 35\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${g^{ab}}{\partial _ a}\tau {\partial _ b}\tau = {g^{\tau \tau}} = - {(1 + {y^2})^{- 1}}{\gamma ^{- 1}}(\theta) <0.$$\end{document} hence, there are no closed timelike curves . One can show the existence of an attractor mechanism for extremal spinning black holes, which are solutions of the action (1). According to, the complete near - horizon solution is generically independent of the asymptotic data and depends only on the electric charges \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}${\mathcal q}_e^i$\end{document}, magnetic charges \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}${\mathcal q}_m^i$\end{document} 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}${\mathcal j}$\end{document} carried by the black hole, but in special cases there may be some dependence of the near horizon background on this asymptotic data . In all cases, the entropy only depends on the conserved electromagnetic charges and the angular momentum of the black hole and might only jump discontinuously upon changing the asymptotic values of the scalar fields, as it does for static charged black holes [227, 118]. One can generalize the construction of near - horizon extremal geometries to higher dimensions . In five dimensions, there are two independent planes of rotation since the rotation group is a direct product so(4) so(3) so(3). Assuming the presence of two axial u(1) symmetries \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}${\partial _ {{\phi _ i}}}$\end{document}, i = 1, 2 (with fixed points at the poles), one can prove that the near - horizon geometry of a stationary, extremal black - hole solution of the five - dimensional action (1) possibly supplemented by chern - simons terms (4) is given by 36\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} {d{s^2} = \gamma (\theta)\left [{- {r^2}d{t^2} + {{d{r^2}} \over {{r^2}}} + \alpha {{(\theta)}^2}d{\theta ^2} + \sum\limits_{i, j = 1}^2 {{\gamma _ {ij}}} {{(\theta)}^2}(d{\phi ^i} + {k_i}r\;dt)(d{\phi ^j} + {k_j}r\;dt)} \right]}, \\ {{\chi ^a} = {\chi ^a}(\theta),\quad \quad {a^i} = \sum\limits_{i = 1}^2 {f_i^i} (\theta)(d{\phi ^i} + {k_i}r\;dt) - {{{e_i}} \over {{k^i}}}d{\phi ^i}}. \\ \end{array}$$\end{document} in particular, the solutions obtained from the uplift (2)(3) fall into this class . In general, these solutions can be obtained starting from both black holes (with s horizon topology) and black rings (with s s horizon topology). We will discuss the cases of the extremal kerr and reissner - nordstrm black holes as well as the extremal kerr - newman and kerr - newman - ads black holes . Other near - horizon geometries of interest can be found, e.g., in [88, 121, 203]. The near - horizon geometry of extremal kerr with angular momentum \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}${\mathcal j} = j$\end{document} can be obtained by the above procedure, starting from the extremal kerr metric written in usual boyer - lindquist coordinates; see the original derivation in as well as in [156, 54]. The result is the nhek geometry, which is written as (25) without matter fields and with 37\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\begin{array}{*{20}c} {\alpha (\theta) = 1,\quad \quad \gamma (\theta) = j(1 + {{\cos}^2}\theta)}, \\ {\gamma (\theta) = {{2\sin \theta} \over {1 + {{\cos}^2}\theta}},\quad \quad k = 1}. There is a value \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}${\theta _ \ast} = \arcsin (\sqrt 3 - 1) \sim 47$\end{document} degrees for which t becomes null . For this feature is a consequence of the presence of the ergoregion in the original kerr geometry . Near the equator ads3 self - dual orbifold (as the s fiber is streched), while near the poles we have a squashed the extremal reissner - nordstrm black hole is determined by only one parameter: the electric charge q. the mass is \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}${\mathcal m} = q$\end{document} and the horizon radius is r+ = r = q. this black hole is static and, therefore, its near - horizon geometry takes the form (21). We have explicitly 38\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${\nu _ 1} = {q^2},\quad {\nu _ 2} = {q^2},\quad e = q,\quad p = 0.$$\end{document} it is useful to collect the different functions characterizing the near - horizon limit of the extremal kerr - newman black hole . We use the normalization of the gauge field such that the lagrangian is proportional to r fabf . The black hole has mass \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}${\mathcal m} = \sqrt {{a^2} + {q^2}}$\end{document}. The horizon radius 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}${r _ +} = {r _ -} = \sqrt {{a^2} + {q^2}}$\end{document}. One finds 39\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\begin{array}{*{20}c} {\alpha (\theta) = 1,\quad \quad \gamma (\theta) = r _ + ^2 + {a^2}{{\cos}^2}\theta}, \\ {\gamma (\theta) = {{(r _ + ^2 + {a^2})\sin \theta} \over {r _ + ^2 + {a^2}{{\cos}^2}\theta}},\quad \quad k = {{2a{r _ +}} \over {r _ + ^2 + {a^2}}}}, \\ {f(\theta) = q\left({{{r _ + ^2 + {a^2}} \over {2a{r _ +}}}} \right){{r _ + ^2 - {a^2}{{\cos}^2}\theta} \over {r _ + ^2 + {a^2}{{\cos}^2}\theta}},\quad \quad e = {{{q^3}} \over {r _ + ^2 + {a^2}}}}. \\ \end{array}$$\end{document} in the limit q 0, the nhek functions (37) are recovered . The near - horizon geometry of extremal kerr - newman is therefore smoothly connected to the near - horizon geometry of kerr . In the limit a 0 one finds the near - horizon geometry of the reissner - nordstrm black hole (38). The limiting procedure is again smooth . As a last example of near - horizon geometry, let us discuss the extremal spinning charged black hole in ads or kerr - newman - ads black hole in short . The lagrangian is given by l r + 6/l f where l> 0 . It is useful for the following to start by describing a few properties of the non - extremal kerr - newman - ads black hole . The physical mass, angular momentum, electric and magnetic charges at extremality are expressed in terms of the parameters (m, a, qe, qm) of the solution as 40\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\mathcal{m} = {m \over {{\xi ^2}}},\quad \quad \mathcal{j} = {{am} \over {{\xi ^2}}},$$\end{document} 41\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${\mathcal{q}_e} = {{{q_e}} \over \xi},\quad \quad {\mathcal{q}_m} = {{{q_m}} \over \xi},$$\end{document} where = 1 a / l and \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}${q^2} = q_e^2 + q_m^2$\end{document} the horizon radius r+(r) is defined as the largest (smallest) root, respectively, of 42\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${\delta _ r} = ({r^2} + {a^2})(1 + {r^2}/{l^2}) - 2mr + {q^2}.$$\end{document} hence, one can trade the parameter m for r+ . If one expands r up to quadratic order around r+, one finds 43\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${\delta _ r} = {\delta _ 0}({r _ +} - {r _ \star})(r - {r _ +}) + {\delta _ 0}{(r - {r _ +}) ^2} + o{(r - {r _ +}) ^{\rm{3}}},$$\end{document} where 0 and r are defined by 44\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\begin{array}{*{20}c} {{\delta _ 0} = 1 + {a^2}/{l^2} + 6r _ + ^2/{l^2}}, \\ {{\delta _ 0}({r _ +} - {r _ \star}) = {r _ +} \left({1 + {{{a^2}} \over {{l^2}}} + {{3r _ + ^2} \over {{l^2}}} - {{{a^2} + {q^2}} \over {r _ + ^2}}} \right)}. \\ \end{array}$$\end{document} in ads, the parameter r obeys r r r+, and coincides with r and r+ only at extremality . In the flat limit l, we have 0 1 and r r. the hawking temperature is given by 45\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${t_h} = {{{\delta _ 0}({r _ +} - {r _ \star})} \over {4\pi (r _ + ^2 + {a^2})}}.$$\end{document} the extremality condition is then r+ = r = r or, more explicitly, the following constraint on the four parameters (r+, a, qe, qm), 46\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$1 + {{{a^2}} \over {{l^2}}} + {{3r _ + ^2} \over {{l^2}}} - {{{a^2} + {q^2}} \over {r _ + ^2}} = 0.$$\end{document} the near - horizon geometry was obtained in [159, 71] (except the coefficient e given here). The result is 47\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\begin{array}{*{20}c} {\gamma (\theta) = {{\rho _ + ^2} \over {{\delta _ 0}}},\quad \quad \alpha (\theta) = {{\delta _ 0^{1/2}} \over {\delta _ \theta ^{1/2}}},\quad \quad k = {{2a{r _ +} \xi} \over {{\delta _ 0}(r _ + ^2 + {a^2})}}}, \\ {\gamma (\theta) = {{\delta _ \theta ^{1/2}\delta _ 0^{1/2}(r _ + ^2 + {a^2})\sin \theta} \over {\rho _ + ^2\xi}},\quad e = {{{q_e}} \over {{\delta _ 0}}}{{r _ + ^2 - {a^2}} \over {r _ + ^2 + {a^2}}}}, \\ {f(\theta) = {{(r _ + ^2 + {a^2})[{q_e}(r _ + ^2 - {a^2}{{\cos}^2}\theta) + 2{q_m}a{r _ +} \cos \theta]} \over {2\rho _ + ^2\xi a{r _ +}}}}, \\ \end{array}$$\end{document} where we defined 48\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\begin{array}{*{20}c} {{\delta _ \theta} = 1 - {{{a^2}} \over {{l^2}}}{{\cos}^2}\theta, \quad \quad \rho _ + ^2 = r _ + ^2 + {a^2}{{\cos}^2}\theta}, \\ {r_0 ^ 2 = {{r _ + ^2 + {a^2}} \over {{\delta _ 0}}}}. \\ \end{array}$$\end{document} the near - horizon geometry of the extremal kerr - newman black hole is recovered in the limit l . The classical entropy of any black hole in einstein gravity coupled to matter fields such as (1) is given by 49\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\mathcal{s} = {1 \over {4{g_n}\hbar}}\int\nolimits_\sigma {vol} (\sigma),$$\end{document} where is a cross - section of the black - hole horizon and gn is the four - dimensional newton s constant . In the near - horizon geometry, the horizon is formally located at any value of as a consequence of the definition (23). Nevertheless, we can move the surface to any finite value of r without changing the integral, thanks to the scaling symmetry 0 of (29). Evaluating the expression (49), we obtain 50\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\mathcal{s} = {\pi \over {2{g_n}\hbar}}\int\nolimits_0^\pi {d\theta} \;\alpha (\theta)\gamma (\theta)\gamma (\theta).$$\end{document} in particular, the entropy of the extremal kerr black hole is given by 51\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\mathcal{s} = 2\pi \mathcal{j}.$$\end{document} in units of the angular momentum \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}${\mathcal j}$\end{document} is a dimensionless half - integer . The main result [156, 203, 21, 159, 225, 83, 173, 22, 204, 97] of the extremal spinning black hole / cft correspondence that we will review below is the derivation of the entropy (50) using cardy s formula (90). When higher derivative corrections are considered, the entropy does not scale any more like the horizon area . The black - hole entropy at equilibrium can still be defined as the quantity that obeys the first law of black - hole mechanics, where the mass, angular momenta and other extensive quantities are defined with all higher - derivative corrections included . More precisely, the entropy is first defined for non - extremal black holes by integrating the first law, and using properties of non - extremal black holes, such as the existence of a bifurcation surface [262, 176]. The resulting entropy formula is unique and given by 52\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\mathcal{s} = {{2\pi} \over \hbar}\int\nolimits_\sigma {{{{\delta ^{{{\rm cov}}}}l} \over {\delta {r_{abcd}}}}} {\epsilon _ {ab}}{\epsilon _ {cd}}vol(\sigma),$$\end{document} where ab is the binormal to the horizon, i.e., the volume element of the normal bundle to . One can define it simply as ab = nab anb, where is the generator of the horizon and n is an outgoing null normal to the horizon defined by n = 0 and na = 1 . Since the lagrangian is diffeomorphism invariant (possibly up to a boundary term), it can be expressed in terms of the metric, the matter fields and their covariant derivatives, and the riemann tensor and its derivatives . This operator /rabcd acts on the lagrangian while treating the riemann tensor as if it were an independent field . It is defined as a covariant euler - lagrange derivative as 53\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${{{\delta ^{{\rm{cov}}}}} \over {\delta {r_{abcd}}}} = \sum\limits_{i = 0} {{{(- 1)}^i}} {\nabla _ {({e_1} \ldots}}{\nabla _ {{e_i})}}{\partial \over {\partial {\nabla _ {({e_1} \ldots}}{\nabla _ {{e_i})}}{r_{abcd}}}}.$$\end{document} moreover, the entropy formula is conserved away from the bifurcation surface along the future horizon as a consequence of the zeroth law of black - hole mechanics . Therefore, one can take the extremal limit of the entropy formula evaluated on the future horizon in order to define entropy at extremality . Quite remarkably, the iyer - wald entropy (52) can also be reproduced using cardy s formula as we will detail below . In five - dimensional einstein gravity coupled to matter, the entropy of extremal black holes can be expressed as 54\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\mathcal{s} = {{{\pi ^2}} \over {\hbar {g_n}}}\int\nolimits_0^\pi {d\theta} \;\alpha (\theta)\gamma (\theta)\gamma (\theta),$$\end{document} where () and () have been defined in (36) and () = det(ij()). From the attractor mechanism for four - dimensional extremal spinning black holes, the entropy at extremality can be expressed as an extremum of the functional 55\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$f(\gamma (\theta),\gamma (\theta),{f^i}(\theta),{\chi ^a}(\theta),k,{e_i}) = {{2\pi} \over {{g_n}\hbar}}(k\mathcal{j} + {e_i}{\mathcal{q}^i} - \int {d\theta} d\phi \sqrt {- g} \mathcal{l}),$$\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}${\mathcal l}$\end{document} is the lagrangian . \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}${\mathcal j}$\end{document} and the conserved charges \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}${\mathcal q}_{e, m}^i$\end{document}, 56\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\mathcal{s} = \mathcal{s}_{{\rm{ext}}}(\mathcal{j},\mathcal{q}_e^i,\mathcal{q}_m^i),$$\end{document} and depend in a discontinuous fashion on the scalar moduli . The result holds for any lagrangian in the class (1), including higher - derivative corrections, and the result can be generalized straightforwardly to five dimensions . When quantum effects are taken into account, the entropy formula also gets modified in a nonuniversal way, which depends on the matter present in quantum loops . In einstein gravity, the logarithmic corrections to the entropy of extremal rotating black holes can be obtained using the quantum entropy function formalism . Even though the hawking temperature is zero at extremality, quantum states just outside the horizon are not pure states when one defines the vacuum using the generator of the horizon . We will drop the index i distinguishing different gauge fields since this detail is irrelevant to the present arguments . From the expression of the entropy in terms of the charges \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}${{\mathcal s}_{{\rm{ext}}}}({\mathcal j},{{\mathcal q}_e},{{\mathcal q}_m})$\end{document}, one can define the chemical potentials \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${1 \over {{t_\phi}}} = {\left({{{\partial {\mathcal{s}_{{\rm{ext}}}}} \over {\partial \mathcal{j}}}} \right)_{{\mathcal{q}_{e, m}}}},\quad {1 \over {{t_e}}} = {\left({{{\partial {\mathcal{s}_{{\rm{ext}}}}} \over {\partial {\mathcal{q}_e}}}} \right)_{\mathcal{j},{\mathcal{q}_m}}},\quad {1 \over {{t_m}}} = {\left({{{\partial {\mathcal{s}_{{\rm{ext}}}}} \over {\partial {\mathcal{q}_m}}}} \right)_{\mathcal{j},{\mathcal{q}_e}}}.$$\end{document}. Note that electromagnetic charges are quantized, but when the charges are large one can use the continuous thermodynamic limit . These potentials obey the balance equation 57\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\delta {\mathcal{s}_{{\rm{ext}}}} = {1 \over {{t_\phi}}}\delta \mathcal{j} + {1 \over {{t_e}}}\delta {\mathcal{q}_e} + {1 \over {{t_m}}}\delta {\mathcal{q}_m}.$$\end{document} another way to obtain these potentials is as follows . At extremality, any fluctuation obeys 58\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$0 = {t_h}\delta \mathcal{s} = \delta \mathcal{m} - (\omega _ j^{{\rm{ext}}}\delta \mathcal{j} + \phi _ e^{{\rm{ext}}}\delta {\mathcal{q}_e} + \phi _ m^{{\rm{ext}}}\delta {\mathcal{q}_m}),$$\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}$\omega _ j^{{\rm{ext}}}$\end{document} is the angular potential at extremality and \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$\phi _ {e, m}^{{\rm{ext}}}$\end{document} are electric and magnetic potentials at extremality; see section 2.1 for a review of these concepts . One can express the first law at extremality (58) as follows: any variation in \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}${\mathcal j}$\end{document} or \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}${{\mathcal q}_{m, e}}$\end{document} is accompanied by an energy variation . One can then solve for \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}${\mathcal m} = {{\mathcal m}_{{\rm{ext}}}}({\mathcal j},{{\mathcal q}_e},{{\mathcal q}_m})$\end{document}. The first law for a non - extremal black hole can be written as 59\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\delta{\mathcal s} = {1 \over {{t_h}}}(\delta {\mathcal m} - ({\omega _ {j}}\delta {\mathcal j} + {\phi _ e}\delta {{\mathcal q}_e} + {\phi _ m}\delta {{\mathcal q}_m})).$$\end{document} let us now take the extremal limit using the following ordering . We first take extremal variations with \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$\delta {\mathcal m} = \delta {{\mathcal m}_{{\rm{ext}}}}({\mathcal j},{{\mathcal q}_e},{{\mathcal q}_m})$\end{document}. Then, we take the extremal limit of the background configuration . We obtain (57) with 60\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${\left . {{t_\phi} = \underset{{t_h} \rightarrow 0}{\lim} {{{t_h}} \over {\omega _ j^{{\rm{ext}}} - {\omega _ j}}} = - {{\partial {t_h}/\partial {r _ +}} \over {\partial {\omega _ j}/\partial {r _ +}}}} \right\vert _ {{r _ +} = {r_{{\rm{ext}}}}}},$$\end{document} 61\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${\left . {{t_{e, m}} = \underset{{t_h} \rightarrow 0}{\lim} {{{t_h}} \over {\omega _ {e, m}^{{\rm{ext}}} - {\phi _ {e, m}}}} = - {{\partial {t_h}/\partial {r _ +}} \over {\partial {\phi _ {e, m}}/\partial {r _ +}}}} \right\vert _ {{r _ +} = {r_{{\rm{ext}}}}}},$$\end{document} where the extremal limit can be practically implemented by taking the limit of the horizon radius r+ to the extremal horizon radius rext . The interpretation of these chemical potentials can be made in the context of quantum field theories in curved spacetimes; see for an introduction . The hartle - hawking vacuum for a schwarzschild black hole, restricted to the region outside the horizon, is a density matrix \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$\rho = {e^{- \omega/{t_h}}}$\end{document} at the hawking temperature th . For spacetimes that do not admit a global timelike killing vector, such as the kerr geometry, the hartle - hawking vacuum does not exist, but one can use the generator of the horizon to define positive frequency modes and, therefore, define the vacuum in the region where the generator is timelike (close enough to the horizon). One can take a suitable limit of the definition of the frolov - thorne vacuum to provide a definition of the vacuum state for any spinning or charged extremal black hole . Quantum fields for non - extremal black holes can be expanded in eigenstates with asymptotic energy \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}${\hat \omega}$\end{document} 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}${\hat m}$\end{document} with \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}${\hat t}$\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}${\hat \phi}$\end{document} dependence as \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}${e^{- i\hat \omega \hat t + i\hat m\hat \phi}}$\end{document}. When approaching extremality, one can perform the change of coordinates (23) in order to zoom close to the horizon . By definition, the scalar field in the new coordinate system x = (t,,, r) reads in terms of the scalar field \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}${\hat \phi}$\end{document} in the asymptotic coordinate system \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}${{\hat x}^a} = (\hat t,\hat \phi, \theta, \hat r)$\end{document} as \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$\phi ({x^a}) = \hat \phi ({{\hat x}^a})$\end{document}. We can then express 62\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${e^{- i\omega t + im\phi}} = {e^{- i\hat \omega \hat t + i\hat m\phi}},$$\end{document} and the near - horizon parameters are 63\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$m = \hat m,\quad \omega = {{\hat \omega - m{\omega _ j}} \over \lambda}.$$\end{document} when no electromagnetic field is present, any finite energy in the near - horizon limit at extremality 0 corresponds to eigenstates with \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$\hat \omega = \hat m\omega _ j^{{\rm{ext}}}$\end{document}. When electric fields are present, zooming in on the near - horizon geometry from a near - extremal solution requires one to perform the gauge transformation a(x) a(x) + d(x) with gauge parameter given in (19), which will transform the minimally - coupled charged scalar wavefunction by multiplying it by \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}${e^{i{q_e}\lambda (x)}}$\end{document}. Finite energy excitations in the near - horizon region then require \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$\hat \omega = m\omega _ e^{{\rm{ext}}} + {q_e}\phi _ e^{{\rm{ext}}}$\end{document}. Invoking (classical) electromagnetic duality, the magnetic contribution has the same form as the electric contribution . In summary, the general finite - energy extremal excitation has the form 64\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\hat \omega = m\omega _ j^{{\rm{ext}}} + {q_e}\phi _ e^{{\rm{ext}}} + {q_m}\phi _ m^{{\rm{ext}}}.$$\end{document} following frolov and thorne, we assume that quantum fields in the non - extremal geometry are populated with the boltzmann factor 65\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\exp = \left({\hbar {{\hat \omega - \hat m{\omega _ j} - {{\hat q}_e}{\phi _ e} - {{\hat q}_m}{\phi _ m}} \over {{t_h}}}} \right),$$\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}${{\hat q}_{e, m}}$\end{document} are the electric and magnetic charge operators . We also assume that modes obey (64) at extremality . Using the definitions (60)(61), we obtain the non - trivial extremal boltzmann factor in the extremal and near - horizon limit 66\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\exp = \left({- \hbar {m \over {{t_\phi}}} - \hbar {{{q_e}} \over {{t_e}}} - - \hbar {{{q_m}} \over {{t_m}}}} \right),$$\end{document} where the mode number m and charges qe, m in the near - horizon region are equal to the original mode number and charges \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$\hat m,{{\hat q}_{e, m}}$\end{document}. This completes the argument that the frolov - thorne vacuum is non - trivially populated in the extremal limit . Now, as noted in, there is a caveat in the previous argument for the kerr black hole and, as a trivial generalization, for all black holes that do not possess a global timelike killing vector . For any non - extremal black hole, if there is no global timelike killing vector, this vector field should become null on some surface at some distance away from the horizon ., this timelike killing field defines a positive conserved quantity for excitations in the near - horizon region, ruling out instabilities . However, when approaching extremality, it might turn out that the velocity of light surface approaches asymptotically the horizon . In that case, the horizon - generating killing field of the extreme black hole may not be everywhere timelike . This causes serious difficulties in defining quantum fields directly in the near - horizon geometry [183, 229, 228]. However, (at least classically) dynamical instabilities might appear only if there are actual bulk degrees of freedom in the near - horizon geometries . We will argue that this is not the case in section 2.9 . As a conclusion, extremal frolov - thorne temperatures can be formally and uniquely defined as the extremal limit of non - extremal temperatures and chemical potentials . However, the physical interpretation of these quantities is better understood finitely away from extremality . The condition for having a global timelike killing vector this condition is violated for the extremal kerr black hole or for any extremal kerr - newman black hole with \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$a \geq q/\sqrt 3$\end{document}, as can be shown by using the explicit values defined in (2.4). (the extremal kerr - newman near - horizon geometry does possess a global timelike killing vector when \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$a <q/\sqrt 3$\end{document} and the kerr - newman - ads black holes do as well when \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$4{a^2}/({\delta _ 0}r _ + ^2) <1$\end{document}, which is true for large black holes with r+ l. nevertheless, there might be other instabilities due to the electric superradiant effect .) The extremal frolov - thorne temperatures should also be directly encoded in the metric (25). More precisely, these quantities should only depend on the metric and matter fields and not on their equations of motion . Indeed, from the derivation (60)(61), one can derive these quantities from the angular velocity, electromagnetic potentials and surface gravity, which are kinematical quantities . More physically, the hawking temperature arises from the analysis of free fields on the curved background, and thus depends on the metric but not on the equations of motion that the metric solves . It should also be the case for the extremal frolov - thorne temperatures . Using a reasonable ansatz for the general black - hole solution of (1), including possible higher - order corrections, one can derive [83, 20] the very simple formula 67\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${t_\phi} = {1 \over {2\pi k}}.$$\end{document} from similar considerations, it should also be possible to derive a formula for te in terms of the functions appearing in (25). We propose simply that 68\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${t_e} = {1 \over {2\pi e}}.$$\end{document} while we do not have a direct proof of the equivalence between (68) and (61), the formula is consistent with the thermodynamics of (ads)-kerr - newman black holes as one can check from the formulae in section 2.4 . It would be interesting to generalize the arguments of [83, 20] to prove the equivalence . Similarly, one can work out the thermodynamics of five - dimensional rotating black holes . Since there are two independent angular momenta \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}${{\mathcal j}_1},{{\mathcal j}_2}$\end{document}, there are also two independent chemical potentials \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}${t_{{\phi _ 1}}},{t_{{\phi _ 2}}}$\end{document} associated with the angular momenta . The same arguments lead to 69\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${t_{{\phi _ 1}}} = {1 \over {2\pi {k_1}}},\quad {t_{{\phi _ 2}}} = {1 \over {2\pi {k_2}}},$$\end{document} where k1 and k2 are defined in the near - horizon solution (36). When considering the uplift (2) of the gauge field along a compact direction of length 2r, one can use the definition (69) to define the chemical potential associated with the direction . Since the circle has a length 2r, the extremal frolov - thorne temperature is expressed in units of r, 70\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${t_\chi} \equiv {t_e}{r_\chi} = {{{r_\chi}} \over {2\pi e}},$$\end{document} where te is defined in (68). The entropy of the extremal kerr black hole is \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}${{\mathcal s}_{{\rm{ext}}}} = 2\pi j$\end{document}. Integrating (57) or using the explicit near - horizon geometry and using (67), we find 71\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${t_\phi} = {1 \over {2\pi}},$$\end{document} and te is not defined . The entropy of the extremal reissner - nordstrm black hole is \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}${{\mathcal s}_{{\rm{ext}}}} = \pi {q^2}$\end{document}. Integrating (57), we obtain 72\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${t_e} = {1 \over {2\pi q}},$$\end{document} while t is not defined . For the electrically - charged kerr - newman black hole, the extremal entropy reads as \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}${{\mathcal s}_{{\rm{ext}}}} = \pi ({a^2} + {r^2})$\end{document}. Expressing the entropy in terms of the physical charges \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$q = \sqrt {r _ + ^2 - {a^2}}$\end{document} and j = ar+, we obtain 73\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${{\mathcal s}_{{\rm{ext}}}} = {\pi \over 2}\left({{{4{j^2}} \over {\sqrt {{q^4} + 4{j^2}} - {q^2}}} + \sqrt {{q^4} + 4{j^2}} - {q^2}} \right).$$\end{document} using (57) and re - expressing in terms of the parameters (a, r+) we find 74\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${t_\phi} = {{{a^2} + r _ + ^2} \over {4\pi a{r _ +}}}, \quad {t_e} = {{{a^2} + r _ + ^2} \over {2\pi {{(r _ + ^2 - {a^2})}^{3/2}}}}.$$\end{document} we can also derive t from (67) and the explicit near - horizon geometry (39). Te is consistent with (68). For the extremal kerr - newman - ads black hole, the simplest way to obtain the thermodynamics at extremality is to compute (60)(61). Using the extremality constraint (46), we obtain 75\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${t_\phi} = {{({a^2} + r _ + ^2){\delta _ 0}} \over {4\pi a{r _ +} \xi}},\quad {t_e} = {{({a^2} + r _ + ^2){\delta _ 0}} \over {2\pi {q_e}(r _ + ^2 - {a^2})}},$$\end{document} where we used the definitions (48). These quantities reduce to (74) in the limit of no cosmological constant when there is no magnetic charge, qm = 0 . The extremal 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}${{\mathcal s}_{{\rm{ext}}}} = \pi (r _ + ^2 + {a^2})/\xi$\end{document}. An important question about near - horizon geometries is the following: how much dynamics of gravity coupled to matter fields is left in a near - horizon limit such as (23)? We will explore in the following sections 2.8 and 2.9 several aspects of the dynamics in the near - horizon limit . In this section, we will discuss the existence of near - extremal solutions obtained from a combined near - horizon limit and zero temperature limit . We will discuss in section 2.8 the absence of non - perturbative solutions in the near - horizon geometries, such as black holes . In section 2.9, we will argue for the absence of local bulk degrees of freedom, and finally in section 4.4 we will discuss non - trivial boundary dynamics generated by large diffeomorphisms . Let us first study infinitesimal perturbations of the near - horizon geometry (25). As a consequence of the change of coordinates and the necessary shift of the gauge field (23), the near - horizon energy \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$\delta {{\mathcal q}_{{\partial _ t}}}$\end{document} of an infinitesimal perturbation is related to the charge associated with the generator of the horizon \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}${\xi _ {{\rm{tot}}}} \equiv (\xi, \lambda) = ({\partial _ t} + \omega _ j^{{\rm{ext}}}{\partial _ \phi},\phi _ e^{{\rm{ext}}})$\end{document} as follows, 76\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\delta {{\mathcal q}_{{\xi _ {{\rm{tot}}}}}} = {\lambda \over {{r_0}}}\delta {{\mathcal q}_{{\xi _ assuming no magnetic charges for simplicity, the conserved charge \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$\delta {{\mathcal q}_{{\xi _ {{\rm{tot}}}}}}$\end{document} is given by \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$\delta {\mathcal m} - \omega _ j^{{\rm{ext}}}\delta {\mathcal j} - \phi _ e^{{\rm{ext}}}\delta {{\mathcal q}_e}$\end{document}.10 using the first law of thermodynamics valid for arbitrary (not necessarily stationary) perturbations, the left - hand side of (76) can be expressed as 77\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${t_h}\delta {{\mathcal s}_{{\rm{ext}}}} = \delta {{\mathcal q}_{{\xi _ {{\rm{tot}}}}}}.$$\end{document} any geometry that asymptotes to (25) will have finite near - horizon energy \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}${{\mathcal q}_{{\partial _ t}}}$\end{document}. Indeed, an infinite near - horizon energy would be the sign of infrared divergences in the near - horizon geometry and it would destabilize the geometry . It then follows from (76)(77) that any infinitesimal perturbation of the near - horizon geometry (25) will correspond to an extremal black - hole solution with vanishing hawking temperature, at least such that th = o(). Common usage refers to black - hole solutions, where th as near - extremal black holes . Nevertheless, it should be emphasized that after the exact limit 0 is taken the hawking temperature of such a solution is exactly zero . Starting from a stationary non - extremal black hole of mass m in boyer - lindquist coordinates, we perform the near - horizon scaling limit (23) together with the scaling of the temperature 78\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${t_h} \rightarrow {\lambda \over {{r_0}}}{t^{{\rm{near}} - {\rm{ext}}}}.$$\end{document} while the form of the general non - extremal solution would be required to perform that limit in detail, all examples so far in the class of theories (1), such as the kerr - newman - ads black hole, lead to the following metric 79\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\begin{array}{*{20}c} {d{s^2} = \gamma (\theta)\left [{- r(r + 4\pi {t^{{\rm{near}} - {\rm{ext}}}})d{t^2}{{d{r^2}} \over {r(r + 4\pi {t^{{\rm{near}} - {\rm{ext}}}})}} + \alpha (\theta)d{\theta ^2} + \gamma (\theta){{(d\phi + krdt)}^2}} \right],}\\ {{\chi ^a} = {\chi ^a}(\theta),\quad \quad {a^i} = {f^i}(\theta)(d\phi + krdt) - {{{e_i}} \over k}d\phi . }\\ \end{array}$$\end{document} the near - extremal near - horizon solution (79) is diffeomorphic to the near - horizon geometry in poincar coordinates (25). Denoting the finite temperature coordinates by a subscript t and the poincar coordinates by a subscript p, the change of coordinates reads as [207, 247, 4, 53] 80\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${t_p} = {1 \over 2}({\tau ^ +} + {\tau ^ -}),$$\end{document} 81\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${r_p} = {2 \over {{\tau ^ -} - {\tau ^ +}}}, $$\end{document} 82\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${\phi _ p} = {\phi _ f} - 2\pi {t^{{\rm{near}} - {\rm{ext}}}}k{t_f} + {k \over 2}\log \left({{{1 - {{({\tau ^ +})} ^2}} \over {1 - {{({\tau ^ -})}^2}}}} \right),$$\end{document} where 83\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${\tau ^ \pm} = \tanh \left [{\pi {t^{{\rm{near}} - {\rm{ext}}}}{t_f} \pm {1 \over 4}\log \left({{{{r_f}} \over {{r_f} + 4\pi {t^{{\rm{near}} - {\rm{ext}}}}}}} \right)} \right].$$\end{document} therefore, the classical geometries are equivalent . However, since the diffeomorphism is singular at the boundary rf, there is a distinction at the quantum level . Since the asymptotic time in near - extremal geometries (79) is different than in extremal geometries (25), fields will be quantized in a different manner in the two geometries . (76) by r0/ and using (77) and (78), we get that the energy variation around the near - extremal geometry is given by 84\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${{\not \delta/}{\mathcal q}_{{\partial _ t}}} = {t^{{\rm{near}} - {\rm{ext}}}}\delta {{\mathcal s}_{{\rm{ext}}}}\,,$$\end{document} where the extremal entropy \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}${{\mathcal s}_{{\rm{ext}}}}$\end{document} can be expressed in terms of the near - horizon quantities as (50). We denote the variation by \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$\not \delta$\end{document} to emphasize that the energy is not the exact variation of a quantity unless t is constant or \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}${{\mathcal s}_{{\rm{ext}}}}$\end{document} is fixed (which would then lead to zero energy). Therefore, the charge \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$\not \delta{{\mathcal q}_{{\partial _ since our derivation of the formula (84) was rather indirect, we check that it is correct for the kerr - newman - ads family of black holes by computing the energy variation directly using the lagrangian charges defined in [36, 90, 97]. We reviewed in section 2.3 that for any stationary extremal spinning black hole one can isolate a geometry in the vicinity of the horizon, which has enhanced symmetry and universal properties . We discussed in section 2.7 that another class of stationary near - horizon geometries can be defined, which are, however, related to the extremal near - horizon geometries via a diffeomorphism . It is natural to ask how unique the stationary near - horizon geometries are . In the case of einstein gravity, one can prove that the nhek (near - horizon extremal kerr geometry) is the unique (up to diffeomorphisms) regular stationary and axisymmetric solution asymptotic to the nhek geometry with a smooth horizon . This can be paraphrased by the statement that there are no black holes inside of the nhek geometry . One can also prove that there is a near - horizon geometry in the class (25), which is the unique (up to diffeomorphisms) near - horizon stationary and axisymmetric solution of ads - einstein - maxwell theory [192, 193, 191]. It is then natural to conjecture that any stationary solution of the more general action (1), which asymptotes to a near - horizon geometry of the form (25) is diffeomorphic to it . This conjecture remains to be proven . In this section, we will review arguments pointing to the absence of local degrees of freedom in the near - horizon geometries (25), following the arguments of [4, 122] for einstein gravity in the nhek geometry . The only non - trivial dynamics can be argued to appear at the boundary of the near - horizon geometries due to the action of non - trivial diffeomorphisms . One usually expects that conserved charges are captured by highly - symmetric solutions . From the theorems presented in section 2.8, we infer that in (ads)-einstein - maxwell theory there is no candidate non - trivial near - horizon solution charged under the sl(2,) u(1) symmetry (u(1) symmetry when electric charge is present), except for a solution related via a diffeomorphism to the near - horizon geometry . If the conjecture presented in section 2.8 is correct, there is no non - trivial candidate in the whole theory (1). One can then argue that there will be no solution even non - stationary with non - zero mass or angular momentum (or electric charge when a maxwell field is present) above the background near - horizon geometry, except solutions related via a diffeomorphism . In order to test whether or not there exist any local bulk dynamics in the class of geometries, which asymptote to the near - horizon geometries (25), one can perform a linear analysis and study which modes survive at the non - linear level after backreaction is taken into account . This analysis has been performed with care for the spin 2 field around the nhek geometry in [4, 122] under the assumption that all non - linear solutions have vanishing sl(2,) u(1) charges (which is justified by the existence of a birkoff theorem as mentioned in section 2.8). The conclusion is that there is no linear mode that is the linearization of a non - linear solution . In other words . It would be interesting to investigate if these arguments could be generalized to scalars, gauge fields and gravitons propagating on the general class of near - horizon solutions (25) of the action (1), but such an analysis has not been done at that level of generality . This lack of dynamics is familiar from the ads2 s geometry, which, as we have seen in sections 2.22.3, is the static limit of the spinning near - horizon geometries . In the above arguments, conversely, in the case of non - compact horizons, such as the extremal planar ads - reissner - nordstrm black hole, flux can leak out the boundary and the arguments do not generalize straightforwardly . There are indeed interesting quantum critical dynamics around ads2 near - horizon geometries, but we will not touch upon this topic here since we concentrate exclusively on compact black holes . Since we aim at drawing parallels between black holes and two - dimensional cfts (2d cfts), it is useful to describe some key properties of 2d cfts . Background material can be found, e.g., in [120, 149, 234]. An important caveat to keep in mind is that there are only sparse results in gravity that can be interpreted in terms of a 2d cft . Only future research will tell if 2d cfts are the right theories to be considered (if a holographic correspondence can be precisely formulated at all) or if generalized field theories with conformal invariance are needed . For progress in this direction, see [130, 169]. A 2d cft is defined as a local quantum field theory with local conformal invariance . In two - dimensions, the local conformal group is an infinite - dimensional extension of the globally - defined conformal group sl(2,) sl(2,) on the plane or on the cylinder . It is generated by two sets of vector fields ln, \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}${l_n},{{\bar l}_n},n \in {\mathbb z}$\end{document}, n obeying the lie bracket algebra 85\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} {[{l_m},{l_n}] = (m - n){l_{m + n}},}\\ {[{l_m},{{\bar l}_n}] = 0},\quad \quad \quad \quad\\ {[{{\bar l}_m},{{\bar l}_n}] = (m - n){{\bar l}_{m + n}}}.\\ \end{array}$$\end{document} from noether s theorem, each symmetry is associated to a quantum operator . The local conformal symmetry is associated with the conserved and traceless stress - energy tensor operator, which can be decomposed into left and right moving modes \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}${{\mathcal l}_n}$\end{document} and \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}${{\bar {\mathcal l}}_n}$\end{document}, n . The operators \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}${{\mathcal l}_n},{{\bar {\mathcal l}}_n}$\end{document} form two copies of the virasoro algebra 86\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\begin{array}{*{20}c} {[{{\mathcal l}_m},{{\mathcal l}_n}] = (m - n){{\mathcal l}_{m + n}} + {{{c_l}} \over {12}}m({m^2} - {a_l}){\delta _ {m + n,0}},} \\ {[{{\mathcal l}_m},{{\overline {\mathcal l}}_n}] = 0,\quad \quad \quad \quad \quad \quad \quad \quad \quad \quad \quad \quad \quad} \\ {[{{\overline {\mathcal l}}_m},{{\overline {\mathcal l}}_n}] = (m - n){{\overline {\mathcal l}}_{m + n}} + {{{c_r}} \over {12}}m({m^2} - {a_r}){\delta _ {m + n,0}},} \\ \end{array}$$\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 l}_{- 1}},{{\mathcal l}_0},{{\mathcal l}_1}$\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}${{\bar {\mathcal l}}_{- 1}},{{\bar {\mathcal l}}_0},{{\bar {\mathcal l}}_1}$\end{document}) span a sl(2,) subalgebra . The pure numbers cl and cr are the left and right - moving central charges of the cft . The auxiliary parameters al, ar depend if the cft is defined on the plane or on the cylinder . They correspond to shifts of the background value of the zero eigenmodes \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}${{\mathcal l}_0},{{\bar {\mathcal l}}_0}$\end{document}. In many examples of cfts, additional symmetries are present in addition to the two sets of virasoro algebras . A 2d cft can be uniquely characterized by a list of (primary) operators \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}${\mathcal o}$\end{document}, the conformal dimensions of these operators (their eigenvalue under \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}${{\mathcal l}_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}${{\bar {\mathcal l}}_0}$\end{document}) and the operator product expansions between all operators . Since we will only be concerned with universal properties of cfts here, such detailed data of individual cfts will not be important for our considerations . We will describe in the next short sections 3.1, 3.2 and 3.3 some properties of cfts that are conjectured to be relevant to the kerr / cft correspondence and its extensions: the cardy formula, some properties of the discrete light - cone quantization (dlcq) and some properties of symmetric product orbifold cfts . In any unitary and modular invariant cft, the asymptotic growth of states in the microcanonical ensemble is determined only by the left and right central charge and the left and right eigenvalues \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}${{\mathcal l}_0},{{\bar {\mathcal l}}_0}$\end{document} as 87\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${\mathcal{s}_{{\text{cft}}}} = 2\pi \left ({\sqrt {\frac{{{c_l}{\mathcal{l}_0}}}{6}} + \sqrt {\frac{{{c_r}{{\overline {\mathcal{l}}} _ 0}}}{6}}} \right),$$\end{document} when \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}${{\mathcal l}_0} \gg {c_l},{{\bar {\mathcal l}}_0} \gg {c_r}$\end{document}. This is known as cardy s formula derived originally in [61, 48] using modular invariance of the cft transforming to the canonical ensemble using the definition of the left and right temperatures, 88\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${\left({{{\partial {{\mathcal s}_{{\rm{cft}}}}} \over {\partial {{\mathcal l}_0}}}} \right)_{{{\bar {\mathcal l}}_0}}} = {1 \over {{t_l}}},\quad \quad {\left({{{\partial {{\mathcal s}_{{\rm{cft}}}}} \over {\partial {{\mathcal l}_0}}}} \right)_{{{\mathcal l}_0}}} = {1 \over {{t_r}}},$$\end{document} we get 89\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${{\mathcal l}_0} = {{{\pi ^2}} \over 6}{c_l}t_l^2,\quad \quad {\bar {\mathcal l}_0} = {{{\pi ^2}} \over 6}{c_r}t_r^2,$$\end{document} and, therefore, we obtain an equivalent form of cardy s formula, 90\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${{\mathcal s}_{{\rm{cft}}}} = {{{\pi ^2}} \over 3}({c_l}{t_l} + {c_r}{t_r}),$$\end{document} valid when tl 1, tr 1 . The role of the dlcq of cfts in the context of the kerr / cft correspondence was suggested in (for closely related work see). Here, we will review how a dlcq is performed and how it leads to a chiral half of a cft . A chiral half of a cft is here defined as a sector of a 2d cft defined on the cylinder, where the right - movers are set to the ground state after the limiting dlcq procedure . Let us start with a cft defined on a cylinder of radius r, 91\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$d{s^2} = - d{t^2} + d{\phi ^2} = - du\;dv,\quad u = t - \phi, \quad v = t + \phi .$$\end{document} here the coordinates are identified as (t,) (t, + 2r), which amounts to 92\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$(u, v) \sim (u - 2\pi r, v + 2\pi r).$$\end{document} the momentum operators p and p along the u and v directions are l0 and \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}${{\bar l}_0}$\end{document}, respectively . They have a spectrum 93\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${p^v}\vert o\rangle = {l_0}\vert o\rangle = (h + n){1 \over r}\vert o\rangle, $$\end{document} 94\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${p^u}\vert o\rangle = {\bar l_0}\vert o\rangle = (\bar h + \bar n){1 \over r}\vert o\rangle, $$\end{document} where the conformal dimensions obey h, \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$\bar h \geq 0$\end{document} and n, \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$\bar n \neq 0$\end{document} are quantized left and right momenta . Following seiberg, consider a boost with rapidity 95\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$u{\prime} = {e^\gamma}u,\quad v{\prime} = {e^{- \gamma}}v.$$\end{document} the boost leaves the flat metric invariant . The discrete light - cone quantization of the cft is then defined as the limit with r re fixed . In that limit, the identification (92) becomes 96\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$(u{\prime},v{\prime}) \sim (u{\prime} - 2\pi r{\prime},v{\prime}).$$\end{document} therefore, the resulting theory is defined on a null cylinder . Because of the boosted kinematics, we have 97\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${p^v}\vert o\rangle = (h + n){1 \over {r{e^\gamma}}}\vert o\rangle, $$\end{document} 98\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${p^{u{\prime}}}\vert o\rangle = (\bar h + \bar n){{{e^\gamma}} \over r}\vert o\rangle .$$\end{document} keeping p (the momentum along v) finite in the limit requires \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$\bar h = 0$\end{document} and \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$\bar n = 0$\end{document}. Therefore, the dlcq limit requires one to freeze the right - moving sector to the vacuum state . All physical finite - energy states in this limit only carry momentum along the compact null direction u. therefore, the dlcq limit defines a hilbert space \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}${\mathcal h}$\end{document}, 99\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${\mathcal h} = {\{\vert {\rm{anything}}\rangle _ l} \otimes \vert 0{\rangle _ r}\}$$\end{document} with left chiral excitations around the sl(2,) sl(2,) invariant vacuum of the cft |0l |0r . As a consequence,, the dlcq of a 2d cft leads to a chiral half of the cft with central charge c = cl . How much dynamics is left in a chiral half of a cft is an important question that is left to be examined in detail in the future . Given a set of virasoro generators \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}${{\mathcal l}_n}$\end{document} and a non - zero integer n 0, one can always redefine a subset or an extension of the generators, which results in a different central charge (see, e.g.,). One can easily check that the generators 100\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${\mathcal l}_n^{{\rm{short}}} = {1 \over n}{{\mathcal l}_{n\;n}}$$\end{document} obey the virasoro algebra with a larger central charge c = n c. conversely, one might define 101\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${\mathcal l}_n^{{\rm{short}}} = n{{\mathcal l}_{n / n}}.$$\end{document} in general, the generators \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}${\mathcal l}_n^{{\rm{long}}}$\end{document} with n n k, k do not make sense because there are no fractionalized virasoro generators in the cft . Such generators would be associated with multivalued modes e on the cylinder (t,) (t, + 2). However, in some cases, as we review below, the virasoro algebra (101) can be defined . The resulting central charge is smaller and given by c = c / n . If a cft with generators (101) can be defined such that it still captures the entropy of the original cft, the cardy formula (90) applied in the original cft could then be used outside of the usual cardy regime tl 1 . Indeed, using the cft with left - moving generators (101) and their right - moving analogue, one has 102\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${{\mathcal s}_{{\rm{cft}}}} = {{{\pi ^2}} \over 3}({{{c_l}} \over n}(n{t_l}) + {{{c_r}} \over n}(n{t_r})),$$\end{document} which is valid when ntl 1, ntr 1 . If n is very large, cardy s formula (90) would then always apply . We will use the assumption of the existence of such a long string cft in section 4.4 to justify the validity of cardy s formula outside the usual cardy regime as done originally in . The long string cft can be made more explicit in the context of symmetric product orbifold cfts, which appear in the ads3/cft2 correspondence [206, 114, 123] (see also and references therein). These orbifold cfts can be argued to be relevant in the present context, since the kerr / cft correspondence might be understood as a deformation of the ads3/cft2 correspondence, as argued in [157, 98, 23, 116, 31, 243, 246, 115, 130]. Let us then briefly review the construction of symmetric product orbifold cfts . Given a conformally - invariant sigma - model with target space manifold \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}${\mathcal m}$\end{document}, one can construct the symmetric product orbifold by considering the sigma - model with identical copies of the target space manifold \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}${\mathcal m}$\end{document}, identified up to permutations, 103\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${\rm{sy}}{{\rm{m}}^n}({\mathcal m}) \equiv ({\otimes ^n}{\mathcal m})/{s_n},$$\end{document} where sn is the permutation group on n objects . The low energy (infrared) dynamics is a cft with central charge csym = nc if the central charge of the low energy cft of the original sigma model is c. the virasoro generators of the resulting infrared cft can then be formally constructed from the generators \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}${{\mathcal l}_m}$\end{document} of the original infrared cft as (100). Conversely, if one starts with a symmetric product orbifold, one can isolate the long string sector, which contains the long twisted operators . One can argue that such a sector can be effectively described in the infrared by a cft, which has a virasoro algebra expressed as (101) in terms of the virasoro algebra of the low energy cft of the symmetric product orbifold . The role of these constructions for the kerr / cft correspondence remains to be fully understood . We discussed that near - horizon geometries of compact extremal black holes are isolated systems with universal properties and we reviewed that in all analyzed cases they have no local bulk dynamics . Given the non - trivial thermodynamic properties of these systems even at extremality, one can suspect that some non - trivial dynamics are left . It turns out that such non - trivial dynamics appears at the boundary of the near - horizon geometry . We now show that near - horizon geometries can be extended to a large class describing extremal boundary excitations . The set of all near - horizon geometries will admit additional symmetries at their boundary asymptotic symmetries which will turn out to be given by one copy of the virasoro algebra . We will then argue that these near - horizon geometries are described by chiral limits of two - dimensional cfts, which we will use to microscopically derive the entropy of any charged or spinning extremal black hole . Let us discuss the existence and the construction of a consistent set of boundary conditions that would define the set of solutions in the near - horizon region of extremal black holes . Since the near - horizon region is not asymptotically flat or asymptotically anti - de sitter, one cannot use previous results in those spacetimes to derive the boundary conditions in the near - horizon region . A large literature on the theory of boundary conditions and asymptotic charges exists, see [9, 237, 59, 196, 35, 36] (see also for a review). We will use the lagrangian methods [35, 36] to address the current problem . A set of boundary conditions always comes equipped with an asymptotic symmetry algebra . Restricting our discussion to the fields appearing in (1), the boundary conditions are preserved by a set of allowed diffeomorphisms and u(1) gauge transformations (,), which act on the fields as 104\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 _ {(\zeta, \lambda)}}{g_{\mu \nu}} = {{\mathcal l}_\zeta}{g_{\mu \nu}},\quad {\delta _ {(\zeta, \lambda)}}{a_\mu} = {{\mathcal l}_\zeta}{a_\mu} + {\partial _ \mu}\lambda,} \\ {{\delta _ {(\zeta, \lambda)}}{\chi ^a} = {{\mathcal l}_\zeta}{\chi ^a}.\quad \quad \quad \quad \quad \quad \quad \quad \quad \quad \quad} \\ \end{array}$$\end{document} asymptotic symmetries are the set of all these allowed transformations that are associated with non - trivial conserved charges . The set of allowed transformations that are associated with zero charges are pure gauge or trivial transformations . The set of asymptotic symmetries inherits a lie algebra structure from the lie commutator of diffeomorphisms and u(1) gauge transformations . Therefore, the asymptotic symmetries form an algebra, 105\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$[({\zeta _ m},{\lambda _ m}),({\zeta _ n},{\lambda _ n})] \equiv ([{\zeta _ m},{\zeta _ n}],{[{\lambda _ m},{\lambda _ n}]_\zeta}),$$\end{document} where [m, n] is the lie commutator and 106\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${[{\lambda _ m},{\lambda _ n}]_\zeta} \equiv \zeta _ m^\mu {\partial _ \mu}{\lambda _ n} - \zeta _ n^\mu {\partial _ \mu}{\lambda _ m}.$$\end{document} consistency requires that the charge associated with each element of the asymptotic symmetry algebra be finite and well defined . Moreover, as we are dealing with a spatial boundary, the charges are required to be conserved in time . By construction, \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$\delta {\mathcal q}$\end{document} from infinitesimal variations of the fields around a solution . If \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$\delta {\mathcal q}$\end{document} is the exact variation of a quantity \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}, the quantity \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 the well - defined charge and the charges are said to be integrable . Imposing consistent boundary conditions and obtaining the associated asymptotic symmetry algebra requires a careful analysis of the asymptotic dynamics of the theory . If the boundary conditions are too strong, all interesting excitations are ruled out and the asymptotic symmetry algebra is trivial . If they are too weak, the boundary conditions are inconsistent because transformations preserving the boundary conditions are associated to infinite or ill - defined charges . In general there is no universal algorithm to define the boundary conditions and the set of asymptotic symmetries . One standard algorithm used, for example, in [168, 167] consists in first promoting all exact symmetries of the background solution as asymptotic symmetries and second acting on solutions of interest with the asymptotic symmetries in order to generate tentative boundary conditions . The boundary conditions are then restricted in order to admit consistent finite, well defined and conserved charges . Finally, the set of asymptotic diffeomorphisms and gauge transformations, which preserve the boundary conditions are computed and one deduces the full asymptotic symmetry algebra after computing the associated conserved charges . As an illustration, asymptotically anti - de sitter spacetimes in spacetime dimensions d + 1 admit the so(2, d) asymptotic symmetry algebra for d 3 [1, 15, 168, 167] and two copies of the virasoro algebra for d = 2 . Asymptotically - flat spacetimes admit as asymptotic symmetry algebra the poincar algebra or an extension thereof depending on the precise choice of boundary conditions [9, 231, 147, 237, 13, 12, 16, 37, 38, 92, 261]. From these examples, we learn that the asymptotic symmetry algebra can be larger than the exact symmetry algebra of the background spacetime and it might in some cases contain an infinite number of generators . We also notice that several choices of boundary conditions, motivated from different physical considerations, might lead to different asymptotic symmetry algebras . Let us now motivate boundary conditions for the near - horizon geometry of extremal black holes . There are two boundaries at r = and r = . It was proposed in [156, 159] to build boundary conditions on the boundary r = such that the asymptotic symmetry algebra contains one copy of the virasoro algebra generated by 107\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${\zeta _ \epsilon} = \epsilon (\phi){\partial _ \phi} - {r^{\epsilon{\prime}}}(\phi){\partial _ r} + ({\rm{subleading}}\;{\rm{terms}}),$$\end{document} 108\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${\lambda _ \epsilon} = (f(\theta) - {e \over k}\epsilon (\phi) + ({\rm{subleading}}\;{\rm{terms}}).$$\end{document} part of the physical motivation behind this ansatz is the existence of a non - zero temperature t associated with modes corotating with the black hole, as detailed in section 2.6 . This temperature suggests the existence of excitations along . The ansatz for will be motivated in (117). The subleading terms might be chosen such that the generator is regular at the poles = 0, . This ansatz has to be validated by checking if boundary conditions preserved by this algebra exist such that all charges are finite, well defined and conserved . 109\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\epsilon(\phi) = - {e^{- in\phi}},$$\end{document} the generators ln (n, n) obey the virasoro algebra with no central extension 110\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$i[{l_m},{l_n}] = (m - n){l_{m + n}},$$\end{document} where the bracket has been defined in (105) finding consistent boundary conditions that admit finite, conserved and integrable virasoro charges and that are preserved by the action of the virasoro generators is a non - trivial task . The details of these boundary conditions depend on the specific theory at hand because the expression for the conserved charges depend on the theory . (for the action (1), the conserved charges can be found in). Specializing in the case of the extremal kerr black hole in einstein gravity, the problem of finding consistent boundary conditions becomes more manageable but is still intricate (see discussions in). In, the following fall - off conditions 111\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\begin{array}{*{20}c} {{g_{tt}} = {\mathcal o}({r^2})\quad {g_{t\phi}} = k\gamma (\theta)\gamma {{(\theta)}^2}r + {\mathcal o}(1),}\\ {{g_{t\theta}} = {\mathcal o}({1 \over r}),\quad {g_{tr}} = {\mathcal o}({1 \over {{r^2}}}),\quad {g_{\phi \phi}} = {\mathcal o}1,}\\ {{g_{\phi \theta}} = {\mathcal o}({1 \over r}),\quad {g_{\phi r}} = {\mathcal o}({1 \over r}),\quad {g_{\theta r}} = {\mathcal o}({1 \over {{r^2}}}),}\\ {{g_{\theta \theta}} = \gamma (\theta)\alpha {{(\theta)}^2} + {\mathcal o}({1 \over r}),\quad {g_{rr}} = {{\gamma (\theta)} \over {{r^2}}} + {\mathcal o}({1 \over {{r^3}}}),}\\ \end{array}$$\end{document} were proposed as a part of the definition of boundary conditions . The zero energy excitation condition 112\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\delta {{\mathcal q}_{{\partial _ t}}} = 0,$$\end{document} was imposed as a supplementary condition . A non - trivial feature of the boundary conditions (111)(112) is that they are preserved precisely by the virasoro algebra (107), by t and the generator (24) (as pointed out in) and subleading generators . (note that these boundary conditions are not preserved by the action of the third sl(2,) generator (29).) It was shown in that the virasoro generators are finite given the fall - off conditions and well defined around the background nhek geometry . It was shown in that the virasoro generators are conserved and well defined around any asymptotic solution given that one additionally regularizes the charges using counter - term methods . Therefore, up to some technical details that remain to be fully understood, it can be claimed that consistent boundary conditions admitting (at least) a virasoro algebra as asymptotic symmetry algebra exist . The set of trivial asymptotic symmetries comprise two of the sl(2,) generators . It is not clear if the boundary conditions could be enhanced in order to admit all sl(2,) generators as trivial asymptotic symmetries . Let us now generalize these arguments to the electrically - charged kerr - newman black hole in einstein - maxwell theory . First, the presence of the chemical potential te suggests that some dynamics are also present along the gauge field . The associated conserved electric charge \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}${{\mathcal q}_e}$\end{document} can be shown to be canonically associated with the zero - mode generator j0= (0, 1) with gauge parameter = 1 . It is then natural to define the current ansatz 113\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${j_n} = (0, - {e^{- in\phi}}),$$\end{document} which obeys the commutation relations 114\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$i\;[{l_m},{j_n}] = - n{j_{m + n}},\quad i\;[{j_m},{j_n}] = 0.$$\end{document} the non - trivial step consists in establishing the existence of boundary conditions such that the virasoro and the current charges are well defined and conserved . Ongoing work is in progress in that direction.12 one can simplify the problem of constructing boundary conditions by imposing the following additional constraints 115\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\delta {{\mathcal q}_{{\partial _ t}}} = 0,\quad \delta {{\mathcal q}_e} = 0,$$\end{document} which discard the current algebra . Such a simplification was used in and the following boundary conditions were proposed (up to the term e / k, which was omitted in) 116\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\begin{array}{*{20}c} {{a_t} = {\mathcal o}(r),\quad \quad {a_\phi} = f(\theta) - {e \over k} + {\mathcal o}({1 \over r}),}\\ {{a_\theta} = {\mathcal o}(1),\quad \quad {a_r} = {\mathcal o}({1 \over {{r^2}}}),}\\ \end{array}$$\end{document} which are preserved upon acting with the virasoro generator (107)(108). In particular, the choice of the compensating gauge transformation (108) is made such that 117\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${{\mathcal l}_{{\zeta _ \varepsilon}}}{a_\phi} + {\partial _ \phi}{\lambda _ \varepsilon} = o({r^{- 1}}).$$\end{document} it can be shown that the virasoro generators are finite under these boundary conditions . The presence of several independent planes of rotation allows for the construction of one virasoro ansatz and an associated frolov - thorne temperature for each plane of rotation [203, 173, 21, 225, 83]. More precisely, given n compact commuting killing vectors, one can consider an sl(n,) family of virasoro anstze by considering all modular transformations on the u(1) torus [201, 76]. However, preliminary results show that there is no boundary condition that allows simultaneously two different virasoro algebras in the asymptotic symmetry algebra . Rather, there are mutually - incompatible boundary conditions for each choice of virasoro ansatz . Since two u(1) circles form a torus invariant under sl(2,) modular transformations, one can then form an ansatz for a virasoro algebra for any circle defined by a modular transformation of the 1 and 2-circles . More precisely, we define 118\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\phi _ 1\prime= {p_1}{\phi _ 1} + {p_2}{\phi _ 2},\quad \quad \phi _ 2\prime = {p_3}{\phi _ 1} + {p_4}{\phi _ 2},$$\end{document} where p1p4 p2p3 = 1 and we consider the vector fields 119\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$l_n^{({p_1},{p_2})} = - {e^{- in\phi _ 1\prime}}{\partial _ {\phi _ 1\prime}} - ir{e^{- in\phi _ 1\prime}}{\partial _ r} + ({\rm{subleading}}\;{\rm{terms}}).$$\end{document} the resulting boundary conditions have not been thoroughly constructed, but evidence points to their existence [21, 201]. The occurrence of multiple choices of boundary conditions in the presence of multiple u(1) symmetries raises the question of whether or not the (ads)-reissner - nordstrom black hole admits interesting boundary conditions where the u(1) gauge symmetry (which is canonically associated to the conserved electric charge q) plays the prominent role . One can also ask these questions for the general class of (ads)-kerr - newman black holes . It was argued in [159, 204] that such boundary conditions indeed exist when the u(1) gauge field can be promoted to be a kaluza - klein direction of a higher - dimensional spacetime, or at least when such an effective description captures the physics . Denoting the additional direction by with + 2r, the problem amounts to constructing boundary conditions in five dimensions . As mentioned earlier, evidence points to the existence of such boundary conditions [21, 201]. The virasoro asymptotic - symmetry algebra is then defined using the ansatz 120\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$l_n^q = - {r_\chi}{e^{- {{i{n_\chi}} \over {{r_\chi}}}}}{\partial _ \chi} - ir{e^{- {{i{n_\chi}} \over {{r_\chi}}}}}{\partial _ r} + ({\rm{subleading}}\;{\rm{terms}})$$\end{document} along the gauge kaluza - klein direction . The same reasoning leading to the sl(2,) family of virasoro generators (119) would then apply as well . The existence of such a virasoro symmetry around the kerr - newman black holes is corroborated by near - extremal scattering amplitudes as we will discuss in section 5, and by the hidden conformal symmetry of probes, as we will discuss in section 6 . The boundary conditions discussed so far do not admit solutions with non - trivial charges under the sl(2,) exact symmetry group of the background geometry generated by 0,1 (29). In fact, the boundary conditions are not even invariant under the action of the generator 1 . One could ask the question if such an enlargement of boundary conditions is possible, which would open the possibility of enlarging the asymptotic - symmetry group to include the sl(2,) group and even a virasoro extension thereof . We will now argue that such enlargement would result in trivial charges, which would not belong to the asymptotic - symmetry group . First, we saw in section 2.7 that there is a class of near - extremal solutions (79) obeying the boundary conditions (111)(116) with near - horizon energy \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$\not \delta{{\mathcal q}_{{\partial _ t}}} = {t^{{\rm{near - ext}}}}\delta {{\mathcal s}_{{\rm{ext}}}}$\end{document}. However, the charge \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$\not \delta{{\mathcal q}_{{\partial _ t}}}$\end{document} is a heat term, which is not integrable when both t and \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}${{\mathcal s}_{{\rm{ext}}}}$\end{document} can be varied . Moreover, upon scaling the coordinates as t t/ and r r using the sl(2,) generator (24), one obtains the same metric as (79) with t t/. If one would allow the class of near - extremal solutions (79) and the presence of sl(2,) symmetries in a consistent set of boundary conditions, one would be forced to fix the entropy \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}${{\mathcal s}_{{\rm{ext}}}}$\end{document} to a constant, in order to define integrable charges . Since there is no other obvious candidate for a solution with non - zero near - horizon energy, we argued in section 2.9 that there is no such solution at all . If that assumption is correct, the sl(2,) algebra would always be associated with zero charges and would not belong to the asymptotic symmetry group . Hence, no additional non - vanishing virasoro algebra could be derived in a consistent set of boundary conditions . For alternative points of view, second, as far as extremal geometries are concerned, there is no need for a non - trivial sl(2,) or second virasoro algebra . As we will see in section 4.4, the entropy of extremal black holes will be matched using a single copy of the virasoro algebra, using the assumption that cardy s formula applies . Matching the entropy of non - extremal black holes and justifying cardy s formula requires two virasoro algebras, as we will discuss in section 6.6 . However, non - extremal black holes do not admit a near - horizon limit and, therefore, are not dynamical objects described by a consistent class of near - horizon boundary conditions . At most, one could construct the near horizon region of non - extremal black holes in perturbation theory as a large deformation of the extremal near - horizon geometry . This line of thought was explored in . In the context of the near - extremal kerr black hole, it was obtained using a dimensionally - reduced model such that the algebra of diffeomorphisms, which extends the sl(2,) algebra, is represented on the renormalized stress - energy tensor as a virasoro algebra . It would be interesting to further define and extend these arguments (which go beyond a standard asymptotic - symmetry analysis) to non - dimensionally - reduced models and to other near - extremal black holes . Finally, let us also note that the current discussion closely parallels the lower dimensional example of the near - horizon limit of the extremal btz black hole discussed in . There it was shown that the near - horizon geometry of the extremal btz black hole of angular momentum j is a geometry with sl(2,) u(1) isometry 121\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$d{s^2} = {{{l^2}} \over 4}\left({- {r^2}d{t^2} + {{d{r^2}} \over {{r^2}}} + 2j{{(d\phi + {r \over {\sqrt {2j}}}dt)}^2}} \right),$$\end{document} with + 2, which is known as the self - dual ads3 orbifold . It was found in that the asymptotic symmetry group consists of one chiral virasoro algebra extending the u(1) symmetry along, while the charges associated with the sl(2,) symmetry group are identically zero . These observations are consistent with the analysis of four - dimensional near - horizon geometries (25), whose constant sections share similar qualitative features with the three - dimensional geometries (121). It was also shown that an extension of the boundary conditions exists that is preserved by a second virasoro algebra extending the sl(2,) exact - symmetry algebra . All associated charges can be argued to be zero, but a non - trivial central extension still appears as a background charge when a suitable regularization is introduced . However, the regularization procedure does not generalize to the four - dimensional geometries mainly because two features of the three - dimensional geometry (121) are not true in general (t is null in (121) and t with 1 is a global timelike killing vector). Let us now assume in the context of the general theory (1) that a consistent set of boundary conditions exists that admit the virasoro algebra generated by (107)(108) as asymptotic - symmetry algebra . Current results are consistent with that assumption but, as emphasized earlier, boundary conditions have been checked only partially [156, 5, 21]. Let us define the dirac bracket between two charges as 122\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\{{{\mathcal q}_{({\zeta _ n})}}\} \equiv - {\delta _ {({\zeta _ m},{\lambda _ m})}}{{\mathcal q}_{({\zeta _ n},{\lambda _ n})}}.$$\end{document} here, the operator \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}${\delta _ {({\zeta _ m},{\lambda _ m})}}$\end{document} is a derivative in phase space that acts on the fields \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}${g_{\mu \nu}},a_\mu ^i,{\chi ^a}$\end{document} appearing in the charge \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}${\mathcal q}$\end{document} as (104). From general theorems in the theory of asymptotic - symmetry algebras [59, 35, 36], the dirac bracket represents the asymptotic symmetry algebra up to a central term, which commutes with each element of the algebra . Namely, one has 123\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\{{{\mathcal q}_{({\zeta _ m},{\lambda _ n})}}\} = {\mathcal q}_{[(\zeta _ {m},\lambda _ {m}),(\zeta _ {n},\lambda _ {n})]} + {{\mathcal k}_{({\zeta _ n})},$$\end{document} where the bracket between two generators has been defined in (105) and \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}${\mathcal k}$\end{document} is the central term, which is anti - symmetric in its arguments . Furthermore, using the correspondence principle in semi - classical quantization, dirac brackets between generators translate into commutators of quantum operators as \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$\{\ldots \} \rightarrow - {i \over \hbar} [\ldots] $\end{document}. Note that, according to this rule, the central terms in the algebra aquire a factor of 1/ when operator eigenvalues are expressed in units of (or equivalently, when one performs \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}${\mathcal q} \rightarrow \hbar {\mathcal q}$\end{document} and divide both sides of (123) by . ). For the case of the virasoro algebra (110), it is well known that possible central extensions are classified by two numbers c and a. the general result has the form 124\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$[{{\mathcal l}_m},{{\mathcal l}_n}] = (m - n){{\mathcal l}_{m + n}} + {c \over {12}}m({m^2} - a){\delta _ {m, - n}},$$\end{document} where a is a trivial central extension that can be set to 1 by shifting the background value of the charge \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}${{\mathcal l}_0}$\end{document}. The non - trivial central extension c is a number that is called the central charge of the virasoro algebra . From the theorems [59, 35, 36], the central term in (123) can be expressed as a specific and known functional of the lagrangian \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}${\mathcal l}$\end{document} (or equivalently of the hamiltonian), the background solution \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$\bar \phi = ({{\bar g}_{\mu \nu}},\bar a_\mu ^i,{{\bar \chi}^a})$\end{document} (the near - horizon geometry in this case) and the virasoro generator (,) around the background 125\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$c - c({\mathcal l},\bar \phi, (\zeta, \lambda)).$$\end{document} in particular, the central charge does not depend on the choice of boundary conditions . The representation theorem leading to (124) only requires that such boundary conditions exist . The representation theorem for asymptotic hamiltonian charges was famously first applied to einstein s gravity in three dimensions around ads, where the two copies of the virasoro asymptotic - symmetry algebra were shown to be centrally extended with central charge \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$c = {{3l} \over {2{g_n}\hbar}}$\end{document}, where l is the ads radius and gn newton s constant . For the general near - horizon solution (25) of the lagrangian (1) and the virasoro ansatz (107)(108), one can prove [159, 97] that the matter part of the lagrangian (including the cosmological constant) does not contribute directly to the central charge, but only influences the value of the central charge through the functions (), (), () and k, which solve the equations of motion . The central charge (125) is then given as the m factor of the following expression defined in terms of the fundamental charge formula of einstein gravity as 126\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${\left . {{c_j} = 12i\underset{r \rightarrow \infty}{\lim}{\mathcal q}_{{l_m}}^{{\rm{einstein}}}[{{\mathcal l}_{{l_{- m}}}}\bar g;\bar g]} \right\vert _ {{m^3}}},$$\end{document} where \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}${{\mathcal l}_{{l_{- m}}}}\bar g$\end{document} is the lie derivative of the metric along lm and 127\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\begin{array}{*{20}c} {{\mathcal q}_{{l_m}}^{{\rm{einstein}}}[h;g] \equiv {1 \over {8\pi {g_n}}}\int\nolimits_s {d{s_{\mu \nu}}\left({{\xi ^\nu}{d^\mu}h} \right . + {\xi ^\mu}{d_\sigma}{h^{\sigma \nu}} + {\xi _ \sigma}{d^\nu}{h^{\sigma \mu}} + {1 \over 2}h{d^\nu}{\xi ^\mu}}}\\ {\left . {\quad \quad \quad \quad \quad \quad \quad \quad \quad \; + {1 \over 2}{h^{\mu \sigma}}{d_\sigma}{\xi ^\nu} + {1 \over 2}{h^{\nu \sigma}}{d^\mu}{\xi _ \sigma}} \right). }\\ \end{array}$$\end{document} here, \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$d{s_{\mu \nu}} = {1 \over {2(d - 2)! }}\sqrt {- g} {\varepsilon _ {\mu \nu {\alpha _ {1 \cdots {\alpha _ {d - 2}}}}}}d{x^{{\alpha _ 1}}} \wedge \cdots \wedge d{x^{{\alpha _ {d - 2}}}}$\end{document} is the integration measure in d dimensions and indices are raised with the metric g, h gmh and s is a surface at fixed time and radius r. physically, \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$q_\xi ^{{\rm{einstein}}}[h;g]$\end{document} is defined as the charge of the linearized metric h around the background g associated with the killing vector, obtained from einstein s equations . Substituting the general near - horizon solution (25) and the virasoro ansatz (107)(108), one obtains 128\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${c_j} = {{3k} \over {{g_n}\hbar}}\int\nolimits_0^\pi {d\theta} \;\alpha (\theta)\gamma (\theta)\gamma (\theta).$$\end{document} we will drop the factors of gn and from now on . In the case of the nhek geometry in einstein gravity, substituting (37), one finds the simple result 129\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${c_j} = 12j.$$\end{document} the central charge of the virasoro ansatz (107)(108) around the kerr - newman black hole turns out to be identical to (129). We note in passing that the central charge cj of extremal kerr or kerr - newman is a multiple of six, since the angular momentum is quantized as a half - integer multiple of . The central charge can be obtained for the kerr - newman - ads solution as well and the result is 130\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${c_j} = {{12a{r _ +}} \over {{\delta _ 0}}},$$\end{document} where 0 has been defined in (44). When higher - derivative corrections are considered, the central charge can still be computed exactly, using as crucial ingredients the sl(2,) u(1) symmetry and the (t,) reversal symmetry of the near - horizon solution . The result is given by 131\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${c_j} = - 12k\int\nolimits_\sigma {{{{\delta ^{{\rm cov}}}l} \over {\delta {r_{abcd}}}}{\epsilon _ {ab}}{\epsilon _ {cd}}vol(\sigma),}$$\end{document} where the covariant variational derivative /rabcd has been defined in (53) in section 2.5 . Are introduced in order to rewrite the arbitrary diffeomorphism - invariant action in a form involving at most two derivatives of the fields . It was independently observed in that the formalism of [35, 36] applied to the gauss - bonnet theory formulated using the metric only cannot reproduce the central charge (131) and, therefore, the black - hole entropy as will be developed in section 4.4 . One consequence of these two computations is that the formalism of [35, 36, 90] is not invariant under field redefinitions . In view of the cohomological results of, this ambiguity can appear only in the asymptotic context and when certain asymptotic linearity constraints are not obeyed . Nevertheless, it has been acknowledged that boundary terms in the action should be taken into account [237, 164]. Adding supplementary terms to a well - defined variational principle amount to deforming the boundary conditions [56, 266, 213] and modifying the symplectic structure of the theory through its coupling to the boundary dynamics . Therefore, it remains to be checked if the prescription of to include boundary effects would allow one to reconcile the work of with that of . In five - dimensional einstein gravity coupled to u(1) gauge fields and scalars, the central charge associated with the virasoro generators along the direction \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}${\partial _ {{\phi _ i}}},i = 1,2$\end{document} can be obtained as a straightforward extension of (128) [159, 97]. One has 132\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${c_{{\phi _ i}}} = 6\pi {k_i}\int\nolimits_0^\pi {d\theta} \;\alpha (\theta)\gamma (\theta)\gamma (\theta),$$\end{document} where the extra factor of 2 with respect to (128) originates from integration around the extra circle (see also [151, 166] for some higher derivative corrections). Since the entropy (54) is invariant under a sl(2,) change of basis of the torus coordinates (1, 2) as (118), \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}${c_{{\phi _ i}}}$\end{document} transforms under a modular transformation as ki . Now, ki transforms in the same fashion as the coordinate i, as can be deduced from the form of the near - horizon geometry (36). Then, the central charge for the virasoro ansatz (119) is given by 133\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${c_{({p_1},{p_2})}} = {p_1}{c_{{\phi _ 1}}} + {p_2}{c_{{\phi _ 2}}}.$$\end{document} let us now discuss the central extension of the alternative virasoro ansatz (120) for the extremal reissner - nordstrm black hole of electric charge q and mass q. first, the central charge is inversely proportional to the scale r set by the kaluza - klein direction that geometrizes the gauge field . The central charge is bilinear in the virasoro generator and, therefore, it gets a factor of (r). Also, the central charge consists of the n term of the formula (127), it then contains terms admitting three derivatives along of e and, therefore, it contains a factor of \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$r_\chi ^{- 3}$\end{document}. Also, the central charge is defined as an integration along and, therefore, it should contain one factor r from the integration measure . Finally, the charge is inversely proportional to the five - dimensional newton s constant g5 = (2r)g4 . Multiplying this complete set of scalings, one obtains that the central charge is inversely proportional to the scale r. using the simple embedding of the metric and the gauge fields in a higher - dimensional spacetime (2), as discussed in section 1.2, and using the virasoro ansatz (120), it was shown [159, 146, 77] that the central charge formula (126) gives 134\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${c_q} = {{6{q^3}} \over {{r_\chi}}}.$$\end{document} one might object that (2) is not a consistent higher - dimensional supergravity uplift . Indeed, as we discussed in section 1.2, one should supplement matter fields such as (3). However, since matter fields such as scalars and gauge fields do not contribute to the central charge (125), the result (134) holds for any such consistent embedding . Similarly, we can uplift the kerr - newman black hole to five - dimensions, using the uplift (2)(3) and the four - dimensional fields (25)(39). Computing the central charge (132) for the virasoro ansatz (120), we find again13 135\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${c_q} = {{6{q^3}} \over {{r_\chi}}}.$$\end{document} under the assumption that the u(1) gauge field can be uplifted to a kaluza - klein direction, we can also formulate the virasoro algebra (119) and associated boundary conditions for any circle related by an sl(2,) transformation of the torus u(1). Applying the relation (133) we obtain the central charge 136\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${c_{({p_1},{p_2})}} = {p_1}{c_j} + {p_2}{c_q} = 6\left({{p_1}(2j) + {p_2}{{{q^3}} \over {{r_\chi}}}} \right).$$\end{document} let us discuss the generalization to ads black holes . As discussed in section 1.2, one cannot use the ansatz (2) to uplift the u(1) gauge field . One can then argue, as in, that the only contribution to the central charge comes from the gravitational action . Even though no formal proof is available, it is expected that it will be the case given the results for scalar and gauge fields in four and five dimensions . Applying the charge formula (126) accounting for the gravitational contribution of the complete higher - dimensional spacetime, one obtains the central charge for the virasoro algebra (120) as 137\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${c_q} = 6{q_e}{{r _ + ^2 - {a^2}} \over {\xi {\delta _ 0}{r_\chi}}},$$\end{document} where parameters have been defined in section 2.4.4 and 2r the values of the central charges (129), (130), (131), (132), (133), (135), (136), (137) are the main results of this section . In section 4.3 we have shown the existence of an asymptotic virasoro algebra at the boundary r = of the near - horizon geometry . We also discussed that the sl(2,) symmetry is associated with zero charges . Following semi - classical quantization rules, the operators that define quantum gravity with the boundary conditions (111), (116), (115) form a representation of the virasoro algebra and are in a ground state with respect to the representation of the sl(2,) symmetry [251, 156]. A consistent theory of quantum gravity in the near - horizon region, if it can be defined at all, is therefore either a chiral cft or a chiral half of a two - dimensional cft . A chiral cft is defined as a holomorphically - factorized cft with zero central charge in one sector, while a chiral half of a 2d cft can be obtained, e.g., after a chiral limit of a 2d cft, see section 3.2 . We will see in sections 5 and 6 that the description of non - extremal black holes favors the interpretation of quantum gravity in extremal black holes as the chiral half of a full - fledged two - dimensional cft . Moreover, the applicability of cardy s formula as detailed later on also favors the existence of a two - dimensional cft . Since the near - horizon geometry is obtained as a strict near - horizon limit of the original geometry, the cft might be thought of as describing the degrees of freedom of the black - hole horizon . Before moving further on, let us step back and first review an analogous reasoning in ads3 . In the case of also, one can define a two - dimensional flat cylinder at the boundary of ads3 using the fefferman - graham theorem . One is then led to identify quantum gravity in ads3 space - times with a two - dimensional cft defined on the cylinder . The known examples of ads / cft correspondences involving ads3 factors can be understood as a correspondence between an ultraviolet completion of quantum gravity on ads3 and a specific cft . The vacuum ads3 spacetime is more precisely identified with the sl(2,) sl(2,) invariant vacuum of the cft, which is separated with a mass gap of c/24 from the zero - mass black holes . Extremal black holes with ads3 asymptotics, the extremal btz black holes, are thermal states in the dual cft with one chiral sector excited and the other sector set to zero temperature . It was further understood in that taking the near - horizon limit of the extremal btz black hole corresponds to taking the dlcq of the dual cft (see section 3.2 for a review of the dlcq procedure and [31, 151] for further supportive studies). The resulting cft is chiral and has a frozen sl(2,) right sector . Given the close parallels between the near - horizon geometry of the extremal btz black hole (121) and the near - horizon geometries of four - dimensional extremal black holes (25), it has been suggested in that extremal black holes are described by a chiral limit of two - dimensional cft . This assumption nicely accounts for the fact that only one virasoro algebra appears in the asymptotic symmetry algebra and it is consistent with the conjecture that no non - extremal excitations are allowed in the near - horizon limit as we discussed earlier . Moreover, the assumption that the chiral half of the cft originates from a limiting dlcq procedure is consistent with the fact that there is no natural sl(2,) sl(2,) invariant geometry in the boundary conditions (111), which would be dual to the vacuum state of the cft . Indeed, even in the three - dimensional example, the geometric dual to the vacuum state (the ads3 geometry) does not belong to the phase space defined in the near - horizon limit of extremal black holes . It remains an enigma why there is no natural sl(2,) sl(2,) invariant geometry in gravity at all that is dual to the vacuum state . Let us now take as an assumption that the near - horizon geometry of the extremal kerr black hole is described by the left - sector of a 2d cft that we will denote as cftj . The details of this cft will depend on the ultraviolet completion of gravity, but these details will be (fortunately) unimportant here . Instead, we will show that one can account for the entropy using the universal properties of that cft . We saw in section 2.6 that scalar quantum fields in the analogue of the frolov - thorne vacuum restricted to extremal excitations have the temperature (67). Individual modes are co - rotating with the black hole along . Since we identify the left - sector of the cft with excitations along and the right sl(2,)r sector is frozen, the cft left - moving states are described by a thermal density matrix with temperatures 138\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${t_l} = {t_\phi},\quad \quad {t_r} = 0,$$\end{document} where t is given in (67). The other quantities te and tm defined in (61) are then better interpreted as being proportional to auxiliary chemical potentials . One can indeed rewrite the boltzman factor (66) as 139\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\exp \left({- \hbar {{m - {q_e}\mu _ l^{j, e} - {q_m}\mu _ l^{j, m}} \over {{t_l}}}} \right),$$\end{document} where the left chemical potentials are defined as 140\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\mu _ l^{j, e} \equiv - {{{t_\phi}} \over {{t_e}}},\quad \mu _ l^{j, m} \equiv - {{{t_\phi}} \over {{t_m}}}.$$\end{document} it is remarkable that applying blindly cardy s formula (90) using the central charge cl = cj given in (129) and using the temperatures (138), one reproduces the extremal bekenstein - hawking black - hole entropy 141\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${{\mathcal s}_{{\rm{cft}}}} \overset{! }{=} {{\mathcal s}_{{\rm{ext}}}},$$\end{document} as first shown in . This matching is clearly not a numerical coincidence . For any spinning extremal black hole of the theory (1), one can associate a left - moving virasoro algebra of central charge cl = cj given in (128). The black - hole entropy (50) is then similarly reproduced by cardy s formula (141). As remarkably, taking any higher curvature correction to the gravitational lagrangian into account, one also reproduces the iyer - wald entropy (52) using cardy s formula, while the central charge (131) is computed (apparently) completely independently from the entropy! One can easily be puzzled by the incredible matching (141) valid for virtually any extremal black hole and outside the usual cardy regime, as discussed in section 3.1 . Indeed, there are no arguments for unitarity and modular invariance of the dual cft . It might suggest that cardy s formula has a larger range of applicability than what has been proven so far . Alternatively, this might suggest the existence of a long string cft, as reviewed in section 3.3 . Note also that the central charge depends on the black - hole parameters, such as the angular momentum or the electric charge . This is not too surprising since, in known ads / cft correspondences where the black hole contains an ads3 factor in the near - horizon geometry, the brown - henneaux central charge c = 3l/2g3 also depends on the parameters of the black hole because the ads length l is a function of the black hole s charge . It turns out that when electromagnetic fields are present, another cft description is available . Instead of assigning the left - moving temperature as (138), one might instead emphasize that electrically - charged particles are immersed in a thermal bath with temperature t = rte, as derived in (70) in section 2.6 . Identifying the left sector of the dual field theory with a density matrix at temperature t and assuming again no right excitations at extremality, we make the following assignment 142\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${t_l} = {t_\chi} = {r_\chi}{t_e},\quad \quad {t_r} = 0.$$\end{document} the other quantities t and tm defined in (61) are then better interpreted as being proportional to auxiliary chemical potentials . One can indeed rewrite the boltzman factor (66) as 143\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\exp \left({- \hbar {{{q_\chi} - m\mu _ l^{q,\phi} - {q_m}\mu _ l^{q, m}} \over {{t_\chi}}}} \right),$$\end{document} where q = rqe is the probe electric charge in units of the kaluza - klein length and the left chemical potentials are defined as 144\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\mu _ l^{q,\phi} \equiv - {{{t_\chi}} \over {{t_\phi}}},\quad \mu _ l^{q, m} \equiv - {{{t_\chi}} \over {{t_m}}}.$$\end{document} we argued above that in the near - horizon region, excitations along the gauge - field direction fall into representations of the virasoro algebra defined in (120). As supported by non - extremal extensions of the correspondence discussed in sections 5 and 6, the left sector of the dual field theory can be argued to be the chiral half of a 2d cft . Remarkably, cardy s formula (90) with temperatures (142) and central charge (134) also reproduces the entropy of the kerr - newman black hole . When the angular momentum is identically zero, the black - hole entropy of the reissner - nordstrm black hole \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}${{\mathcal s}_{{\rm{ext}}}} = \pi {q^2}$\end{document} is then reproduced from cardy s formula with left central charge cl = cq given in (134) and left temperature tl = r/(2q) as originally obtained in . As one can easily check, the entropy of the general kerr - newman - ads black hole can be similarily reproduced, as shown in [79, 71, 78, 76, 82]. We will refer to the class of cfts with virasoro algebra (120) by the acronym cftq . Note that the entropy matching does not depend on the scale of the kaluza - klein dimension r, which is arbitrary in our analysis . Finally, when two u(1) symmetries are present, one can apply a modular transformation mixing the two u(1) and one obtains a different cft description for each choice of sl(2,) element . Indeed, we argued that the set of generators (119) obeys the virasoro algebra with central charge (136). After performing an sl(2,) change of basis in the boltzman factor (66), we deduce the temperature of the cft and cardy s formula is similarly reproduced . We will denote the corresponding class of cfts by the acronym \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}${\rm{cf}}{{\rm{t}}_{({p_1},{p_2},{p_{3)}}}}$\end{document}. In section 4, we presented how the entropy of any extremal black hole can be reproduced microscopically from one chiral half of one (or several) two - dimensional cft(s). In this section, we will present arguments supporting the conjecture that this duality can be extended to near - extremal black holes dual to a cft with a second sector slightly excited, following [53, 106, 160]. We will show that the derivation of [53, 106, 160] is supporting evidence for all cfts presented in section 4, as noted in [79, 71]. In the case of the cftj dual to near - extremal spinning black holes, one can think intuitively that the second cft sector is excited for the following reason: lights cones do not quite coalesce at the horizon, so microscopic degrees of freedom do not rotate at the speed of light along the single axial direction . The intuition for the other cfts (cftq, \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}${\rm{(cf}}{{\rm{t}}_q}{\rm{,cf}}{{\rm{t}}_{({p_1},{p_2},{p_{3)}}}})$\end{document}) is less immediate . Near - extremal black holes are defined as black holes with a hawking temperature that is very small compared with their inverse mass 145\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$m{t_h} \ll 1.$$\end{document} at finite energy away from extremality, one cannot isolate a decoupled near - extremal near - horizon geometry . As we discussed in section 4, the extremal near - horizon geometry then suffers from infrared divergences, which destabilize the near - horizon geometry . This prevents one to formulate boundary conditions la brown - henneaux to describe non - chiral excitations . If near - extremal black holes are described by a dual field theory, it means that all properties of these black holes classical or quantum can be derived from a computation in the dual theory, after it has been properly coupled to the surrounding spacetime . We now turn our attention to the study of one of the simplest dynamical processes around black holes: the scattering of a probe field . This route was originally followed for static extremal black holes in [208, 209]. In this approach, moreover, since gravitational backreaction is a higher - order effect, it can be neglected . One simply computes the black - hole - scattering amplitudes on the black - hole background . In order to test the near - extremal black hole / cft correspondence, one then has to determine whether or not the black hole reacts like a two - dimensional cft to external perturbations originating from the asymptotic region far from the black hole . We will only consider fields that probe the near - horizon region of near - extremal black holes . These probe fields have energy and angular momentum m close to the superradiant bound \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$\omega \sim m\omega _ j^{{\rm{ext}}} + {q_e}\phi _ e^{{\rm{ext}}}$\end{document}, 146\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$m(\omega - m\omega _ j^{{\rm{ext}}} - {q_e}\phi _ e^{{\rm{ext}}}) \ll 1.$$\end{document} in order to simplify the notation, in this section we will drop all hats on quantities defined in the asymptotic region far from the black hole . Since no general scattering theory around near - extremal black - hole solutions of (1) has been proposed so far, we will concentrate our discussion on near - extremal asymptotically - flat kerr - newman black holes, as discussed in [53, 160] (see also [79, 72, 81, 74, 77, 3]). Extensions to the kerr - newman - ads black hole or other specific black holes in four and higher dimensions in gauged or ungauged supergravity can be found in [53, 106, 73, 242, 46] (see also [71, 80, 129, 224]). Near - extremal kerr - newman black holes are characterized by their mass m, angular momentum j = ma and electric charge q. (we take a, q 0 without loss of generality .) They contain near - extremal kerr and reissner - nordstrm black holes as particular instances . The metric and thermodynamic quantities can be found in many references and will not be reproduced here . The near - extremality condition (145) is equivalent to the condition that the reduced hawking temperature is small, 147\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${\tau _ h} \equiv {{{r _ +} - {r _ -}} \over {{r _ +}}} \ll 1.$$\end{document} indeed, one has h = m th[4((r+/m) + (a / m))/(r+/m)] and the term in between the brackets is of order one since 0 a / m 1, 0 q / m 1 and 1 r+/m 2 . Therefore, we can use interchangeably the conditions (145) and (147). Since there is both angular momentum and electric charge, extremality can be reached both in the regime of vanishing angular momentum and vanishing electric charge q. when angular momentum is present, we expect that the dynamics could be described by the cftj, while when electric charge is present the dynamics could be described by the cftq . It is interesting to remark that the condition 148\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${{{t_h}} \over {{\omega _ j}}} \ll 1,$$\end{document} implies (145)(147) since \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}${\tau _ h} = {{{t_h}} \over {{\omega _ j}}}(4\pi a / m) \ll 1$\end{document} but it also implies a> 0 . Similarly, the condition 149\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$m{{{t_h}} \over {{\phi _ e}}} \ll 1$$\end{document} implies (145)(147), since h = 4qth/e, but it also implies q> 0 . In the following, we will need only the near - extremality condition (145), and not the more stringent conditions (148) or (149). This is the first clue that the near - extremal scattering will be describable by both the cftj and the cftq . Near - extremal black holes are characterized by an approximative near - horizon geometry, which controls the behavior of probe fields in the window (146). Upon taking th = o() and taking the limit 0 the near - horizon geometry decouples, as we saw in section 2.7 . Probes will penetrate the near - horizon region close to the superradiant bound (146). When th = o() we need 150\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\omega = m\omega _ j^{{\rm{ext}}} + {q_e}\phi _ e^{{\rm{ext}}} + o(\lambda).$$\end{document} indeed, repeating the reasoning of section 2.6, we find that the boltzman factor defined in the near - horizon vacuum (defined using the horizon generator) takes the following form 151\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${e^{\hbar {{\omega - m{\omega _ j} - {q_e}{\phi _ e}} \over {{t_h}}}}} = {e^{- \hbar n}}{e^{- \hbar {m \over {{t_\phi}}} - \hbar {{{q_e}} \over {{t_e}}}}},$$\end{document} where, m and qe are the quantum numbers defined in the exterior asymptotic region and 152\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$n \equiv {{\omega - m\omega _ j^{{\rm{ext}}} - {q_e}\phi _ e^{{\rm{ext}}}} \over {{t_h}}},$$\end{document} is finite upon choosing (150). The conclusion of this section is that the geometries (79) control the behavior of probes in the near - extremal regime (145)(146). We will now turn our attention to how to solve the equations of motion of probes close to extremality . The problem of scattering of a general spin field from a kerr black hole was solved in a series of classic papers by starobinsky, starobinsky and churilov and press and teukolsky [255, 256, 235, 257] in the early 1970s (see also [145, 4, 122]). The scattering of a spin 0 and 1/2 field from a kerr - newman black hole has also been solved, while the scattering of spins 1 and 2 from the kerr - newman black hole has not been solved to date . First, one has to realize that the kerr - newman black hole enjoys a remarkable property: it admits a killing - yano tensor [269, 232, 142]. (for a review and some surprising connections between killing - yano tensors and fermionic symmetries, see .) A killing - yano tensor is an anti - symmetric tensor fh = f, which obeys 153\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${\nabla _ {(\lambda}}{f_{\mu)}}_\nu = 0.$$\end{document} this tensor can be used to construct a symmetric killing tensor 154\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${k_{\mu \nu}} = f_\mu ^{\;\lambda}{f_{\lambda \nu}},\quad \quad {\nabla _ {(\lambda}}{k_{\mu \nu)}} = 0,$$\end{document} which is a natural generalization of the concept of killing vector k (obeying (k) = 0). This killing tensor was first used by carter in order to define an additional conserved charge for geodesics 155\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$q = {k_{\mu \nu}}{\dot x^\mu}{\dot x^\nu},$$\end{document} and thereby reduce the geodesic equations in kerr to first - order equations . More importantly for our purposes, the killing tensor allows one to construct a second - order differential operator k, which commutes with the laplacian . This allows one to separate the solutions of the scalar wave equation = 0 as 156\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${\psi ^{s = 0}} = {e^{- i\omega t + im\phi}}{s_{\omega, a, m}}(\theta){r_{\omega, a, m}}(r),$$\end{document} where is the real separation constant present in both equations for s() and r(r). The underlying killing - yano tensor structure also leads to the separability of the dirac equation for a probe fermionic field . For simplicity, we will not discuss further fermionic fields here and we refer the interested reader to the original reference (see also). The equations for spin 1 and 2 probes in kerr can also be shown to be separable after one has conveniently reduced the dynamics to a master equation for a master scalar, which governs the entire probe dynamics . As a result, one has 157\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${\psi ^s} = {e^{- i\omega t + im\phi}}s_{\omega, a, m}^s(\theta)r_{\omega, a, m}^s(r).$$\end{document} the master scalar is constructed from the field strength and from the weyl tensor for spin 1 (s = 1) and spin 2 (s = 2) fields, respectively, using the newman - penrose formalism . For the kerr - newman black hole, all attempts to separate the equations for spin 1 and spin 2 probes have failed . Hence, there is no known analytic method to solve those equations (for details, see). Going back to kerr, given a solution to the master scalar field equation, one can then in principle reconstruct the gauge field and the metric from the teukolsky functions . This non - trivial problem was778ikm solved right after teukolsky s work [89, 87]; see appendix c of for a modern review (with further details and original typos corrected). In summary, for all separable cases, the dynamics of probes in the kerr - newman geometry can be reduced to a second - order equation for the angular part of the master scalar \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$s_{\omega, a, m}^{\mathcal s}(\theta)$\end{document} and a second - order equation for the radial part of the master scalar \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$r_{\omega, a, m}^{\mathcal s}(r)$\end{document}. Let us now discuss their solutions after imposing regularity as boundary conditions, which include ingoing boundary conditions at the horizon . We will limit our discussion to the non - negative integer spins s = 0, 1, 2 in what follows . The angular functions \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$s_{\omega, a, m}^{\mathcal s}(\theta)$\end{document} obey the spin - weighted spheroidal harmonic equation 158\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} {\left [{{1 \over {\sin \theta}}{\partial _ \theta}(\sin \theta {\partial _ \theta})} \right . - {a^2}({\omega ^2} - {\mu ^2}{\delta _ {s,0}}){{\sin}^2}\theta - 2a\omega s\cos \theta - {{{{(m + s\cos \theta)}^2}} \over {{{\sin}^2}\theta}}}\\ {\left . {\quad \quad \quad \quad + a} \right]s_{\omega, a, m}^s(\theta) = 0. }\\ \end{array}$$\end{document} (the kronecker s0 is introduced so that the multiplicative term only appears for a massive scalar field of mass .) All harmonics that are regular at the poles can be obtained numerically and can be classified by the usual integer number l with l |m| and l |s| . In general, the separation constant \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$a = a_{a\omega, l, m}^{\mathcal s}$\end{document} depends on the product a, on the integer l, on the angular momentum of the probe and on the spin s. at zero energy (= 0), the equation reduces to the standard spin - weighted spherical - harmonic equation and one simply has \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$a_{0,l, m}^{\mathcal s} = l(l + 1) - {{\mathcal s}^2}$\end{document}. For a summary of analytic and numerical results, see . Let us now take the values \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$a_{a\omega, l, m}^{\mathcal s}$\end{document} as granted and turn to the radial equation . The radial equation reduces to the following sturm - liouville equation 159\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\left [{{\delta ^{- s}}{\partial _ r}({\delta ^{s + 1}}{\partial _ r}) - {v^s}(r)} \right]{r^s}(r) = 0,$$\end{document} where (r) = (r r+)(r r) = r 2mr + a + q in a potential v(r). The form of the potential is pretty intricate . For a scalar field of mass, the potential v(r) is real and is given by 160\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${v^0}(r) = - {{{h^2}(r)} \over {\delta (r)}} - 2am\omega + a_{a\omega, l, m}^0 + {\mu ^2}({r^2} + {a^2}),$$\end{document} where h(r) = (r + a) qeqr am . For a field of general spin on the kerr geometry, the potential is, in general, complex and reads as 161\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${v^s}(r) = - {{{h^2}(r) - 2is(r - m)h(r)} \over {\delta (r)}} - 4is\omega r - 2am\omega + a_{a\omega, l, m}^s - s(s + 1),$$\end{document} where h(r) = (r + a) am . This radial equation obeys the following physical boundary condition: we require that the radial wave has an ingoing group velocity or, in other words, is purely ingoing at the horizon . The solution is then unique up to an overall normalization . For generic parameters, the sturm - liouville equation (159) cannot be solved analytically and one has to use numerical methods . For each frequency and spheroidal harmonic (l, m), the scalar field can be extended at infinity into an incoming wave and an outgoing wave . The absorption probability abs or macroscopic greybody factor is then defined as the ratio between the absorbed flux of energy at the horizon and the incoming flux of energy from infinity, 162\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${\sigma _ {{\rm{abs}}}}(\omega, l, m, s;m, a, q) = {{d{e_{{\rm{abs}}}}/dt} \over {d{e_{{\rm{in}}}}/dt}}.$$\end{document} an important feature is that in the superradiant range (13) the absorption probability turns out to be negative, which results in stimulated as well as spontaneous emission of energy, as we reviewed in section 2.1 . The sturm - liouville problem (159) cannot be solved analytically . However, in the regime of near - extremal excitations (145)(146) an approximative solution can be obtained analytically using asymptotic matched expansions: the wave equation is solved in the near - horizon region and in the far asymptotically - flat region and then matched along their common overlap region . For that purpose, it is useful to define the dimensionless horizon radius x = (r r+)/r+ such that the outer horizon is at x = 0 . The two other singular points of the radial equation (159) are the inner horizon x = h and spatial infinity x = . One then simply partitions the radial axis into two regions with a large overlap as near - horizon region: x 1,far region: x h, overlap region: h x 1 . Near - horizon region: x 1, overlap region: h x 1 . In the near - extremal regime, the absorption probability abs gets a contribution from each region as 163\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${\sigma _ {{\rm{abs}}}} = \sigma _ {{\rm{abs}}}^{{\rm{near}}}\sigma _ {{\rm{abs}}}^{{\rm{match}}},$$\end{document} 164\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\sigma _ {{\rm{abs}}}^{{\rm{near}}} = {{d{e_{{\rm{abs}}}}/dt} \over {\vert \psi (x = {x_b})\vert ^{2}}},$$\end{document} 165\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\sigma _ {{\rm{abs}}}^{{\rm{match}}} = {{\vert \psi (x = {x_b})\vert ^{2}} \over {d{e_{{\rm{in}}}}/dt}},$$\end{document} where |(x = xb)| is the norm of the scalar field in the overlap region with h xb 1 . One can conveniently normalize the scalar field such that it has unit incoming flux dein / dt = 1 . The contribution \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$\sigma _ {{\rm{abs}}}^{{\rm{match}}}$\end{document} is then simply a normalization that depends on the coupling of the near - horizon region to the far region . In the near - horizon region, the radial equation reduces to a much simpler hypergeometric equation . One can in fact directly obtain the same equation from solving for a probe in a near - extremal near - horizon geometry of the type (79), which is, as detailed in section 2.3, a warped and twisted product of ads2 s. the presence of poles in the hypergeometric equation at x = 0 and x = h requires one to choose the ads2 base of the near - horizon geometry to be 166\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$ds_{(2)}^2 = - x(x + {\tau _ h})d{t^2} + {{d{x^2}} \over {x(x + {\tau _ h})}}.$$\end{document} one can consider the non - diagonal term 2()()kr dtd appearing in the geometry (79) as a u(1) electric field twisted along the fiber spanned by d over the ads2 base space . It may then not be surprising that the dynamics of a probe scalar on that geometry can be expressed equivalently as a charged massive scalar on ads2 with two electric fields: one coming from the u(1) twist in the four - dimensional geometry, and one coming from the original u(1) gauge field . By sl(2,) invariance, these two gauge fields are given by 167\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${a_1} = {\alpha _ 1}x\;dt,\quad \quad {a_2} = {\alpha _ 2}x\;dt.$$\end{document} the coupling between the gauge fields and the charged scalar is dictated by the covariant derivative 168\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${\mathcal d} = \nabla - i{q_1}{a_1} - i{q_2}{a_2} = \nabla - i{q_{{\rm{eff}}}}a,$$\end{document} where is the levi - civita connection on ads2 and q1 and q2 are the electric charge couplings . One can rewrite more simply the connection as qeffa, where qeff = q11 + q22 is the effective total charge coupling and a = xdt is a canonically - normalized effective gauge field . The equation for a charged scalar field (t, x) with mass eff is then 169\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${{\mathcal d}^2}\phi - \mu _ {{\rm{eff}}}^2\phi = 0.$$\end{document} taking \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$\phi (t, r) = {e^{- i{\omega _ {{\rm{ef}}{{\rm{f}}^\tau}{h^t}}}}}\phi (x)$\end{document}, we then obtain the following equation for (x), \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\left [{{\partial _ x}(x(x + {\tau _ h}){\partial _ x}) + {{{{({\omega _ {{\rm{eff}}}}{\tau _ h} + {q_{{\rm{eff}}}}x)}^2}} \over {x(x + {\tau _ h})}} - \mu _ {{\rm{eff}}}^2} \right]\phi (x) = 0.$$\end{document} using the field redefinition 170\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\phi (x) = {x^{s/2}}{({x \over {{\tau _ h}}} + 1)^{s/2}}{r^s}(x),$$\end{document} we obtain the equivalent equation, 171\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$x(x + {\tau _ h})\partial _ x^2{r^s} + (1 + s)(2x + {\tau _ h}){\partial _ x}{r^s} + v(x){r^s} = 0,$$\end{document} where the potential is 172\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$v(x) = {{{{(ax + b{\tau _ h})}^2} - is(2x + {\tau _ h})(ax + b{\tau _ h})} \over {x(x + {\tau _ h})}} - c.$$\end{document} here, the parameters a, b, c are related to eff, qeff and eff as14 173\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$a = {q_{{\rm{eff}}}} + is,\quad b = {\omega _ {{\rm{eff}}}} + {{is} \over 2},\quad c = \mu _ {{\rm{eff}}}^2 - s.$$\end{document} finally, comparing eq . (171) with (159), where the potential v(r) is approximated by the near - horizon potential, we obtain that these equations are identical, as previously announced, after identifying the parameters as 174\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\begin{array}{*{20}c} {{\omega _ {{\rm{eff}}}} = {n \over {4\pi}} - {{is} \over 2},\quad \quad \quad \quad \quad \quad \quad \quad \quad \quad \quad \quad \quad \quad} \\ {{q_{{\rm{eff}}}} = 2{r _ +} \omega - {q_e}q - is,\quad \,\quad \quad \quad \quad \quad \quad \quad \quad \quad \quad \,} \\ {\mu _ {{\rm{eff}}}^2 = a_{a\omega, l, m}^s - 2am\omega - {s^2} + {\mu ^2}(r_{\,\, +} ^2 + {a^2}) - 2ims\quad \,\,\,} \\ \end{array}.$$\end{document} moreover, using the expression of the frequency (150) near extremality, one can write the effective charge in the convenient form 175\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${q_{{\rm{eff}}}} = {m \over {2\pi {t_\phi}}} + {{{q_e}} \over {2\pi {t_e}}} - is,$$\end{document} where the extremal frolov - thorne temperatures te and t are defined in (74). We can now understand that there are two qualitatively distinct solutions for the radial field r(x). Uncharged fields in ads2 below a critical mass are unstable or tachyonic, as shown by breitenlohner and freedman . Charged particles in an electric field on ads2 have a modified breitenlohner - freedman bound 176\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$m_{{\rm{bf}}}^2 = - {1 \over 4} + q_{{\rm{eff}}}^2,$$\end{document} in which the square mass is lifted up by the square charge . Below the critical mass, let us define 177\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${\beta ^2} \equiv \mu _ {{\rm{eff}}}^2 - m_{{\rm{bf}}}^2.$$\end{document} stable modes will be characterized by a real 0, while unstable modes will be characterized by an imaginary . This distinction between modes is distinct from superradiant and non - superradiant modes . Indeed, from the definition of n (152), superradiance happens at near - extremality when n <0 . We can now solve the equation, impose the boundary conditions, compute the flux at the horizon and finally obtain the near - horizon absorption probability . A massive, charge e, spin s = 0, field with energy and angular momentum m and real> 0 scattered against a kerr - newman black hole with mass and charge has near - region absorption probability 178\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\begin{array}{*{20}c} {\sigma _ {{\rm{abs}}}^{{\rm{near}}} \sim {{{{({t_h})}^{2\beta}}({e^{{n \over 2}}} - {{(- 1)}^{2s}}{e^{- {n \over 2}}})} \over {\gamma {{(2\beta)}^2}}}\vert \gamma \left({{1 \over 2} + \beta - s + i{{\rm re}} ({q_{{\rm{eff}}}})} \right)\vert ^{2}}\\ {\quad \quad \vert \gamma \left({{1 \over 2} + \beta + i\left({{n \over {2\pi}} - {{\rm re}} ({q_{{\rm{eff}}}})} \right)} \right)\vert ^{2}. }\\ \end{array}$$\end{document} for a massless spin s = 1, 2 field scattered against a kerr black hole, exactly the same formula applies, but with e = q = 0 . The absorption probability in the case where is imaginary can be found in the original papers [235, 257]. We will now show that the formulae (178) are fourier transforms of cft correlation functions . We will not consider the scattering of unstable fields with imaginary in this review . We refer the reader to for arguments on how the scattering absorption probability of unstable spin 0 modes around the kerr black hole match with dual cft expectations . In this section we model the emission amplitudes from a microscopic point of view . We will first discuss near - extremal spinning black holes and we will extend our discussion to general charged and/or spinning black holes at the end of this section . The working assumption of the microscopic model is that the near - horizon region of any near - extremal spinning black hole can be described and therefore effectively replaced by a dual two - dimensional cft . In the dual cft picture, the near - horizon region is removed from the spacetime and replaced by a cft glued along the boundary . Therefore, it is the near - horizon region contribution alone that we expect to be reproduced by the cft . The normalization \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$\sigma _ {{\rm{abs}}}^{{\rm{match}}}$\end{document} defined in (164) will then be dictated by the explicit coupling between the cft and the asymptotically - flat region . Remember from the asymptotic symmetry group analysis in section 4.1 and 4.3 that boundary conditions were found where the exact symmetry of the near - horizon extremal geometry can be extended to a virasoro algebra as 179\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$u{(1)_l} \times sl{(2,{\mathbb r})_r} \rightarrow {\rm{vi}}{{\rm{r}}_l} \times sl{(2,{\mathbb r})_r}.$$\end{document} the right sector was taken to be frozen at extremality . The resulting chiral limit of the cft with central charge cj = 12j sufficed to account for the extremal black - hole entropy . We will now assume that quantum gravity states form a representation of both a left and a right - moving virasoro algebra with generators ln and \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}${{\bar l}_n}$\end{document}. The value of the right - moving central charge will be irrelevant for our present considerations . At near - extremality, the left sector is thermally excited at the extremal left - moving temperature (67). We take as an assumption that the right - moving temperature is on the order of the infinitesimal reduced hawking temperature . As discussed in sections 2.9 and 4.2, the presence of right - movers destabilize the near - horizon geometry . For the kerr - newman black hole, we have 180\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${t_l} = {{{m^2} + {a^2}} \over {4\pi j}},\quad \quad {t_r} \sim {\tau _ h}.$$\end{document} in order to match the bulk scattering amplitude for near - extremal kerr - newman black holes, the presence of an additional left - moving current algebra is required [106, 160]. This current algebra is expected from the thermodynamic analysis of charged rotating extremal black holes . We indeed obtained in section 2.6 and in section 4.4 that such black holes are characterized by the chemical potential \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$\mu _ l^{j, e}$\end{document} defined in (140) associated with the u(1)e electric current . Using the expressions (74), we find for the kerr - newman black hole the value 181\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\mu _ l^e = - {{{q^3}} \over {2j}}.$$\end{document} as done in, we also assume the presence of a right - moving u(1) current algebra, whose zero eigenmode \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}${{\bar j}_0}$\end{document} is constrained by the level matching condition 182\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${\bar j_0} = {l_0}.$$\end{document} the level matching condition is consistent with the fact that the excitations are labeled by three (, m, qe) instead of four conserved quantities . This right - moving current algebra cannot be detected in the extremal near - horizon geometry in the same way that the right - moving virasoro algebra cannot be detected, so its existence is conjectural (see, however,). This right - moving current algebra and the matching condition (182) will turn out to be adequate to match the gravitational result, as detailed below . Note that three - dimensional analogues of this level matching condition appeared in logically independent analyses [95, 94]. Therefore, under these assumptions, the symmetry group of the cft dual to the near - extremal kerr - newman black hole is given by the product of a u(1) current and a virasoro algebra in both sectors, 183\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$({\rm{vi}}{{\rm{r}}_l} \times {\rm{cur}}{{\rm{r}}_l}) \times ({\rm{vi}}{{\rm{r}}_r} \times {\rm{cur}}{{\rm{r}}_r}).$$\end{document} in the description where the near - horizon region of the black hole is replaced by a cft, the emission of quanta is due to couplings 184\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${\phi _ {{\rm{bulk}}}}{\mathcal o}$$\end{document} between bulk modes bulk and operators \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}${\mathcal o}$\end{document} in the cft . The structure of the scattering cross section depends on the conformal weights (hl, hr) and charges (ql, qr) of the operator . The normalization of the coupling is also important for the normalization of the cross section . The conformal weight hr can be deduced from the transformation of the probe field under the scaling \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}${{\bar l}_0} = t{\partial _ t} - r{\partial _ r}$\end{document} (24) in the overlap region h x 1 . The scalar field in the overlap region is \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$\phi \sim {\phi _ 0}(t,\theta, \phi){r^{- {1 \over 2} + \beta}} + {\phi _ 1}(t,\theta, \phi){r^{- {1 \over 2} - \beta}}$\end{document}. Using the rules of the ads / cft dictionary, this behavior is related to the conformal weight as \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$\phi \sim {r^{{h_{r - 1}}}},{r^{- {h_r}}}$\end{document}. One then infers that 185\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${h_r} = {1 \over 2} + \beta .$$\end{document} the values of the charges (ql, qr) are simply the u(1) charges of the probe, 186\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${q_l} = {q_e},\quad {q_r} = m,$$\end{document} where the charge qr = m follows from the matching condition (182). We do nt know any first - principle argument leading to the values of the right - moving chemical potential r, the right - moving temperature tr and the left - moving conformal weight hl . We will deduce those values from matching the cft absorption probability with the gravitational result . In general, the weight (185) will be complex and real weight will not be integers . However, a curious fact, described in [129, 224], is that for any axisymmetric perturbation (m = 0) of any integer spin of the kerr black hole, the conformal weight (185) is an integer 187\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${h_r} = 1 + l,$$\end{document} where l = 0, 1, . One can generalize this result to any axisymmetric perturbation of any vacuum five - dimensional near - horizon geometry . Throwing the scalar bulk at the black hole is dual to exciting the cft by acting with the operator \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}${\mathcal o}$\end{document}. Reemission is represented by the action of the hermitian conjugate operator . Therefore, the absorption probability is related to the thermal cft two - point function 188\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$g({t^ +}, {t^ -}) = \langle {{\mathcal o}^\dagger}({t^ +}, {t^ -}){\mathcal o}(0)\rangle, $$\end{document} where t are the coordinates of the left and right moving sectors of the cft . At left and right temperatures (tl, tr) and at chemical potentials (l, r) an operator with conformal dimensions (hl, hr) and charges (ql, qr) has the two - point function 189\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$g \sim {(- 1)^{{h_l} + {h_r}}}{\left({{{\pi {t_l}} \over {\sinh (\pi {t_l}{t^ +})}}} \right)^{2{h_l}}}{\left({{{\pi {t_r}} \over {\sinh (\pi {t_r}{t^ -})}}} \right)^{2{h_r}}}{e^{i{q_l}{\mu _ l}{t^ +} + i{q_r}{\mu _ r}{t^ -}}},$$\end{document} which is determined by conformal invariance . From fermi s golden rule, the absorption cross section is [53, 106, 160] 190\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\begin{array}{*{20}c} {{\sigma _ {{\rm{abs}}}}({\omega _ l},{\omega _ r})\sim \int {d{t^ +} d{t^ -}{e^{- i{\omega _ r}{t^ -} - i{\omega _ l}{t^ +}}} \left [{g({t^ +} - i\epsilon, {t^ -} - i\epsilon)} \right .}} \\ {\left . {\quad \quad \quad \quad \quad \quad - g({t^ +} - i\epsilon, {t^ -} + i\epsilon)} \right].\quad \quad \quad \quad \quad \,\,\,\,\,} \\ \end{array}$$\end{document} performing the integral in (190), we obtain15 191\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${\sigma _ {{\rm{abs}}}}\sim t_l^{2{h_l} - 1}t_r^{{2_r} - 1}\left({{e^{\pi {{\tilde \omega}_l} + \pi {{\tilde \omega}_r}}} \pm {e^{- \pi {{\tilde \omega}_l} - \pi {{\tilde \omega}_r}}}} \right)\vert \gamma ({h_l} + i{\tilde \omega _ l})\vert ^{2}\vert \gamma ({h_r} + i{\tilde \omega _ r})\vert ^{2},$$\end{document} where 192\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${\tilde \omega _ l} = {{{\omega _ l} - {q_l}{\mu _ l}} \over {2\pi {t_l}}},\quad {\tilde \omega _ r} = {{{\omega _ r} - {q_r}{\mu _ r}} \over {2\pi {t_r}}}.$$\end{document} in order to compare the bulk computations to the cft result (191), we must match the conformal weights and the reduced momenta \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$({{\tilde \omega}_l},{{\tilde \omega}_r})$\end{document}. The gravity result (178) agrees with the cft result (191) if and only if we choose 193\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\begin{array}{*{20}c} {{h_l} = {1 \over 2} + \beta - \vert s\vert, \quad \quad {h_r} = {1 \over 2} + \beta,} \\ {{{\tilde \omega}_l} = {{\rm re}} ({q_{{\rm{eff}}}}),\quad \quad {{\tilde \omega}_r} = {n \over {2\pi}} - {{\rm re}} ({q_{{\rm{eff}}}}). }\\ \end{array}$$\end{document} the right conformal weight matches with (185), consistent with sl(2,)r conformal invariance . The left conformal weight is natural for a spin s field since |hl hr| = |s| . The value for \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}${{\tilde \omega}_l}$\end{document} is consistent with the temperature (180) and chemical potential (181). Indeed, since the left - movers span the direction of the black hole, we have l = m. we then obtain 194\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${\tilde \omega _ l} = {{2mj + {q_e}{q^3}} \over {r _ + ^2 + {a^2}}}={\rm{re}}({q_{{\rm{eff}}}}),$$\end{document} after using the value (175). The value of \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}${{\tilde \omega}_r}$\end{document} is fixed by the matching . It determines one constraint between r, r and tr . However, there is a subtlety in the above matching procedure . The conformal weights hl and hr depend on m through . This m dependence cannot originate from l = m since l is introduced after the fourier transform (190), while hl, hr are already defined in (189). One way to introduce this m dependence is to assume that there is a right - moving current algebra and that the dual operator \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}${\mathcal o}$\end{document} has the zero - mode charge qr = m, which amounts to imposing the condition (182). (it is then also natural to assume that the chemical potential is r j, but the matching does not depend on any particular value for r .) The dependence of the conformal weights in qe is similarly made possible thanks to the existence of the left - moving current with ql = qe . The matching is finally complete . Now, let us notice that the matching conditions (193)(194) are democratic in that the roles of angular momentum and electric charge are put on an equal footing, as noted in [79, 71]. One can then also obtain the conformal weights and reduced left and right frequencies \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}${{\tilde \omega}_l},{{\tilde \omega}_r}$\end{document} using alternative cft descriptions such as the cftq with virasoro algebra along the gauge field direction, and the mixed sl(2,) family of cfts . We can indeed rewrite (192) in the alternative form 195\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${\tilde \omega _ l} = {{m{t_e} + {q_e}{t_\phi}} \over {2\pi {t_\phi}{t_e}}} = {{{q_\chi} - \mu _ l^{\phi, q}m} \over {2\pi t_l^q}},$$\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}$t_l^q = {r_\chi}{t_e}$\end{document} is the left - moving temperature of the cftq, \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$\mu _ l^{\phi, q}$\end{document} is the chemical potential defined in (144) and q = rqe is the probe electric charge in units of the kaluza - klein circle length . The identification of the right - moving sector is unchanged except that now qr = qe . One can trivially extend the matching with the sl(2,) family of cfts conjectured to describe the (near-)extremal kerr - newman black hole . In summary, near - superradiant absorption probabilities of probes in the near - horizon region of near - extremal black holes are exactly reproduced by conformal field theory two - point functions . This shows the consistency of a cft description (or multiple cft descriptions in the case where several u(1) symmetries are present) of part of the dynamics of near - extremal black holes . We expect that a general scattering theory around any near - extremal black - hole solution of (1) will also be consistent with a cft description, as supported by all cases studied beyond the kerr - newman black hole [106, 73, 242, 71, 80, 46]. Finally, let us note finally that the dynamics of the cfts dual to the kerr - newman geometry close to extremality can be further investigated by computing three - point correlation functions in the near - horizon geometry, as initiated in [40, 39]. We mentioned in section 2.1 that extremal spinning black holes that do not admit a globally - defined timelike killing vector spontaneously emit quanta in the range of frequencies (11). This quantum effect is related by detailed balance to the classical effect of superradiant wave emission, which occur in the same range of frequencies . It has been argued that the bound (11) essentially follows from fermi - dirac statistics of the fermionic spin - carrying degrees of freedom in the dual two - dimensional cft (see also). These arguments were made for specific black holes in string theory but one expects that they can be applied to generic extremal spinning black holes, at least qualitatively . One starts with the assumption that extremal spinning black holes are modeled by a 2d cft, where the left and right sectors are coupled only very weakly . Therefore, the total energy and entropy are approximately the sum of the left and right energies and entropies . The state corresponding to an extremal spinning black hole is modeled as a filled fermi sea on the right sector with zero entropy and a thermal state on the left sector, which accounts for the black - hole entropy . The right - moving fermions form a condensate of aligned spins s = + 1/2, which accounts for the macroscopic angular momentum . It is expected from details of emission rates in several parametric regimes that fermions are only present on the right sector, while bosons are present in both sectors [105, 106]. Superradiant spontaneous emission is then modeled as the emission of quanta resulting from interaction of a left and a right - moving mode . Using details of the model such as the fact that the fermi energy should be proportional to the angular velocity j . It would be interesting to better compare these arguments to the present setup, and to see how these arguments could be generalized to the description of the bound (12) for static extremal rotating black holes . Let us finally argue that the existence of a qualitative process of superradiant emission in these models further supports the conjecture that the dual theory to extremal black holes is a chiral limit of a 2d cft instead of a chiral cft with no right - moving sector . In section 4 we described evidence showing that the asymptotic growth of states of extremal rotating or charged black holes is controlled by a chiral half of a two - dimensional cft, at least in the semi - classical limit . We also reviewed in section 5 how the near - horizon dynamics of probes can be reproduced by manipulating near - chiral cft two - point functions in the near - extremal limit . These analyses strongly rely on the existence of a decoupled near - horizon geometry for all extremal or near - extremal black holes . Away from extremality therefore, it is unclear whether any of the previous considerations will be useful in describing non - extremal geometries . It might then come as a surprise that even away from extremality, conformal invariance is present in the dynamics of probe scalar fields around the kerr black hole in a specific regime (at low energy and close enough to the black hole as we will make more precise below). In that regime, the probe scalar field equation can be written in a sl(2,) sl(2,) invariant fashion in a region close enough to the horizon . Such a local hidden symmetry is non - geometric but appears in the probe dynamics . The 2 periodic identification of the azimuthal angle breaks globally - conformal symmetry . Using the properties of this representation of conformal invariance, one can then argue that the kerr black hole is described by a cft with specific left and right - moving temperatures 196\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${t_l} = {{{m^2}} \over {2\pi j}},\quad \quad {t_r} = {{\sqrt {{m^4} - {j^2}}} \over {2\pi j}}.$$\end{document} the well - known low - energy scattering amplitudes coincide with correlators of a two - dimensional cft with these temperatures . Finally, quite remarkably, the entropy of the kerr black hole is then reproduced by cardy s formula if one assumes that the cft has left and right - moving central charges equal to the value cl = cr = 12j, which matches with the value for the left - moving central charge (129) derived at extremality.16 these observations are consistent with the interpretation of a 2d cft dual to the kerr black hole, but the existence of such a cft is conjectural . For example, there is no known derivation of two virasoro algebras with central charges cl = cr = 12j from the non - extremal kerr geometry . Asymptotic symmetry group methods are not directly applicable here because the horizon is not an isolated system . However, as argued in, the resulting picture shows a remarkable cohesiveness and only future research can prove or disprove such a cft interpretation . Given the successful generalization of the extremal kerr / cft correspondence to several independent extremal black hole / cft correspondences in gravity coupled to matter, as we reviewed above, it is natural to test the ideas proposed in to more general black holes than the kerr geometry . First, hidden symmetry can be found around the non - extremal reissner - nordstrm black hole [81, 77] under the assumption that the gauge field can be understood as a kaluza - klein gauge field, as done in the extremal case . One can also generalize the analysis to the kerr - newman black hole [263, 74, 78]. In complete parallel with the existence of an sl(2,) family of cft descriptions, there is a class of hidden sl(2,) sl(2,) symmetries of the kerr - newman black hole related with sl(2,) transformations . What has not been noted in the literature so far is that each member of the sl(2,) family of cfts describes only probes with a fixed ratio of probe angular momentum to probe charge as we will discuss in detail in section 6.4 . Therefore, one needs a family of cfts to fully describe the dynamics of low energy, low charge and low mass probes . Remarkably, for all cases where a hidden local conformal invariance can be described, the non - extremal black - hole entropy matches with cardy s formula using the central charges cr = cl and using the value cl in terms of the quantized conserved charges derived at extremality . Five - dimensional asymptotically - flat black holes were also discussed in [189, 80]. In attempting to generalize the hidden symmetry arguments to four - dimensional black holes in ads it is expected that hidden symmetries are present at least close to extremality, as illustrated by five - dimensional analogues . However, the structure of the wave equation is more intricate far from extremality because of the presence of complex poles, which might have a role to play in microscopic models . Quite surprisingly, one can also find a single copy of hidden sl(2,) symmetry around the schwarzschild black hole, which turns out to be globally defined . As a consequence this hidden symmetry can be understood as a special case of a generalized notion of hidden conformal symmetry around the kerr geometry . At present, it is unclear how these hidden symmetries fit in the general picture of the kerr / cft correspondence since the derivations of the central charges of the cft dual to kerr, reissner - nordstrm or kerr - newman black holes are done at extremality, which clearly cannot be done in the schwarzschild case . All arguments presented in the literature so far have been derived for a probe scalar field . It is not clear if any of these arguments can be generalized to higher - spin fields, and, if such, a generalization would give the same values for the left and right - moving cft temperatures . It would certainly be interesting to understand whether this is a technical obstruction that can be overcome or whether it is a fundamental limitation in the cft descriptions . Hidden symmetries in asymptotically - flat spacetimes only appear in a region close enough to the black hole . It has been suggested that one deform the geometry far from the black hole such that hidden symmetries appear in the entire resulting geometry [108, 107]. The resulting subtracted geometries are not asymptotically flat and are supported by additional matter fields [108, 107, 101]. The nature of these geometries and their role in the kerr / cft correspondence remains to be clarified . In what follows, we present a summary of the derivation of the hidden symmetries of the kerr - newman black hole and we discuss their cft interpretation . We will limit our presentation to the approach of but we will generalize the discussion to the kerr - newman black hole, which contains several new interesting features . In particular, we will show that each member of the conjectured sl(2,) family of cfts controls part of the dynamics of low energy, low charge and low mass probes . This matching is very similar to the analysis already performed in section 5 at near - extremality and it follows from local conformal invariance . As noted in, the only difference is that in the present context the region close enough to the horizon is not geometrically a near - horizon region, but it does not affect the discussion . The klein - gordon equation for a charged massive spin 0 field of mass and charge qe was analyzed in section 5.2 . Expanding in eigenmodes and using the fact that the equation is separable, we have 197\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\phi (t, r,\theta, \phi) = {e^{- i\omega t + im\phi}}s(\theta)r(r).$$\end{document} the equations for the functions s() and r(r) were written in (158) and (159). Substituting \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$a_{a\omega, l, m}^0 = {k_l} + {a^2}({\omega ^2} - {\mu ^2})$\end{document}, the equations can be written in a convenient way as 198\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\left [{{1 \over {\sin \theta}}{\partial _ \theta}(\sin \theta {\partial _ \theta}) - {{{m^2}} \over {{{\sin}^2}\theta}} + {a^2}({\omega ^2} - {\mu ^2}){{\cos}^2}\theta + {k_l}} \right]s(\theta) = 0,$$\end{document} 199\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\left [{{\partial _ r}(\delta {\partial _ r}) + {{\alpha {{({r _ +})} ^2}} \over {(r - {r _ +}) ({r _ +} - {r _ -})}} - {{\alpha {{({r _ -})}^2}} \over {(r - {r _ -})({r _ +} - {r _ -})}} - {k_l} + v(r)} \right]r(r) = 0,$$\end{document} where (r) = (r r+)(r r). The function (r) is defined as 200\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\alpha (r) = (2mr - {q^2})\omega - am - qr{q_e},$$\end{document} and is evaluated either at r+ or r and 201\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$v(r) = ({\omega ^2} - {\mu ^2}){r^2} + 2\omega (m\omega - {q_e}q)r - {\omega ^2}{q^2} + {(2m\omega - {q_e}q)^2}.$$\end{document} these equations can be solved by heun functions, which are not among the usual special functions . A solution can be found only numerically . The equations for probe scalars fields on the kerr - newman - ads black hole can be obtained straightforwardly . Using again the decomposition (197), the klein - gordon equation is decoupled into an angular equation 202\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\left [{{1 \over {\sin \theta}}{\partial _ \theta}(\sin \theta {\delta _ \theta}{\partial _ \theta}) - {{{m^2}{\xi ^2}} \over {{{\sin}^2}\theta {\delta _ \theta}}} + {{2ma\xi \omega - {a^2}{\omega ^2}{{\sin}^2}\theta} \over {{\delta _ \theta}}} + {c_l}} \right]s(\theta) = 0,$$\end{document} and a radial equation 203\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\left [{{\partial _ r}(\delta {\partial _ r}) + {{{{[\omega ({r^2} + {a^2}) - ma\xi - {q_e}{q_e}r]}^2}} \over {{\delta _ r}}} - {c_l}} \right]r(r) = 0,$$\end{document} where cl is a separation constant and the various functions and parameters in the equations have been defined in section 2.4.4 . In the flat limit, eqs . (198)(199) are recovered with kl = cl + 2ma a. the radial equation has a more involved form than the corresponding flat equation (199) due to the fact that r is a quartic instead of a quadratic polynomial in r; see (42). More precisely, the quartic polynomial r can be written as 204\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${\delta _ r} = {l^{- 2}}(r - {r _ +}) (r - {r _ -})(r - {r_c})(r - r_c^{\ast}),$$\end{document} where rc is a complex root . The radial equation is a general heun s equation due to the presence of two conjugate complex poles in (203) in addition to the two real poles corresponding to the inner and outer horizons and the pole at infinity . It has been suggested that all these poles have a role to play in the microscopic description of the ads black hole . It is an open problem to unravel the structure of the hidden symmetries, if any, of the full non - extremal radial equation (203). It has been shown that in the context of five - dimensional black holes, one can find hidden conformal symmetry in the near - horizon region close to extremality . It is expected that one could similarly neglect the two complex poles in the near - horizon region of near - extremal black holes, but this remains to be checked in detail.17 since much remains to be understood, we will not discuss ads black holes further . Let us go back to the scalar wave equation around the kerr - newman black hole . We will now study a particular range of parameters, where the wave equations simplify . We will assume that the wave has low energy and low mass as compared to the black hole mass and low electric charge as compared to the black hole charge, 205\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\omega m = o(\epsilon),\quad \mu m = o(\epsilon),\quad {q_e}q = o(\epsilon),$$\end{document} where 1 . From these approximations, we deduce that a, r+, q and a = o() as well . We will only look at a specific region of the spacetime the near region defined by 206\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\omega r = o(\epsilon),\quad \mu r = o(\epsilon){. }$$\end{document} note that the near region is a distinct concept from the near - horizon region r r+ m. indeed, for sufficiently small and, the value of r defined by the near region can be arbitrarily large . Using the approximations (205) it can be solved both in the near region and in the far region r m in terms of special functions . A complete solution can then be obtained by matching near and far solutions together along a surface in the matching region m r . As noted in, conformal invariance results from the freedom to locally choose the radius of the matching surface within the matching region . More precisely, using (205), the angular equation (198) reduces to the standard laplacian on the two - sphere 207\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\left [{{1 \over {\sin \theta}}{\partial _ \theta}(\sin \theta {\partial _ \theta}) - {{{m^2}} \over {{{\sin}^2}\theta}} + {k_l}} \right]s(\theta) = o({\epsilon ^2}).$$\end{document} the solutions es() are spherical harmonics and the separation constants are 208\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${k_l} = l(l + 1) + o({\epsilon ^2}){. }$$\end{document} in the near region, the function v(r) defined in (201) is very small, v(r) = o(). The near region scalar - wave equation can then be written as 209\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\left [{{\partial _ r}(\delta {\partial _ r}) + {{\alpha {{({r _ +})} ^2}} \over {(r - {r _ +}) ({r _ +} - {r _ -})}} - {{\alpha {{({r _ -})}^2}} \over {(r - {r _ -})({r _ +} - {r _ -})}} - l(l + 1)} \right]r(r) = 0,$$\end{document} where (r) has been defined in (200). We will now make explicit the local sl(2,) sl(2,) symmetries of the near - horizon scalar field equations (209). Conformal coordinates (, y) defined in terms of coordinates (t, r,) by (see and for earlier relevant work) 210\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\begin{array}{*{20}c} {{\omega ^ +} = \sqrt {{{r - {r _ +}} \over {r - {r _ -}}}} {e^{2\pi {t_r}(\phi {\prime} - {\omega _ r}t)}},}\\ {{\omega ^ -} = \sqrt {{{r - {r _ +}} \over {r - {r _ -}}}} {e^{2\pi {t_l}(\phi {\prime} - {\omega _ l}t)}},}\\ {\;\;y = \sqrt {{{{r _ +} - {r _ -}} \over {r - {r _ -}}}} {e^{\pi {t_l}(\phi {\prime} - {\omega _ l}t) + \pi {t_r}(\phi {\prime} - {\omega _ r}t)}}. }\\ \end{array}$$\end{document} the change of coordinates is locally invertible if = l r 0 . We choose the chirality> 0, as it will turn out to match the chirality convention in the description of extremal black holes in section 4.4 . Several choices of coordinate + 2 will lead to independent sl(2,) sl(2,) symmetries . For the kerr black hole, there is only one meaningful choice: = . For the reissner - nordstrm black hole, we identify = /r, where is the kaluza - klein coordinate that allows one to lift the gauge field to higher dimensions, as done in section 4.3 . For the kerr - newman black hole, we use, in general, a coordinate system (,) (, + 2) (+ 2,) parameterized by a sl(2,) transformation 211\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\begin{array}{*{20}c} {\phi {\prime} = {p_1}\phi + {p_2}\chi/{r_\chi},}\\ {\chi {\prime} = {p_3}\phi + {p_4}\chi/{r_\chi},}\\ \end{array}$$\end{document} with p1p4 p2p3 = 1 so that 212\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${\partial _ {\phi {\prime}}} = {p_4}{\partial _ \phi} - {p_3}{r_\chi}{\partial _ \chi},$$\end{document} 213\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${\partial _ {\chi {\prime}}} = - {p_2}{\partial _ \phi} + {p_1}{r_\chi}{\partial _ \chi}.$$\end{document} let us define locally the vector fields 214\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\begin{array}{*{20}c} {{h_1} = i{\partial _ +}, \,\quad \quad \quad \quad \quad \quad \quad \quad \quad \quad} \\ {\,{h_0} = i({\omega ^ +} {\partial _ +} + {1 \over 2}y{\partial _ y}),\,\quad \quad \quad \quad \quad} \\ {\,{h_{- 1}} = i({\omega ^{+ 2}}{\partial _ +} + {\omega ^ +} y{\partial _ y} - {y^2}{\partial _ -}),\quad \quad} \\ \end{array}$$\end{document} and 215\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\begin{array}{*{20}c} {{{\bar h}_1} = i{\partial _ -},}\\ {\;{{\bar h}_0} = i({\omega ^ -}{\partial _ -} + {1 \over 2}y{\partial _ y}),}\\ {{{\bar h}_{- 1}} = i({\omega ^{- 2}}{\partial _ -} + {\omega ^ -}y{\partial _ y} - {y^2}{\partial _ +}). }\\ \end{array}$$\end{document} these vector fields obey the sl(2,) lie bracket algebra, 216\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$[{h_0},{h_{\pm 1}}] = \mp i{h_{\pm 1}},\quad [{h_{- 1}},{h_1}] = - 2i{h_0},$$\end{document} and similarly for \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$({{\bar h}_0},{{\bar h}_{\pm 1}})$\end{document}. Note that 217\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${t_l}{\bar h_0} + {t_r}{h_0} = {i \over {2\pi}}{\partial _ {\phi {\prime}}}.$$\end{document} the sl(2,) quadratic casimir is 218\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${{\mathcal h}^2} = {\bar {\mathcal h}^2} = - h_0 ^ 2 + {1 \over 2}({h_1}{h_{- 1}} + {h_{- 1}}{h_1})$$\end{document} 219\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$= {1 \over 4}({y^2}\partial _ y^2 - y{\partial _ y}) + {y^2}{\partial _ +} {\partial _ -}.$$\end{document} in terms of the coordinates (r, t,), the casimir becomes \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\begin{array}{*{20}c} {{{\mathcal h}^2} = - {{{r _ +} - {r _ -}} \over {(r - {r _ +}) {{(4\pi {t_r})}^2}}}{{\left({{\partial _ {\phi {\prime}}} + {{{t_l} + {t_r}} \over {{t_l}\delta \omega}}({\partial _ t} + {\omega _ r}{\partial _ {\phi {\prime}}})} \right)}^2}}\\ {\quad \quad \; + {{{r _ +} - {r _ -}} \over {(r - {r _ -}){{(4\pi {t_r})}^2}}}{{\left({{\partial _ {\phi {\prime}}} + {{{t_l} - {t_r}} \over {{t_l}\delta \omega}}({\partial _ t} + {\omega _ r}{\partial _ {\phi {\prime}}})} \right)}^2} + {\partial _ r}\delta {\partial _ r},}\\ \end{array}$$\end{document} where (r) = (r r+)(r r). We will now match the radial wave equation around the kerr - newman black hole in the near region (209) with the eigenvalue equation 220\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${{\mathcal h}^2}\phi = l(l + 1)\phi .$$\end{document} the scalar field has the following eigenvalues t = i and = im. In the case where an electromagnetic field is present, one can perform the uplift (2) and consider the five - dimensional gauge field (6). In that case, the eigenvalue of the five - dimensional gauge field under is the electric charge = iqe. Let us denote the eigenvalue along as im i(p4 m p3qer). (209) and (220) will match if and only if the two following equations are obeyed 221\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\alpha ({r _ \pm}) = {{{r _ +} - {r _ -}} \over {4\pi {t_r}}}\left({- {m\prime} + {{{t_l} \pm {t_r}} \over {{t_r}\delta \omega}}(\omega - {\omega _ r}m\prime)} \right),$$\end{document} where (r) has been defined in (200). For simplicity, let us first discuss the case of zero probe charge qe = 0 and non - zero probe angular momentum m 0 . The matching equations then admit a unique solution 222\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\begin{array}{*{20}c} {{\omega _ r} = 0,\quad {\omega _ l} = {a \over {2{m^2} - {q^2}}},\quad \quad \quad \quad} \\ {{t_l} = {{2{m^2} - {q^2}} \over {4\pi j}},\quad {t_r} = {{m({r _ +} - {r _ -})} \over {4\pi j}},} \\ \end{array}$$\end{document} upon choosing = (and = /r). This shows in particular that the kerr black hole has a hidden symmetry, as derived originally in . For probes with zero angular momentum m = 0, but electric charge qe 0, there is also a unique solution, 223\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\begin{array}{*{20}c} {{\omega _ r} = {q \over {2m{r_\chi}}},\quad {\omega _ l} = {{mq} \over {(2{m^2} - {q^2}){r_\chi}}},}\\ {{t_l} = {{(2{m^2} - {q^2}){r_\chi}} \over {2\pi {q^3}}},\quad {t_r} = {{m({r _ +} - {r _ -}){r_\chi}} \over {2\pi {q^3}}},}\\ \end{array}$$\end{document} upon choosing = /r (and =). This shows, in particular, that the reissner - nordstrm black hole admits a hidden symmetry, as pointed out in [81, 77]. Finally, one can more generally solve the matching equation for any probe scalar field whose probe angular momentum and probe charge are related by 224\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${p_2}m - {p_1}{q_e}{r_\chi} = 0.$$\end{document} in that case, one chooses the coordinate system (211) and the unique solution is then 225\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\begin{array}{*{20}c} {{\omega _ r} = {{{p_2}q} \over {2m{r_\chi}}},\quad {\omega _ l} = {{{p_1}a + {p_2}mq/{r_\chi}} \over {2{m^2} - {q^2}}},}\\ {{t_l} = {{2{m^2} - {q^2}} \over {2\pi (2{p_1}j + {p_2}{q^3}/{r_\chi})}},\quad {t_r} = {{m({r _ +} - {r _ -})} \over {2\pi (2{p_1}j + {p_2}{q^3}/{r_\chi})}}. }\\ \end{array}$$\end{document} when p1 = 0 and q 0 or p2 = 0 and j 0, one recovers the two previous particular cases . The condition (224) is equivalent to the fact that the scalar field has zero eigenvalue along . Since m and qer are quantized, as derived in (7), there is always (at least) one solution to (224) with integers p1 and p2 . In conclusion, any low energy and low mass scalar probe in the near region (206) of the kerr black hole admits a local hidden sl(2,) sl(2,) symmetry . Similarly, any low energy, low mass and low charge scalar probe in the near region (206) of the reissner - nordstrm black hole admits a local hidden sl(2,) sl(2,) symmetry . In the case of the kerr - newman black hole, we noticed that probes obeying (205) also admit an sl(2,) sl(2,) hidden symmetry, whose precise realization depends on the ratio between the angular momentum and the electric charge of the probe . For a given ratio (224), hidden symmetries can be constructed using the coordinate = p1 + p2/r. Different choices of coordinate are relevant to describe different sectors of the low energy, low mass and low charge dynamics of scalar probes in the near region of the kerr - newman black hole . The union of these descriptions cover the entire dynamical phase space in the near region under the approximations (205)(206). The vector fields that generate the sl(2,) sl(2,) symmetries are not globally defined . They are not periodic under the angular identification 226\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\phi {\prime} \sim \phi {\prime} + 2\pi .$$\end{document} therefore, the sl(2,) symmetries cannot be used to generate new global solutions from old ones . In other words, solutions to the wave equation in the near region do not form sl(2,) sl(2,) representations . In the (,) plane defined in (210), the identification (226) is generated by the sl(2,)l sl(2,)r group element 227\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${e^{- i4{\pi ^2}{t_r}{h_0} - i4{\pi ^2}{t_l}{{\bar h}_0}}},$$\end{document} as can be deduced from (217). This can be interpreted as the statement that the sl(2,)l sl(2,)r symmetry is spontaneously broken to the u(1)l u(1)r symmetry generated by \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$({{\bar h}_0},{{\bar h}_0})$\end{document}. The situation is similar to the btz black hole in 2 + 1 gravity that has a sl(2,)l sl(2,)r symmetry, which is spontaneously broken by the identification of the angular coordinate . This breaking of symmetry can be interpreted in that case as placing the dual cft to the btz black hole in a density matrix with left and right - moving temperatures dictated by the sl(2,)l sl(2,)r group element generating the 2 identification of the geometry . In the case of non - extremal black - hole geometries, one can similarly interpret the symmetry breaking using a cft as follows . First, we need to assume that before the identification, the near region dynamics is described by a dual two - dimensional cft, which possesses a ground state that is invariant under the full sl(2,)l sl(2,)r symmetry . This is a strong assumption, since there are several (apparent) obstacles to the existence of a ground state, as we already discussed in the case of extremal black holes; see section 4.4 . Nevertheless, assuming the existence of this vacuum state, the two conformal coordinates (,) can be interpreted as the two null coordinates on the plane where the cft vacuum state can be defined . At fixed r, the relation between conformal coordinates (,) and boyer - lindquist (, t) coordinates is, up to an r - dependent scaling, 228\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${\omega ^ \pm} = {e^{\pm {t^ \pm}}},$$\end{document} where 229\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${t^ +} = 2\pi {t_r}({\phi \prime} - {\omega _ r}t),$$\end{document} 230\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${t^ -} = - 2\pi {t_l}({\phi \prime} - {\omega _ l}t).$$\end{document} this is precisely the relation between minkowski () and rindler (t) coordinates . The periodic identification (226) then requires that the rindler domain be restricted to a fundamental domain under the identification 231\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${t^ +} \sim {t^ +} + 4{\pi ^2}{t_r},\quad \;\;{t^ -} \sim {t^ -} - 4{\pi ^2}{t_l},$$\end{document} generated by the group element (227). The quantum state describing this accelerating strip of minkowski spacetime is obtained from the sl(2,)l sl(2,)r invariant minkowski vacuum by tracing over the quantum state in the region outside the strip . The result is a thermal density matrix at temperatures (tl, tr). Hence, under the assumption of the existence of a cft with a vacuum state, non - extremal black holes can be described as a finite temperature (tl, tr) mixed state in a dual cft . It is familiar from the three - dimensional btz black hole that the identifications required to obtain extremal black holes are different than the ones required to obtain non - extremal black holes [27, 210]. Here as well, the vector fields (214)(215) are not defined in the extremal limit because the change of coordinates (210) breaks down . Nevertheless, the extremal limit of the temperatures tl and tr match with the temperatures defined at extremality in section 5.4 . More precisely, the temperatures tl and tr defined in (222), (223) and (225) match with the temperatures defined at extremality t, rte and \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}${({p_1}t_\phi ^{- 1} + {p_2}{({r_\chi}{t_e})^{- 1}})^{- 1}}$\end{document}, respectively, where t and te are defined in (74). This is consistent with the interpretation that states corresponding to extremal black holes in the cft can be defined as a limit of states corresponding to non - extremal black holes . We will now argue that the temperatures tl and tr obtained in section 6 combined with the analysis at extremality in section 4 lead to a (several) microscopic counting(s) of the black hole entropy of the kerr, reissner - nordstrm and kerr - newman black holes . Let us assume that there is a two - dimensional cft (cftj) describing the kerr black hole, a two - dimensional cft (cftq) describing the reissner - nordstrm black hole and a sl(2,) family of two - dimensional cfts \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$({\rm{cf}}{{\rm{t}}_{({p_1},{p_2},{p_3})}},{p_1},{p_2},{p_2} \in {\mathbb z})$\end{document} describing the kerr - newman black hole . If these cfts are dual to the black hole, the entropy is reproduced by cardy s formula 232\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${{\mathcal s}_{{\rm{cft}}}} = {{{\pi ^2}} \over 3}({c_l}{t_l} + {c_r}{t_r}),$$\end{document} which is valid when tl 1, tr 1 . As already mentioned in section 4.4 and argued in, the regime tl 1, tr 1 is not a necessary condition for cardy s formula to be valid if these cfts have special properties such as admitting a long string picture, as reviewed in section 3.3 . The difference cr cl is proportional to the diffeomorphism anomaly of the cft [188, 187]. One can then argue from diffeomorphism invariance that the two left and right sectors should have the same value for the central charge, 233\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${c_r} = {c_l}.$$\end{document} we obtained the value cl at extremality in section 4.3 and checked that cardy s formula reproduces the extremal black - hole entropy . One way to uniquely fix the value cl away from extremality would consist in matching cardy s formula (232) with the kerr - newman black - hole entropy 234\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${{\mathcal s}_{{\rm{kn}}}}({\mathcal m},{\mathcal j},{\mathcal q}) = \pi (r _ + ^2 + {a^2}),$$\end{document} using (233) and the values for the temperatures derived in section 6.4 . Therefore, the matching of black - hole entropy is true by construction, which is clearly unsatisfactory . It would be more satisfactory to have an independent computation of cl away from extremality, but such a computation is currently not available . However, the resulting central charge cl is, however, non - trivial . For the cftj, we obtain cl = 12 j. for cftq, we have cq = 6q / r and for the \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}${\rm{cf}}{{\rm{t}}_{({p_1},{p_2},{p_3})}}$\end{document}, we find \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}${c_{({p_1},{p_2})}} = 6({p_1}(2j) + {p_2}{q^3}/{r_\chi})$\end{document}. Quite remarkably, these central charges are expressed solely in terms of quantized charges . The presence of several cfts dual to the kerr - newman black hole is curious but not inconsistent . Each cft describes part of the low - energy dynamics of probe scalar fields and multiple cfts are needed in order to reproduce the full dynamics for arbitrary ratios of the probe angular momentum to probe electric charge . Any extremal black hole containing a compact u(1) axial symmetry admits a virasoro algebra in its near - horizon geometry with a nontrivial central charge . The black - hole entropy is reproduced by a chiral half of cardy s formula . This result is robust for any diffeomorphism - invariant theory and holds even including scalar and gauge field couplings and higher - derivative corrections . Moreover, if a u(1) gauge field can be geometrized into a kaluza - klein vector in a higher - dimensional spacetime, a virasoro algebra can be defined along the kaluza - klein compact u(1) direction and all analysis goes through in a similar fashion as for the axial u(1) symmetry . The deep similarity between the effects of rotation and electric charge can be understood from the fact that these charges are on a similar footing in the higher - dimensional geometry . When two u(1) symmetries are present, one can mix up the compact directions using a modular transformation and the construction of virasoro algebras can still be made . Independent of these constructions, the scattering probabilities of probes around the near - extremal kerr - newman black hole can be reproduced near the superradiant bound by manipulating near - chiral thermal two - point functions of a two - dimensional cft . The result extends straightforwardly to other asymptotically - flat or ads black holes in various gravity theories . Finally away from extremality, hidden sl(2,) sl(2,) symmetries are present in some scalar probes around the kerr - newman black hole close enough to the horizon . We showed that several cfts are required to account for the entire probe dynamics in the near region in the regime of small mass, small energy and small charge . These results obtained in gravity coupled to matter are naturally accounted for by assuming that the microstates of asymptotically - flat black holes, at extremality and away from extremality, can be described by 2d cfts and that the microstates of asymptotically - ads black holes at extremality can be described by chiral halves of 2d cfts . Scattering amplitudes and hidden symmetries are also accounted for by assuming that part of the dynamics of black holes can be mapped to the dynamics of these cfts once they are suitably coupled to the exterior black - hole region . By consistency with the gravitational analysis, the existence of such cfts is conjectural and only future research will tell how far these kerr / cft correspondences and their extensions can be made more precise . A fair concluding remark would be that our understanding of the kerr, reissner - nordstrm and kerr - newman black hole has increased over the last four years, but there is still a long road ahead of us to comprehend what these cfts really are and what they are telling us about the nature of quantum black holes . We hope that the interested reader will tackle them with the aim of shedding more light on the kerr / cft correspondence . We tried to order the problems with increasing difficulty but the evaluation is rough and highly subjective . Discuss hidden symmetries for a probe gauge field or a probe graviton on kerr or kerr - newman . Does one obtain the same temperatures tl and tr as in the scalar probe case?a black hole in de sitter spacetime can be extremal in the sense that its outer radius coincides with the cosmological horizon . The resulting geometry, called the rotating narirai geometry, has many similarities with the near - horizon geometries of extremal black holes in flat spacetime or in ads spacetime . The main difference is that the near - horizon geometry is a warped product of ds2 with s instead of ads2 with s. it has been conjectured that these extremal black holes are dual to the chiral half of a euclidean cft . Test the conjecture by generalizing all arguments of the kerr / cft correspondence to this cosmological setting.away from extremality, it is curious that the right - moving temperature is given by r = th/j for the kerr - newman black hole . Also, for all known asymptotically - flat extremal black holes in einstein gravity coupled to matter, the product of the horizon areas of the inner and outer horizon can be expressed in terms of quantized charges (j, q,) and fundamental constants only [195, 103, 106]. Explain this feature from a fundamental perspective.in the analysis of near - extremal superradiant scattering for any spin, we discarded the unstable modes that are below the breitenlohner - freedman bound . Clarify the match between these modes and cft expectations for the kerr - newman black hole.the probe scalar wave equation in kerr - newman - ads has two complex poles in addition to poles corresponding to the inner and outer horizon and infinity . This prevented a straightforward generalization of the hidden sl(2,) sl(2,) symmetry . Also explain why the product of all horizon areas (inner, outer and complex horizons) seems in general not to depend on the mass of the black hole .near - horizon geometries of black - hole solutions of (1) have been classified . Classify the four - dimensional near - horizon geometries of extremal black holes for gravity coupled to charged scalars, massive vectors, p - forms and non - abelian gauge fields.compute the central charges cl and cr away from extremality . Also, compute the quantum corrections to the central charge cl and investigate the matching between the quantum - corrected entropy of extremal black holes derived in and the asymptotic growth of states in the dual cft.understand how the extension of the kerr / cft correspondence to extremal ads black holes fits within the ads / cft correspondence . As discussed in, the extremal ads - kerr / cft correspondence suggests that one can identify a non - trivial virasoro algebra acting on the low - energy states of strongly coupled large n super - yang - mills theory in an extremal thermal ensemble . Try to make this picture more precise.from the point of view of 2d cfts, study if a sl(2,) action exists that transforms a cft into another cft this would clarify the existence of an sl(2,) family of cfts dual to the kerr - newman black hole . Note that this can be done for three - dimensional cfts with a u(1) current .compute the superradiant scattering amplitude of probe scalar fields on the kerr - newman geometry with first - order backreaction . Compare the result with the scattering amplitude defined in the cft at one loop order (using two and three - point correlation functions).formulate a general scattering theory around near - extremal black - hole solutions of (1). This would require one to classify the geometries admiting a killing - yano tensor so that the wave equation could be separated . A long - standing problem already consists in separating and decoupling the wave e quation of a probe spin 1 or spin 2 field in the kerr - newman geometry.construct one example in string theory of an exact quantum field theory dual to (an embedding in string theory of) the kerr black hole . Characterize whether that field theory is a cft, a limit of a cft, or a deformation thereof . Discuss hidden symmetries for a probe gauge field or a probe graviton on kerr or kerr - newman . Does one obtain the same temperatures tl and tr as in the scalar probe case? A black hole in de sitter spacetime can be extremal in the sense that its outer radius coincides with the cosmological horizon . The resulting geometry, called the rotating narirai geometry, has many similarities with the near - horizon geometries of extremal black holes in flat spacetime or in ads spacetime . The main difference is that the near - horizon geometry is a warped product of ds2 with s instead of ads2 with s. it has been conjectured that these extremal black holes are dual to the chiral half of a euclidean cft . Test the conjecture by generalizing all arguments of the kerr / cft correspondence to this cosmological setting . Away from extremality, it is curious that the right - moving temperature is given by r = th/j for the kerr - newman black hole . Also, for all known asymptotically - flat extremal black holes in einstein gravity coupled to matter, the product of the horizon areas of the inner and outer horizon can be expressed in terms of quantized charges (j, q,) and fundamental constants only [195, 103, 106]. Explain this feature from a fundamental perspective . In the analysis of near - extremal superradiant scattering for any spin clarify the match between these modes and cft expectations for the kerr - newman black hole . The probe scalar wave equation in kerr - newman - ads has two complex poles in addition to poles corresponding to the inner and outer horizon and infinity . This prevented a straightforward generalization of the hidden sl(2,) sl(2,) symmetry . Also explain why the product of all horizon areas (inner, outer and complex horizons) seems in general not to depend on the mass of the black hole . Near - horizon geometries of black - hole solutions of (1) have been classified . Classify the four - dimensional near - horizon geometries of extremal black holes for gravity coupled to charged scalars, massive vectors, p - forms and non - abelian gauge fields . Compute the central charges cl and cr away from extremality . Also, compute the quantum corrections to the central charge cl and investigate the matching between the quantum - corrected entropy of extremal black holes derived in and the asymptotic growth of states in the dual cft . Understand how the extension of the kerr / cft correspondence to extremal ads black holes fits within the ads / cft correspondence . As discussed in, the extremal ads - kerr / cft correspondence suggests that one can identify a non - trivial virasoro algebra acting on the low - energy states of strongly coupled large n super - yang - mills theory in an extremal thermal ensemble . Try to make this picture more precise . From the point of view of 2d cfts, study if a sl(2,) action exists that transforms a cft into another cft . This would clarify the existence of an sl(2,) family of cfts dual to the kerr - newman black hole . Note that this can be done for three - dimensional cfts with a u(1) current . Compute the superradiant scattering amplitude of probe scalar fields on the kerr - newman geometry with first - order backreaction . Compare the result with the scattering amplitude defined in the cft at one loop order (using two and three - point correlation functions). Formulate a general scattering theory around near - extremal black - hole solutions of (1). This would require one to classify the geometries admiting a killing - yano tensor so that the wave equation could be separated . A long - standing problem already consists in separating and decoupling the wave e quation of a probe spin 1 or spin 2 field in the kerr - newman geometry . Construct one example in string theory of an exact quantum field theory dual to (an embedding in string theory of) the kerr black hole . Characterize whether that field theory is a cft, a limit of a cft, or a deformation thereof.
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According to the euro heart survey on valvular heart disease, aortic stenosis (as) is the most frequent and mitral stenosis (ms) is the least frequent among native single - sided valve diseases in europe (43.1% vs. 12.1%). In as, the most common etiology is degenerative, and the least common etiology is rheumatic (81.9% vs. 11.2%), whereas in the case of ms, the situation is just the opposite, rheumatic being the most common and degenerative being the least common etiology (85.4% vs. 12.5%). Patients with the combination of severe and symptomatic degenerative as and severe symptomatic degenerative ms are very rare, and there is a lack of data about their frequency in the literature . According to the current european guidelines on the management of valvular heart disease, transcatheter aortic valve implantation (tavi) is recommended in patients with severe symptomatic as, who are considered unsuitable for conventional surgery because of severe comorbidities . Likewise, percutaneous mitral balloon commissurotomy (pmc) is recommended in patients with severe symptomatic ms, who are unsuitable for surgery . We discuss a rare case of an elderly patient with the combination of severe degenerative symptomatic as and symptomatic ms, who was found unsuitable for surgical treatment by the heart team due to several comorbidities including severe obesity . She was considered also unsuitable for two - time general anesthesia in the case of two - step single - valve percutaneous approach by the anesthesiologists and was subsequently offered a percutaneous dual - valve treatment in a single intervention (tavi and pmc). According to our knowledge, this is the first case of this kind described in the literature . A 76-year - old female patient with the combination of severe symptomatic degenerative as and severe symptomatic degenerative ms was admitted because of acute worsening of heart failure . At the time of presentation, the patient was new york heart association (nyha iii) with bilateral peripheral edema and the signs of left - heart failure . Moreover, the patient suffered from chronic ischemic heart disease (myocardial infarction 20 years earlier), underwent coronary artery bypass grafting (17 years earlier), stent implantation in saphenous vein graft to first diagonal branch, and the left anterior descending artery (7 years earlier). The patient was also diabetic (oral treatment) and suffered from chronic kidney failure (kdigo type iiia), dyslipoproteinemia, sleep - apnea syndrome, and had severe obesity (body mass index of 53.3). Baseline diagnostics included coronary angiography [figure 1], transesophageal echocardiography (tee) [figures 2 and 3], and computed tomography angiography (cta). (a) left anterior descending occlusion, 80% ostial intermediate and left circumflflex stenosis, patent saphenous vein graft to intermediate and first obtuse marginal . Lad: left anterior descending, i m: intermediate, lcx: left circumflflex, svg: saphenous vein graft, om1: first obtuse marginal, rca: right coronary artery, cto: chronic total occlusion, pda: posterior descending artery, d1: first diagonal branch transesophageal echocardiography before intervention - aortic valve . (b) peak aortic jet = 4.5 m / s, mean pressure gradient = 49 mmhg, aortic valve area = 0.78 cm (ejection fraction = 50%) transesophageal echocardiography before intervention - mitral valve . (c) three - dimensional echocardiography of joined leaflflets creating two orifices (a1 and a2). Al: anterior leaflflets, pl: posterior leaflflets, la: left atrium, lv: left ventricle, mv: mitral valve with an euroscore ii of 16%, the patient was classified as high risk and due to concomitant comorbidities, the heart team found her unsuitable for surgery and repeated general anesthesias . Before intervention, the patient was scheduled for computed tomography for detailed analysis of aortic root and peripheral arteries . The analysis and measurements were done using the 3mensio software, valve and vascular modules (3mensio medical imaging bv, bilthoven, the netherlands). Based on the detailed analysis of all imaging modalities, the strategy was to perform dual - valve single - intervention with pmc - inou balloon (toray, tokyo, japan) as the first step, followed by tavi - right femoral approach and 26 mm corevalve (medtronic, minneapolis, mn, usa). Pmc and tavi were performed under the angiographic and tee guidance [figure 4] with no periprocedural complications . Postintervention tee showed an excellent result of pmc with a residual transmitral gradient of 2 mmhg and mild regurgitation [figure 5], and an excellent result of tavi (normal pressure gradient over the corevalve and no remaining aortic regurgitation). The proper position and apposition of corevalve were also confirmed with postinterventional cta [figure 6]. Postinterventional course was uneventful, free of complication, and the patient was discharged on day 4 . At 6-months clinical follow - up, the patient is stable, compensated and nyha i. dual - valve single - intervention . Lv: left ventricle, la: left atrium, mv: mitral valve, ao: aorta, *: inou balloon transesophageal echocardiography control . (a) successful resolution of mitral stenosis . (d) absence of corevalve regurgitation (arrow) computed tomography angiography control after dual - valve intervention . (d) three - dimensional reconstruction of the thorax with a clearly visible corevalve. Ao: aortic the first - in - man case of tavi for the treatment of severe degenerative as was described by cribier et al . In 2002 . A 57-year - old very high - risk patient (cardiogenic shock, subacute leg ischemia, and several other noncardiac comorbidities) was found unsuitable for surgery and underwent successful tavi . Four - month follow - up showed satisfactory valvular function; however, the patient died soon after for noncardiac reasons . Implantation techniques and devices improved since then and tavi became the standard of treatment for the patients with severe as, who are unsuitable for surgery . With the aging population and the fact that degenerative as is the most frequent type of valvular disease, we can expect the rise in the number of tavi procedures in the near future . Moreover, cribier et al . Have shown noninferiority of tavi to surgery and the first reports of the corevalve us pivotal study 2-year results in high - risk patient population show superior results to surgery, which suggests in the direction of possible superiority of tavi in all subsets of patients . Clinical trend to treat low- or intermediate - risk patients is already established in many experienced centers, however, for tavi to become the method of choice also in patients with severe as, who are otherwise suitable surgical candidates, we need to wait for long - term results of the partner ii and surtavi trials . On the other hand, severe ms is a very rare disease in the western world, and most of the cases are rheumatic in etiology . According to the guidelines, pmc should remain the first choice of treatment in symptomatic patients who are unsuitable for surgery and in younger patients where postponing the surgery is desirable . Have confirmed good long - term results of pmc in the group of patients with rheumatic ms; on the other hand, tuzcu et al . Have shown that pmc is not the best option of treatment for nonrheumatic ms . In the case of our high - risk patient, who was unsuitable for surgery or two - time anesthesia in the case of two - step single - valve percutaneous approach, dual - valve single - intervention (pmc and tavi) proved to be a feasible and successful choice of treatment, allowing the patient to improve from nyha iii at the time of presentation to nyha i at the time of discharge and to remain in the same good condition at a 6-month follow - up.
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Chemically, it is a hydrophilic base which exists at physiological ph as the cationic species . This drug was observed to reduce hypergly - cemia, improve glucose utilization, reduce free fatty acid utilization, gluconeogenesis, serum lipids, insulin, insulin - like growth factor 1 (igf-1), reduce body weight, and decrease metabolic immunodepression both in humans and rodents. [14] various studies revealed that mf is more than a simple antidiabetic agent . Indeed, for an equivalent effect on glycemic control after a long time of treatment, mf was found to be obviously superior to other therapeutic measures for reducing vessel diseases and all - cause related mortality . It was suggested that insulin and mf treatments, y improving glycoxidative, inflammatory, and fibrotic renal damage markers, play a key role in the prevention of diabetic nephropathy . The role of mitochondria in programmed cell death is associated with the release of apoptotic signaling molecules . The production of reactive oxygen species (ros) by mitochondria also contributes to cell degradation process . Mitochondria are believed to have a role in a various renal diseases, and mitochondrial dysfunction could have a major role in nephrotoxicity . Gentamicin (gm) is probably the most the commonly used and studied of all the aminoglycosides . Hence, gm - enhanced ros formation in isolated cortical mitochondria[1117] and ros - induced cell death were found to have a role in gm - mediated acute renal failure . Apart from the superiority of mf to other antidiabetic drugs, various investigations strongly suggest that this antidiabetic agent prevented oxidative stress - induced death in several cell types through a mechanism dependent on the mitochondrial permeability transition pore (ptp) opening. [68131719] thus, mf may afford protection against gm - induced tubular injury by affecting the mitochondria through a mechanism dependent on the mitochondrial ptp opening . However, less data are present regarding the renoprotective effects of mf, and previous studies on attenuation of gm tubular toxicity by mf need to be re - tested . Therefore, we aimed to test the potential properties of mf to protect the kidney from gm - induced acute renal failure . Also, finding whether delayed treatment with mf exerts similar benefits on gm - induced renal toxicity in rats was the second aim of this study . Mf (metformin hexal; germany) was supplied as a white powder, soluble in distilled water, and freshly prepared as an aqueous solution to be given as a single daily oral dose of 100 mg / kg / day . Gm treatment protocols used in the present study have been reported earlier . In a preclinical study, 50 male wistar rats with a weight range of 200250 g, purchased from jundishapur university of medical sciences, ahvaz, iran, were transferred to the animal house of shahrekord university of medical sciences, shahrekord, iran . Animals were housed at an environment of controlled temperature (25 3c) and humidity (5060%) with a 12-hour dark light cycle (lights on at 7 am) and free access to pelleted diet and tap water for 2 weeks to acclimatize to the new environment . The animal experimentation was conducted in accordance with the national institute of health guide for the careful use of laboratory animals . The animals were divided into five groups (10 rats each) as follows: on the first day (before experiment) and the final day (day of sacrificing), serum samples were obtained to measure blood urea nitrogen (bun) and serum creatinine (cr) for all the rats . Bun and cr levels were measured by a colorimetric method using commercial kits on an autoanalyzer . The kidneys of each animal were dissected out, then fixed in buffered formalin for 12 h and processed for histopathologic examination . Three - micrometer - thick paraffin sections were stained with hematoxylin and eosin (h and e) for light microscope examination using conventional protocol . Slides were coded and examined by a histopathologist who was blinded to the treatment groups . All specimens were examined for six morphologic parameters including epithelial cell vacuolization, degeneration, tubular cell flattening, hyaline cast, tubular dilatation, and debris materials in tubular lumen on a semi - quantitative score from 1 to 5, while the score of zero was assigned to the normal tissue without damage . The t - paired test was used to compare the serum bun and cr levels before and after the experiments . One - way analysis of variance (anova) was applied to compare the serum bun and cr levels between the groups . To compare the pathology damage score between the groups, kruskal mf (metformin hexal; germany) was supplied as a white powder, soluble in distilled water, and freshly prepared as an aqueous solution to be given as a single daily oral dose of 100 mg / kg / day . In a preclinical study, 50 male wistar rats with a weight range of 200250 g, purchased from jundishapur university of medical sciences, ahvaz, iran, were transferred to the animal house of shahrekord university of medical sciences, shahrekord, iran . Animals were housed at an environment of controlled temperature (25 3c) and humidity (5060%) with a 12-hour dark light cycle (lights on at 7 am) and free access to pelleted diet and tap water for 2 weeks to acclimatize to the new environment . The animal experimentation was conducted in accordance with the national institute of health guide for the careful use of laboratory animals . The animals were divided into five groups (10 rats each) as follows: on the first day (before experiment) and the final day (day of sacrificing), serum samples were obtained to measure blood urea nitrogen (bun) and serum creatinine (cr) for all the rats . Bun and cr levels were measured by a colorimetric method using commercial kits on an autoanalyzer . The kidneys of each animal were dissected out, then fixed in buffered formalin for 12 h and processed for histopathologic examination . Three - micrometer - thick paraffin sections were stained with hematoxylin and eosin (h and e) for light microscope examination using conventional protocol . Slides were coded and examined by a histopathologist who was blinded to the treatment groups . All specimens were examined for six morphologic parameters including epithelial cell vacuolization, degeneration, tubular cell flattening, hyaline cast, tubular dilatation, and debris materials in tubular lumen on a semi - quantitative score from 1 to 5, while the score of zero was assigned to the normal tissue without damage . The t - paired test was used to compare the serum bun and cr levels before and after the experiments . One - way analysis of variance (anova) was applied to compare the serum bun and cr levels between the groups . To compare the pathology damage score between the groups, kruskal wallis and mann whitney u - test were applied . The levels of bun in groups ii, iv, and v, and the serum levels of cr in groups ii and v were increased significantly after the experiment (p <0.05). The serum levels of bun and cr before and after the experiment in five groups of animals . Group i, sham group; group ii, positive control group treated with gentamicin; group iii, treated with metformin; group iv, treated with gentamicin for 10 days and post - treatment with metformin for the next 10 days; and group v, co - administration of metformin and gentamicin for 10 days . The symbols (*) and () stand for significant difference before the experiment (p <0.05) and significant difference from positive control group (p <0.05), respectively the increase in serum bun and cr levels was highly significant in group 2 versus groups 4 and 5, therefore mf has a protective effect . The pathology damage score indicated a higher score for all gm treated groups, which was significantly different from non - gm treated groups (p <0.05). However, post - administration of mf after 10 days of gm treatment (group iv) and co - administration of mf and gm for 10 days attenuated the damage score significantly (p <0.05), when compared with group ii . As it is demonstrated in figure 2, co - administration of gm and mf for 10 days or post - treatment with mf after 10 days of treatment with gm could reduce the damage induced by gm . The pathology damage score in five groups of animals . Group i, sham group; group ii, positive control group treated with gentamicin; group iii, treated with metformin; group iv, treated with gentamicin for 10 days and post - treatment with metformin for the next 10 days; and group v, co - administration of metformin; and gentamicin for 10 days . The symbols (#) and () stand for significant difference from group i (p <0.05) and group ii (p <0.05), respectively the levels of bun in groups ii, iv, and v, and the serum levels of cr in groups ii and v were increased significantly after the experiment (p <0.05). The serum levels of bun and cr before and after the experiment in five groups of animals . Group i, sham group; group ii, positive control group treated with gentamicin; group iii, treated with metformin; group iv, treated with gentamicin for 10 days and post - treatment with metformin for the next 10 days; and group v, co - administration of metformin and gentamicin for 10 days . The symbols (*) and () stand for significant difference before the experiment (p <0.05) and significant difference from positive control group (p <0.05), respectively the increase in serum bun and cr levels was highly significant in group 2 versus groups 4 and 5, therefore mf has a protective effect . The pathology damage score indicated a higher score for all gm treated groups, which was significantly different from non - gm treated groups (p <0.05). However, post - administration of mf after 10 days of gm treatment (group iv) and co - administration of mf and gm for 10 days attenuated the damage score significantly (p <0.05), when compared with group ii . As it is demonstrated in figure 2, co - administration of gm and mf for 10 days or post - treatment with mf after 10 days of treatment with gm could reduce the damage induced by gm . The pathology damage score in five groups of animals . Group i, sham group; group ii, positive control group treated with gentamicin; group iii, treated with metformin; group iv, treated with gentamicin for 10 days and post - treatment with metformin for the next 10 days; and group v, co - administration of metformin; and gentamicin for 10 days . The symbols (#) and () stand for significant difference from group i (p <0.05) and group ii (p <0.05), respectively in this study, we found that mf could ameliorate gm - induced kidney tissue toxicity . We also found that mf was still effective when the drug was administered after progression of tubular damage by gm . Mf is used for the treatment of diabetes as a sugar - lowering agent . In addition, mf is recommended as the drug of first choice in type 2 diabetes. [1619] there are potential survival benefits associated with the use of mf, in addition to the benefits in respect to cardiovascular outcomes and metabolic parameters as suggested by recent studies. [25] mf exerts its metabolic activity through the induction of the adenosine monophosphate (amp)-activated protein kinase (ampk) pathway which acts as a sensor detecting variations of intracellular energy levels . It was suggested that hypoxia - induced hypoxia - inducible factor (hif)-1 accumulation in diabetic nephropathy could be suppressed by gm through the repression of oxygen consumption. [92327] hif-1 plays an important role in chronic hypoxia and tubulointerstitial fibrosis, which are presently considered to be the common pathways for various progressive kidney diseases, including diabetic nephropathy. [92327] alterations in epithelial cell polarity and in the subcellular distributions of epithelial ion transport proteins are key molecular consequences of acute kidney injury and intracellular energy depletion . Ampk, a cellular energy sensor, is rapidly activated in response to renal ischemia, and ampk activity may influence the maintenance or recovery of epithelial cell organization in mammalian renal epithelial cells subjected to energy depletion . At a molecular level, energy deprivation causes key energy - dependent membrane proteins to become displaced and dysfunctional . Specially, in the proximal tubule, the na k - atpase is internalized from the basolateral membrane, disrupting the cell's capacity to maintain normal transepithelial sodium transport . Preservation of a polarized plasma membrane distribution of na k - atpase in renal epithelia is essential for the maintenance of both solute reabsorption and volume homeostasis . Atp depletion also perturbs the distribution of tight junction proteins, further disrupting epithelial cell polarity and organization and leading to back leak of extracellular fluid into the urinary space . Mf treatment increases detectable p - ampk in a dose - dependent manner, and mf - induced ampk activation occurs in proximal tubules as well as in distal segments . Mitochondria represents one of the major cellular sources of ros generation and mitochondrial toxicity can also be mediated by ros . Indeed, after gm treatment, intracellular ros content can reach a toxic level, thus causing cell death and malfunctioning of the organ. [113033] literature review shows that the renal protective effects of mf in diabetic nephropathy have not been fully evaluated . Meanwhile, in accordance with our results, prevention of histologic changes due to gm toxicity by mf was shown by morales et al . They found that control and mf - treated rats showed no structural alterations in renal tissues, while massive and diffuse cell necrosis was observed in the proximal tubules of kidneys from rats injected with gm . They could show that tubular lumen was frequently filled with hyaline casts or heterogeneous cellular debris, while in rats treated with gm and mf, most of the proximal tubules were saved and manifestations of necrosis were observed in less than 10% of cells . Based on the above in vivo results, it was concluded that mf ameliorates gm - induced acute renal failure . Also, the present study shows that delayed treatment of mf for gm - induced acute renal failure was still effective as when mf was given in combination with the gm . Hence, mf is a nephroprotective drug to prevent or attenuate the tubular damage caused by gm or other nephrotoxic agents which act through the same mechanisms as this aminoglycoside.
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(www.eia.org) requiring water for cooling in thermoelectric (steam electric) power plants, it is important to understand how much water is used for thermoelectric generation to assess vulnerability of power generation to water shortages and determine potential impacts of generation on water resources . Life cycle analysis indicates that most water in thermoelectric power plants is used for cooling steam rather than in fuel extraction, atmospheric emissions controls, or other purposes . Large - scale expansion of natural gas production using hydraulic fracturing and increased use of natural gas in thermoelectric generation may have important implications for water resources, . Because of the long lifespan of power plants (3050 years), the current fleet represents a legacy of past fuel choices, generator technologies, and cooling systems . There seems to be a lack of generic understanding on water use for thermoelectric generation . Water use is a general term that can refer to water withdrawals, defined by the u.s . Geological survey (usgs) as water removed from a water source, most of which is returned to the source, or water consumption, defined as the portion of water removed that is lost to the system by evaporation and unavailable to other users . Many studies emphasize the large water withdrawals associated with thermoelectric generation (41% of u.s . Water withdrawals in 2005); however, 98% of this water is returned to the source . Equating water withdrawals for thermoelectric generation to the number of cities that could be supplied with water (6770 new york cities) seems to ignore the fact that 98% of the water is available to other users downstream of the return, although the water is not available to others between points of withdrawal and return . Some studies suggest that thermoelectric generation is also a major consumer of freshwater resources; however, u.s .- based estimates indicate that consumption for thermoelectric generation was 3% of total freshwater consumption in the u.s . While much emphasis has been placed upon reducing water withdrawals by converting cooling systems from once - through cooling to wet cooling towers, a recent report highlights the increased water consumption rates of cooling towers . There are many concerns about data availability and quality for quantifying cooling water requirements for thermoelectric generation . The government accountability office (gao) recommended that the department of energy - energy information administration (doe - eia) and usgs collaborate to improve water use estimates . The eia relies on self - reporting from power plant operators on electricity generation and water use and has revised its forms twice since 2009 to improve the data . The usgs is using various approaches to estimate consumption and withdrawals, including analysis of eia data and a new linked water and heat balance . However, usgs suggests that accurate measurement and reporting of water use is required to develop reliable estimates of water use at local scales . If we want to reduce water use for thermoelectric generation, we need to understand the primary drivers or controls on water consumption and withdrawal . Because of long lifespans of power plants, current power plant choices may impact water resources for decades into the future . Many studies show that different aspects of power plants, such as energy source, generator technology, or cooling system have varying water requirements . For example, switching fuel sources from coal to natural gas saves water and switching from once - through cooling to cooling towers increases water consumption . Therefore, it is important to understand the basic components of thermoelectric power plants and how they relate to water consumption and withdrawal . Major components of thermoelectric power plants include the (a) energy source, (b) generator technology or prime mover to generate electricity, and (c) cooling system (figure 1). Thermoelectric power plants primarily generate electricity by either burning fossil fuels (coal or natural gas) or using nuclear fission to boil pure water, creating steam that drives turbines that generate electricity (figure 2). Thermoelectric plants generated 86% of net electricity in texas and 89% in the u.s . In 2010 primary energy sources in thermoelectric power plants in texas include nuclear, coal, and natural gas sources (www.eia.org) (figure 1).figure 1basic components of power plant systems based on net electricity generation for texas in 2010 (411 million mwh or twh), including thermoelectric energy sources: nuclear (10%) and fossil fuels [coal (37%), natural gas (46%)], and nonthermoelectric energy sources, mostly wind (6%), and other minor fuel sources (total 1%, not shown), including hydroelectric, petroleum, and biomass; generator technologies or prime movers (pm) (steam turbines, st, 53%), combined cycle (cc, 35%), gas, or combustion turbines (ct, 5%), wind turbines (wt, 6%), and other minor generator types (total 1%, not shown), including hydroelectric turbines and internal combustion engines; and cooling systems, once - through (ot, 42%), wet cooling towers (45%), dry cooling towers (1%), and no cooling (12%). Steam turbines use rankine cycle to generate electricity and are not fuel specific: they are used with nuclear, coal, and natural gas energy sources . Combined cycle plants use combustion turbines (ct) and steam turbines (st) and are fuel specific, used with natural gas . Integrated gasification combined cycle (igcc) can be used with coal, but these plants are very rare in the u.s . And globally . Combustion turbines are also referred to as gas turbines and are similar to jet engines . They use natural gas as a fuel source and the brayton cycle to generate electricity . Basic components of power plant systems based on net electricity generation for texas in 2010 (411 million mwh or twh), including thermoelectric energy sources: nuclear (10%) and fossil fuels [coal (37%), natural gas (46%)], and nonthermoelectric energy sources, mostly wind (6%), and other minor fuel sources (total 1%, not shown), including hydroelectric, petroleum, and biomass; generator technologies or prime movers (pm) (steam turbines, st, 53%), combined cycle (cc, 35%), gas, or combustion turbines (ct, 5%), wind turbines (wt, 6%), and other minor generator types (total 1%, not shown), including hydroelectric turbines and internal combustion engines; and cooling systems, once - through (ot, 42%), wet cooling towers (45%), dry cooling towers (1%), and no cooling (12%). Steam turbines use rankine cycle to generate electricity and are not fuel specific: they are used with nuclear, coal, and natural gas energy sources . Combined cycle plants use combustion turbines (ct) and steam turbines (st) and are fuel specific, used with natural gas . Integrated gasification combined cycle (igcc) can be used with coal, but these plants are very rare in the u.s . And globally . Combustion turbines are also referred to as gas turbines and are similar to jet engines . They use natural gas as a fuel source and the brayton cycle to generate electricity . Generator technologies, or prime movers, for thermoelectric power plants for texas include steam turbines (st), combustion turbines (ct), sometimes referred to as gas turbines, and combined cycle (cc). Traditional cts have no cooling water requirements; however, newer models use a relatively minor amount of water to prechill inlet air . A combined cycle plant combines cts with sts in sequence, generally in a ratio of 2 cts to 1 st . Because cooling water is not required for traditional cts, the rule - of - thumb is that cc plants require about a third of the water that st plants require because of the general ratio of 2 cts/1 st . Cooling systems in thermoelectric power plants are required to condense steam . These cooling systems can be based on water, air, or both (hybrid system). Cooling water forms a separate system from the closed - loop steam cycle . Water - based cooling systems are broadly classified as (1) once - through or open - loop systems (e.g., rivers, lakes, reservoirs, ponds) (figure 2a); and (2) recirculating or closed - loop systems (wet cooling towers) (figure 2b). Schematic diagrams of power plant steam turbine generation and cooling systems: (a) steam turbine (coal, nuclear, or natural gas) with once - through recirculating pond cooling system . The internal closed - loop steam cycle is shown, including boiler, turbine, and condenser, and the external cooling cycle is also shown from pond to condenser, returning to pond . The footprint could be defined for the power plant with water circulation / withdrawal for cooling based on the cycle labeled 2; alternatively, a larger footprint could be defined including the pond with water withdrawal from the river to the pond (labeled 1) and representing the amount of water required to replenish the pond water (termed makeup water). (b) wet cooling tower with dominant circulation between the power plant and the tower and low withdrawal from the pond or other water source to the tower . Water can be returned to the pond (discharge) or the system may have zero discharge . For classification of cooling systems by eia, the term once - through technically refers to water moving once through the condenser . Once - through systems in the eastern u.s . Generally consist of power plants that withdraw water directly from a river and then discharge that water back to the river farther downstream at a higher temperature . Dissipation of waste heat from the power plant in water bodies (rivers or ponds) occurs by evaporation, long wave radiation, conduction (as sensible heat), and convection . It is difficult to apply the once - through concept to ponds where water is continuously recirculated between the pond and the power plant (labeled 2a in figure 2a). In many analyses, if the plant boundary is redrawn to include the pond, withdrawals might instead refer to water transfers from another river / lake / reservoir to the cooling pond (typically termed diversion, labeled 1 in figure 2a) to replace evaporative losses in the cooling pond, in which case circulation between the pond and the plant would not be accounted for separately . Classifications of cooling systems according to eia are shown in supporting information, si, figure s1 . In wet cooling towers, water moves through the condenser and then to the tower, where it evaporates (figure 2b). Evaporation is the only significant heat - dissipation process in wet cooling towers; therefore, consumption is up to 50% greater for cooling towers relative to once - through systems that can also dissipate heat using radiation, conduction, and convection . Several recent studies provide reviews or meta - analysis of water use estimates from the literature . However, many estimates in the literature are based on dated information of uncertain quality . Multiple regression was used to identify important determinants of water use for power generation based on 19962000 eia data . However, reliable estimates of water consumption were particularly difficult to obtain from earlier data . Many of the values provide representative water use estimates for common thermal power plants and cooling system types rather than an assessment of original data from eia or other sources . Some recent studies are based on analysis of original data from eia, for example, 2006 eia data for texas and 2008 eia data for the u.s . Many of the studies focus on future projections of water use for various environmental regulations, climate change scenarios, and carbon - based policies . The primary objectives of this study were to quantify water consumption and withdrawal for thermoelectric generation and to better understand controls on water use . Texas provides an excellent case study because of data availability on water use from multiple sources (eia, texas commission on environmental quality, tceq, and texas water development board, twdb). The detailed analysis and cross - checking and verification of water - use data from multiple sources conducted in this study and meetings with power plant operators to assess data quality represent one of the most intensive examinations of raw data to date . Additionally, texas climatic conditions range from humid in the east to arid in the west, providing an analog for u.s . Electricity generation . Heavy reliance on surface water for cooling in texas is also typical of that in the u.s . The range of fuel sources (coal, natural gas, and nuclear) and variety of generator technologies and cooling systems are typical of those in the u.s . The understanding of controls on water use developed in this study should be applicable to all thermoelectric power generation . Recent trends in water use related to expansion of natural gas (ng) plants in texas is much more advanced that what is occurring in the u.s . But may provide some ideas of what is projected to occur throughout the u.s . In the near future, with large - scale expansion of ng production through hydraulic fracturing . Data from 2010 were used because this year represents near normal climatic conditions (precipitation 35.4 in . [899 mm], 26% higher than long - term mean of 28.1 in . This work builds on a previous study quantifying water consumption for thermoelectricity in texas in 2006 by taking advantage of improvements in eia data since 2006 and focusing on controls rather than on future projections . Quantitative information on water use and understanding of controls provided by this study should be very valuable for future management of water and energy to ensure reliable electric generation . Water use for thermoelectric cooling was obtained from the eia, tceq, and twdb databases . Power generation is organized by generator technology and water use is associated with cooling systems in eia form 860, but direct links between generators and cooling systems are not provided . Generators are associated with boilers and boilers are, in turn, associated with cooling systems . Each connection may represent a one - to - one, one - to - many, or many - to - one association . Operational information, including monthly power generation and water use, are reported in eia form 923 . Water use is categorized as rates of diversion (labeled 1 in figure 2a), withdrawal (2a), discharge (2b), and consumption (si, figure s1). The tceq water rights database includes reported monthly total diversion (assumed to represent eia withdrawal), return, and consumption volumes from surface water . The twdb database includes reported annual total power plant water intakes (purchased or self - produced) by source (surface water, groundwater, salt water, or reuse) and reported as water withdrawal and water use . For comparison with eia data, twdb water use was assumed to represent consumption; however, it may reflect diversion . Data from tceq and twdb, unlike that from eia, represent total plant values for all uses and may not be cooling system specific because information on cooling systems is not included in these databases . Water consumption or withdrawal intensities or rates (gal / kwh or l / kwh) were calculated by dividing water consumption or withdrawal by net generation . Power plant capacity factors were calculated by dividing actual power plant output (net generation) by theoretical output if operated at full nameplate capacity . Thermal efficiencies were calculated according to fuel source and generator technology by dividing electricity output (net generation mwh) by heat input of fuel source (gigajoule, gj), using the conversion factor (1 mwh = 3.6 gj). Cooling water was required for 264 generators at 143 operational power plants in texas in 2010, representing 354 twh of cooled net generation, and accounting for 86% of total net generation (411 twh). Data on water consumption was reported by eia for 71 plants (65% of net generation), by tceq for 43 plants (56% of net generation), and by twdb for 62 plants (58% of net generation) (si table s1). Data on water withdrawal was reported by eia for 82 plants (73% of net generation), tceq for 45 plants (55% of net generation), and twdb for 64 plants (61% of net generation). There was no water consumption or withdrawal data for 39 plants (10% of net generation). Eia data on water consumption / withdrawal were used as the default data and represent the only data source for most regions in the u.s . Comparison of water use from eia and tceq, where both are available, indicates that water use values agree to within 20% for 80% of overlapping consumption rates and 57% of overlapping withdrawal rates for once - through systems (figure 3). Many of the consumption and withdrawal rates for power plants with cooling towers reported to tceq, though reasonable, tend to be somewhat greater than those reported to eia, possibly reflecting additional consumption for purposes other than cooling . Some of the outliers are up to an order of magnitude different, suggesting reporting of consumption for withdrawal or vice versa . Although tceq reports diversion, the values are often close to withdrawals reported by eia but sometimes may reflect true diversion . For example, the south texas project nuclear plant reports diversion, which is close to consumption for that plant, whereas the comanche peak nuclear plant reports withdrawal values . Where eia data differ markedly from tceq data and were judged less reliable, that is, highly inconsistent with other plants within the same generator / cooling category, then tceq estimates were used . In a limited number of cases, twdb data were used where data were not available from eia or tceq, or twdb data were most consistent with other power plants in similar categories . More detailed information on data comparisons is provided in si, section 2 . In the absence of any water use data or for systems with questionable data, the category weighted average values of similarly configured plants were assigned (representing 18% of consumption and 24% of withdrawal total volume). Comparison of (a) water consumption and (b) water withdrawal intensities for power plants reported by eia and by tceq . Solid symbols represent eia data that were selected, while open symbols indicate tceq data that were selected . Statewide withdrawal required for cooling was calculated separately for once - through systems by multiplying hours of plant operation required to generate reported electricity output at full nameplate capacity by cooling system pump capacity rating specification and represented 66% of reported actual withdrawal derived from the various databases . This comparison indicates that about one - third of water withdrawn for thermoelectric generation in the state is not actually required for cooling . Plants with high capacity factors (nuclear, 92%; coal, 73%) are considered baseload plants and reported withdrawals are equal to (for nuclear st) or slightly higher than (12% for coal st) cooling water requirements . However, plants with low capacity factors that operate as peaking plants often have low electrical output levels, that is, are idling in anticipation of meeting a near - term peak demand; however, their cooling circulation pumps can only operate at full capacity, resulting in overestimation of water withdrawal requirements for cooling, particularly for ngst (24% capacity factor) and ngcc (40% capacity factor) plants with once - through cooling systems . Estimated withdrawals required for cooling in these plants were only 16% (ngst) to 33% (st part of cc plants) of reported withdrawals . Water consumption totaled 0.43 maf (0.53 km) for thermoelectric generation in texas in 2010, representing 4.2% of the states water consumption (10.1 maf, 12.5 km; 2010) (tables 1 and 2, figure 4). This water consumption is much lower than consumption for irrigation (65%), municipal use (17%), and manufacturing (9.2%). Indirectly, water consumption for thermoelectric cooling represents 5% of typical household water withdrawals (si, section 3, figure s3). Although water consumption for thermoelectric cooling represented only 4.2% of total statewide water consumption, locally it can be important if water availability is low . Water consumption for thermoelectric generation (te) in texas relative to water consumption for other sectors based on 2010 data from the texas water development board (www.twdb.state.tx.us). Water consumption was estimated from water withdrawal reported by twdb by assuming 85% of irrigation water and 85% of manufacturing water was consumed . These percentages are based on comparison of water consumption and withdrawal data for texas for 1995 from the usgs data . The only sector with low water consumption is municipal, with an estimated 30% consumed for residential irrigation and 10% lost to leakage in texas . Values in bold indicate dominant control of generator technology on water consumption, with combined cycle plants having 63% lower consumption intensities than steam turbines and cooling technology on water withdrawal with once - through cooling ponds having about two orders of magnitude higher withdrawal than wet cooling towers . The statewide estimates are represented by three different approaches: (1) fuel source, (2) generator technology, and (3) cooling system . Netg: net generation in twh = million mwh; ng: natural gas; nuc: nuclear; oth: other; gen tech: generator technology or prime mover; cool: cooling system type; st: steam turbine . Total cooled net generation of 354 twh represents 86% of 411 twh total net generation . Consumption of 427 thousand ac - ft (kaf) is equivalent to 0.526 km . Total water withdrawal of 26 184 kaf is equivalent to 32.3 km . Because water consumption is affected by generator technology and cooling systems, totals and rates do not only reflect the reported category but are also impacted by other categories . For example, sts are predominantly once - through systems and ccs are mostly cooling towers, both of which affect water consumption . Note water consumption intensity or rate (gal / kwh) is controlled primarily by generator technology (cc 63% lower than st, shown in bold), and withdrawal intensity or rate (gal / kwh) is controlled primarily by cooling system (towers 2 orders of magnitude lower than ponds, shown in bold). Number of generators (n), fuel sources include nuclear (nuc), coal, natural gas (ng), other (oth). Generator technologies (gen tech) include steam turbines (st) and combined cycle (cc). Cooling systems (cool) include once - through (ot), wet cooling towers (t), and cogeneration (combined heat and power, c). Assessing impacts of generator technologies on water consumption rates with similar cooling systems: once - through ponds, cc 73% lower consumption intensity than st (0.12 vs 0.44 gal / kwh); wet cooling towers, cc 66% lower than st (0.23 vs 0.68 gal / kwh). Assessing impacts of cooling systems on water consumption rates with similar generator technologies: coal st, towers 8% higher than once - through ponds (0.56 vs 0.52 gal / kwh); ngst, towers 55% higher than once - through ponds (0.68 vs 0.44 gal / kwh); ngcc, towers 92% higher than once - through ponds (0.23 vs 0.11 gal / kwh). Note: there are not many examples of ngcc with once - through ponds, accounting for only 2.4% of all net generation and the category represents only 0.8% of all water consumption and 7% of all water withdrawals . Assessing the impacts of generator technology on water withdrawal rates with similar cooling systems: once - through ponds, water withdrawal intensities are 58% lower for ngcc plants relative to ngst plants (59 vs 141 gal / kwh); cooling towers, water withdrawal intensities are 63% lower for ngcc plants relative to ngst plants (0.26 vs 0.71 gal / kwh). All fuel, all generator technologies, and all cooling systems refer to both thermoelectric and nonthermoelectric generation . Total water consumption of 427 thousand acre feet (kaf) = 0.43 million acre feet = 0.53 km = 526 mm water consumption intensity of 0.34 gal / kwh = 1.3 l / kwh statewide . Water withdrawals for thermoelectric generation vary depending on where the system boundary is defined . Eia classifies cooling systems as once - through, recirculating ponds, and cooling towers . Only two power plants with once - through cooling systems withdraw water directly from a river in texas (the guadalupe river, si, section 4, figure s4), similar to typical once - through systems in the eastern u.s . (si figure s5). The remaining once - through power plants in texas withdraw water from ponds / reservoirs, which are also found throughout the eastern u.s . There is no systematic variation in terms of storage volume and recirculation estimates (pond storage / withdrawal rate) for systems classified by eia as once - through systems versus recirculating ponds in texas (si figure s6), suggesting that the classification is somewhat arbitrary and generally lacks physical meaning . Examining google earth maps of most cooling ponds / reservoirs shows structures designed to increase recirculation efficiency (si figure s7); therefore, we classified all these systems as recirculating ponds / reservoirs for simplicity . Withdrawals are generally calculated assuming the system boundary is at the power plant, with withdrawals representing water circulation between the cooling pond and power plant (figure 2a). The withdrawal value from analysis of the eia / twdb / tceq databases for 2010 totaled 26.2 maf (32.3 km). Withdrawals for thermoelectric generation represent 66% of the state total (26.2 maf + 13.4 maf [nonthermoelectric withdrawals] = 39.6 maf, 48.8 km) (table 1). However, only about 2% (0.43/26.2 maf) of this water is lost to evaporation from cooling towers or forced evaporation from ponds / reservoirs due to increased temperatures . The system boundary could also be defined to include the cooling pond (figure 2a), which may be appropriate for recirculating ponds and in this case withdrawal from the river / lake to the recirculating cooling pond, sometimes referred to as diversion (si figure s1), would be much lower, reflecting the net watershed balance for the pond (rainfall + runoff into pond minus natural + forced evaporation). These withdrawals to replenish the pond, termed makeup water, would be closer to water consumption . With the larger footprint (including the pond), water consumption should increase because it would include natural evaporation from the pond . Although initial reservoir impoundment removes a significant amount of water from the river circulation, withdrawals from rivers / reservoirs afterward are only required to replenish the cooling pond . A variety of factors can impact water consumption / withdrawal, including fuel type, generator technology, cooling system, thermal efficiency, capacity factor, and age of plant . Water consumption is controlled primarily by (a) generator technology, which is related to thermal efficiency, and by (b) cooling system design with up to 50% higher consumption in cooling towers relative to ponds (figure 5). (a) generator technologies impact water consumption through their thermal efficiency, which is defined as the fraction of heat from fossil fuels or nuclear fission that is converted to electricity (electricity output / heat or energy input) (si, section 5). Increasing the thermal efficiency of a system means that more of the energy from the fuel source is converted to electricity, reducing residual or waste heat requiring cooling . Combined cycle plants have the highest thermal efficiency (ngcc, 44%) because residual heat from the ct part is used by the st part of cc plants through a heat recovery steam generator (hrsg) system (figure s8, table s2). In contrast, thermal efficiencies of coal and nuclear st plants are lower (both 36%) than that of ngcc plants . Increasing temperatures of cooling ponds would also reduce thermal efficiency; however, this effect is much lower than temperature differentials between different generator technologies (st, ct, cc). Relationship between water consumption and water withdrawal for different power plants according to fuel type generator cc: combined cycle . Note the log scales for consumption and withdrawal intensities or rates . Withdrawal rates are controlled primarily by the cooling systems as shown by the large difference between once - through cooling (mostly recirculating ponds) on the left and cooling towers on the right . Note the trade - off between water withdrawal and consumption rates, with higher withdrawal and lower consumption for once - through systems and lower withdrawal and higher consumption for wet cooling towers . The low withdrawal rates for some ngcc plants with once - through cooling reflect plants that mostly operated the ct parts of cc plants with low capacity factors . Within each cooling system, total water consumption by generator technology is 82% for sts and 18% for ccs in texas (table 1). All traditional st generation requires cooling, whereas, statewide, generation from cc plants is 71% ct, which does not require cooling, and 29% st, which requires cooling . Therefore, cc plants should have about 70% lower cooling water requirements than st plants on average statewide, similar to the 63% found for the 2010 data (cc 0.19 gal / kwh vs st 0.52 gal / kwh) (table 1). Comparing similar cooling technologies (once - through and cooling towers) results in water consumption intensities for cc generation being 6673% lower than for st generation (table 2, figure 5), consistent with that expected from the noted ct to st ratio in cc plants . The impact of cooling systems on water consumption was evaluated by comparing cooling systems for plants with similar generator technology . One would expect up to 50% higher water consumption for wet cooling towers relative to once - through ponds, if everything else is the same . Results show that, for ngst generators alone, water consumption intensity is 55% higher for wet cooling towers relative to once - through ponds (table 2). Similarly, for ngcc generators alone, water consumption intensity for towers is 92% higher than that for ponds . For coal plants, the difference is only 8% higher . The large range in water consumption intensities for different cooling systems may reflect limited data for some categories (table 2), possible operational differences, (for example, relative use of ct [no cooling] versus st in cc plants with different cooling systems), or reporting problems . Although wet cooling towers generally consume more water than once - through ponds for a given generator technology, the average statewide consumptive water intensity for all cooling towers (0.31 gal / kwh) is contrarily 35% lower than that of all cooling ponds (0.48 gal / kwh) because most cooling tower generation is associated with cc generators (table 1), with much lower cooling requirements per kwh than other generator technologies (71% of cc net generation is from cts, requiring no cooling). These statewide comparisons of once - through systems and towers underscore the dominant role of generator technology (st, ct, cc) over cooling system technology in controlling water consumption . Water consumption rates from this analysis according to fuel, generator, and cooling system (table 2) are generally within the range of those reported from the literature (si table s3). The age of a plant is also a factor in thermal efficiency and water use . Cc plants were the only plants to show a distinct increase in thermal efficiency based on analysis of decadal data, with less efficient plants constructed before 1990 (36% efficient) compared to those that have come on - line since (46% efficient). Traditionally water withdrawals are calculated from the water source (pond / reservoir) to the power plant, resulting in statewide water withdrawal intensities for 2010 of 49 gal / kwh for once - through systems, more than 2 orders of magnitude higher than withdrawal intensities for wet cooling towers (0.35 gal / kwh) (table 1). Water withdrawals are also affected to a lesser extent by generator technology or thermal efficiency . For similar cooling systems, water withdrawal intensities are 5863% lower for ngcc plants relative to ngst plants (table 2). Withdrawal intensities from this study are generally within the range of reported withdrawals from the literature (si table s3). Water withdrawal volumes are much higher in the eastern part of the state, where ponds and reservoirs are concentrated (si figure s9). (26 maf) and 1% for wet cooling towers (0.2 maf) for 2010 data (table 1). There are several issues associated with water withdrawals, including (a) water storage requirements to support withdrawals, (b) impacts of withdrawals on other water users withdrawing water from the same pond / reservoir or on downstream users, (c) discharge temperature issues (clean water act 316a), and (d) effects of cooling water intake structures on aquatic species (cwis, clean water act 316b). The issues vary depending on whether it is an industrial pond or multipurpose reservoir that is used for power plants and water supply . Of the 43 ponds / reservoirs supplying water directly to power plants in texas, 25 are industrial ponds and 18 are multipurpose, primarily water supply . The industrial ponds represent only 1 maf (1.2 km) of the associated water storage but account for 80% of the net electricity generation from ponds, whereas the multipurpose ponds / reservoirs represent 3.4 maf (4.2 km) of water storage but only 20% of the net electricity generation . Industrial ponds generally have smaller drainage areas (300 mi, mean 42 mi) than multipurpose reservoirs / ponds (> 150 mi, mean 3700 mi) (si figure s10). Although reservoir storage must be available to support the large water withdrawals at power plants with once - through cooling systems, less water is required to replenish industrial ponds supporting once - through cooling systems than those supporting wet cooling towers, other factors being equal, because of up to 50% to 100% higher water consumption associated with towers relative to once - through cooling systems . Once - through systems supported by multipurpose reservoirs may reduce water availability for municipal water supplies abstracting water from the same reservoir but can actually increase water available for downstream users because of lower consumption relative to wet cooling towers . Many industrial cooling ponds often need only to meet discharge temperature regulations where the pond physically discharges back to the supplying river / reservoir system (figure 2a). However, industrial ponds only discharge such water during very high flow periods; therefore, discharge temperatures may not a concern for many industrial ponds . In fact, industrial ponds may protect larger reservoir / river systems from high temperatures . Cooling water intake structures (cwis) because industrial ponds are man - made, they may or may not be stocked with fish . Impacts on fish can be addressed using best technology available, such as screens etc ., where there are concerns about cwis . Therefore, there are trade - offs between once - through systems, with at least order of magnitude higher water withdrawals and impacts on aquatic species and thermal discharges, relative to wet cooling towers with much lower water withdrawals but up to 50%- to 100%-higher water consumption . Attributes of industrial ponds and multipurpose ponds / reservoirs should be considered when promoting or mandating a particular cooling technology . The long lifespan of power plants (3050 years) results in the current texas power plant fleet representing a legacy of past variations in fuel availability and costs, water availability and water rights, and advances in technologies for example, ngcc systems, which impact trends in water consumption (figure 6). Fuel sources varied over time, from predominantly natural gas in the 1960s and early 1970s to coal and nuclear sources following the 1973 oil embargo and more recently to large increases in natural gas generation (85% increase 19982004) in response to hydraulic fracturing and low natural gas prices . The dominant generator technology was sts until the early 1990s; however, cc plants markedly increased in the late 1990s (400% increase 19982004). Proliferation of ponds, mostly in the 1970s and 1980s (340% increase) reflects availability of large quantities of unappropriated surface water and increases in water rights permitting during this time and lower cost and higher cooling efficiency of ponds relative to wet cooling towers . Cumulative generation capacity in 1000s of mw or gw of the existing texas power plant fleet by (a) fuel type, (b) generator technology, and (c) cooling system type . Hindcasts were developed from the current power plant fleet based on information on when the systems came online (www.eia.org). The original fleet consisted mostly of ngst with increasing coal st and also increasing nuclear st in the late 1980s in response to the 1973 oil embargo . Natural gas increased markedly from 19982004 (85% increase) and consists mostly of combined cycle generators (400% increase 19982004) and cooling towers (118% increase). Wind energy increased significantly in mid-2000s related to the texas renewable portfolio standard and the federal production tax credit for wind energy, renewed in 2013 . If future epa regulations mandate conversion of existing once - through pond cooling systems to tower cooling systems, water consumption would increase by up to 50100% if everything else (e.g., generator technology etc) remained the same . What we have seen in the past decade and a half is that new plants are mostly ngcc with cooling towers . Reduced cooling requirements associated with cc plants should more than compensate for increased consumption intensity associated with cooling towers because the latter only applies to the st part of cc generation and should result in net reduction in water consumption intensity . Future projections of water use are based on electric reliability council of texas (ercot) projections of future net generation for texas . Historically, annual energy for 20032012 grew at an average annual growth rate (aagr) of 1.5% . Future projections of water use presented here are based on extending the current power plant fleet to 2030 with no retirements except for ngst plants, for which the declining generation rate of 2 mwh / year that occurred between 2004 and 2010 is projected forward to result in no ngst generation by 2023 . Finally, applying the forecasted aagrs results in 30% total growth between 2010 and 2030, with all new generation from ngcc plants with wet cooling towers . Based on these assumptions, water consumption in terms of total volume is projected to grow at an average annual rate of 0.2%, resulting in total water consumption by power plants increasing 4.6% by 2030 relative to current levels . However, water consumption intensity is projected to decrease by 19% relative to the overall 2010 rate because of the assumed new power plant type (ngcc with cooling towers). Withdrawal volumes are projected to decline slowly (average 0.8% annually) until 2023, reflecting the ngst retirements, and because the assumed new systems use cooling towers that withdraw at least an order of magnitude less water than ponds, and withdrawal intensity is projected to decrease by 31% overall by 2030 . These projections of water consumption and withdrawal are considered upper bounds in terms of ercot projections because retirements of current plants (except ngst plants) were not considered and new generation was assumed to be ngcc, whereas ercot (28) estimates 36% from ngct (no cooling requirement) and 64% from ngcc in a business as usual (bau) projection and much more power from renewables in other scenarios (bau with updated wind shapes, ngcc 10%, ngct, 19%, solar, 27%, and wind, 45%). However, ercot projections might change markedly if prices for natural gas increase and generation from coal and nuclear sources increase . In addition, water use would increase if carbon capture is required in the future . While the analysis in this study focused on texas, the results have broader implications . The understanding of controls on water consumption and withdrawal can apply to power plants anywhere and should be considered as various regulatory and economic forces shift emphasis to different technologies, allowing impacts on water resources to be considered . The importance of generator technology controlling water consumption with up to 63% reduction in consumption intensity for ngcc plants relative to traditional sts (0.19 vs 0.52 gal / kwh) is very important, with projected future expansion of ngcc plants in texas and throughout the u.s . The use of cooling towers instead of once - through cooling ponds or rivers has been decreasing water withdrawal rates and is expected to continue in the future . This study emphasized the evolution of the power plant fleet in texas and the legacy infrastructure that cannot be modified in a short period . Use of once - through cooling ponds is unlikely to expand in texas in the future as use of ponds essentially leveled off in the early 1990s . Retirement of existing power plants with once - through cooling ponds may be replaced by plants with similar cooling systems because the power plant operators have the water rights and the ponds are being maintained, as seen in the proposed new ferguson plant on lake lbj . Even when power plants are retired and not replaced, the ponds / reservoirs are often maintained for recreational purposes (e.g., lake lady bird johnson in austin). If replacement plants change generator technology from st to cc along with cooling systems (cooling towers replacing once - through ponds), both water consumption and withdrawal rates will decrease . Projected increases in ngcc plants with cooling towers in texas based on ercot forecasts should further reduce water consumption and withdrawal rates in the state . In general, with recent increases in ng generation (120% increase 19962010) and increasing use of cooling towers reducing water withdrawals in the u.s . Understanding fundamental controls on water consumption and withdrawal of thermoelectric generation within the context of power plant fleets with long lifespans and legacy infrastructure is an essential prerequisite to assessing impacts of water availability on thermoelectric generation and impacts of thermoelectric generation on water resources.
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Extralobar pulmonary sequestration is defined as a mass of pulmonary parenchyma anatomically separate from the normal lung . In extralobar pulmonary sequestration, the tissue has a distinct pleural covering and may be supplied by an aberrant artery from the aorta or its branch, in rare instances, extralobar pulmonary sequestration communicates with the esophagus or stomach . The pulmonary sequestration is found lying mostly at the left lung base but can be found in the right lung and in the mediastinum at any level from the neck to below the diaphragm . Extralobar pulmonary sequestration is often discovered at the time of routine roentgenogram of the chest or at the time of repair of associated congenital anomalies such as diaphragmatic hernia and fistulous communication with the gastrointestinal tract because extralobar sequestration rarely presents like the intralobar form with recurrent pulmonary infection . A 27-year - old man was admitted to yong dong severance hospital, yonsei university medical center, for evaluation and management of mediastinal mass . Three years prior to admission, he found mediastinal mass on computerized tomographic (ct) scan performed in another institution . At admission, he was noted to be with generalized weakness and right chest pain, but he denied cough, sputum and dyspnea . The temperature was 38.6c, the pulse rate 72/min and the blood pressure 110/70 mmhg . On examination, he appeared chronically ill, the skin was warm and dry, the head and neck were normal and cervical lymphadenopathy was not found, the lungs were clear and the heart was normal . The urine was normal and the hemoglobin was 13.5gm / dl; hematocrit 41.9%; white cell count 5,500/mm with 64 percent neutrophils, 35 percent lymphocytes and 1 percent eosinophils, the serum electrolytes and liver function tests were normal . T3: 150 ng / dl, t4: 7.1 ug / dl, ft4: 1.6ng / dl and tsh was 1.5 uu / ml . The electrocardiogram showed right axis deviation and tall r wave in right precordial leads, and the sputum exanination did not suggest malignancy or tuberculosis . Chest ct scan revealed large multi - cystic mass lesion on the right paratracheal area which was attached to the lower pole of right thyroid gland, displaced the superior vena cava anteriorly and extended to the level of the azygous arch vein (fig . The lung perfusion scan and whole body bone scan were normal and the thyroid scan revealed cold area in the lower pole of right thyroid gland . The pulmonary function studies showed mild restriction with fev1:2.85 l / sec and fvc: 3.49 l / sec . 88 cm sized mass composed of multi - cystic and solid part was located in the pericardium and excised easily from the surrounding structures and the feeding vessel maybe originated from the subclavian artery . Grossly, the mass was composed of several cystic lesions and yellow solid lesion, resembling unexpanded lung tissues (fig . Mlicroscopically, the cyst wall showed a bronchial tissue which was composed of ciliated columanar epithelium, submucosal gland and cartilage (fig . 5) and solid portion of the mass showed alveolar space filled with inflammatory cells such as macrophages or lymphocytes (fig . Pulmonary sequestration is an uncommon congenital malformation characterized by the presence of nonfunctioning lung tissue which usually has no communication with the normal bronchial tree and which receives its blood supply from an anomalous systemic artery instead of a pulmonary arterial branch . In intralobar sequestration, the abnormal pulmonary tissue is incorporated within the normal lung and shares a common covering of visceral pleura, and in extralobar sequestration, the abnormal pulmonary tissue is separate from the normal lung and has its own pleural covering . Is generally accepted and their spectrum of bronchopulmonary foregut malformation includes intralobar and extralobar sequestration, esophageal diverticuli, foregut duplication cysts (esophageal cysts) and maybe expanded to include bronchogenic cysts . The incidence rate was 1.1 to 1.8 percent of all lobectomied patients by carter and 0.15 to 6.4 percent of all congenital pulmonary malformation by savic . Savic found 133 cases of extralobar sequestration in a review of 547 cases of pulmonary sequestration . The male predominance of patients with extralobar pulmonary sequestration (3:1) was described by carter and savic described that extralobar sequstration was located between the diaphragm and lower lobe in 77.4% and on left lung in 78.9% of the cases . Most of the published cases of extralobar pulmonary sequestration were located outside the pericardium, only four reported cases were intrapericardial . Since an extralobar sequestered segment is enveloped in its own pleural sac, the chance of becoming infected are very small, unless there is communication with the gastrointestinal tract . The extralobar sequestration (42%) is more often associated with other anomalies than intralobar type (14%) such as diaphragmatic hernia and fistulous communication with the gastrointestinal tract . Consequently, extralobar sequestration is usually an incidental finding on routine roentgenograms of the chest or during management of some other congenital anomaly and infrequently, extralobar sequestration presents with symptoms similar to those seen with the intralobar form and with a picture of congestive heart failure or pulmonary overcirculation . In all cases of intrapericardial extralobar pulmonary sequestration, a large quantity of pericardial fluid was found, but no sign of congestive heart failure or cardiac tamponade was noticed . A correct preoperative diagnosis is made in 39 percent of intralobar lung sequestration and only in 9 percent of extralobar lung sequstration by savic and correct preoperative diagnosis is more difficult in the cases of unusual location . Preoperative angiography is important to confirm the diagnosis and to afford a safe operative approach . The sequestratered segment is typically made up of multiple cysts or noncystic mass containing branching bronchi which run in the direction of the aberrant artery . Histologically, the sequestration consists of normal lung elements in an abnormal and disorderly arrangement with a variable presence of cartilage, bronchial glands and alveolar parenchyma . Extralobar pulmonary sequestration with symptoms or discovered at operation for another problem should be excised and it is feasible to observe asymptomatic and definite extralobar pulmonary sequestration without operation.
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The collection of medical image data for research can be an expensive and time - consuming task . Positron emission tomography (pet), x - ray computed tomography (ct), and magnetic resonance imaging (mri) systems can easily cost over a million dollars . They may require dedicated staff, maintenance contracts, and access to expensive supporting equipment . The process is complicated by equipment schedules, organization of volunteers / subjects, and involvement of potentially harmful electromagnetic radiation, radiopharmaceuticals, and contrast agents, as well as patient privacy rights . These difficulties limit the availability of clinical data, especially for smaller academic research programs . Creating software models of the human anatomy and imaging systems and modeling the medical physics of the imaging acquisition process can provide a means to generate realistic synthetic data sets . In many cases, synthetic data sets can be used, reducing the time and cost of collecting real images and making data sets available to institutions without clinical imaging systems . Synthetic data sets can be used for training purposes and as evaluation data for image processing and analysis algorithms . One additional advantage of synthetic data sets, that can make them an invaluable tool, is that they have a known ground truth . Ground truth is, in many cases, nearly impossible to obtain for real images of living humans . In addition, system models can be used to improve system design and study imaging parameter selection and acquisition protocols . While medical image simulation software has been under development since the 1980s, until recently the complexity of the procedures and long computation times have limited the realism and accuracy of artificially generated images . Advancements in processor architecture, increases in speed and amount of memory, and development of large storage systems have enabled computers to be used for increasingly complex problems . The use of distributed systems and technologies provides unparalleled computational capabilities . With these technological improvements and an increased understanding of human anatomy and medical physics, three - dimensional high - resolution realistic synthetic medical data sets in addition, the large quantity of images often necessary for studies, hundreds or thousands of images, can be quickly generated and made available . In some cases the remainder of this paper is dedicated to the generation of mr image sets . Mr images are generally the most computationally complex images to generate, with simulations usually being performed in 2d or low - resolution 3d . What follows is, at least to the authors' knowledge, an overview of the first system capable of successfully generating high - resolution 3d mri images . Simulators can be classified according to the procedure used to estimate the radio frequency response of the phantom (digital anatomy being imaged). In general, existing simulators fall into one of three categories: signal equation - based simulators [1, 2], k - space - based simulators, and isochromat summation - based simulators [410]. Synthetic images are created using known tissue properties (i.e., spin density, spin lattice relaxation time, and spin - spin relaxation time) and the signal intensity equation for the pulse sequence being simulated . By substituting the known tissue properties into the signal equation for example, (1) is the signal equation for the standard spin - echo pulse sequence where is the spin density, t1 is the spin lattice relaxation time, t2 is the spin - spin relaxation time, and te and tr are the pulse sequence parameters representing the echo time and repetition time, respectively (1)s=(1etr / t1)ete / t2 . Since the simulation is a simple mathematical calculation, simulated images can be created nearly instantaneously . Simulation is however limited to pulse sequences with a known signal equation, and generally more complex phenomena and artifacts cannot easily be simulated using this method . A second, rarely used, approach to generate synthetic data sets leverages the k - space representation of the data . This simulation begins by approximating k - space by taking the fourier transform of the phantom being imaged . The signal is then adjusted for each pulse event, for relaxation, and for the artifacts being simulated . While this technique provides a relatively computationally efficient way to obtain the mri signal, it is difficult to simulate certain effects, such as field inhomogeneities . In addition, an understanding of how each event and artifact manifests itself in k - space is necessary requiring expert knowledge and making simulation of more complex artifacts difficult . The third simulator type, and by far the most robust, arrives at the mri signal by summing a large number of isochromats . The phantom is treated as a set of spin packets, and the evolution of the magnetization of each packet is governed by the bloch equations . The rotation of the magnetization due to radio - frequency (rf) pulses, gradients, and relaxation is modeled . The final k - space signal is generated by summing the signal provided by each isochromat (isochromat summation), and the image is obtained by fourier transforming the k - space data . These simulators are robust and in theory can simulate any pulse sequence and most phenomena . Limitations exist only due to the assumptions made by the bloch equations and by computation time . This work attempts to ameliorate the situation by lessening the limitations imposed by computational complexity . Each isochromat is assigned a location within the phantom and is represented by a three - dimensional magnetization vector ([mx(t) my(t) mz(t)]), which specifies the isochromat's orientation and magnitude at time t. the location of the isochromat within the magnet bore is used to identify the magnetic field strength at the isochromat (due to the main static magnetic field, gradients, and any inhomogeneities such as from susceptibility). The local magnetic field strength (b) and the gyromagnetic ratio () of the isochromat are used to find the frequency at which the isochromat precesses within the main magnetic field (larmor frequency,) as follows: (2)=b . After a period of time (t) has passed, the larmor frequency can be used to find the new orientation of the isochromat by modeling the precession as a rotation about the main field (usually oriented along the z - axis). This rotation is modeled as follows: (3)[mx(t+t)my(t+t)mz(t+t)]=[cos (t)sin (t)0sin (t)cos (t)0001][mx(t)my(t)mz(t)]. In addition to precession within the main magnetic field, the magnitude of the isochromat changes due to relaxation effects . The transverse spin - spin relaxation is a result of molecular interactions causing a dephasing of the spins and a loss of signal coherence . The rate of relaxation is governed by the tissue - specific spin - spin relaxation time constant (t2) and affects the magnitude of the magnetization vector orthogonal to the main magnetic field . The effect of this dephasing on the magnetization vector can be modeled with (4)[mx(t+t)my(t+t)mz(t+t)]=[et / t2000et / t20001][mx(t)my(t)mz(t)]. Longitudinal spin lattice relaxation occurs as the isochromats return to equilibrium (realign with the static magnetic field). Longitudinal relaxation is governed by the spin lattice relaxation time constant (t1) and affects the magnitude of the magnetization vector in the direction of the main magnetic field, as calculated from (5)mz(t+t)=mz(t)et / t1 + m0(1et / t1). Here, the magnitude of the isochromat's magnetization at equilibrium within the main magnetic field is given by m0 . This magnitude is proportional to the isochromat's spin density . Applying both relaxation effects simultaneously yields (6)[mx(t+t)my(t+t)mz(t+t)]=[et / t2000et / t2000et / t1][mx(t)my(t)mz(t)] + [00m0(1et / t1)]. Including the precession within the main static field yields (7) used to iteratively update the isochromat magnetization vector (7)[mx(t+t)my(t+t)mz(t+t)]=[et / t2000et / t2000et / t1] [cos (t)sin (t)0sin (t)cos (t)0001][mx(t)my(t)mz(t)] + [00m0(1et / t1)]. Rf pulses are used to excite the isochromats causing a deviation of their magnetic moments from equilibrium . An alternating current is run through a coil placed around the x - axis . Turning on and off the current at the larmor frequency results in a pulsed magnetic field (b1) orthogonal to the main static magnetic field (b0). If we consider a frame of reference which rotates about the z - axis at the lamor frequency, b1 will be a constant field along the x - axis . In this rotating frame of reference, the energy from the pulsed field will be absorbed by the isochromats causing them to rotate about the direction of the b1 field . Frame of reference, the isochromats will follow a helical path making increasingly larger circles down towards the xy - plane . The frequency of the pulse is selected, such that it targets specific isochromats . The local magnetic field strength (bt) and the gyromagnetic ratio (t) of the targeted isochromat are used to determine the frequency of the pulse (p) according to (8)p=tbt . The amount of rotation is dependent on the pulse duration and the strength of the b1 field . Isochromats precessing at p will experience a rotation of t degrees when a pulse with a magnitude of b1 is applied for a duration of, as given by (9). (9)t=tb1. In reality, the rectangular pulse will contain other frequencies as well . Isochromats precessing at other frequencies may still absorb some of the energy causing them to experience smaller perturbations . The observed rotation will depend on the deviation of the isochromat's magnetic field strength (bi) from bt and occur around the direction of the effective magnetic field b1. B 1 deviates from b1 by an angle i, found using (10)i = tan1 (btbib1). The angle of rotation will depend on the pulse duration and strength of the b1 magnetic field, as given by (11)i=i|b1|=i(btbi)2+(b1)2 . First, a rotation of i around the y - axis aligns b1 with the x - axis . The excitation by i degrees can then be implemented by a rotation about the x - axis . Finally, a rotation around the y - axis of i will recover the proper orientation . I0cos i][1000cos isin i0sin icos i] [cos i0sin i010sin i0cos i][mx(t)my(t)mz(t)]. More complex pulses, such as sin (x)/x, can be represented as a series of short rectangular pulses . Generally, a restriction is placed that is much smaller than t1 and t2, so that no relaxation or dephasing occurs as the pulse is being applied . In this case, the final result of applying the pulse in the rotating frame of reference is equivalent to applying the pulse in the laboratory frame . As previously mentioned, each isochromat is under the influence of the local magnetic field (b) present at its spatial location within the scanner . This field determines the precessing frequency of the isochromat as well as its behavior during the application of an rf pulse . The local magnetic field strength is the sum of the main static magnetic field (b0), along with any variation due to tissue susceptibility, any inhomogeneities in the main field that have not been corrected by proper shimming, and any gradients that are currently being applied . Gradients are applied linearly across the x, y, and z - axes of the system . The change in local magnetic field strength due to the gradients (bg) can be calculated with (13), where (x, y, z) is the spatial location of the isochromat, and gx, gy, and gz are the strengths of the applied gradients along the x, y, and z - axes of the system, respectively (13)bg = xgx+ ygy+zgz . The gradients applied during image acquisition are defined by the pulse sequence and serve purposes such as phase and frequency encoding and slice selection . They can be applied anytime during acquisition including during precession, during the application of a pulse, or during signal acquisition . This signal is typically represented in complex notation where the real component is the signal observed by the first coil, and the imaginary component is the signal observed by the second coil . The observed signal is calculated by summing the magnetization of all the isochromats along the direction of observation . For example, if the coils are placed along the x- and y - axes, the real component can be the sum of the magnetization along the x - axis, and the imaginary component can be the sum of the magnetization along the y - axis . The observed signal consists of multiple samples collected over a short period of time which will define one line of k - space . A typical acquisition process will consist of repeatedly applying a pulse sequence (with varying parameters such as the phase - encoding gradient strength) and collecting signal samples to fill a line of k - space . This will be repeated until samples have been collected for all of k - space . An image reconstruction algorithm is then applied to transform the k - space data into an image . One area in which isochromat simulators vary is in the way they accurately simulate the spin - echo phenomenon . Accurate echo simulation requires the use of a large number of isochromats per voxel of the reconstructed image, greatly increasing execution time . Alternative methods have been proposed including using a time variable to track when rephasing should occur, using gradient magnitudes to predict the amount of dephasing that occurs in the region represented by an isochromat, and calculating intravoxel magnetization gradients . Simri is a bloch equation - based magnetic resonance image simulator, developed in creatis, lyons, france . It includes a few pulse sequences, and the framework allows for the implementation of ad hoc ones . Given a digital phantom of an object, the magnetic resonance signal can be generated if the fractional tissue components of each voxel are known . It supports static field inhomogeneities due to improper shimming and tissue susceptibility, features efficient modeling of intravoxel inhomogeneities, and properly models the main artifacts such as susceptibility, wrap around, chemical shift, and partial volume effects . The design of simri is shown in figure 2 . Simri is designed to take advantage of small clusters supporting the message passing interface (mpi) communications protocol, but modifications are necessary to take advantage of larger parallel computing solutions . Research at mcgill university has resulted in a set of high - resolution voxel - based brain phantoms [1517]. These phantoms were created by semiautomatically segmenting real brain data sets . In order to accurately identify all of the tissues of interest, the segmentation was based on t1, t2, and proton density weighted magnetic resonance images, magnetic resonance angiography (mra), and computed tomography (ct) images . The resulting high - resolution phantom consists of 11 tissue types: grey matter, white matter, cerebrospinal fluid, skull, marrow within the bone, dura, fat, tissue around the fat, muscles, skin / muscles, and vessels . The phantom is presented as a set of fuzzy volumes, one volume for each tissue type, where the voxel intensities represent the amount of that tissue in the given voxel . Blue gene is a massively parallel computer developed by ibm in collaboration with several partners including the lawrence livermore national laboratory . For this work, two blue gene / l systems available in new york were used . New york blue is the system hosted at brookhaven national laboratory consisting of 18,432 dual 700 mhz powerpc 440 nodes with 1024 mb of memory . Rensselaer's computational center for nanotechnology hosts another system consisting of 16,384 dual 700 mhz powerpc 440 nodes divided equally between 512 mb and 1024 mb configurations . Blue gene / l utilizes system - on - a - chip technology, integrating all functionality of a node, except for main memory, into a single integrated circuit . The use of lower clock speed processors in addition to this high level of integration allows a highly scalable architecture at low cost and low power consumption . Compute nodes consist of two double floating point core processors, supporting an enhanced instruction set including single instruction and multiple data (simd) operations . Two operation modes are provided: communication coprocessor mode in which one cpu is used for computation and the other for communication and virtual node mode in which both cpu are leveraged for computation . In virtual node mode nodes are connected into a 3d torus, with the smallest unit being 4 4 2 . Dedicated i / o nodes with gigabit ethernet provide an interface between the compute nodes and external systems including the file systems . I / o node to compute node ratios of 1: 8 to 1: 256 is supported . Applications built on the blue gene / l system utilize message passing interface (mpi) for communication between nodes . The most notable difference is that blue gene / l nodes do not support virtual paging . This means that unlike the 2 or 3 gb of virtual memory typically available to any processes running on a 32-bit system, regardless of physical memory amounts, a blue gene / l node is limited by the actual physical memory . The application, kernel, and mpi communication buffers must total less than the 256 mb, 512 mb, or 1024 mb of physical memory available to the node . This requires applications to have a very low memory footprint per node, carefully manage communication buffers, and be clean of any memory leaks that can quickly consume the available memory . Several modifications needed to be made in order for simri to run successfully on the blue gene / l systems . The modified version of simri can be requested by contacting the corresponding author . The most significant modifications included redistribution of the workload and optimization of the memory . Since each isochromat progresses independently, parallelization is straightforward . The progression of the isochromats can then be independently modeled and the resulting acquired signals summed together at the end . In simri, this was done by assigning one slice (i.e., plane) of the phantom object to each node . The number of divisions (and participating compute nodes) is limited by the dimension of the image . This division of labor is appropriate for small clusters, but not for massively parallel systems like the blue gene / l . In order to leverage the additional compute nodes, modifications were made, so assignment of tasks no longer needs to occur around slice boundaries and that arbitrary groups of isochromats can be assigned to each node . (1) when launching the application, a number of manager nodes are specified . Each worker is assigned to one of the manager nodes in a manner, such that each manager has the same number of workers . (3) the isochromats to be modeled are divided up evenly between the manager nodes . (4) each manager node further divides up the isochromats amongst its workers . (5) the worker nodes perform the simulation and return back to their manager the mr signal from the isochromats they were responsible for modeling . (6) the manager nodes combine the signals from each of its workers . (7) the accumulated signal from each manager is then combined to create the final mr signal . (8) this mr signal can then be reconstructed to produce the simulated image . This tree - like distribution network allows efficient use of the large number of nodes in the blue gene / l systems . Initial test runs required approximately 2.7 gb of memory per node for a 256 voxel simulation . This is well in excess of the 1 gb maximum of the blue gene / l nodes . The memory usage pattern was modified to retain memory only as long as necessary and to reuse previously allocated blocks . This is important as the buffers internally allocated by mpi can consume considerable memory if not careful . Finally, the code executed on each node was made more specialized for the task the node would be performing . The increased specialization of each node results in memory being allocated conditionally based on the node's role . As a result of these modifications, the memory use is sufficiently below the 1 gb that is shared between the application and the kernel on the bluegene / l systems . This 256 voxel spin - echo simulation took approximately 4.3 hours executing on 8192 nodes of the bluegene / l system benchmark runs were performed using the montreal brain phantom and a spin - echo protocol . A 128 voxel simulation was performed using 512, 1024, 2048, 4096, and 8192 nodes . Table 1 shows the run times for each simulation and the scalability (run time versus number of nodes) is plotted in figure 4 . As expected, the run time is found to be inversely proportional to the number of nodes used . The efficiency (speedup divided by number of nodes), plotted in figure 6, is approximately unity tapering off only slightly as the number of nodes approaches the number of isochromats and the communication / computation ratio increases . A set of simulations were run on a single workstation in order to emphasize the benefit of a distributed approach . A workstation equipped with an intel xeon x5260 (3.33 ghz) processor was used to run two - dimensional and three - dimensional spin - echo simulations of a phantom consisting of a single spherical object . Using a single workstation in it is possible to perform two - dimensional and small three - dimensional simulations . Higher - resolution simulations may however require a prohibitive amount of time, and it is may not be feasible to create a large set of simulated data . Modern distributed systems provide unparalleled computational capabilities allowing full resolution data sets to be generated in reasonable amounts of time . The growing availability of these systems, and the public funding for them, has resulted in them being readily available to a large number of academic researchers . Simri has proved to be a robust modeling package that can easily be modified to target a variety of distributed computing environments . As shown here, it can be used to model the image acquisition process allowing the generation of realistic high - resolution images . In addition, large quantities of simulated images can be generated enabling their use in research studies (for example, see). Collections of realistic simulated data sets can be used for validation of image processing algorithms, perception studies, and training . Historically computational complexity has limited the accuracy of medical image simulations . As technology continues to evolve, the realism of the simulated data will be limited by the accuracy of the image acquisition process model and the accuracy of the anatomical model being imaged.
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Restrictive cardiomyopathy (rcm) is very rare in children and it is characterized by dilated atria, elevated end - ventricular diastolic pressure, and severe diastolic dysfunction resulting from increased stiffness of the myocardium, . Rcm carries a poor prognosis with a low survival rate and ultimately requires heart transplantation. Pediatric rcm is most commonly idiopathic and its molecular basis is still unclear . Recently, mutations in the sarcomeric protein genes (cardiac troponin i, tnni3; cardiac troponin t, tnnt2; -cardiac actin, actc; -myosin heavy chain, myh7) have been identified in pediatric rcm, which suggests that sarcomeric protein mutations may be important causes of rcm. Here, we performed genetic investigations of candidate genes that have been reported in rcm and identified a missense mutation in the tnni3 gene in a 12-year - old girl with rcm . The patient and her family were recruited at nanjing children's hospital into an ongoing research protocol approved by the institution's ethics committee . All participants gave informed consents and were evaluated by family history, physical examination, electrocardiogram and echocardiogram . Genomic dna was extracted from peripheral blood samples using a puregene dna purification kit (gentra systems, minneapolis, mn, usa) according to the manufacturer's instructions . Genetic analysis was performed for 4 candidate genes (tnni3, tnnt2, actc, myh7) known to associate with rcm by bidirectional sequencing of all the coding exons . The patient and her family were recruited at nanjing children's hospital into an ongoing research protocol approved by the institution's ethics committee . All participants gave informed consents and were evaluated by family history, physical examination, electrocardiogram and echocardiogram . Genomic dna was extracted from peripheral blood samples using a puregene dna purification kit (gentra systems, minneapolis, mn, usa) according to the manufacturer's instructions . Genetic analysis was performed for 4 candidate genes (tnni3, tnnt2, actc, myh7) known to associate with rcm by bidirectional sequencing of all the coding exons . The patient was a 12-year - old chinese girl and experienced her first episode of dyspnea on exertion . An echocardiogram disclosed that the girl had a structurally normal heart with normal biventricular dimensions, normal biventricular wall thickness and normal systolic functions, but with massive biatrial enlargement (fig 1), markedly left ventricular (lv) diastolic dysfunction, which is consistent with restrictive lv filling pattern: a decreased e - wave deceleration time (102 ms, normal> 150 ms), an abnormal e / a wave ratio of 2.29 (normal <2), and a decreased isovolumic relaxation time (30 ms, normal> 70 ms). Electrocardiogram (ecg) showed sinus rhythm, and biatrial enlargement, and diffuse t - wave changes (fig 2). The case had a mild short pr interval, but showed no evidence of neuromuscular diseases and metabolic diseases . Magnetic resonance imaging (mri) revealed no delayed gadolinium enhancement and demonstrated enlargement in both atria . Etiologic investigations revealed normal karyotype, normal plasma amino acids and urine organic acids, and normal plasma autoantibody . The results of the major laboratory tests were within normal range except for a marked elevation in plasma pro - bnp level (1023 ng / l) (the upper limit of normal range, 115 ng / l). Endomyocardial biopsy was not performed in this case due to refusal by the parents . A 2-dimensional echocardiographic image (apical 4-chamber view) showing a markedly enlarged la and ra with normal biventricular size . Ecg showing sinus rhythm, incomplete right bundle branch block, st segment depression in the inferior leads, and marked biatrial enlargement . Genetic analysis was performed for 4 candidate genes (tnni3, tnnt2, actc, and myh7) known to associate with restrictive cardiomyopathy by bidirectional sequencing of all the coding exons . A heterozygous 575 g> a mutation in exon 8 of tnni3 was identified in the patient . The mutation resulted in the substitution of histidine for arginine at amino acid 192 (r192h). Amino acid 192 (arginine) is highly conserved across species and this missense mutation has previously been described to be associated with both restrictive and hypertrophic cardiomyopathy, (fig . 3). This mutation was absent in her parents and 100 healthy subjects screened at our institution . The patient was a 12-year - old chinese girl and experienced her first episode of dyspnea on exertion . An echocardiogram disclosed that the girl had a structurally normal heart with normal biventricular dimensions, normal biventricular wall thickness and normal systolic functions, but with massive biatrial enlargement (fig 1), markedly left ventricular (lv) diastolic dysfunction, which is consistent with restrictive lv filling pattern: a decreased e - wave deceleration time (102 ms, normal> 150 ms), an abnormal e / a wave ratio of 2.29 (normal <2), and a decreased isovolumic relaxation time (30 ms, normal> 70 ms). Electrocardiogram (ecg) showed sinus rhythm, and biatrial enlargement, and diffuse t - wave changes (fig 2). The case had a mild short pr interval, but showed no evidence of neuromuscular diseases and metabolic diseases . Magnetic resonance imaging (mri) revealed no delayed gadolinium enhancement and demonstrated enlargement in both atria . Etiologic investigations revealed normal karyotype, normal plasma amino acids and urine organic acids, and normal plasma autoantibody . The results of the major laboratory tests were within normal range except for a marked elevation in plasma pro - bnp level (1023 ng / l) (the upper limit of normal range, 115 ng / l). Endomyocardial biopsy was not performed in this case due to refusal by the parents . A 2-dimensional echocardiographic image (apical 4-chamber view) showing a markedly enlarged la and ra with normal biventricular size . Ecg showing sinus rhythm, incomplete right bundle branch block, st segment depression in the inferior leads, and marked biatrial enlargement . Genetic analysis was performed for 4 candidate genes (tnni3, tnnt2, actc, and myh7) known to associate with restrictive cardiomyopathy by bidirectional sequencing of all the coding exons . A heterozygous 575 g> a mutation in exon 8 of tnni3 was identified in the patient . The mutation resulted in the substitution of histidine for arginine at amino acid 192 (r192h). Amino acid 192 (arginine) is highly conserved across species and this missense mutation has previously been described to be associated with both restrictive and hypertrophic cardiomyopathy, (fig . 3). This mutation was absent in her parents and 100 healthy subjects screened at our institution . Over the past decade, molecular genetic analyses have revealed dozens of mutations in sarcomeric protein genes, which encode the contractile unit of cardiac muscles, in both dcm and hcm . These findings have shown that mutations in specific functional regions of sarcomeric protein genes result in different phenotypes . An interesting feature of cardiomyopathies is that rcm may overlap with hcm, especially in many familial cases . Up to now, more than 900 mutations have been confirmed to be associated with hcm . Since the first sarcomere gene mutation was identified in the tnni3 gene in rcm in 2003, several heterozygous mutations in tnni3, tnnt2, actc, and myh7 have been reported to be associated with rcm, indicating that rcm may also be caused by single heterozygous mutations in the genes encoding sarcomeric proteins . In this report, we identified a known heterozygous missense mutation in exon 8 of tnni3 (r192h) in a 12-year - old chinese girl . According to our knowledge, the r192h ctni mutation was firstly reported by mogensen in 2003 in a 19-year - old rcm patient, who died of heart failure . This mutation was also found in a rcm family with hypertrophic physiology, in which all the three persons affected had a early onset of rcm from the first to third decades . All those reports suggest that this mutation may have a good genotype - phenotype correlation with early age of disease onset . Rcm and hcm may occur in one family with the same sarcomere gene mutation, which shows phenotypic heterogeneity in cardiomyopathies, . The r192h mutation is located in the highly conserved c - terminal region in tnni3 and in vitro studies showed that this mutation disrupts interactions within the troponin complex . Found that the r192h mutation increased the ca sensitivity of force development in skinned fibers . Furthermore, du et al . Showed that the transgenic r193h mice, similar to human r192h, demonstrated rcm characteristics . Cardiomyocyte contraction is regulated primarily by the interactions between the intracellular calcium concentration and its major senor - the troponin complex . Tnni3 is the inhibitory component of the troponin complex and it can bind to actin - tropomyosin and prevent muscle contraction by inhibition of actin - tropomyosin - activated myosin atpase activity, . The occurrence of tnni3 mutations in rcm was first reported by mogensen, who found 6 novel mutations located in the conserved and functional important regions of the gene . To date, at least eight tnni3 mutations including seven substitutions and one deletion were identified in rcm that occupied several critical functional domains: the inhibitory region, the switch region and the c - terminus,, . Studies found that the c - terminus of ctni plays an important role in maintaining the diastolic parameters of the heart . Three of the eight mutations are located in the conserved c - terminal region of tnni3 protein . Tnni3 c - terminus is required for normal inhibitory function and studies using transgenic mice demonstrated that mutations existing in this region had serious consequences for cardiac function . In vitro studies also have revealed rcm - linked tnni3 c - terminus mutations sensitized the myofilaments to ca, slowed relaxation and ca transient decay rate . However, most identified hcm linked tnni3 mutations clustered within the same functional domains shared by rcm, and the molecular mechanism whereby mutations in the same functional region of tnni3 result in diverse phenotypic expression needs further studies . In summary, we report a tnni3 missense mutation (r192h) in idiopathic rcm in a 12-year - old chinese girl . This case further improves our knowledge of the causes of cardiomyopathic disease and shows that the spectrum of sarcomeric gene mutations may be involved in pediatric rcm.
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Depressive disorder (dd, including recurrent depressive disorder [rdd]) is a severe psychiatric illness, which at its worst can lead to suicide . It is characterized by persistent low mood, loss of interest or pleasure, feeling of tiredness, loss of appetite, and disturbed sleeping . According to the world health organization, 350 million dd is a growing problem in western countries, where it may affect larger percentages of people than in the worldwide population up to 10% of the people in some western countries . Some estimates show that by 2020 the disease will be the second - most serious illness in terms of social and economic burden, exceeded only by ischemic heart disease . Moreover, there are some reports suggesting that depression may coexist with other diseases, like cardiovascular disease . To make matters worse, approximately one - third of dd patients do not respond to traditional pharmacological medications such as monoamine reuptake inhibitors . Despite the common occurrence and extensive research of depression, its pathophysiology is still not fully understood . People with dd have changes in the expression of neurotransmitters, alterations to the hypothalamus - adrenal - pituitary (hpa) axis, genes, and structural changes within the brain . Increasing patients with dd had elevated levels of interleukin-1 (il-1), interleukin-6 (il-6), interleukin-8, and tnf, which are inflammatory cytokines activate in pro - inflammatory pathways [810]. Moreover, it was shown that anti - inflammatory drugs act as antidepressants, and medication used in dd therapy has anti - inflammatory effects [1114]. This protein complex contains an intracellular sensor, most often one of the nod - like receptors (nlrs), which is a precursor of procaspase-1 and apoptosis - associated speck - like protein containing a card (asc). Increased gene expression of nlrp3 was found in peripheral blood mononuclear cells (pbmcs) of dd patients, and elevated levels of il-1 and il-8 in the patients serum . Nlrp3 is one of the most versatile nlrs, activated by a large spectrum of stressors . It was shown that alternations of mitochondria and reactive oxygen species (ros) generated in mitochondria are the main activators of nlrp3 . In agreement with this, activation of nlrp3 in pbmcs of the patients was accompanied by increased lipid peroxidation, which can be attributed to increased oxidative stress and elevated mitochondrial ros (mtros) production . The nlrp3 inflammasome was also found to be involved in dna damage response (ddr). After oxidative and genotoxic stress, the wild - type murine dendritic cells (dcs) had lower expression of genes involved in double - strand and base - excision repair (ber), and increased apoptosis rate, when compared to nlrp3 knock - out dcs . Currently, the extent of dna damage in depression has only been estimated only by measurement of the level of 8-oxoguanine (8-oxog) a marker of oxidative dna damage and the results are contradictory . Increased concentration of serum and urinary 8-oxog was found in patients with clinical depression, depressed patients with gastric adenocarcinoma, chronic heart failure, myalgic encephalomyelitis, and chronic fatigue syndrome [1822]. Moreover, higher levels of 8-oxog was detected in peripheral blood lymphocytes in depressed japanese office workers when compared to controls . However, there was no association between occurrence of depressive symptoms in japanese office workers and urinary levels of 8-oxog . Since the findings described above are inconsistent, we wanted to determine if the oxidative modification of purines, like 8-oxog, and pyrimidines are present in a higher degree in patients with depression than in controls . Moreover, we also wanted to know if the patients have elevated levels of other kinds of dna damage, such as strand breaks . Finally, we wanted to determine if the increased dna damage is caused, at least partly, by impairments in dna damage repair . Elucidation of those molecular factors and mechanisms may be helpful in development of new methods for diagnosing depression in the future . To achieve these objectives, we measured and compared the extent of endogenous dna damage single- and double - strand breaks, alkali - labile sites, and oxidative damage of the pyrimidines and purines in pbmcs isolated from dd patients and healthy controls . Moreover, we induced oxidative dna damage in those pbmcs by incubating them with hydrogen peroxide, measured the kinetics of removing of such damage, and compared the results between the patients and the controls . The study was carried out in a group of 86 subjects aged 2265 (m=40.75 years, sd=15.02). The participants were divided into 2 groups: patients with rdd (n=40) and healthy subjects (a control group, cg, n=46). Respondents, before deciding to participate in the study, had been informed of its purpose, ensured that the participation was voluntary, and were guaranteed that personal data and test results would not be distributed, but only used in the overall statistics . Patients were selected for the study according to the inclusion criteria of icd-10 (f32.07.32.2, f33.0f33.8). The presence of axis i and ii disorders other than depressive episode, and the diagnosis of somatic diseases and injuries of the central nervous system (cns) were regarded as exclusion criteria . Other exclusion criteria were inflammatory or autoimmune disorders and unwilling to give informed consent . In all the included subjects, case history was obtained prior to main study procedure, using the standardized composite international diagnostic interview (cidi). During hospitalization informed, written consent for participation in the study was obtained from each subject, according to the protocol approved by the bioethics committee of the medical university of lodz (no . Pbmcs were isolated from 3 ml of fresh blood diluted with 3 ml of phosphate - buffered saline (pbs) by isopycnic centrifugation (30 min, 400 g, 4c) in histopaque-1077 (sigma - aldrich, st . Then, the cells were washed twice with pbs and counted using a brcker chamber . Dna damage was induced by exposing pbmcs to 20 m h2o2 (poch s.a ., dna damage induced by h2o2 was measured in the cells, which were immediately subjected to comet assay after the exposure . To estimate the efficiency of dna damage repair, the cells were washed with pbs, suspended in fresh rpmi-1640 medium, and kept at 37c for 15, 30, 60, and 120 min . After each time period we used the alkaline version (ph> 13) of the comet assay according to the procedure described by singh et al . With later modifications [2729]. Although this technique recognizes both double- and single - strand breaks along with alkali - labile sites, it does not detect oxidative dna damage . Thus, the level of this kind of dna damage was assessed by the use of 2 dna glycosylases: human 8-oxoguanine dna glycosylase or nth (hoog1 and endonuclease iii, respectively; new england biolabs, ipswich, ma, usa) according to the procedure described earlier . Both enzymes are bifunctional glycosylases and they possess ap - lyase activity . Therefore, after the recognition and removal of the damage bases, they introduce single - strand breaks, which can be detected by the alkaline version of the comet assay . Hoog1 recognizes and removes 8-oxoadenine when paired with cytosine, 7,8-dihydro-8-oxoguanine (8-oxoguanine) when paired with cytosine, methyfapy - guanine and foramidopyrimidine (fapy)-guanine . Nth recognizes and removes urea, thymine glycol, 5,6-dihydroxythymine, uracil glycol, 5-hydroxy-5-methylhydanton, 6-hydroxy-5,6-dihdrothimine, and methyltartronylurea . For each sample, aliquots of 510 pbmcs were centrifuged (250 g, 15 min 4c), the supernatant was removed and the pellet was re - suspended in 40 l of 1.13% low gelling temperature agarose type xi (sigma - aldrich, st . Then, the suspension was cast on a microscope slide, which was earlier coated with 0.5% low electroendosmosis agarose type i (sigma - aldrich, st . Louis, mo, usa) in distillated water, covered with a cover glass, and gelled on a cold plate for 10 min . After this time, the cover glass was removed, the slide was submerged in a chilled lysis solution (100 mm edta, 2.5 mm nacl, 10 mm tris, 1% triton x-100, ph 10) and stored at 4c overnight . On the next day, the enzyme - treated samples were washed with enzyme buffer (0.5 mm edta, 40 mm hepes, 0.1 m kcl, 0.2 mg / ml bovine serum albumin; ph 8). Then, 100 l of the buffer containing 1 u of either hogg1 or nth enzymes, or, as a control, the buffer alone, were placed onto gels . Those samples were covered with cover glasses and incubated at 37c for 60 min in a moist chamber . After the incubation, cover slips were removed, the slides were submerged in a electrophoresis buffer (1 mm edta and 300 mm naoh, ph> 13), and left for 20 min for unwinding of dna . Then, electrophoresis was conducted at an electric field strength of 0.73 v / cm (300 ma) for 20 min . Approximately 60 min before analysis, gels were stained with dapi (1 g / ml). The analysis was done using an eclipse fluorescence microscope (nikon, tokyo, japan) at 200 magnification . Fifty images of the comets were randomly selected from each sample, captured using a cohu 4910 video camera (cohu, san diego, ca, usa) and their parameters were measured with the lucia - comet image analysis system (laboratory imaging, praha, czech republic). The level of dna damage of the samples was assessed by calculating the mean of the percentage of dna in the tail of the comets . Results for samples digested with the dna repair enzymes were normalized by subtracting the level of dna damage evoked by enzyme buffer only . Data shown in this paper are presented as a mean sem from 2 separate experiments . We applied the shapiro - wilk test to assess normality of distribution of the studied group . If it was normally distributed, we used the t test to determine differences between means, if not, we used the mann - whitney test . Analysis of the data was done using statistica (statsoft, tulsa, ok). The study was carried out in a group of 86 subjects aged 2265 (m=40.75 years, sd=15.02). The participants were divided into 2 groups: patients with rdd (n=40) and healthy subjects (a control group, cg, n=46). Respondents, before deciding to participate in the study, had been informed of its purpose, ensured that the participation was voluntary, and were guaranteed that personal data and test results would not be distributed, but only used in the overall statistics . Patients were selected for the study according to the inclusion criteria of icd-10 (f32.07.32.2, f33.0f33.8). The presence of axis i and ii disorders other than depressive episode, and the diagnosis of somatic diseases and injuries of the central nervous system (cns) were regarded as exclusion criteria . Other exclusion criteria were inflammatory or autoimmune disorders and unwilling to give informed consent . In all the included subjects, case history was obtained prior to main study procedure, using the standardized composite international diagnostic interview (cidi). During hospitalization informed, written consent for participation in the study was obtained from each subject, according to the protocol approved by the bioethics committee of the medical university of lodz (no . Pbmcs were isolated from 3 ml of fresh blood diluted with 3 ml of phosphate - buffered saline (pbs) by isopycnic centrifugation (30 min, 400 g, 4c) in histopaque-1077 (sigma - aldrich, st . Then, the cells were washed twice with pbs and counted using a brcker chamber . Dna damage was induced by exposing pbmcs to 20 m h2o2 (poch s.a ., dna damage induced by h2o2 was measured in the cells, which were immediately subjected to comet assay after the exposure . To estimate the efficiency of dna damage repair, the cells were washed with pbs, suspended in fresh rpmi-1640 medium, and kept at 37c for 15, 30, 60, and 120 min . After each time period in this study we used the alkaline version (ph> 13) of the comet assay according to the procedure described by singh et al . With later modifications [2729]. Although this technique recognizes both double- and single - strand breaks along with alkali - labile sites, it does not detect oxidative dna damage . Thus, the level of this kind of dna damage was assessed by the use of 2 dna glycosylases: human 8-oxoguanine dna glycosylase or nth (hoog1 and endonuclease iii, respectively; new england biolabs, ipswich, ma, usa) according to the procedure described earlier . Therefore, after the recognition and removal of the damage bases, they introduce single - strand breaks, which can be detected by the alkaline version of the comet assay . Hoog1 recognizes and removes 8-oxoadenine when paired with cytosine, 7,8-dihydro-8-oxoguanine (8-oxoguanine) when paired with cytosine, methyfapy - guanine and foramidopyrimidine (fapy)-guanine . Nth recognizes and removes urea, thymine glycol, 5,6-dihydroxythymine, uracil glycol, 5-hydroxy-5-methylhydanton, 6-hydroxy-5,6-dihdrothimine, and methyltartronylurea . For each sample, aliquots of 510 pbmcs were centrifuged (250 g, 15 min 4c), the supernatant was removed and the pellet was re - suspended in 40 l of 1.13% low gelling temperature agarose type xi (sigma - aldrich, st . Then, the suspension was cast on a microscope slide, which was earlier coated with 0.5% low electroendosmosis agarose type i (sigma - aldrich, st . Louis, mo, usa) in distillated water, covered with a cover glass, and gelled on a cold plate for 10 min . After this time, the cover glass was removed, the slide was submerged in a chilled lysis solution (100 mm edta, 2.5 mm nacl, 10 mm tris, 1% triton x-100, ph 10) and stored at 4c overnight . On the next day, the enzyme - treated samples were washed with enzyme buffer (0.5 mm edta, 40 mm hepes, 0.1 m kcl, 0.2 mg / ml bovine serum albumin; ph 8). Then, 100 l of the buffer containing 1 u of either hogg1 or nth enzymes, or, as a control, the buffer alone, were placed onto gels . Those samples were covered with cover glasses and incubated at 37c for 60 min in a moist chamber . After the incubation, cover slips were removed, the slides were submerged in a electrophoresis buffer (1 mm edta and 300 mm naoh, ph> 13), and left for 20 min for unwinding of dna . Then, electrophoresis was conducted at an electric field strength of 0.73 v / cm (300 ma) for 20 min . Approximately 60 min before analysis, gels were stained with dapi (1 g / ml). The analysis was done using an eclipse fluorescence microscope (nikon, tokyo, japan) at 200 magnification . Fifty images of the comets were randomly selected from each sample, captured using a cohu 4910 video camera (cohu, san diego, ca, usa) and their parameters were measured with the lucia - comet image analysis system (laboratory imaging, praha, czech republic). The level of dna damage of the samples was assessed by calculating the mean of the percentage of dna in the tail of the comets . Results for samples digested with the dna repair enzymes were normalized by subtracting the level of dna damage evoked by enzyme buffer only . Data shown in this paper are presented as a mean sem from 2 separate experiments . We applied the shapiro - wilk test to assess normality of distribution of the studied group . If it was normally distributed, we used the t test to determine differences between means, if not, we used the mann - whitney test . Analysis of the data was done using statistica (statsoft, tulsa, ok). We evaluated the level of basal endogenous dna damage by subjecting pbmcs to comet assay procedure immediately after isolation from blood . We used an alkaline version of comet assay to measure the amount of dna alkali label sites and strand breaks, and the results are presented in the figure 1 . We found that this kind of dna damage was significantly higher in the rdd patients than in the controls (p<0.001). Moreover, we estimated the extent of oxidative dna damage by employing modified comet assay with 2 glycosylases: nth removing oxidized pyrimidines and hogg1 excising oxidized purines . The results obtained using nth and hogg1 are presented in figure 1 . In both cases the damage was significantly higher in the patients than in the controls (p<0.001). Figure 2 shows basal endogenous dna damage and the damage induced after 10-min incubation with 20 m h2o2 in pbmcs isolated from the patients and controls without psychiatric disturbances . For both groups there was no difference between increase of the damage in the patients and the controls (p=0.090), which equaled 197.29% and 228.46%, respectively . Additionally, dna damage after treatment with h2o2 was significantly higher in pbmcs of the patients than in those of the controls (p<0.001). After the induction of dna damage, h2o2 was removed and the cells were suspended in fresh medium and left for 120 min of repair incubation . The extent of dna damage was assessed using the alkaline version of comet assay at the beginning of the incubation, and after 15, 30, 60, and 120 min . Figure 3 shows mean dna damage changes in pbmcs of the patients with depression and the controls without psychiatric disturbances during the repair incubation . At each time, dna damage was higher in the patients than in the controls (p<0.001). The observed initial elevation of the dna damage in the repair kinetics can be explained by the actions of enzymes involved in dna repair . The latter ones, mainly repaired by base excision repair (ber), are not recognized by the alkaline version of comet assay . One of the earliest steps of this repair pathway is excision of the damaged base, which creates structure recognized by the alkaline version of comet assay, thus causing increased dna damage . Figure 4 presents comparison of dna damage at the beginning (0 min) and after 60 min of the repair incubation in pbmcs of the patients and the controls measured by the alkaline version of comet assay . In the controls, dna damage was lower after 60 min than at the beginning of the incubation (p<0.001), and in the patients it was at the same level (p=0.231). Figure 5 compares basal endogenous dna damage and the level of this parameter at the end of the repair incubation in pbmcs of the patients and the controls measured by the alkaline version of comet assay . In the controls, dna damage induced by h2o2 was fully repaired after 120 min of incubation, since the dna damage at this point was at the same level as an endogenous dna damage (p=0.812). In contrast, in patients, dna damage at the end of the repair was higher than the endogenous levels of this parameter (p<0.001). We evaluated the level of basal endogenous dna damage by subjecting pbmcs to comet assay procedure immediately after isolation from blood . We used an alkaline version of comet assay to measure the amount of dna alkali label sites and strand breaks, and the results are presented in the figure 1 . We found that this kind of dna damage was significantly higher in the rdd patients than in the controls (p<0.001). Moreover, we estimated the extent of oxidative dna damage by employing modified comet assay with 2 glycosylases: nth removing oxidized pyrimidines and hogg1 excising oxidized purines . The results obtained using nth and hogg1 are presented in figure 1 . In both cases the damage was significantly higher in the patients than in the controls (p<0.001). Figure 2 shows basal endogenous dna damage and the damage induced after 10-min incubation with 20 m h2o2 in pbmcs isolated from the patients and controls without psychiatric disturbances . For both groups the incubation caused a significant increase of the damage (p<0.001). There was no difference between increase of the damage in the patients and the controls (p=0.090), which equaled 197.29% and 228.46%, respectively . Additionally, dna damage after treatment with h2o2 was significantly higher in pbmcs of the patients than in those of the controls (p<0.001). After the induction of dna damage, h2o2 was removed and the cells were suspended in fresh medium and left for 120 min of repair incubation . The extent of dna damage was assessed using the alkaline version of comet assay at the beginning of the incubation, and after 15, 30, 60, and 120 min . Figure 3 shows mean dna damage changes in pbmcs of the patients with depression and the controls without psychiatric disturbances during the repair incubation . At each time, dna damage was higher in the patients than in the controls (p<0.001). The observed initial elevation of the dna damage in the repair kinetics can be explained by the actions of enzymes involved in dna repair . The latter ones, mainly repaired by base excision repair (ber), are not recognized by the alkaline version of comet assay . One of the earliest steps of this repair pathway is excision of the damaged base, which creates structure recognized by the alkaline version of comet assay, thus causing increased dna damage . Figure 4 presents comparison of dna damage at the beginning (0 min) and after 60 min of the repair incubation in pbmcs of the patients and the controls measured by the alkaline version of comet assay . In the controls, dna damage was lower after 60 min than at the beginning of the incubation (p<0.001), and in the patients it was at the same level (p=0.231). Figure 5 compares basal endogenous dna damage and the level of this parameter at the end of the repair incubation in pbmcs of the patients and the controls measured by the alkaline version of comet assay . In the controls, dna damage induced by h2o2 was fully repaired after 120 min of incubation, since the dna damage at this point was at the same level as an endogenous dna damage (p=0.812). In contrast, in patients, dna damage at the end of the repair was higher than the endogenous levels of this parameter (p<0.001). The goal of our research was to examine the susceptibility of rdd patients to dna damage induced by oxidative stress by measuring the level of endogenous dna damage, including oxidative dna damage, the amount of dna damage induced by h2o2, and efficiency of dna damage repair in the patients as compared to the controls without psychological disturbances . Although we are fully aware that these cells are not affected by depression, there some reasons why we have chosen to do our experiments on these cells . Firstly, in polish conditions it is not possible to carry out large - scale studies based on the analysis of preparations taken from the brains of patients with dd, because such samples can be obtained only post - mortem . Secondly, even though the results acquired from experiments done on pbmcs cannot be simply extrapolated to the cells of the central nervous system (cns), pbmcs are affected by the same environmental conditions as cns cells, thus any disturbances found in the former may reflect the condition of the latter . Finally, results obtained from pbmcs may reveal evidence of inherited defects in the process of repairing dna damage, because the genetic background of those cells reflects the constitution of the whole body, including cns cells . We found that patients with depression, when compared to controls without psychiatric disturbances, had more single- and double - strand breaks and alkali labile sites as measured by the alkaline version of comet assay (figure 1). Moreover, we showed, using modified comet assay with hogg1 and nth, that the patients in comparison to the controls had elevated levels of oxidative dna damage, particularly oxidative modifications of the purines and pyrimidines (figure 2). As mentioned above, to date, the study of dna damage in the context of depression has been limited to measurements of 8-oxog levels in serum, urine, or peripheral blood lymphocytes . It is worth noting that this technic measures only levels of free, not incorporated into dna 8-oxog, thus it does not measure oxidative dna damage per se . The exception is work done by yi et al ., who found no significant correlation between the amount of urinary 8-oxog and occurrence of depressive symptoms . Those discrepancies can be explained by the fact, as also emphasized by other authors, that study populations consisted of rather healthy people with milder forms of depressive syndromes and not the more severe, clinically diagnosed depression cases, as in our work . It is possible that increased oxidative dna damage occurs only in patients with more severe forms of depression, or in later stages of the disease development . Apart from measuring the extent of endogenous dna damage, we also estimated the amount of dna damage induced by the incubation of pbmcs with h2o2 and efficiency of its repair . We found that this incubation caused a significant increase of dna damage, both in the patients and the controls (figure 2). The increase was similar in both groups, and higher dna damage after exposure to h2o2 in the patient when compared to the controls was rather a result of the differences in endogenous dna damage levels, thus we can conclude that dna of the cells isolated from the patients and the controls had similar susceptibility to h2o2 . Additionally, we monitored the repair efficiency of the induced dna damage . Kinetics of the repair were similar for both studied groups, except that the damage levels were higher in the patients than in the controls (figure 3). We observed initial increase of dna damage caused by the action of the repair enzymes, mainly glycosylates, which excise damaged bases and create structures recognized by the alkaline version of comet assay . The maximum of dna damage was observed after 15 min of repair incubation in both patients and controls, and after this point the amount of dna damage was systematically reduced . Because of the significantly higher levels of dna damage after incubation with h2o2 in the patients than in the controls, the efficiency of dna repair cannot be evaluated simply by comparing the extent of dna damage between the groups . We found that in the controls, after 60 min of repair, incubation dna damage was reduced below the level of dna damage induced by h2o2, when in the patients it was still at the same level (figure 4). Moreover, at the end of the incubation, in the controls the dna damage was repaired and matched to the extent of endogenous dna damage, in contrast to the patients, where dna damage after 120 min was higher than the endogenous levels of this parameter (figure 5). These results indicate that in the patients, oxidative dna damage is less efficiently removed than in the controls . Our results do not show whether the increased dna damage and less efficient dna repair are the hallmarks of rdd patients and at some point they contribute to development of the diseases, or rather they are caused by the depression itself . On the one hand, the latter hypothesis is proved by the results obtained in the subject with milder depressive syndromes, where levels of 8-oxog were not elevated . Moreover, nlrp3 inflammasome, activation of which was detected in the patients pbmcs, was also found to inhibit dna repair after induction of oxidative stress . Additionally, depression was also found to be associated with activation of innate immune response, which may cause oxidation of lipids and dna [3537]. On the other hand, correlation analysis done in the patients with depression between levels of 8-iso - pgf2 a marker of oxidative damage to lipids apart from this, a growing body of evidence shows that disturbances of mitochondria, mainly increased ros production, are present in depression . Because most of enzymes involved in mitochondrial ber are splicing variants or are the same proteins as those used in nuclear ber, one can speculate that disturbances of the nuclear dna repair pathway may reflect impairments of its mitochondrial counterpart . When at some point mitochondrial dna damage levels cross a specific threshold, they may interfere with the electron transport chain, causing increased production of mtros . Mtros were found to be the main activators of nlrp3, which could suggest that dna damage triggers the inflammatory state and by this may contribute to development of depression . It must be noted that the study group was relatively small and was ethnically homologous . That is why, if the hypothesis that amount of dna damage in patients with depression can be associated with severity of the disease is going to be confirmed, such an association could provide a valuable diagnostic tool . We emphasize that there is a necessity for more advance research, including clinical, molecular, and epidemiological studies . For the first time, we showed that patients with depression had elevated levels of dna breaks, alkali - labile sites, and oxidative dna damage, and that these lesions may be accumulated by impairments of dna repair pathways . There is a need for further studies to define the role of nuclear and mitochondrial dna damage and repair in people with depression, and their implications for clinical outcome.
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When tooth extraction is performed for the management of diseases or trauma, complete hemostasis should be achieved in order to prevent infection at the extraction site and sutures may be used to allow natural healing . After extraction, the bone resolves and remodels itself due to the nature of the alveolar bone . Tallgren et al.1 observed alveolar bone loss for 25 years and reported that most bone loss occurred during the first year after extraction and that the bone continues to resolve afterwards at a slow rate . Therefore, bone graft is performed at the extraction site or at the resolved alveolar ridge in order to prepare the site for an implant, to prevent bone loss for aesthetic reasons, or to preserve the extraction socket.2,3 in the treatment of periodontal defects, current practice includes the use of alloplastic materials, such as -tricalcium phosphate (-tcp) and hydroxyapatite (ha), which are synthetic osteoconductive materials . A recent systematic review has also indicated that bone replacement grafts lead to significant clinical improvements in periodontal osseous defects.4 although -tcp / ha does not form new connective tissue, it provides not only a scaffold for new bone formation but also facilitates the stabilization of blood clot.5 as -tcp is porous, it entraps growth factors within its micropores, thereby prolonging their activity.6 bmp, a protein derived from a subgroup of the transforming growth factor family,7 accelerates ossification by controlling the essential factors of the bone induction cascade, resulting in the proliferation of osteoblasts from mesenchymal stem cells and the biosynthesis of bone matrices.8,9 recombinant human bmps are currently produced by bmp gene - transfected mammalian cell (cho) cultures,10,11 and rhbmp-2 and bmp-7 are commercially available for the treatment of bony defects.12,13 one of the problems associated with clinical application of cho - cell - derived rhbmp-2 (crhbmp-2) is its high costs due to high dose requirements . One possible way of solving this problem is to produce monomer rhbmps from bmp - gene - transfected escherichia coli (e. coli) with a high efficiency and low costs . Bessho et al.14 examined the bone - inducing ability of an e. coli - derived rhbmp-2 (erhbmp-2) variant with an n - terminal sequence and compared it with crhbmp-2 . Quantitative analysis indicated that the activity of erhbmp-2 is similar to that of crhbmp-2 . However, it is unclear whether the characteristics of erhbmp-2 are appropriate for clinical application . In particular, there have been few studies about the efficacy and safety of erhbmp-2 and -tcp graft materials in osseous defects, such as tooth extraction sockets . Therefore, this randomized clinical trial was conducted to assess the safety and effectiveness of the erhbmp-2 in alveolar bone regeneration as well as preservation of the -tcp bone graft material that contains erhbmp-2 . A double - blind, active - controlled, randomized, parallel, multicenter, prospective, phase iii study was conducted with the approval of the korean food and drug association at 3 centers in the republic of korea from april 2009 to march 2010, in order to assess the efficacy of erhbmp-2 + -tcp / ha in comparison with -tcp / ha alone for the treatment of tooth extracted sockets . The institutional review board at each of the 3 study centers approved the study protocol . This study initially involved 72 patients aged from 35 to 65 years at the 3 study centers, whose premolars or molars were indicated for extraction with less than 50% of localized alveolar vertical bone loss (table 1). However, patients with the following conditions were excluded from the study: (1) those who had severe periodontitis with localized alveolar vertical bone loss of more than 50%, (2) those who were currently pregnant or planned to get pregnant within 1 year of the experiment, (3) those who were older than 65 years, (4) those who had recent myocardial infarction or uncontrolled bleeding disorders, (5) those who were contraindicated to minor surgeries, (6) those who had mental illness or suspected mental illness or hypersensitivity to bone graft materials, and (7) those who were classified as inappropriate for clinical trial participation by the clinician due to ethical reasons or other possible impacts on the results of clinical trials . Patients were divided into 2 groups: the experiment group who had erhbmp-2 coated tcp / ha (cowellmedi co, pusan, korea; 1.5 mg / ml) and the control group who had tcp / ha graft material alone transplanted immediately into the socket of tooth extraction . At the first visit, the purpose of the study was explained, written consent was obtained, and patient blood samples were taken to check pre - existing antibodies against erhbmp-2 . At the second visit, the indicated tooth of the patients, who provided written consent, was extracted and the socket was filled with the bone graft and sutured . The first ct scan the fifth visit was made 1 month after the second visit, and a blood sample was drawn and an intraoral examination was performed . The final visit was made 3 months after the second visit, and a computed tomography (ct) and an intraoral examination were performed . To verify the effectiveness of these bone grafts, ct was performed before the transplantation and 3 months after the transplantation and healing status was compared between the two . The efficacy endpoints that were used to measure the degree of bone induction included alveolar bone height (1 measurement) and 3 measurements of bone width (1 measurement each at 25%, 50% and 75% of the extraction socket length [esl]). This method followed a previous similar study.15 in addition, maxillary teeth extraction sockets were measured by the same way as in a previous study.15 mandibular tooth extraction sockets were measured by the method depicted in fig . Alveolar bone heights at baseline and 3 months post - transplantation were compared between the control and experiment groups . The mean and standard deviation of the test parameters were calculated using spss (ver . The paired t test was used to determine the significance of the changes . To assess the minor effects of the bone graft materials, changes in alveolar bone width at 25% esl, 50% esl and 75% esl at the baseline and 3 months post - treatment a double - blind, active - controlled, randomized, parallel, multicenter, prospective, phase iii study was conducted with the approval of the korean food and drug association at 3 centers in the republic of korea from april 2009 to march 2010, in order to assess the efficacy of erhbmp-2 + -tcp / ha in comparison with -tcp / ha alone for the treatment of tooth extracted sockets . The institutional review board at each of the 3 study centers approved the study protocol . This study initially involved 72 patients aged from 35 to 65 years at the 3 study centers, whose premolars or molars were indicated for extraction with less than 50% of localized alveolar vertical bone loss (table 1). However, patients with the following conditions were excluded from the study: (1) those who had severe periodontitis with localized alveolar vertical bone loss of more than 50%, (2) those who were currently pregnant or planned to get pregnant within 1 year of the experiment, (3) those who were older than 65 years, (4) those who had recent myocardial infarction or uncontrolled bleeding disorders, (5) those who were contraindicated to minor surgeries, (6) those who had mental illness or suspected mental illness or hypersensitivity to bone graft materials, and (7) those who were classified as inappropriate for clinical trial participation by the clinician due to ethical reasons or other possible impacts on the results of clinical trials . Patients were divided into 2 groups: the experiment group who had erhbmp-2 coated tcp / ha (cowellmedi co, pusan, korea; 1.5 mg / ml) and the control group who had tcp / ha graft material alone transplanted immediately into the socket of tooth extraction . At the first visit, the purpose of the study was explained, written consent was obtained, and patient blood samples were taken to check pre - existing antibodies against erhbmp-2 . At the second visit, the indicated tooth of the patients, who provided written consent, was extracted and the socket was filled with the bone graft and sutured . The first ct scan the fifth visit was made 1 month after the second visit, and a blood sample was drawn and an intraoral examination was performed . The final visit was made 3 months after the second visit, and a computed tomography (ct) and an intraoral examination were performed . To verify the effectiveness of these bone grafts, ct was performed before the transplantation and 3 months after the transplantation and healing status was compared between the two . The efficacy endpoints that were used to measure the degree of bone induction included alveolar bone height (1 measurement) and 3 measurements of bone width (1 measurement each at 25%, 50% and 75% of the extraction socket length [esl]). This method followed a previous similar study.15 in addition, maxillary teeth extraction sockets were measured by the same way as in a previous study.15 mandibular tooth extraction sockets were measured by the method depicted in fig . 1 . In order to assess the major effects of the bone graft materials, alveolar bone heights at baseline and 3 months post - transplantation were compared between the control and experiment groups . The mean and standard deviation of the test parameters were calculated using spss (ver . The paired t test was used to determine the significance of the changes . To assess the minor effects of the bone graft materials, changes in alveolar bone width at 25% esl, 50% esl and 75% esl at the baseline and 3 months post - treatment changes in alveolar bone height were examined using ct scans taken before and 3 months after treatment, which turned out to be -1.0871.413 mm in the control group and -0.0590.960 mm in the experimental group . The paired student t test was used to compare the mean change between the 2 groups in alveolar bone height preservation, and the difference was statistically significant (p<.01) (table 2). Changes in alveolar bone width were also measured to determine the minor effects of bone grafts on the preservation of alveolar bone . At 25% esl, the changes were 0.0061.149 mm in the control group and 1.2791.387 mm in the experimental group . At 50% esl, the changes were 0.5421.157 mm and 1.2391.249 mm, respectively, and at 75% esl, the changes were 1.4051.753 mm and 1.8632.310 mm, respectively . The paired student t - test was used to compare the changes between the 2 groups, and the differences were statistically significant (p<.01 for 25% esl, and p<.05 for 50% esl) (table 3). Preclinical studies have evaluated induction and repair of bony defects in a variety of indications.16,17 a previous study of utilizing rhbmp-2 in humans showed the safety and technical feasibility . Howell et al.18 reported that in local ridge preservation and augmentation, 0.43 mg / ml rhbmp / absorbable collagen sponge (acs) was well tolerated locally and systemically, with no adverse events . In a pivotal study by fiorellini et al.15 assessment of alveolar bone indicated that patients treated with 1.50 mg / ml crhbmp-2/acs had significantly better results after bone augmentation than control patients (p.05). The adequacy of bone for the placement of a dental implant was approximately twice greater in the rhbmp-2/acs group than in the non - treatment or placebo group . This randomized and double - blind clinical trial was designed to assess the safety and bone regenerative ability of the erhbmp-2 coated -tcp / ha bone graft material, which is coated with erhbmp-2 . After the completion of the clinical trial, the anova test was performed to evaluate whether the evaluation parameters had homogeneity according to institutional and demographic data . Since there were no significant variations in these parameters, the results of this study on the safety and effectiveness of the bone graft materials are considered valid . In this study, to assess the major effects of the bone graft material in preserving the alveolar bone, alveolar bone height at baseline and 3 months post - treatment were compared by measuring bone height in the cross sectional ct images . To assess the minor effects of the bone graft material, changes in alveolar bone width at 25% esl, 50% esl and 75% esl were compared using cross - sectional ct images at baseline and 3 months post - treatment . In addition, clinical observation was performed to evaluate the safety of the graft material, and antibody against rhbmp-2 was tested to evaluate its immunological safety . No clinical adverse reactions or even inflammatory responses were observed in patients who received the bone graft . Basedon the immunological evaluations of the blood tests at the fifth visit of 72 patients (36 in the control group and 36 in the experiment group), none of the patients were suspected of having developed antibodies against the bone graft material . Ct images were analyzed using the same software (ondemend, cybermed inc, la, ca, usa) at the same site immediately after transplantation and 3 months post - transplantation . Changes in alveolar bone height were considered as a major evaluation variable in determining the effectiveness of the bone graft for alveolar bone regeneration . Based on ct scans at baseline and 3 months post - transplantation, the changing rate of the alveolar bone height, was higher in the control group than in the experimental group (p<.05). Changes in alveolar bone width were considered a minor evaluation variable in determining the effectiveness of the bone graft in alveolar bone regeneration . In this study, materials other than the subject's gingiva (e.g., collagen membrane and others) were excluded in order to eliminate the effects of other factors . However, one crucial factor that affects the outcome of the conventional guided - bone regeneration (gbr) procedure is the fact that the treatment area must be protected from the surrounding soft tissue . Therefore, because of chewing, brushing and other oral habits, as well as differences in remaining gingival levels, the tested bone graft material did not survive at the treatment site over a sufficient time period . Therefore, it is conceivable that if the bone graft material is transplanted after allowing the gingival tissue to heal to a certain extent after the extraction, rather than being transplanted immediately after extraction, the alveolar bone preservation effect of the bone graft material may increase . In this study, -tcp / ha bone grafts coated with erhbmp-2 were found to be more effective in preserving alveolar bone than conventional -tcp / ha alloplastic bone grafts . Furthermore, during the experiment, no adverse reactions to the graft material were observed . Thus, this alloplastic bone graft coated with erhbmp-2 is considered to be an effective bone graft material.
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Patients with a preexisting episcleral encircling element who underwent implantation of a silicone agv to treat intractable glaucoma during january 2009 to september 2010 at a large, tertiary care ophthalmic hospital were identified by means of operative records . Postsurgical follow - up of less than 6 weeks and inability to measure intraocular pressure (iop) were the exclusion criteria . The conjunctival incision was made at the corneal limbus or at 4 - 5 mm behind and parallel to the limbus, at the operating surgeon's discretion, for approximately 100 in the supero - temporal quadrant . The agv (model fp7, new world medical, rancho cucamonga, la, usa) was primed by injecting 1 - 2 ml balanced salt solution . The plate of agv was placed over the episcleral encircling element; either encircling band or scleral buckle [figure 1]. The plate was secured to the underlying sclera with 8 - 0 nylon suture material (m / s gn corporation ltd ., yamanashi, japan) such that its anterior edge lied 8 mm behind the corneal limbus . This was followed by placement of the silicone tube into anterior chamber or pars plana region through a 23-gauge needle track . The anterior part of the tube was covered with previously prepared human donor scleral patch graft . Fibrin sealant (tisseel kit, baxter ag, vienna, austria) or 8 - 0 nylon suture material was used to secure the scleral patch graft . The overlying conjunctiva was sutured with 8 - 0 polyglactin suture material (ethicon inc ., if there was limited mobility of the conjunctiva due to prior surgeries, relaxing incisions were given in the conjunctiva and/or amniotic membrane transplantation was considered . The eye was inspected for any leaks after the anterior chamber was inflated using balanced salt solution . The base plate of ahmed glaucoma valve being placed over the episcleral encircling element that is indicated by arrows the donor scleral tissue preserved in absolute alcohol was used in every case . The tissue was cleaned of all the uveal tissue attachments, washed thoroughly with balanced salt solution and cut into the desired size (4 - 5 4 - 5 mm). The data collection included information on patient demography, diagnosis of glaucoma, prior ocular surgeries, measurement of visual acuity, iop, number of antiglaucoma medications at pre - agv and every post - agv follow - up visit, duration between placement of episcleral encircling element and agv, and complications, if any . We measured iop either by applanation tonometer, namely, goldmann tonometer (haag - streit, switzerland), a hand - held perkin's tonometer (haag - streit, essex, uk), or by tonopen xl (reichert ophthalmic instruments, walden ave . The cause (s) for low vision and postoperative reduction in visual acuity, if any, were also recorded . Surgical success was defined as a final iop between 5 and 22 mmhg without (complete success) or with topical antiglaucoma medication (s) (qualified success) and without any vision threatening complication . Paired t - test was used to compare measurements of iop and number of antiglaucoma medications at the preoperative and the final visits . Data analysis was done using spss software version 15.0 for windows (spss inc ., patients with a preexisting episcleral encircling element who underwent implantation of a silicone agv to treat intractable glaucoma during january 2009 to september 2010 at a large, tertiary care ophthalmic hospital were identified by means of operative records . Postsurgical follow - up of less than 6 weeks and inability to measure intraocular pressure (iop) were the exclusion criteria . The conjunctival incision was made at the corneal limbus or at 4 - 5 mm behind and parallel to the limbus, at the operating surgeon's discretion, for approximately 100 in the supero - temporal quadrant . The agv (model fp7, new world medical, rancho cucamonga, la, usa) was primed by injecting 1 - 2 ml balanced salt solution . The plate of agv was placed over the episcleral encircling element; either encircling band or scleral buckle [figure 1]. The plate was secured to the underlying sclera with 8 - 0 nylon suture material (m / s gn corporation ltd ., yamanashi, japan) such that its anterior edge lied 8 mm behind the corneal limbus . This was followed by placement of the silicone tube into anterior chamber or pars plana region through a 23-gauge needle track . The anterior part of the tube was covered with previously prepared human donor scleral patch graft . Fibrin sealant (tisseel kit, baxter ag, vienna, austria) or 8 - 0 nylon suture material was used to secure the scleral patch graft . The overlying conjunctiva was sutured with 8 - 0 polyglactin suture material (ethicon inc ., aurangabad, india). If there was limited mobility of the conjunctiva due to prior surgeries, relaxing incisions were given in the conjunctiva and/or amniotic membrane transplantation was considered . The eye was inspected for any leaks after the anterior chamber was inflated using balanced salt solution . The base plate of ahmed glaucoma valve being placed over the episcleral encircling element that is indicated by arrows the tissue was cleaned of all the uveal tissue attachments, washed thoroughly with balanced salt solution and cut into the desired size (4 - 5 4 - 5 mm). The data collection included information on patient demography, diagnosis of glaucoma, prior ocular surgeries, measurement of visual acuity, iop, number of antiglaucoma medications at pre - agv and every post - agv follow - up visit, duration between placement of episcleral encircling element and agv, and complications, if any . We measured iop either by applanation tonometer, namely, goldmann tonometer (haag - streit, switzerland), a hand - held perkin's tonometer (haag - streit, essex, uk), or by tonopen xl (reichert ophthalmic instruments, walden ave . The cause (s) for low vision and postoperative reduction in visual acuity, if any, were also recorded . Surgical success was defined as a final iop between 5 and 22 mmhg without (complete success) or with topical antiglaucoma medication (s) (qualified success) and without any vision threatening complication . Paired t - test was used to compare measurements of iop and number of antiglaucoma medications at the preoperative and the final visits . Data analysis was done using spss software version 15.0 for windows (spss inc ., fourteen patients underwent implantation of agv in eyes with a preexisting episcleral encircling element during the study period . Two eyes (16.6%) had undergone placement of a circumferential scleral buckle in the supero - temporal quadrant . In one of these eyes with scleral buckle the shunt plate was additionally covered with two layers of cryo - preserved human amniotic membrane to reduce the chances of exposure of this implant in the postoperative period . The mean (standard deviation) duration between placement of episcleral encircling element and implantation of agv was 30.5 (33.8) months; median, 11.5 months . The mean follow - up was 37.4 (22.9) weeks; median, 33.5 weeks . The mean preoperative iop was 31.4 (7.9) mmhg; median, 32mmhg with an average of 2.8; median, 3 medications . At last postoperative follow - up, the mean iop decreased to 12.5 (3.5) mmhg the postoperative reduction in iop and in number of antiglaucoma medications was statistically significant (p <0.001, paired t - test). Demographic / pre - operative data corneal graft failure occurred in three eyes in late postoperative period following implantation of agv (cases 4, 7, 9). In one of these eyes, the agv tube was placed in the pars plana region . In the remaining two eyes, the agv tube was placed in the anterior segment, however, there was no tube - cornea touch . The mean duration between placement of agv and corneal graft failure was 7.3 (5.3) months; median, 8.5 months . Tube erosion occurred in another two eyes in late postoperative period (cases 10, 11). Case 12 reported reduced vision at sixth postoperative week and was found to have a rhegmatogenous retinal detachment . In this series, we describe implantation of the silicone plate agv in eyes with a preexisting episcleral encircling element . This novel use of ahmed valve can be helpful in the management of intractable glaucoma after the encircling procedure . Described the use of a silicone tube to shunt aqueous from the anterior segment to the fibrous capsule surrounding a previously placed episcleral encircling element . Using the existent fibrous capsule around the encircling element as an aqueous reservoir obviated the need for temporary restriction of the aqueous flow to allow encapsulation . Early postoperative complications attributable to hypotony were few and self - limiting . However, eight instances of fibrous obstruction of the distal tube opening occurred within 4 months of the initial procedure . Suh et al . Also reported high overall success rate with the same technique, but they studied only seven eyes . Modified the technique by using a valved (plate less) krupin - denver tube . No case of early postoperative hypotony occurred, although two (29%) of seven eyes experienced distal tube occlusion necessitating surgical revision . Scott et al . Described an alternative approach to manage refractory glaucoma in eyes with a preexisting encircling band . They inserted 250- (7 eyes [44%]) or 350-mm (9 eyes [56%]) baerveldt drainage devices behind or over a preexisting encircling band and over the adjacent recti muscles . The fibrous capsule overlying the encircling band was excised in the quadrant of surgical implantation . Nine patients (56%) achieved complete success and seven patients (44%) achieved qualified success at 1 year postoperatively . Implant migration or exposure and diplopia did not develop in any patient over the median follow - up of 22.9 months . However, it might be unsafe to insert a large sized implant in the presence of an encircling band in all four quadrants due to the effects of crowding . It may be that this study with limited number of patients does not reflect the true outcome . The incidence of postoperative diplopia has been shown to be significantly higher with the baerveldt than any other implant . This is the reason smith et al . Trimmed the wings of baerveldt implants to debulk them before insertion in eyes with a preexisting episcleral encircling element . The valve abutted against the anterior portion of the scleral band and was sutured into position . The valve function of the implant was retained to reduce postoperative hypotony . However, the reduction in the surface area of the trimmed drainage implant is of concern as the extent of iop reduction with glaucoma drainage implants has been demonstrated to be directly proportional to the surface area of the fibrous capsule . Additionally, it may be difficult to mobilize adequate conjunctiva to cover the implant in eyes with a preexisting episcleral encircling element, especially a scleral buckle . An implant with minimum - sized end - plate that is sufficient to achieve iop control is preferable in such eyes . Increasing the surface area of a randomized controlled clinical trial showed comparable failure rates at 1-year for both agv and 350 mm baerveldt implants . Therefore, agv should be preferred over baerveldt implant in eyes with a preexisting episcleral encircling element . Moreover, the base plate of the newer silicone ahmed valve (model fp7) is thinner (0.9 mm) and has the same surface area (184 mm) compared with the earlier polypropylene model s2 (thickness 1.9 mm). The silicone base plate is also flexible and has a tapered profile for easy insertion . These features allowed us to insert a silicone agv over the episcleral element in our patients . One can also make use of a long, curved surgical scissor for better access to the supero - temporal quadrant, and guide the base plate of the agv into position over the partly open blades of the scissor . Two patients in our series had preexisting scleral buckle in the supero - temporal quadrant (cases 8 and 12). However, the procedure was difficult in one eye (case 12) due to multiple prior surgeries . We gave a couple of relaxing incisions in the surrounding conjunctiva for adequate wound closure . The shunt plate was additionally covered with two layers of cryo - preserved human amniotic membrane, as described by amini et al . To reduce the chances of exposure of the implant in the postoperative period . Despite these efforts, the anterior edge of the scleral patch graft covering the implant tube did expose in the early postoperative period one of them had prior corneal graft failure (case 9). In two of these three eyes, the agv tube was placed in the anterior segment, however, there was no demonstrable tube - cornea touch . Found corneal graft failure in 24 (43.6%) out of 55 eyes that had undergone both penetrating keratoplasty (pk) and either baerveldt or ahmed glaucoma implant . Also found poor long - term survival of corneal grafts in their study of 77 eyes that underwent pk following agv implantation . The grafted corneas failed in 46 [59.1%, (95% confidence interval: 47.5 - 71.2%)] eyes at 3 years . Prior pk and stromal vessels, but not tube - cornea touch, did increase the risk of graft failure . Erosion of drainage tube through the overlying conjunctiva was seen in two eyes despite meticulous conjunctival closure and the use of a scleral patch graft . Both eyes underwent early repair as tube erosion carries with it increased risk of endophthalmitis . He was a high myope, suffered from posttraumatic retinal detachment and developed secondary glaucoma following retinal detachment repair . Retinal detachment as a complication of glaucoma drainage device has been reported in as many as 16% cases with varied risk factors . Nevertheless, considering the presence of multiple risk factors for retinal detachment, the complication may not necessarily be related to the implantation of agv in our case . Conventional filtration surgery such as trabeculectomy was either not possible or carried an unacceptably high risk of failure in these eyes . Known frequent complications such as early postoperative hypotony, shallow anterior chamber, tube obstruction, and conjunctival wound leak were not detected in this series . Even so, the overall postsurgical complications do appear frequent in this series; however, many patients maintained useful vision that they could potentially have lost since all had uncontrolled glaucoma prior to surgery . The retrospective design and the heterogeneity of the cases are major limitations of this study . The sample size is small and therefore, we did not analyze complete and qualified success separately . However, the clinical situations in which the procedures were done were extremely severe and until now none of the proposed treatments have gained general acceptance . This study, for the first time, shows that the silicone, flexi - plate ahmed valve can be effectively implanted in eyes with a preexisting episcleral encircling element keeping in mind the possibility of significant postoperative complications.
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The main drawback with the traditional stainless steel files is their inability to follow the canal curvature with a tendency to straighten, particularly at the start of the curvature . Ni - ti instruments introduced in 1988 to endodontics showed super - elasticity and shape memory . The main problems with instruments used in continuous rotary motion are the instrument separation caused by cyclic fatigue and inability of the instrument to maintain canal curvature . An alternative for continuous rotary is a reciprocating rotary instrument that travels shorter angular distance in one direction and reverses its direction before completing a full rotation, thereby subjecting the instrument to lower stress values . As a result, an instrument has an extended fatigue life when used in reciprocation as opposed to rotary motion . Cone beam computed tomography (cbct) is a recent advancement with advantages of image accuracy, acquisition of images in three planes (coronal, sagittal and axial) and reduced image artifacts . The null hypothesis tested was that increase in root canal surface area and canal transportation may not vary between rotary systems with different designs in either continuous rotary or reciprocating rotary motions . Sixty freshly extracted human mandibular molars from patients of age group 40 to 50 years with fully formed apices, absence of root caries, cracks, and structural defects with mesial root canal curvatures in distal direction between 20 and 30 (schneider's method) and those with initial minimum working width of 0.1 mm were selected for analysis so that apical enlargement can be done uniformly to the same size in all the teeth . Modeling wax was manipulated and adapted into a stock tray . Into each wax mounting, the radicular portions of five teeth were embedded, leaving the crowns oriented upward so that they are accessible for the endodontic procedure . Pre- and post - instrumentation scans were performed using cbct (kodak 9000, kodak carestream health, trophy, france) and all the scans were conducted with a 4 cm field of view, at 0.125 mm voxel resolution with 60 kv and 2.5 ma . After preoperative scanning, access cavities were prepared and working length (wl) was established 0.5 mm short of the apex . Manual instrumentation was not done prior to the use of mechanical instrumentation as the purpose of this study was to measure the canal transportation with different rotary systems . Recapitulation was done with one size smaller hand k - file corresponding to the rotary file . During instrumentation, india) was used as a lubricant and, after every instrumentation, irrigation was done with 2 ml of 3% naocl (prime dental products pvt ltd, india) in all groups . Instrumentation was started with sx file upto half of the working length (wl) and proceeded with s1 file upto two thirds the working length, and s2 file to the complete wl . The sx and s1 instruments were used at 300 rpm and 3 ncm torque, followed by instruments s2, f1, and f2 upto the working length, at 250 rpm and 2 ncm torque, avoiding apical pressure and applying gentle strokes against the canal walls . Group 1: protaper group, 20 teeth were made into two subgroups (n = 10) subgroup a: instrumentation of mesial canals was done by continuous rotary technique with protaper universal rotary files (dentsply maillefer, ballaigues, switzerland) connected to x - smart endomotor (dentsply maillefer, switzerland) according to the manufacturer's instructionssubgroup b: instrumentation was done by reciprocating rotary technique with protaper rotary files connected to waveone endomotor (dentsply maillefer, switzerland) according to the manufacturer's instructions following the same irrigation protocol as in subgroup a. group 2: k3xf group, 20 teeth were made into two subgroups (n = 10) subgroup a: instrumentation was done by continuous rotary technique with k3xf rotary files (sybronendo, glendora, ca, usa) connected to x - smart endomotor according to the manufacturer's instructions up to iso size 25 and 06% tapersubgroup b: instrumentation was done by reciprocating rotary technique with k3xf rotary files connected to waveone endomotor following the manufacturer's instructions up to iso size 25 and 06% taper . Group 3: lsx group, 20 teeth were made into two subgroups (n = 10) subgroup a: instrumentation was done in crown down method by continuous rotary technique with lsx rotary files (lightspeed technology inc ., san antonio, tx, usa) connected to tcm endo v endomotor (nouvag). Initially, #15, #20 lsx files were used, and then #25 lsx file was used up to the wl . All the teeth were selected with initial minimum working width of 0.1 mm and apical enlargement was done upto three sizes larger than the initial apical file i.e., upto iso size 25subgroup b: instrumentation was done by reciprocating rotary technique with lsx rotary files connected to waveone endomotor following the same protocol as in subgroup a. group 1: protaper group, 20 teeth were made into two subgroups (n = 10) subgroup a: instrumentation of mesial canals was done by continuous rotary technique with protaper universal rotary files (dentsply maillefer, ballaigues, switzerland) connected to x - smart endomotor (dentsply maillefer, switzerland) according to the manufacturer's instructionssubgroup b: instrumentation was done by reciprocating rotary technique with protaper rotary files connected to waveone endomotor (dentsply maillefer, switzerland) according to the manufacturer's instructions following the same irrigation protocol as in subgroup a. subgroup a: instrumentation of mesial canals was done by continuous rotary technique with protaper universal rotary files (dentsply maillefer, ballaigues, switzerland) connected to x - smart endomotor (dentsply maillefer, switzerland) according to the manufacturer's instructions subgroup b: instrumentation was done by reciprocating rotary technique with protaper rotary files connected to waveone endomotor (dentsply maillefer, switzerland) according to the manufacturer's instructions following the same irrigation protocol as in subgroup a. group 2: k3xf group, 20 teeth were made into two subgroups (n = 10) subgroup a: instrumentation was done by continuous rotary technique with k3xf rotary files (sybronendo, glendora, ca, usa) connected to x - smart endomotor according to the manufacturer's instructions up to iso size 25 and 06% tapersubgroup b: instrumentation was done by reciprocating rotary technique with k3xf rotary files connected to waveone endomotor following the manufacturer's instructions up to iso size 25 and 06% taper . Subgroup a: instrumentation was done by continuous rotary technique with k3xf rotary files (sybronendo, glendora, ca, usa) connected to x - smart endomotor according to the manufacturer's instructions up to iso size 25 and 06% taper subgroup b: instrumentation was done by reciprocating rotary technique with k3xf rotary files connected to waveone endomotor following the manufacturer's instructions up to iso size 25 and 06% taper . Group 3: lsx group, 20 teeth were made into two subgroups (n = 10) subgroup a: instrumentation was done in crown down method by continuous rotary technique with lsx rotary files (lightspeed technology inc ., san antonio, tx, usa) connected to tcm endo v endomotor (nouvag). Initially, #15, #20 lsx files were used, and then #25 lsx file was used up to the wl . All the teeth were selected with initial minimum working width of 0.1 mm and apical enlargement was done upto three sizes larger than the initial apical file i.e., upto iso size 25subgroup b: instrumentation was done by reciprocating rotary technique with lsx rotary files connected to waveone endomotor following the same protocol as in subgroup a. subgroup a: instrumentation was done in crown down method by continuous rotary technique with lsx rotary files (lightspeed technology inc ., san antonio, tx, usa) connected to tcm endo v endomotor (nouvag). Initially, #15, #20 lsx files were used, and then #25 lsx file was used up to the wl . All the teeth were selected with initial minimum working width of 0.1 mm and apical enlargement was done upto three sizes larger than the initial apical file i.e., upto iso size 25 subgroup b: instrumentation was done by reciprocating rotary technique with lsx rotary files connected to waveone endomotor following the same protocol as in subgroup a. adobe photoshop software [figure 1a] was used to export and evaluate the preoperative [figure 1b] and postoperative cbct images [figure 1c] (axial sections). The surface area of root canals at three different levels, i.e., at 1, 3, and 5 mm cross - sections was measured according to the protocol given by zer . The difference between the post- and pre - operative values represented the increase in root canal surface area at that cross - section [figure 1d]. (a) preoperative marking of root canal outline using magic wand tool of adobe photoshop software to measure the root canal surface area; (b) preoperative root canal outline to measure the root canal surface area; (c) postoperative root canal outline to measure the root canal surface area; (d) diagrammatic representation of increase in root canal surface area for mesiobuccal canal at 5 mm; (e) diagrammatic representation of root canal transportation for mesiobuccal canal at 5 mm; and (f) preoperative distance measurements for mesiobuccal canal of a sample from root canal wall to external root surface using cone - beam computed tomography software the distances from the root canal outline to the external surface of root were measured in all the four directions, both preoperatively and postoperatively as per the method suggested by gambill et al . [figure 1e and f]. To standardize the procedure and avoid variations in results, a single operator conducted the study . Differences in increase of root canal surface area and canal transportation between groups were analyzed by one - way analysis of variance, followed by tukey pairwise multiple comparison tests . Pre- and post - instrumentation scans were performed using cbct (kodak 9000, kodak carestream health, trophy, france) and all the scans were conducted with a 4 cm field of view, at 0.125 mm voxel resolution with 60 kv and 2.5 ma . After preoperative scanning, access cavities were prepared and working length (wl) was established 0.5 mm short of the apex . Manual instrumentation was not done prior to the use of mechanical instrumentation as the purpose of this study was to measure the canal transportation with different rotary systems . Recapitulation was done with one size smaller hand k - file corresponding to the rotary file . During instrumentation, 17% ethylenediaminetetraacetic acid (prime dental products pvt . Ltd ., india) was used as a lubricant and, after every instrumentation, irrigation was done with 2 ml of 3% naocl (prime dental products pvt ltd, india) in all groups . Instrumentation was started with sx file upto half of the working length (wl) and proceeded with s1 file upto two thirds the working length, and s2 file to the complete wl . The sx and s1 instruments were used at 300 rpm and 3 ncm torque, followed by instruments s2, f1, and f2 upto the working length, at 250 rpm and 2 ncm torque, avoiding apical pressure and applying gentle strokes against the canal walls . Group 1: protaper group, 20 teeth were made into two subgroups (n = 10) subgroup a: instrumentation of mesial canals was done by continuous rotary technique with protaper universal rotary files (dentsply maillefer, ballaigues, switzerland) connected to x - smart endomotor (dentsply maillefer, switzerland) according to the manufacturer's instructionssubgroup b: instrumentation was done by reciprocating rotary technique with protaper rotary files connected to waveone endomotor (dentsply maillefer, switzerland) according to the manufacturer's instructions following the same irrigation protocol as in subgroup a. group 2: k3xf group, 20 teeth were made into two subgroups (n = 10) subgroup a: instrumentation was done by continuous rotary technique with k3xf rotary files (sybronendo, glendora, ca, usa) connected to x - smart endomotor according to the manufacturer's instructions up to iso size 25 and 06% tapersubgroup b: instrumentation was done by reciprocating rotary technique with k3xf rotary files connected to waveone endomotor following the manufacturer's instructions up to iso size 25 and 06% taper . Group 3: lsx group, 20 teeth were made into two subgroups (n = 10) subgroup a: instrumentation was done in crown down method by continuous rotary technique with lsx rotary files (lightspeed technology inc ., san antonio, tx, usa) connected to tcm endo v endomotor (nouvag). Initially, #15, #20 lsx files were used, and then #25 lsx file was used up to the wl . All the teeth were selected with initial minimum working width of 0.1 mm and apical enlargement was done upto three sizes larger than the initial apical file i.e., upto iso size 25subgroup b: instrumentation was done by reciprocating rotary technique with lsx rotary files connected to waveone endomotor following the same protocol as in subgroup a. group 1: protaper group, 20 teeth were made into two subgroups (n = 10) subgroup a: instrumentation of mesial canals was done by continuous rotary technique with protaper universal rotary files (dentsply maillefer, ballaigues, switzerland) connected to x - smart endomotor (dentsply maillefer, switzerland) according to the manufacturer's instructionssubgroup b: instrumentation was done by reciprocating rotary technique with protaper rotary files connected to waveone endomotor (dentsply maillefer, switzerland) according to the manufacturer's instructions following the same irrigation protocol as in subgroup a. subgroup a: instrumentation of mesial canals was done by continuous rotary technique with protaper universal rotary files (dentsply maillefer, ballaigues, switzerland) connected to x - smart endomotor (dentsply maillefer, switzerland) according to the manufacturer's instructions subgroup b: instrumentation was done by reciprocating rotary technique with protaper rotary files connected to waveone endomotor (dentsply maillefer, switzerland) according to the manufacturer's instructions following the same irrigation protocol as in subgroup a. group 2: k3xf group, 20 teeth were made into two subgroups (n = 10) subgroup a: instrumentation was done by continuous rotary technique with k3xf rotary files (sybronendo, glendora, ca, usa) connected to x - smart endomotor according to the manufacturer's instructions up to iso size 25 and 06% tapersubgroup b: instrumentation was done by reciprocating rotary technique with k3xf rotary files connected to waveone endomotor following the manufacturer's instructions up to iso size 25 and 06% taper . Subgroup a: instrumentation was done by continuous rotary technique with k3xf rotary files (sybronendo, glendora, ca, usa) connected to x - smart endomotor according to the manufacturer's instructions up to iso size 25 and 06% taper subgroup b: instrumentation was done by reciprocating rotary technique with k3xf rotary files connected to waveone endomotor following the manufacturer's instructions up to iso size 25 and 06% taper . Group 3: lsx group, 20 teeth were made into two subgroups (n = 10) subgroup a: instrumentation was done in crown down method by continuous rotary technique with lsx rotary files (lightspeed technology inc ., san antonio, tx, usa) connected to tcm endo v endomotor (nouvag). Initially, #15, #20 lsx files were used, and then #25 lsx file was used up to the wl . All the teeth were selected with initial minimum working width of 0.1 mm and apical enlargement was done upto three sizes larger than the initial apical file i.e., upto iso size 25subgroup b: instrumentation was done by reciprocating rotary technique with lsx rotary files connected to waveone endomotor following the same protocol as in subgroup a. subgroup a: instrumentation was done in crown down method by continuous rotary technique with lsx rotary files (lightspeed technology inc ., san antonio, tx, usa) connected to tcm endo v endomotor (nouvag). Initially, #15, #20 lsx files were used, and then #25 lsx file was used up to the wl . All the teeth were selected with initial minimum working width of 0.1 mm and apical enlargement was done upto three sizes larger than the initial apical file i.e., upto iso size 25 subgroup b: instrumentation was done by reciprocating rotary technique with lsx rotary files connected to waveone endomotor following the same protocol as in subgroup a. adobe photoshop software [figure 1a] was used to export and evaluate the preoperative [figure 1b] and postoperative cbct images [figure 1c] (axial sections). The surface area of root canals at three different levels, i.e., at 1, 3, and 5 mm cross - sections was measured according to the protocol given by zer . The difference between the post- and pre - operative values represented the increase in root canal surface area at that cross - section [figure 1d]. (a) preoperative marking of root canal outline using magic wand tool of adobe photoshop software to measure the root canal surface area; (b) preoperative root canal outline to measure the root canal surface area; (c) postoperative root canal outline to measure the root canal surface area; (d) diagrammatic representation of increase in root canal surface area for mesiobuccal canal at 5 mm; (e) diagrammatic representation of root canal transportation for mesiobuccal canal at 5 mm; and (f) preoperative distance measurements for mesiobuccal canal of a sample from root canal wall to external root surface using cone - beam computed tomography software the distances from the root canal outline to the external surface of root were measured in all the four directions, both preoperatively and postoperatively as per the method suggested by gambill et al . [figure 1e and f]. To standardize the procedure and avoid variations in results, a single operator conducted the study . Differences in increase of root canal surface area and canal transportation between groups were analyzed by one - way analysis of variance, followed by tukey pairwise multiple comparison tests . Adobe photoshop software [figure 1a] was used to export and evaluate the preoperative [figure 1b] and postoperative cbct images [figure 1c] (axial sections). The surface area of root canals at three different levels, i.e., at 1, 3, and 5 mm cross - sections was measured according to the protocol given by zer . The difference between the post- and pre - operative values represented the increase in root canal surface area at that cross - section [figure 1d]. (a) preoperative marking of root canal outline using magic wand tool of adobe photoshop software to measure the root canal surface area; (b) preoperative root canal outline to measure the root canal surface area; (c) postoperative root canal outline to measure the root canal surface area; (d) diagrammatic representation of increase in root canal surface area for mesiobuccal canal at 5 mm; (e) diagrammatic representation of root canal transportation for mesiobuccal canal at 5 mm; and (f) preoperative distance measurements for mesiobuccal canal of a sample from root canal wall to external root surface using cone - beam computed tomography software the distances from the root canal outline to the external surface of root were measured in all the four directions, both preoperatively and postoperatively as per the method suggested by gambill et al . [figure 1e and f]. To standardize the procedure and avoid variations in results, a single operator conducted the study . Differences in increase of root canal surface area and canal transportation between groups were analyzed by one - way analysis of variance, followed by tukey pairwise multiple comparison tests . In both mesiobuccal and mesiolingual canals, increase in root canal surface area was significantly more between protaper universal and lsx groups, k3xf, and lsx groups . There was no significant difference between protaper universal and k3xf groups at 1, 3, and 5 mm cross - sections at 1 mm, 3 mm and 5 mm cross - sections in both continuous rotary and reciprocating rotary techniques . Increase of root canal surface area was not significant (p = 1.000) for any given rotary system in this study when used in either continuous rotary or reciprocating rotary techniques at all the three levels [table 1]. Tukey pairwise multiple comparison tests to determine the significant increase in root canal surface area between the groups at 5, 3, and 1 mm for mesiolingual canal mesio - buccal and mesio - lingual canals were analysed independently in all the samples . Individual canals vary both in angle of curvature and radius of curvature but were not measured as it was not the aim in the present study . However measurement of canal curvature and radius of curvature in all the three dimensions is possible only with -ct . Increase in root canal surface area and canal transportation in both the mesial canals was clinically significant but was not statistically significant . In both of the mesial canals, canal transportation was significantly more between protaper universal and k3xf groups, k3xf and lsx groups, and protaper universal and lsx at all the three cross - sections both in continuous rotary and reciprocating rotary techniques . Canal transportation was seen in mesial direction in all the samples, i.e., opposite to the direction of canal curvatures for all the rotary systems used . This parameter was not measured as it was not the aim in the present study . Increase in canal transportation was not significant (p = 0.986) for any given rotary system in this study when used in either continuous rotary or reciprocating rotary techniques at all the three levels [table 2]. Tukey pairwise multiple comparison tests to determine the significant increase in root canal transportation between the groups at 5, 3 and 1 mm for mesiolingual canal increase in root canal surface area and canal transportation was measured at 1 mm, 3 mm and 5 mm short of the apex as this was the region most crucial with respect to canal aberrations . Transportation of the root canal is considered to be an important parameter to assess the root canal preparation, particularly in curved root canals . Severe canal transportation associated with an over reduction of sound dentin along the inner aspect in the middle part and along the outer aspect in the apical part of the root canal may result in a marked reduction of the fracture resistance of enlarged root canals . The effect of reciprocating rotary technique on increase of root canal surface area and canal transportation has not been well investigated, and very few studies compared the canal transportation in continuous rotary and reciprocating rotary techniques . Hence, the present study was aimed at determining the increase in root canal surface area and canal transportation in both continuous rotary and reciprocating rotary techniques . On comparison of the increase in root surface area between protaper universal and k3xf groups, there was no significant difference (p> 0.05) at all the three cross - sections . This might be because both groups were enlarged to the same apical diameter (0.25 mm) and both of them had a positive rake angle for efficient cutting . While comparing increase in root canal surface area between protaper universal, k3xf, and lsx groups, it showed that there was a significant increase in root canal surface area (p <0.05) for protaper universal and k3xf groups when compared to lsx group . This might be because the length of cutting portion of lsx files was greatly reduced (0.52 mm) with no taper, presence of small radial lands, and noncutting tip whereas protaper and k3xf systems had positive rake angle that led to increased cutting of dentin . Similar results were reported by rao et al . Where they found that least amount of dentin was removed with lsx files when compared to k3 and mtwo groups . On comparison of the increase in root canal surface area between two subgroups, i.e. In continuous rotary and reciprocating rotary techniques, of a given group, there was no significant difference (p> 0.05) between each other . This might be because the reciprocating rotary technique increases the cyclic fatigue resistance of the instrument but had no extra beneficial effects in the enlargement of the root canal . Significantly more apical transportation in mesial direction in protaper universal group (p <0.05) when compared to k3xf and lsx groups might be because of the variable taper along the length of cutting portion, positive rake angle without radial lands, and the triangular cross - section of protaper rotary system . Schfer and vlassis where they concluded that protaper group showed maximum canal transportation (p <0.05) when compared to k3, mtwo, and race systems . In the present study, canal transportation was significantly less in k3xf group (p <0.05) when compared to protaper universal group . This might be because of the variable core diameter along the length of cutting portion of k3xf that led to maintenance of canal curvature . Least canal transportation in lsx group might be because of the short cutting surface (0.52 mm) with small radial lands, neutral rake angle, smooth, flexible shaft, and noncutting tip that led to minimum canal deviation . The present study included teeth with moderate root canal curvatures (between 20 and 30) as most of the mesiobuccal and mesiolingual root canals of mandibular molars were found to have moderate curvatures . In the present study, all the three groups showed canal transportation toward the outer side of root curvature at all the three apical cross - sections . Similar results were reported by ayar and love and bergmans et al . With protaper and k3 systems . In the present study, there was statistically significant difference in increase of root canal surface area and canal transportation for the three rotary systems in either continuous rotary or reciprocating rotary motions . Limitations of the present study include only teeth with moderate root canal curvatures were included and usage of manual method to measure the root canal surface area by counting the number of pixels using adobe photoshop software . Further research in teeth with severe root curvatures followed by ex vivo and in vivo studies are needed to confirm these results in a clinical scenario . Increase in root canal surface area was significantly more in protaper universal and k3xf groups when compared to lsx group . Canal transportation was significantly more in protaper universal group when compared to k3xf and lsx groups . There was no significant difference in increase of root canal surface area and canal transportation between the continuous rotary and reciprocating rotary techniques in all the three groups . Uniformity in the increase of root canal surface area was better in lsx rotary system when compared to protaper universal and k3xf rotary systems . Lsx rotary system showed minimal canal transportation when compared to protaper universal and k3xf rotary systems.
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Chronic pain is a highly disabling condition, which can significantly reduce patients quality of life . Typically, chronic pain results in depression, anxiety, and loss of independence.14 chronic pain is associated with a wide range of cancer and non - cancer pain conditions including neuropathic pain and osteoarthritis . Prevalence of moderate and severe chronic pain is high in the general population, and in a large survey conducted by breivik et al in more than 46,000 adults in europe and israel, 19% of respondents reported having chronic pain.5 the incidence of pain increases significantly in patients with advanced cancer; in fact, up to 70% of them have been reported to experience chronic pain.6,7 the aim of the treatment of chronic pain is to increase the quality of life of the patient with a multidisciplinary and multimodal approach . The pharmacological agents currently used to treat chronic pain include non - opioid analgesics, in particular paracetamol and nonsteroidal anti - inflammatory drugs (nsaids), and opioids . Guidelines for the management of chronic pain recommend opioids for the treatment of moderate - to - severe pain in patients whose pain is not responsive to initial therapies with paracetamol and/or nsaids.8 despite their analgesic efficacy being well recognized, adverse events (aes) related to the opioid therapy which include headache, dizziness, fatigue, and especially the opioid - induced bowel dysfunction (oibd) can affect daily functioning and patient quality of life.912 opioid - induced constipation (oic), which is part of oibd, occurs in 40% of opioid - treated patients.13 in contrast with the adverse effects mediated through the central opioid receptors, which occur at the start of treatment and usually rapidly disappear, oic is mediated through intestinal opioid receptors and often persists.5,13 laxatives are the most common drugs used to prevent and treat oic . Laxatives do not address the underlying mechanisms of oic, and for this reason, they result ineffective in the majority of patients with oic.14,15 in a survey of oibd conducted by pappagallo14 and kumar et al16 54% of patients treated with laxatives did not report the desired improvement at least 50% of the time . One strategy to minimize or prevent oic while maintaining analgesic efficacy is blocking the intestinal opioid receptor while allowing the activation of the central one.17,18 oxycodone is an opioid receptor agonist commonly used for the treatment of patients with moderate - to - severe pain . Oxycodone stimulates opioid receptors in the gastrointestinal tract, modifying normal bowel activity and reducing gut motility, potentially resulting in constipation.19 naloxone is an opioid receptor antagonist with low systemic bioavailability . When administered orally, naloxone antagonizes the opioid receptors in the gut wall, while its extensive first - pass hepatic metabolism ensures the lack of antagonist influence on the central - mediated analgesic effect.20 a prolonged - release formulation consisting of oxycodone and naloxone (pr oxn) in a 2:1 ratio was developed trying to reduce the incidence of oic maintaining the analgesic effect compared to use of the sole oxycodone.19 in this review, we focus on the specific role of pr oxn in the management of chronic pain and oic in non - cancer patients . A literature search was conducted in the pubmed database using the term oxycodone and naloxone through april 2015 . The pubmed search generated 223 results, with 45 containing clinically relevant information of pr oxn . This review does not contain any new studies with human or animal subjects performed by any of the authors . Opioids have been increasingly being used in the last decade for the treatment of chronic cancer and non - cancer pain.2123 despite proven analgesic efficacy, their use is associated with constipation that can significantly have an impact on patients quality of life even more than the pain itself.24 in a multinational study conducted on more than 300 patients taking daily opioids (probe 1), constipation was the most common ae reported by 81% of patients, and it was often reported as severe.25 oic is attributed to the activation of the mu - opioid receptors present in the gut wall that lead to decreased gastric motility as well as decreased gastrointestinal, biliary, and pancreatic secretions.13,26 additionally, propulsion in the small and large intestines is inhibited, whereas non - propulsive contractions are increased . Bowel dysfunction in patients with oic, factors such as pain, medications other than opioids, diet, and underlying disease factors play an important role.27 the bowel function index (bfi) is a validated, clinician - administered, patient - reported questionnaire . Bfi is designed to evaluate oic in cancer and non - cancer chronic pain patients, and it uses a numerical scale from 0 (easy) to 100 (severe) to record a patient s subjective assessment of three items related to oic: ease of defecation, feeling of incomplete evacuation, and patient personal judgment of constipation . A lower score indicates a better bowel function; a score of <28.8 is considered a normal bowel function with respect to oic, and a bfi change of> 12 points is considered a clinically relevant change.2832 strategies for the management of oic symptoms include the use of laxatives and treatment with opioid antagonists . Laxatives are commonly used medication to reduce the constipation induced by the chronic use of opioids.33 despite the fact that laxatives can be useful in some circumstances, including delayed colonic transit, since oic has a unique etiology, they are not really effective in its treatment.25 the interaction between opioids and opioid receptors present throughout the gut can affect numerous gastrointestinal functions, including neural activity, motility, secretion, resorption of fluid, and blood flow.13,26 consequently, opioids delay gastric emptying and prolong transit time throughout the small and large intestines . For these reasons, laxatives, which predominantly act on the colon, frequently do not address the symptoms of oic . No single laxative is considered optimal for oic . Moreover, there are no direct comparative data on different laxatives in the prevention or treatment of oic, resulting in a lack of generally accepted guidelines regarding laxative use for this condition.34 furthermore, laxatives are associated with potential side effects including bloating, gas, and gastroesophageal reflux and may be associated with tolerability.35 the oibd arise from opioid - mediated actions on the central nervous system (cns) and gastrointestinal tract.36 in the cns, opioids interact with four receptor subtypes (mu, delta, kappa, and opioid receptor - like-1) inducing analgesia and reducing the gastrointestinal propulsion due to an alteration of autonomic outflow from the cns.36 nevertheless, the high density of mu receptors in the enteric system appears to mediate most of opioid gastrointestinal effects.37 studies of the human intestine suggest that delta and kappa receptors make a lesser contribution to oic.37 the incidence of constipation related to the opioid therapy can be reduced using opioid antagonists, such as methylnaltrexone, alvimopan, naloxegol, and naloxone.38 the aim of using opioid antagonist in patients with oic is to try to inhibit the actions of the opioids in the gut without affecting their central effect and maintaining their analgesic action.17 the subcutaneous injection of methylnaltrexone was initially approved only for palliative care in patients with advanced cancer when traditional oral laxatives fail.39 methylnaltrexone is, in fact, approved to treat oic in chronic non - cancer pain too.40 alvimopan is approved and indicated for short - term use to accelerate postoperative recovery of upper and lower gastrointestinal tracts after surgeries, but its use is actually characterized by cardiovascular safety concerns.40 oral naloxegol was approved in 2014 by the us food and drug administration as an add - on to existing pain therapy, and it has shown to be effective in increasing bowel movements.40 naloxegol is a pegylated naloxone molecule with mu - opioid antagonist activity, reduced central permeability, and oral bioavailability . Trials showed long - term safety and at least 12 weeks of effectiveness in patients with oic including patients not previously responding to laxative use.41 naloxone is a competitive opioid antagonist at opioid receptors inside and outside the cns primarily used intravenously for the treatment of opioid overdose . After systemic administration, it reverses both centrally and peripherally mediated opioid effects with a half - life amounting to 11.5 hours . Elimination is primarily by glucuronidation in the liver . When administered orally, naloxone antagonizes the opioid receptors in the gut wall, potentially reducing oic, while its extensive first - pass hepatic metabolism ensures the lack of antagonist influence on the central analgesic effect.42 to minimize or prevent oic while maintaining analgesic efficacy, a prolonged - release tablet consisting of oxycodone and naloxone in a 2:1 ratio was developed.43 several randomized controlled trials (rcts) have reported on the comparable analgesic efficacy of pr oxn and prolonged - release oxycodone (pr oxy), with a clinically relevant improvement in oic in various types of pain.4446 recently, a noninterventional, observational, real - life study evaluating the pain relief and oic with pr oxn treatment in daily practice in patients with chronic severe pain compared with previous pr oxy treatment was published.47 patients enrolled in this study were treated with pr oxy for at least the last 30 days before pr oxn treatment, and in particular, they reported oic despite the use of at least two laxatives with different mechanisms of action . Patients were switched immediately from pr oxy to pr oxn with equal oxycodone doses.47 the study found that pr oxn was superior to pr oxy in terms of pain relief, oic, and quality of life in patients with chronic pain previously treated with pr oxy and experiencing oic despite the use of at least two different laxatives.47 this study confirmed that pr oxn clinically improves oic even in patients experiencing laxative - refractory oic . Furthermore, the average bfi was <28.8 after 6 weeks of pr oxn treatment, indicating that most patients were no longer constipated despite opioid treatment.47 the observed improved pain relief during the study period was not related to an increased dose or increased use of analgesic, and it was probably due to a better adherence to the opioid therapy due to improved oic.47 osteoarthritis and spine pain are two of the leading causes of pain and disability worldwide.48 pain in these patients can reduce function and affects a person s ability to carry out his / her daily activities . Guidelines usually recommend the prescription of paracetamol as the first - line analgesic for these conditions.4953 recently, a systematic review and meta - analysis published by machado et al evaluating the efficacy and safety of paracetamol for spinal pain and osteoarthritis raised doubts related to the place of this medication as a first analgesic choice to treat such conditions.54 machado reports that there is high - quality evidence that paracetamol does not have a clinical effect as pain medication in patients with low back pain or osteoarthritis.54 these findings confirmed what was already underlined in the first draft of the new national institute for health and care excellence guidelines on the management of osteoarthritis; that is, paracetamol should not be routinely offered to patients as it might not be effective and was potentially associated with side effects when used at high doses for a long period of time.55 this decision was reversed due to the fact that the royal college of general practitioners, the primary care rheumatology society, and the british society for rheumatology raised concerns that removing paracetamol as an analgesia option could result in the increased use of oral nsaids and opioids.56 despite the limited availability of strong scientific evidence to support long - term opioid therapy for chronic non - cancer pain, use of opioids has increased substantially through the years.57 in the case of long - term opioid prescription to treat patient with spine pain or osteoarthritis, guidelines are in agreement with regard to providing adequate patient evaluation, judicious opioid dosing, and careful patient monitoring to minimize the risks of aes and abuse.57 oxycodone has been shown to be an effective analgesic in various types of pain, and its combination with naloxone in a fixed 2:1 ratio was tested in terms of analgesic efficacy and gastrointestinal tolerability in a number of clinical studies including rcts.4446 due to the degenerative nature of chronic pain in particular in patients with osteoarthritis, effective pain management often requires prolonged therapy . Consequently, the long - term effects of opioid treatments must be established.57 the effectiveness as pain medication of the fixed combination of oxycodone naloxone prolonged - release tablets was already demonstrated in studies of short duration (12 weeks).43,45,46 recently, a pooled analysis related to two 52-week extension phases evaluating the efficacy and the safety of pr oxn was published.58 the extension phases, during which all the patients received pr oxn, followed two rcts conducted in patients with moderate - to - severe non - cancer pain and oic to compare the efficacy and safety of the combination of oxycodone naloxone versus oxycodone alone . The pooled analysis showed that pain control was maintained with pr oxn throughout the 12-month study period.56 improvement in bowel function, indicated by a decrease in bfi scores, throughout the extension phase was significant in patients who switched from receiving pr oxy in the previous studies to pr oxn at the start of the extension phases . Forty - six percent of the patients experienced treatment - related aes.58 eight percent of the patients experienced constipation that was classified as possibly, probably, or definitely related to study drug . Diarrhea was considered possibly, probably, or definitely related to study drug in only 13 patients, and was considered unlikely related to study medication in five patients.58 chronic pain due to osteoarthritis increases dramatically with age, affecting approximately 60% of people aged over 65.59 opioids are recommended as part of a multipharmacological pain management for older patients to treat moderate - to - severe chronic pain impairing their daily quality of life . Limits of opioids use in this population are represented by a higher incidence of opioid - related side effects.6062 a 4-week, single - center, prospective observational study investigating the analgesic efficacy and tolerability of pr oxn in patients older than 70 years nave to strong opioids was published by guerriero et al.63 the effects of pr oxn on functional and cognitive status, mood, and quality of life were also analyzed . Twenty - six percent of the patients enrolled in this study complained of constipation at baseline, but almost all had bfi values> 29, indicating some degree of bowel dysfunction.63 the administration of pr oxn was associated with a clinically nonsignificant decrease in bfi values throughout the study; otherwise, at the last follow - up visit, fewer patients were still complaining of constipation.63 overall, pr oxn was well tolerated during the 28 days of treatment . Out of the 53 patients who started treatment, only one patient experienced a severe ae leading to pr oxn discontinuation within the first week.63 one of the major concerns of the opioid therapy in the older patients is their potential negative effect on the cognitive function . The impairment of the cognitive function seems to be related to an increase in the daily dose and not with stable doses of opioids.6466 in the study by guerriero et al low dose of pr oxn did not impact negatively on the overall cognitive status of the study population.63 opioids are routinely used in the treatment of moderate - to - severe pain in a wide range of conditions . Despite their analgesic efficacy, chronic management with opioids is often compromised by adverse effects which include nausea, sedation, euphoria / dysphoria, itching, and what is called oibd . Laxatives are the most commonly used treatments for oibd, although they are not very effective, since they are not mechanism based . If administered orally, naloxone antagonizes the opioid receptors in the gut wall, while its extensive first - pass hepatic metabolism ensures the lack of antagonist influence on the central opioids receptor . Naloxone does not appear to impair the opioid analgesic effect in the majority of patients . Treatment of oic improving adherence at the opioids pain therapy and the quality of life even after a 12-month therapy and in the older patients.
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Coronary collateral circulation (ccc) is an adaptive compensatory response to myocardial ischemia . In addition, ccc is an alternative source of blood supply to the myocardium which can be jeopardized by the failure of the original stenotic, or occluded vessel, to provide adequate blood flow to the targeted ischemic region . There are multiple collateral coronary arteries connecting to the normal coronary arteries in individuals without documented coronary artery disease (cad); however, most of these additional vessels are not angiographically visible ., in contrast, patients with coronary stenosis, or occlusion, develop various visible ccc . A well - developed ccc has a favourable impact on the prognosis of patients with cad by minimizing infarct size, reducing evolution of left ventricular aneurysm formation, improving ventricular function, and may lead to improved survival. Epicardial fat volume (efv) is a biomarker of visceral adipose tissue that has been shown to be correlated with adverse cardiovascular events, both short, and long- term. Endothelial dysfunction, and structural changes of the microcirculation are well established features of patients with increased efv. On the other hand, the relationship between efv and ccc is still unclear . Therefore, we sought to investigate the relationship between efv level and ccc in patients with cad . The study population consisted of 400 consecutive patients with stable cad who underwent coronary angiography . The patients with a history of coronary angiogram showing a lesion of <80% stenosis, history of percutaneous coronary intervention, or coronary artery bypass grafting were excluded from the analyses . Finally, 152 patients with stable cad were included in our study . The clinical risk factors for the patients, such as age, gender, hypertension, diabetes mellitus (dm), history of hyperlipidemia, smoking status, and family history, were recorded . Hypertension was defined as systolic blood pressure> 140 mmhg and/or a diastolic pressure> 90 mmhg at least two times, or if the individual was taking antihypertensive medications . The diagnosis of dm was based on the previous history of diabetes treated with, or without, drug therapies . Current smokers were defined as those who had smoked for some period during the past year . Furthermore, on admission, each patient was evaluated for blood pressure, heart rate, previously used drugs, presence of angina, high sensitivity c - reactive protein (hs - crp), serum creatinine, glucose, lipid profile and hematological indices . Also each patient underwent transthoracic echocardiography using the biplane simpson method measuring left ventricular ejection fraction (lvef). Hematological indices were measured as part of the automated complete blood count (cbc) using a coulter lh 780 hematology analyzer (beckman coulter ireland inc, mervue, galway, ireland). The inclusion criteria were the presence of 80%, or greater, degree of diameter stenosis in at least one coronary artery . Therefore, since development of ccc is known to be inadequate in patients not comlpying with this criteria, they were excluded from the index study . Accordingly, grade 0 was classified as no filling; grade 1 classified as filling of side branches via collateral channels without visualization of the epicardial segment; grade 2 classified as a partial filling of epicardial major coronary artery via collateral channels; and grade 3 classified as complete filling of epicardial major coronary artery . In patients with more than one coronary lesion, and when there was more than one ccc, the ccc with the highest reentrop was used . The patients were classified into impaired ccc (group 1, reentrop grades 01) or adequate ccc (group 2, reentrop grades 23). Multivessel disease was defined as the presence of a lesion in two, or more major epicardial arteries . Continuous variables are expressed as mean sd, whereas categorical variables are expressed as percentage . Comparisons between two ccc groups were made using the student t test or mann - whitney u test or chi square tests, as appropriate . Comparison between rentrop grades were made using the analysis of variance, and turkey honestly significant difference test was chosen as a post hoc test . Multiple logistic regression analysis was performed to identify the independent predictors of ccc using variables showing marginal association with it on univariate testing (p> 0.01). Receiver - operating characteristics (roc) analyses were used to detect the cutoff value of efv in the prediction of ccc . All statistical analyses were carried our using spss 16.0 for windows (spss inc, chicago, illinois). Ace: angiotensin - converting enzyme; arb: angiotensin receptor blocker; bmi: body mass index . Cad: coronary artery disease; ccb: calcium channel blocker; ccc: coronary collateral circulation; hs - crp: high sensitivity c - reactive protein; efv: epicardial fat volume; gfr: glomerular filtration rate; hdl: high - density lipoprotein; hs- oad: oral antidiabetic drug; ldl: low - density lipoprotein; lvef: left ventricular ejection fraction . Bmi: body mass index; efv: epicardial fat volume; mpv: mean platelet volume; rdw: red cell distrubution width; tg: triglyceride . The study population consisted of 400 consecutive patients with stable cad who underwent coronary angiography . The patients with a history of coronary angiogram showing a lesion of <80% stenosis, history of percutaneous coronary intervention, or coronary artery bypass grafting were excluded from the analyses . Finally, 152 patients with stable cad were included in our study . The clinical risk factors for the patients, such as age, gender, hypertension, diabetes mellitus (dm), history of hyperlipidemia, smoking status, and family history, were recorded . Hypertension was defined as systolic blood pressure> 140 mmhg and/or a diastolic pressure> 90 mmhg at least two times, or if the individual was taking antihypertensive medications . The diagnosis of dm was based on the previous history of diabetes treated with, or without, drug therapies . Current smokers were defined as those who had smoked for some period during the past year . Furthermore, on admission, each patient was evaluated for blood pressure, heart rate, previously used drugs, presence of angina, high sensitivity c - reactive protein (hs - crp), serum creatinine, glucose, lipid profile and hematological indices . Also each patient underwent transthoracic echocardiography using the biplane simpson method measuring left ventricular ejection fraction (lvef). Hematological indices were measured as part of the automated complete blood count (cbc) using a coulter lh 780 hematology analyzer (beckman coulter ireland inc, mervue, galway, ireland). The inclusion criteria were the presence of 80%, or greater, degree of diameter stenosis in at least one coronary artery . Therefore, since development of ccc is known to be inadequate in patients not comlpying with this criteria, they were excluded from the index study . Accordingly, grade 0 was classified as no filling; grade 1 classified as filling of side branches via collateral channels without visualization of the epicardial segment; grade 2 classified as a partial filling of epicardial major coronary artery via collateral channels; and grade 3 classified as complete filling of epicardial major coronary artery . In patients with more than one coronary lesion, and when there was more than one ccc, the ccc with the highest reentrop was used . The patients were classified into impaired ccc (group 1, reentrop grades 01) or adequate ccc (group 2, reentrop grades 23). Multivessel disease was defined as the presence of a lesion in two, or more major epicardial arteries . Continuous variables are expressed as mean sd, whereas categorical variables are expressed as percentage . Comparisons between two ccc groups were made using the student t test or mann - whitney u test or chi square tests, as appropriate . Comparison between rentrop grades were made using the analysis of variance, and turkey honestly significant difference test was chosen as a post hoc test . Multiple logistic regression analysis was performed to identify the independent predictors of ccc using variables showing marginal association with it on univariate testing (p> 0.01). Receiver - operating characteristics (roc) analyses were used to detect the cutoff value of efv in the prediction of ccc . All statistical analyses were carried our using spss 16.0 for windows (spss inc, chicago, illinois). Ace: angiotensin - converting enzyme; arb: angiotensin receptor blocker; bmi: body mass index . Cad: coronary artery disease; ccb: calcium channel blocker; ccc: coronary collateral circulation; hs - crp: high sensitivity c - reactive protein; efv: epicardial fat volume; gfr: glomerular filtration rate; hdl: high - density lipoprotein; hs- oad: oral antidiabetic drug; ldl: low - density lipoprotein; lvef: left ventricular ejection fraction . Bmi: body mass index; efv: epicardial fat volume; mpv: mean platelet volume; rdw: red cell distrubution width; tg: triglyceride . A total of 152 patients with stable cad (age: 65 10 years, male ratio: 70%) were included in the study . Compared to the patients with adequate ccc, patients with impaired ccc exhibited higher red cell distrubution width (rdw), mean platelet volume (mpv), triglyceride (tg), hs - crp values and frequency of bmi and preinfarction rates . Compared to the patients with impaired ccc, patients with adequate ccc manifested significantly higher efv levels . Furthermore, reentrop grade 2 and 3 patients had significantly higher efv levels when compared to the reentrop grade 0 and 1 patients . Multivariate logistic regression test was employed for determining the independent predictors of impaired ccc (figure 1, table 2). The variables that were found to have significance in the univariate analysis (preinfarction angina, rdw, mpv, tg, body mass ndex (bmi), hs - crp) were included in the multivariate model . Among those, efv (or: 1.059; 95% ci (1.035 1.085); p = 0.00), presence of angina were found to be the independent predictors of adequate ccc . In receiver - operating characteristic curve analysis, the efv value> 106.5 ml yielded an area under the curve value of 0.84, with 49.3% sensitivity and 98.3% specificity (figure 2). Our study demonstrated that the presence of angina and high efv levels may serve to be independent predictors of adequate ccc . The clinical significance of our findings is unclear, but may help in developing prediction models in the near future . Coronary collateral vessels are structures that are normally present in the human heart, though being invisible by angiography ., these are interconecting branches between the main arteries which serve as an alternative conduits for blood flow in obstructive coronary heart disease . The ccc development occurs as a result of new vessel formation and growth of pre - existing arterioles (arteriogenesis). There are many factors influencing the development of the ccc, such as percent diameter of coronary artery stenosis, duration of angina, dm, hypertension smoking status, hypoxia, endothelial dysfunction, exercise, oxidative stress genetic factors, and drugs used . A well - developed ccc has a favourable impact on the prognosis of patients with coronary artery disease by minimizing infarct size, reducing evolution of left ventricular aneurysm formation, improving ventricular function, and leading to improved survival . Epicardial adipose tissue is a type of visceral adipose tissue functioning as a metabolically active endocrine organ . The relationship between efv presence and components of cardiovascular diseases have been described in several studies. Moreover, it has been shown that epicardial adipose tissue is in direct contact with the myocardium, and it is very metabolically active and can secrete a large number of cytokines and vasoactive peptides, including free fatty acids, interleukin-6, tumor necrosis factor (tnf)-, angiotensin ii, and plasminogen activator inhibitor-1 . Although the causes of ccc are completely unknown, one of the responsible mechanisms is thought to be the activation of immune sytems with predominant involvement of cytokines and chemokines . Cytokines, which are one of the important sources of inflamatory mediators, have emerged as key cellular determinants of progression of the ccc . Previous articles alerted the scientific community that hyper activation of cytokines and adhesion molecules which were in concordance with the severity of diseaese may give prognostic data about the severity of the ccc ., recent studies have shown that cytokines, which mediate the immune response to inflamatory disorders, were also secreted from epicardial adipocyte tissues. On the basis of these facts, efv may be associated with the patholophysiological processes causing ccc . There are also several other pathogenic mechanisms thought to be the causative role for the association between increased efv and the severity of the disease . Epicardial fat tissue seems to affect the endothelial function and increase sympathetic activity by its paracrine effect . Aydin et al . Showed that efv correlated with endothelial dysfunction assessed by flow - mediated dilatation in patients with metabolic syndrome . Based on these data, associated endothelial dysfunction can be considered as one of the mechanisms for predicting severity of cad by assessing efv levels . The present study findings do not prove a direct link between increased epicardial fat volume and the development of ccc . Although increased efv could be a marker of cad severity, and also be a predictive factor for adequate ccc, the possible causative effect of epicardial fat accumulation has not been clarified . Qualitative analysis of epicardial fat using biochemical techniques will be required to confirm the effect of accumulated epicardial fat on the progression of coronary atherosclerosis . The relatively small sample size is one of the major limitations of our study and certainly should be confirmed in a larger, better designed, randomized trial . Morover, the cross - sectional design of our study makes it difficult to comment on the causal relationship of efv and impaired ccc, and the observational design preclude us to adequately assess these important links . We conducted the index study in the experimental setting, and applied the observational design, therefore, not drawing any definite pathophysiologic mechanisms for the association between increased efv and the severity of ccc . It is possible that similar mechanisms may be attributable for increase in efv level in patients with ccc . Finally, one of the most important limitations was the failure to measure some parameters, such as fgf (fibroblast growth factor), vascular endothelial growth factor, no, tnf- that could be helpful in evaluating the relationship between efv and impaired ccc in detail . In conclusion, further studies to investigate the cause and effect relationship between efv and ccc are required.
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Nigella sativa l. (ns) is a vegetal specie of the ranunculaceae family, commonly known as black cumin seed, neguilla or ajemuz, that is widely cultivated in the mediterranean region . Its seeds have played an important role over the years in ancient islamic system of herbal medicine and in spain, where they have been traditionally used in folk medicine . Ns seeds have shown several therapeutic effects such as prevention of cancer, antihypertensive effect, anti - inflammatory, analgesic, and antihistaminic actions . The volatile oil from this plant presents a relaxant action on different smooth muscles and tracheal muscles of guinea pigs . There is evidence of anticonvulsant and antioxidant effects against pentylenetetrazol - induced kindling in mice . . Likewise, aqueous extract of this plant suppresses penicillin - induced epileptic activity in rats . This anticonvulsant effect is a consequence of selectively altering the monoamine level in different brain regions . In recent works, histopathological changes of neurodegeneration in the frontal cortex and brain stem in neurons after exposition to toluene have been observed [15, 16]. The administration of ns extract and thymoquinone (major component from ns volatile oil) causes morphologic improvement over apoptosis and indicates that ns therapy is useful as a potential treatment of neurodegeneration prevention . Ns seeds composition includes nutritional components such as carbohydrates (glucose, xylose, rhamnose, and arabinose), vitamins as thiamine, riboflavin, pyridoxine, niacin and folic acid, mineral elements, and proteins . The ns seeds are also a source of calcium, iron and potassium, alkaloids (nigellidine, nigellimine, and nigellicine), 36%38% fixed oil and 0.4%2.5% essential oil . The fixed oil is mainly composed of unsaturated and essential fatty acids (linoleic acid, followed by oleic acid) whereas the volatile oil has been shown to contain 18.424% thymoquinone and 46% monoterpenes such as p - cymene and pinene [5, 20]. Thimoquinone, as indicated above, is thought to be the main active component of ns seeds and suppresses itself epileptic seizures in rats, while a monodesmosidic triterpene saponin, -hederin, has also been isolated from the extract of ns seeds and proved to exert antitumoral activity . Three flavonoid glycosides and triterpene saponins were also identified from nigella sativa, together with four phospholipid classes: phosphatidylcholine phosphatidylethanolamine, phosphatidylserine, and phosphatitdylinositol [24, 25]. In previous studies we demonstrated that aqueous and methanolic extracts of ns seeds exert a potent sedative and depressive effect on cns and induce analgesia . The effect on cns resulted in a significant reduction of spontaneous motility, a decrease in normal body temperature and significant analgesic action against hot - plate and pressure tests . Body temperature reduction can be interpreted as an index of alteration of various central neurotransmitters; anxiety and sedation are mainly mediated by the gaba - a receptor in the cns . Since depressant action was confirmed specially for the methanolic extract, we decided to study whether the addition of this methanolic dry extract in cortical neurons culture could exert any influence on the secretion of the excitatory amino acids aspartate (asp) and glutamate (glu), and the inhibitory amino acids gaba and glycine (gly), as well as the presence of these amino acid neurotransmitters in the extract . We also considered its effect on the amino acids secretion when stimulated by a depolarizing agent and its effect on cultured neurons viability . The viability of the cultured neurons after exposition to ns extract concentrations 2.5, 25, and 250 g / ml during 15 and 60 minutes are shown in figure 1 . Results are expressed as a percentage with respect to control value (100% viability). The amino acids gaba, gly, glu, and asp were measured in ns methanolic extract by hplc . The results are expressed as pmoles / mg of ns extract (table 1). The results showed gly as the most abundant, followed by gaba, glu, and asp, respectively . The amino acids content in the extract was subtracted from the total amino acid content measured in the cellular medium after neuronal stimulation with ns extract so we can state that the final amino acids content in cell culture is a direct consequence of cell release . The release of the four amino acids after stimulation with the chosen nontoxic concentrations of ns extract (2.5, 25, and 250 g / ml) showed a diminished secretion response that was statistically significant (p <.05 for gly and glu; p <.01 for gaba; p <.001 for asp) after 15 minutes of incubation with respect to control (neuronal cells stimulated with normal locke medium during the same period of time and considered as 100% secretion) (figures 2, 3, 4, and 5). Gly and asp release was reduced in a dose dependent manner; glu and gaba showed a tendency to retrieve control values, although their secretion was lower than control . This fall of aminoacids release is greater for higher extract concentration except for glu and gaba, which showed a tendency to recuperation to control values at the same time (figure 2). The hplc analysis revealed the same behavior for all the amino acids, with the exception of gaba, after treatment with ns extract during 60 minutes . The increased presence of this amino acid was statistically significant for 25 and 250 g / ml ns extract . In order to know the response to a depolarizing agent, cortical neurons were stimulated with ns extract at the indicated concentrations, during 15 and 60 minutes previous to depolarization with 60 mm kcl (figures 6 and 7). The neurons treated with ns extract during 15 minutes and subsequently stimulated with kcl showed a dose - dependent decrease in amino acids secretion with respect to control value (neuronal cells stimulated with locke medium), which was considered as 100% . The observed behaviour was more relevant for glu and asp at 25 and 250 g / ml than for gaba and gly under the same conditions (figure 6). Measurement of secretion mediated by kcl during 60 minutes revealed an inhibition of the liberation of these neurotransmitters . In this case, only gaba and glu were released in a dose - dependent manner (figure 7). The aim of the study was to determine the effects of ns methanolic extract on the release of neurotransmitter amino acids by measuring their concentrations in the culture media using hplc precolumn derivatization technique . Three concentrations of ns extract (2.5, 25, and 250 g / ml) and two time points (15 and 60 min) for the determination of the effects were used . This is a preliminary study which shows that exposure of the cultured neurons have a modulatory effect on the release and contents of these aminoacids . The 3-(4,5-dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide (mtt) assay was employed to estimate the cells viability when neurons were treated with ns extract . The three concentrations of dry methanolic extract used in our study did not affect cellular respiratory capacity at any of the two periods of time considered . These results allowed us to consider the adequate approach to the study of amino acid secretion in the conditions selected and to confirm the innocuous characteristics of the chosen extract concentrations . Our previous in vitro findings support the hypothesis that the sedative and depressive effects of nigella sativa (ns) observed in vivo, could be based on changes of inhibitory / excitatory amino acids levels . Several authors attribute the sedative effects of different plant extracts (valeriana officinalis l., scutellaria lateriflora l.) [27, 28] to its endogenous gaba concentration, although they also hypothesize that there exist other components of the vegetal extracts with benzodiazepine - like effects that may account for their in vivo effects . The aim of our research was to determine whether these substances that are present in the ns extract could mediate the specific effects previously observed in vivo through the secretion of asp, glu, gaba, and gly . First of all, the presence and content of the four amino acids in the extract was analyzed by hplc, showing a major presence of inhibitory amino acids (gly and gaba) (table 1). Then, the cell amino acids release was assayed after addition of several concentrations of ns extract to cultured neurons during two different periods of time (15 and 60 minutes). The aim of this approach was to observe whether neuronal secretion could be modified depending on the exposition time . High secretion of gaba was observed after 60 minutes' contact with ns extract at 25 and 250 g / ml . This time is close to the one that allowed the maximum effect in our in vivo study, close to 40 minutes . Under these conditions, similar results have been observed when high gaba concentrations are present in an aqueous extract of valeriana officinalis root which induced in vitro liberation of [h]gaba in rat synaptosomes by reversal of the gaba carrier and inhibition of its reuptake . Both plants are widely prescribed as sedative / anxiolytic ones . As the gaba presence in the methanolic extract of ns was confirmed with our results, it is possible to assume that a high level of gaba in the medium during cellular incubation and a longer period of exposition at this one could exert the same effect above described . Recent findings suggest that ns protects from induced generalized epilepsy in rats by selectively altering the monoamine level in different brain regions . This study pointed that ns possibly facilitates the inhibitory activity of the gabaergic system through a competitive agonist action in the benzodiazepine (bzd) site of the gaba receptor as well as the involvement of dopaminergic and noradrenergic system . The confirmed presence of gaba in the methanolic extract could be directly related to its conduct over the gaba receptor and this could explain the potent sedative and depressive effect on cns as previously reported . Moreover, the important presence of gly in the extract could be also related to its inhibitory action . The binding of both neurotransmitters to their receptors on the neuronal membrane induces hyperpolarization that could be responsible for a significantly lower secretion of amino acids with respect to control values . In addition to this, our results showed a decrease in neuronal excitatory activity derived from a diminished asp and glu secretion, specially the second one . Our results show the behavior of the cortical neurons and confirm what we expected from the previous study performed in vivo . Likewise the methanolic extract composition is able to mediate in the neuronal amino acids release . In this case, the ns extract had an influence on neuronal transmission because it modifies the neurotransmitter amino acids release . Depolarization of neuronal cells by application of high k+ concentration (60 mm kcl) induced a diminished amino acid release in both periods of time assayed . This diminished release was similar to nondepolarizing secretion except for gaba which also diminished its secretion . Although multiple mechanisms of neurotransmitter release evoked by elevated extracellular k+ may be involved [29, 30], we consider that the effect of ns extract over neuronal cells produces a drop in the transmission and is responsible for of the inhibitory effect indicated above . A possible action of ns extract over l - type calcium channels or an opening on potassium channels had been suggested by others authors [3135], and it could contribute to the relaxant activities of this plant . Furthermore, its possible effect over these channels could persist even after the extract was eliminated from the medium before depolarization . With respect to gaba secretion, the only amino acid which differs in its behavior with respect to nondepolarizing liberation, the diminished secretion may derivate from the membrane transporter - reversal for gaba above suggested . In our study, we have considered the possibility that the gaba carrier had been previously affected by the presence of ns extract during 15 or 60 minutes, inhibiting gaba reuptake and favouring its liberation from neurons . Other authors have concluded that extracellular k+ (50 mm kcl) provokes gaba release by reversal transporter of gaba . In our case, we observed a diminished liberation that could be justified by the previous loss of cellular gaba content as the neurons had been treated with the ns extract . In conclusion, this study suggests a sedative effect of ns methanolic extract by modification of neurotransmitter amino acids release, because the ns extract may induce an important release of gaba and gly in the cultured neurons medium and therefore, exert an increase in the agonist action over their receptors . The results explain the sedative and depressive effects observed in vivo by an increase in inhibitory amino acids at the synaptic terminals . Even more, this effect is complemented with a possible decrease of excitatory transmission, as it has been demonstrated in vitro and could contribute to inhibitory response . Minimum essential eagle's medium (emem) was obtained from bio - whittaker, and foetal bovine serum (fbs) and horse serum (hs) were procured from sera - lab (sussex, england). Standards of glutamate, aspartate, gaba and glycine were purchased from sigma (st . Louis, mo, usa). Reagents and solvents for hplc were triethylamine from sigma (st . Louis, mo, usa), acetic acid and methanol ultra gradient grade from merck (darmstadt, germany). Distilled water used for the preparation of buffers and standards was deionized with milli - q purification system . Syringe filters millex - gv were obtained from millipore (milford, ma, usa). Membrane filters (0.45 m pore size) from tecknochroma (barcelona, spain) were used for filtration of the mobile phase and samples . Cell proliferation kit ii (xtt), colorimetric determination, was purchased from roche diagnostics gmbh (mannheim, germany)., usa) coupled with a photodiode array detector shimadzu spd-10a (izasa, madrid, spain) was used for the amino acids isolation and quantification . The analytical system consisted of a waters ods spherisorb 150 4.6 mm i.d . ; 5 m packed column (teknokroma, barcelona, spain) as stationary phase, preceded by a guard column spherisorb rp-18, 5 m, 4 mm 4 mm . A microplate fluorescence reader fl600-biotek spectrofluorimeter was used for the quantification of cell viability at 492 nm . Seeds of nigella sativa l. were supplied by the medicinal and aromatic plants research institute of egypt (el cairo, egypt). Herbarium samples were authenticated by a taxonomist and a voucher specimen was deposited in the herbarium of the faculty of pharmacy, universidad complutense de madrid, with voucher number maf 161043 . The methanolic extract of the plant was prepared according to (science and technology program for development) cyted protocol for vegetal species from countries that are included in this program . Brain neurons were obtained from foetal rat brains of 19 days of gestation as previously described with minor modifications . Isolated neurons were suspended in eagle's minimum essential medium (emem) containing 0.3 g / l glutamine, 0.6% glucose, 5% phosphate buffered saline (pbs), 5% horse serum (hs), 100 u / ml penicillin and 100 g / ml streptomycin . Cells, at a density of 1 10 cells / well, were placed on plastic petri dishes of 24 wells, treated with 10 g / ml poly - l - lysine to aid attachment . The plates were incubated in a humidified incubator in an atmosphere of 5% co2/95% air at 37c . After 72 hours, nonneuronal cells (contaminating glial cells) were mitotically inhibited by exposure to cytosine arabinoside . The incubation medium was replaced by fresh medium to which cytosine arabinoside was added to a final concentration of 10 m . After 3 days, this medium was replaced by fresh medium and experiments were carried out using cultures ranging from 1015 days . Cell purity was checked by both cells staining with cresyl violet to identify neurons and with the specific antiglial fibrillary acidic protein (gfap) antibody to identify glial cells . Cortical neurons, after 7 days in culture, were detached from the culture plates with trypsin solution (0.25% trypsin and 0.02% edta in dulbecco's buffered saline without calcium and magnesium) and then the cells were fixed (during 30 min) with 2% p - formaldehyde . After two washes with 1 ml of pbs, the cells were treated (during 1 h) with anti - gfap antirabbit (at dilution of 1/500). Subsequently, the cells were washed with pbs and treated with antirabbit conjugated igg fitc, at 1/100, for 30 min . The glial cells in the cultures were 8.3 3.6% of the total (neural + glial cells). This assay is used as an index of cell survival or cellular respiratory capacity - based on method of mossmann and improved by weislow et al . And roehm et al . . The tetrazolium assay is based on the mitochondrial dehydrogenases (md) activity and their inactivation after cell death . In live cells, mtt is reduced to a highly water - soluble orange colored product, formazan dye . Neuronal cells were seeded in 96-multiwell plates at a density of 36 10 cells / well (in 200 l medium) and kept in the incubator until 8090% confluence . After this, the medium was removed and the cells were washed twice with pbs and the ns extract previously dissolved in pbs was added to the wells and incubated for 15 minutes or 1 hour . After each one of the treatment periods (15 or 60 min), the medium was removed and incubated with mtt solution (final concentration 0.3 mg / ml), according to the kit specifications . After 2 h incubation at 37c in a humidified atmosphere, orange dye solution was spectrophotometrically quantified using an elisa plate reader at 492 nm . The amount of orange formazan formed, as monitored by the absorbance, directly correlates to the number of living cells . Hplc analysis of amino acids was performed by a previously described method with minor modifications and with the equipment and conditions that were previously developed for the amino acids determination . Prior to hplc amino acid secretion analysis cells were washed twice, at 10 min intervals, with 1 ml of locke medium . After removing the medium, cells were stimulated for 15 min or 1 hour at 37c with 250 l fresh locke medium (control cells) or with 250 l of locke medium containing the dry methanolic extract of ns seeds at different concentrations (2.5, 25 and 250 g / ml). After stimulation, the wells medium (supernatant s1) was taken for amino acid valuation and cells were then stimulated with 250 l of 60 mm kcl for 15 min . After this, the cells secretion (supernatant s2) was removed for their valuation and cells were lysated with 250 l of distilled water for total intracellular amino acids content determination . The different supernatants obtained, s1 and s2, as well as the cell lysated, were lyophilized for their dansylation . The content of the four amino acids present in the methanolic extract was also determined by hplc analysis under the same conditions and these results were subtracted from the total amino acids content in cellular medium in presence of ns extract (s1) in order to obtain cellular secretion value itself . The results were calculated as the amino acid release into the incubation medium with respect to the total amino acid content . These results were expressed as a percentage of secretion with respect to control experiments that were considered as 100% . Data are presented as means sem of four separated experiments from different cell cultures, each one performed in triplicate with different batches of neuronal cells.
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During the epidemic, close contacts were prospectively identified by the hong kong special administrative region government department of health through standardized telephone interviews with all 1,755 confirmed sars patients within 1 week of hospital admission (february 15june 22, 2003). A close contact was defined as a person who had cared for, lived with (in the same household), or came into direct contact with body fluids of the sars patients within 10 days before hospital admission . A total of 3,612 close contacts were recorded; 505 were diagnosed as having sars . Of the remaining 3,107 contacts, 2,805 (90%) had a telephone number available, as reported by the primary patient . We successfully contacted 2,337 (83%) from october 23 to november 30, 2003, and 1,776 (57% of those eligible) consented to a telephone interview after the purpose of the study was explained to them by trained public health nurses . The interview consisted of questions that assessed the relationship between the patients and contacts; the timing, intensity and frequency of contact; precautionary measures adopted during contact with the patient; known contact with other sars patients; clinical symptoms of febrile, respiratory, gastrointestinal, or constitutional illness since february 2003; medical and travel history; and sociodemographic details . $25.00) were given to participants after blood was collected as compensation for time and travel costs . Samples were screened by the government virus unit of the department of health by using viral lysate enzyme - linked immunosorbent assay (elisa) (gbi biotech, beijing). Positive results were confirmed with immunofluorescence assay (ifa) and neutralization tests . For the ifa, microscopic slides coated with sars - cov a positive test is indicated by cytoplasmic fluorescence under uv microscopy . By using ifa as the standard, the elisa detects antibody with ifa titer of> 25 (i.e., sensitivity of 100%) and has a specificity of 95% . Neutralization test was performed by standard virologic method with vero e6 cells and sars - cov isolate 6109 . The reported sensitivity of 100% was for convalescent - phase serum samples taken a few weeks after the onset of infection in sars patients, which should apply to our study . During the early phase of infection therefore, the sensitivity is 80%90% (depending on the number of days after illness onset when the serum samples were taken). However, this sensitivity should not have affected our findings, which were based on tests carried out at least 6 months after the last reported case of sars in hong kong . The study received ethics approval from the department of health ethics committee, which complies with the declaration of helsinki . Of the 1,068 samples analyzed, 2 (0.19%, exact 95% ci 0.02%0.67%) had a positive titer (1:25 to 1:50 on ifa compared to at least 1:100 in most recovered sars cases) for sars - cov igg antibody . Neither participant with a positive sample reported a chronic medical condition or being sick with febrile or respiratory illness from february to august . Both seropositive participants arose from two superspreading events in hong kong, i.e., prince of wales hospital nosocomial outbreak and amoy gardens environmental point source community outbreak (1,3). The contact of the prince of wales hospital seropositive participant reported one other close contact, who was interviewed but declined to be tested . The other seropositive index patient living in amoy gardens was separately identified by three intrafamilial index patients, all of whom lived in the same household and reported only each other as close contacts . The participants who consented to testing were broadly similar to those who declined, except that the first group had relatively fewer children and fewer of the first group were men (table). However, those who consented to testing were more likely to report more frequent contact and closer relationships with sars patients, more febrile or respiratory illness episodes since february, and a travel history to sars - affected regions, which may have biased our seroprevalence estimate upwards . Includes washing hands before touching mouth, eyes, and nose; washing hands with soap; wearing face mask; using serving utensils during meals; adopting precautionary measures when touching possibly contaminated objects, washing hands after touching possibly contaminated objects; adopting home preventive measures (such as maintaining good ventilation and using bleach to clean surfaces and home appliances) against sars; and adopting workplace preventive measures (such as maintaining good ventilation, using bleach to clean surfaces and office furniture, and not allowing staff who are sick to come to work) against sars . The extent of seroconversion in close contacts of confirmed patients should provide the upper limit of sars - cov antibody seroprevalence in the general population, given the relatively intense exposure history of these persons to sars patients . Our finding of the near absence of transmission resulting in asymptomatic infection in this representative high - risk group of close contacts indicates that the prevailing sars - cov strains in hong kong almost always led to clinically apparent disease . Whereas some sars patients, especially healthcare workers, might have been initially admitted to reduce transmission to family members, virtually all sars patients (perhaps with very few exceptions in children) had severe disease requiring inpatient treatment; thus, we can infer that infection with sars - cov inevitably caused severe disease requiring hospitalization . Although our results suggest that sars - cov was a new virus in humans without a close precursor or an antigenically related virus that would have induced at least a small degree of cross - reactivity on serologic testing, a recent study on a select group of 938 healthy hong kong adults, whose serum had been previously stored as part of a hepatitis b serosurvey in 2001, indicated that 1.8% of the sample had acquired a sars - cov related virus infection at least 2 years before the 2003 sars outbreak (5). The investigators speculated that the virus that affected these healthy, seropositive persons was antigenically closer to the recently isolated animal sars - cov like virus (3) than human sars - cov, but interspecies transmission from animals to humans was probably inefficient, as the virus might not have adapted in the new host . This hypothesis would explain why only a few persons became infected and why they were likely to have been asymptomatic . This hypothesis would be compatible with the presumed asymptomatic infection observed in guangdong animal traders, especially in those who handled masked palm civets, who had a seropositivity rate of 72.7% (exact 95% ci 49.8%89.3%) in the absence of prior overt clinical disease (6). The limitations of the study include incomplete contact tracing, especially in the earlier parts of the epidemic, and potential recall bias from underreporting of contacts by some patients who were too sick to answer questions . Another possible shortcoming is the lack of a survey of close contacts who did not report a telephone number, although there is no reason to suspect they had a systematically different serologic profile . In fact, these were mostly nonhousehold contacts who would have had less intense exposure to sars patients . In addition, because peak infectivity, as indicated by viral load, usually occurred during week 2 of illness (7), when most of the patients would have been isolated in hospital (the mean onset - to - admission interval decreased from a maximum of 9.3 days in late february to 1.0 day by mid - may) (8), transmission to close contacts in the later stages of the epidemic was less likely . Finally, contacts who refused to participate (561) or refused to have serologic testing (708) might have done so because they were concerned about having had sars (possibly because of having had sars - like symptoms) and did not want to be identified and stigmatized as having been infected with sars - cov . Surveys in other countries with large - scale outbreaks such as canada, china, singapore, and taiwan should be undertaken to confirm our findings . Of the 1,068 samples analyzed, 2 (0.19%, exact 95% ci 0.02%0.67%) had a positive titer (1:25 to 1:50 on ifa compared to at least 1:100 in most recovered sars cases) for sars - cov igg antibody . Neither participant with a positive sample reported a chronic medical condition or being sick with febrile or respiratory illness from february to august . Both seropositive participants arose from two superspreading events in hong kong, i.e., prince of wales hospital nosocomial outbreak and amoy gardens environmental point source community outbreak (1,3). The contact of the prince of wales hospital seropositive participant reported one other close contact, who was interviewed but declined to be tested . The other seropositive index patient living in amoy gardens was separately identified by three intrafamilial index patients, all of whom lived in the same household and reported only each other as close contacts . The participants who consented to testing were broadly similar to those who declined, except that the first group had relatively fewer children and fewer of the first group were men (table). However, those who consented to testing were more likely to report more frequent contact and closer relationships with sars patients, more febrile or respiratory illness episodes since february, and a travel history to sars - affected regions, which may have biased our seroprevalence estimate upwards . Includes washing hands before touching mouth, eyes, and nose; washing hands with soap; wearing face mask; using serving utensils during meals; adopting precautionary measures when touching possibly contaminated objects, washing hands after touching possibly contaminated objects; adopting home preventive measures (such as maintaining good ventilation and using bleach to clean surfaces and home appliances) against sars; and adopting workplace preventive measures (such as maintaining good ventilation, using bleach to clean surfaces and office furniture, and not allowing staff who are sick to come to work) against sars . The extent of seroconversion in close contacts of confirmed patients should provide the upper limit of sars - cov antibody seroprevalence in the general population, given the relatively intense exposure history of these persons to sars patients . Our finding of the near absence of transmission resulting in asymptomatic infection in this representative high - risk group of close contacts indicates that the prevailing sars - cov strains in hong kong almost always led to clinically apparent disease . Whereas some sars patients, especially healthcare workers, might have been initially admitted to reduce transmission to family members, virtually all sars patients (perhaps with very few exceptions in children) had severe disease requiring inpatient treatment; thus, we can infer that infection with sars - cov inevitably caused severe disease requiring hospitalization . Although our results suggest that sars - cov was a new virus in humans without a close precursor or an antigenically related virus that would have induced at least a small degree of cross - reactivity on serologic testing, a recent study on a select group of 938 healthy hong kong adults, whose serum had been previously stored as part of a hepatitis b serosurvey in 2001, indicated that 1.8% of the sample had acquired a sars - cov related virus infection at least 2 years before the 2003 sars outbreak (5). The investigators speculated that the virus that affected these healthy, seropositive persons was antigenically closer to the recently isolated animal sars - cov like virus (3) than human sars - cov, but interspecies transmission from animals to humans was probably inefficient, as the virus might not have adapted in the new host . This hypothesis would explain why only a few persons became infected and why they were likely to have been asymptomatic . This hypothesis would be compatible with the presumed asymptomatic infection observed in guangdong animal traders, especially in those who handled masked palm civets, who had a seropositivity rate of 72.7% (exact 95% ci 49.8%89.3%) in the absence of prior overt clinical disease (6). The limitations of the study include incomplete contact tracing, especially in the earlier parts of the epidemic, and potential recall bias from underreporting of contacts by some patients who were too sick to answer questions . Another possible shortcoming is the lack of a survey of close contacts who did not report a telephone number, although there is no reason to suspect they had a systematically different serologic profile . In fact, these were mostly nonhousehold contacts who would have had less intense exposure to sars patients . In addition, because peak infectivity, as indicated by viral load, usually occurred during week 2 of illness (7), when most of the patients would have been isolated in hospital (the mean onset - to - admission interval decreased from a maximum of 9.3 days in late february to 1.0 day by mid - may) (8), transmission to close contacts in the later stages of the epidemic was less likely . Finally, contacts who refused to participate (561) or refused to have serologic testing (708) might have done so because they were concerned about having had sars (possibly because of having had sars - like symptoms) and did not want to be identified and stigmatized as having been infected with sars - cov . Surveys in other countries with large - scale outbreaks such as canada, china, singapore, and taiwan should be undertaken to confirm our findings.
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Key pathophysiologic changes observed in the brain of autistic individuals provide compelling evidence that autism begins during fetal development (casanova 2007; bauman and kemper 2005; rodier 2000). While genetic susceptibility undoubtedly underlies autism etiology in many cases (abrahams and geschwind 2008), environmental factors likely play a role as well (cook and scherer 2008). Evidence has recently emerged linking maternal prenatal exposures such as illnesses, medications, environmental toxins, and psychological stress with autism risk (connors et al . 2005; roberts et al . 2007; terbutaline, a 2 adrenergic receptor (b2ar) agonist, has been used off - label for the past 30 years to treat preterm labor . 1984; hsu et al . 1994; slotkin et al . 1994) and has been shown in animals to elicit biochemical alterations and structural damage in the immature brain during critical developmental periods (rhodes et al . 2004). When administered to rats at a neurodevelopmental stage equivalent to the mid - second to early third trimester in humans (rice and barone 2000), terbutaline dysregulates function of the enzyme adenylyl cyclase and leads to abnormal generation (excess and deficiency) of the signaling molecule cyclic adenosine monophosphate (camp) in different brain regions at different stages of development (rhodes et al . Terbutaline exposure during this prenatal window results in changes in the rat central nervous system similar to those found in autism postmortem studies such as cerebellar abnormalities (delays in synaptogenesis and reduction in purkinje cell number), deficient hippocampal development, and neuroimmune activation (rhodes et al . 2004; zerrate et al . 2007; bauman and kemper 1985; vargas et al . 2006). In humans, continuous administration of terbutaline for 2 weeks or more between 16 and 36.5 weeks gestation has been linked to concordance of autism spectrum disorders (asds) in dizygotic twins (connors et al . 2005). The study by connors et al . Ascertained 36 dizygotic twin pairs from autism clinics in which at least one twin had an asd diagnosis and found an association (relative risk = 2.28) between terbutaline exposure for more than 2 weeks and concordance for asds . The relative risk increased to 4.4 (p = 0.035) in those twin sets who were both male and without a family history of asds . Terbutaline and other b2ar agonists are food and drug administration (fda) approved for the treatment of asthma, one of the most common conditions that may complicate pregnancy . The use of medications to control asthma during pregnancy is still recommended since uncontrolled asthma is associated with poor birth outcomes (bracken et al . Previously, we reported that a diagnosis of asthma in the second trimester was associated with a twofold increased risk of autism in the offspring (croen et al . 2005). While factors related to the underlying disease process may put pregnant women with asthma at higher risk for having a child on the autism spectrum, the possibility that asthma treatment with b2ar agonists may explain this link requires investigation . We conducted a case control study to investigate the potential association between maternal exposure to terbutaline and other b2ar agonists during pregnancy and risk of delivering an infant subsequently diagnosed with an asd . Our study population was drawn from the childhood autism perinatal study, a large case control study examining pre-, peri-, and neonatal risk factors for asds among the membership of kaiser permanente of northern california (kpnc; croen et al . Kpnc is a group model integrated health plan that provides care for over 3.2 million northern california residents . The kpnc membership represents approximately 30% of the insured population in the region and is demographically similar to the residents of the counties served by kpnc, except that the very poor and very wealthy are underrepresented (krieger 1992). Cases and controls were identified from the cohort of infants born at a kpnc facility between january 1995 and june 1999 who remained kpnc members for at least 2 years following birth (n = 88,163). Cases were defined as children with at least one diagnosis of an asd, including autism (international classification of diseases 1999; icd-9-cm code 299.0) and asperger s disorder or pervasive developmental disorder not otherwise specified (icd-9-cm code 299.8) recorded anytime between january 1995 and december 2002 in kpnc outpatient clinical databases (n = 420). Children with fragile x syndrome (n = 2), tuberous sclerosis (n = 0), or neurofibromatosis (n = 0) were excluded . One control per case was randomly selected from the cohort of kpnc births without an asd diagnosis, frequency matched to cases on sex, birth year, and delivery hospital . Since we were interested in examining characteristics of the mother in relation to autism risk in the offspring, we randomly sampled one child for inclusion for each woman who contributed two children to the original study sample (13 case mothers, 5 control mothers). Only children whose mothers were kpnc members with pharmacy benefits from at least 1 month before conception through the end of the pregnancy were included (291 cases, 284 controls). Women with pharmacy benefits during this time period were very likely to have purchased all of their prescription medications at a kpnc pharmacy . Information on maternal exposure to b2ar agonists was ascertained from the kpnc pharmacy database, which records all dispensed prescriptions at kp pharmacies, and abstracted from prenatal medical records using a standardized form . All inpatient and outpatient prescriptions were identified in the 30 days before conception through the end of the pregnancy with the study child . B2ar agonists included: advair, albuterol, combivent, foradil, maxair, metaproterenol, ritodrine, serevent, terbutaline, tornalate, and xopenex (table 1). Only two b2ar agonists terbutaline is the generic for brethine, bricanyl, and brethaire, all of which were searched in the pharmacy database along with ritodrine but only prescriptions for terbutaline were found . Since generic terbutaline is inexpensive and comes in a variety of forms for administration, it was the b2ar agonist in use for tocolysis during the time period of this study . Topical, ophthalmic, and otic routes were excluded.table 1b2ar agonists and mimics included in analyses, listed by indicationb2ar agoniststotal (n = 575)n (%) asthma salmeterol xinafoate / fluticasone propionate (advair, advair hfa)0 (0.00) albuterol, albuterol sulfate (proventil, ventolin, accuneb)47 (8.17) ipratropium bromide / albuterol sulfate (combivent)0 (0.00) formoterol fumarate (foradil, foradil aerolizer)0 (0.00) pirbuterol acetate (maxair)0 (0.00) metaproterenol, metaproterenol sulfate (alupent, arm - a - med (metapro), metaprel)2 (0.35) salmeterol xinafoate (serevent)2 (0.35) levalbuterol hcl (xopenex)0 (0.00)pre - term labor ritodrine, ritodrine hydrochloride0 (0.00) terbutaline, terbutaline sulfate (brethaire, breathair, brethine)55 (9.57)b2ar 2-adrenergic receptor b2ar agonists and mimics included in analyses, listed by indication b2ar 2-adrenergic receptor four time periods of exposure were defined: preconception (the 30 days prior to the last menstrual period (lmp)), first trimester (in the 90 days post - lmp), second trimester (90180 days post - lmp), and third trimester (180 days post - lmp to date of delivery). To account for possible inaccuracies in lmp date, the date the prescription was dispensed and the number of days supplied were used to determine exposure status during each time period . Exposure during a given time period was assumed if a prescription was dispensed during the time period or the days supplied overlapped some portion of the time period . Duration of exposure was derived by summing the total number of days during pregnancy for which a b2ar agonist was prescribed . Maternal conditions that were indications for b2ar agonists during pregnancy were identified from inpatient and outpatient databases and abstracted from prenatal records . Clinical indicators included asthma, preterm labor, and conditions that predispose to preterm labor (bicornuate uterus, cervical incompetence, multiple fibroids, hypertension, diabetes, anemia, bacterial vaginosis, pyelonephritis, asymptomatic bacteriuria, chorioamnionitis, urinary tract infection, and sexually transmitted diseases). Information on several maternal and infant characteristics was obtained from health plan and vital statistics databases . Characteristics of cases and controls were compared using contingency tables for categorical variables and comparisons of means for continuous variables . Unconditional logistic regression analysis was conducted to estimate relative risks of asd associated with maternal use of b2ar agonists during pregnancy . Separate models were run for all b2ar agonists combined, terbutaline alone, and albuterol alone, the two most frequently used b2ar agonists . For each exposure definition, we examined time period of exposure as well as duration of exposure . We defined exposure duration categories based on the distribution of days of exposure among the control group . For terbutaline, most use was for 1 or 2 days, reflecting acute exposure, or for 10 or more days, reflecting maintenance tocolysis . We also examined exposure to terbutaline for 2 weeks, the duration found to be associated with asd concordance among dizygotic twins in a previous study . For albuterol, duration was dichotomized as> 21 days vs. 21 days, the cut - point representing the median length of exposure among control mothers . Women with no prescriptions or days supplied for any b2ar agonists for the entire period from 30 days before conception through the end of pregnancy were considered unexposed for all analyses . Maternal and infant characteristics associated with maternal exposure to b2ar agonists or case control status were treated as possible confounders and included as covariates in multivariable models as were the matching variables . All covariates were entered simultaneously into regression models and all statistical tests were two - tailed . Study procedures were approved by the kpnc northern california institutional review board and the california state committee for the protection of human subjects . Children with autism (n = 291) were more likely than controls (n = 284) to be a twin or triplet, second born, weighing <2,500 g at birth, and have a mother of older age and with higher level of education (table 2). Among controls, the frequency of exposure to b2ar agonists during pregnancy was higher among women carrying twins compared with singletons (45.5% vs. 13.6%, p <0.01), women who delivered preterm compared with term (29.4% vs. 13.9%, p = 0.08), and women who delivered low birth weight compared with normal birth weight children (40.0% vs. 13.9%, p = 0.02).table 2characteristics of the study population, kaiser permanente northern california births, 19951999characteristicsno . (%) p valueasd cases (n = 291)controls (n = 284)gender (male)243 (83.51)230 (80.99)0.43plurality singleton273 (93.81)273 (96.13)0.21 twin or triplet18 (6.19)11 (3.87)birth order first born121 (41.58)122 (42.96)0.18 second born113 (38.83)92 (32.39) third or later born57 (19.59)70 (24.65)maternal age <203 (1.03)8 (2.82)0.14 202425 (8.59)29 (10.21) 252964 (21.99)79 (27.82) 3034104 (35.74)99 (34.86) 353984 (28.87)60 (21.13) 4011 (3.78)9 (3.17)maternal education <hs10 (3.44)18 (6.34)<0.001 hs49 (16.84)78 (27.46) college163 (56.01)152 (53.52) post - graduate68 (23.37)34 (11.97) unknown1 (0.34)2 (0.70)maternal race / ethnicity white, non - hispanic157 (53.95)138 (48.59)0.23 white, hispanic40 (13.75)60 (21.13) black27 (9.28)24 (8.45) asian28 (9.62)24 (8.45) other39 (13.4)38 (13.38)gestational age <37 weeks25 (8.59)17 (5.99)0.23 37 weeks266 (91.41)267 (94.01)birthweight <2,500 g24 (8.25)10 (3.52)0.02 2,500 g267 (91.75)274 (96.48)parity no previous births126 (43.3)118 (41.55)0.03 one previous birth114 (39.18)91 (32.04) two or more previous births51 (17.53)75 (26.41)birth year 199585 (29.21)75 (26.41)0.81 199665 (22.34)72 (25.35) 199759 (20.27)57 (20.07) 199859 (20.27)62 (21.83) 199923 (7.90)18 (6.34)hs high schoolchi - square test characteristics of the study population, kaiser permanente northern california births, 19951999 the prevalence of exposure to any b2ar agonist was similar for the mothers of children with autism compared with mothers of control children for the entire pregnancy period (18.9% vs. 14.8%, p = 0.19) as well as for each trimester (table 3). Albuterol was the most prevalent b2ar agonist among mothers of both cases and controls during the preconception period (3.1% vs. 2.1%, p = 0.46), as well as in the first (4.8% vs. 3.2%, p = 0.31) and second (6.2% vs. 4.9%, p = 0.51) trimesters . Prevalence of exposure to any b2ar agonist was highest in the third trimester for both case and control mothers (14.1% vs. 12.0%, p = 0.45). Terbutaline accounted for the majority of thirdtrimester exposures for both cases and controls (11.0% vs. 7.8%, p = 0.18) followed by albuterol (4.1% vs. 4.2%, p = 0.95). After controlling for covariates (birth order, plurality, maternal age, maternal education, and gestational age) and matching variables (gender, birth year, and delivery hospital), the risk of delivering a child later diagnosed with an asd was not significantly elevated for women with any b2ar agonist exposures during pregnancy (table 3).table 3odds ratios (ors) and 95% confidence intervals (cis) for autism spectrum disorders (asds) associated with prenatal exposure to b2ar agonistsexposureno . (%) p valuecrude or (95% ci)adjusted or (95% ci)adjusted or (95% ci)asd cases (n = 291)controls (n = 284)any b2ar agonist preconception period9 (3.09)6 (2.11)0.461.6 (0.64.4)1.9 (0.66.1)2.0 (0.66.4) pregnancy period55 (18.90)42 (14.79)0.191.4 (0.92.1)1.1 (0.71.9)1.2 (0.72.0) first trimester15 (5.15)10 (3.52)0.341.6 (0.73.5)1.4 (0.63.6)1.6 (0.64.0) second trimester19 (6.53)15 (5.28)0.531.3 (0.72.7)1.3 (0.62.8)1.4 (0.63.1) third trimester41 (14.09)34 (11.97)0.451.3 (0.82.0)1.0 (0.61.8)1.1 (0.61.9)terbutaline preconception period0 (0.00)0 (0.00) pregnancy period33 (11.34)22 (7.75)0.141.6 (0.92.8)1.2 (0.62.3)1.2 (0.62.4) first trimester1 (0.34)0 (0.00)0.32 second trimester1 (0.34)1 (0.35)0.991.0 (0.116.7)0.8 (0.029.1)0.8 (0.031.8) third trimester32 (11.00)22 (7.75)0.181.5 (0.92.7)1.2 (0.62.3)1.2 (0.62.4)albuterol preconception period9 (3.09)6 (2.11)0.461.6 (0.64.4)1.9 (0.66.1)2.0 (0.66.4) pregnancy period27 (9.28)20 (7.04)0.331.4 (0.82.6)1.3 (0.72.6)1.5 (0.72.9) first trimester14 (4.81)9 (3.17)0.311.6 (0.73.8)1.6 (0.64.1)1.7 (0.64.4) second trimester18 (6.19)14 (4.93)0.511.3 (0.72.8)1.3 (0.63.0)1.4 (0.63.2) third trimester12 (4.12)12 (4.23)0.951.0 (0.52.4)1.0 (0.42.5)1.1 (0.42.7)b2ar 2-adrenergic receptor; asd autism spectrum disorder; or odds ratio; ci confidence intervalthe reference group had no exposure to any b2ar agonists or mimics from 30 days before conception through deliverychi - square testodds ratios were adjusted for maternal education, maternal age, birth type, gestational age, parity, birth hospital, birth year, and sexodds ratios were adjusted for the variables listed above in addition to asthma and preterm labor indicationsonly five case mothers and seven control mothers had exposure to any b2ar agonist during each trimester of pregnancy odds ratios (ors) and 95% confidence intervals (cis) for autism spectrum disorders (asds) associated with prenatal exposure to b2ar agonists b2ar 2-adrenergic receptor; asd autism spectrum disorder; or odds ratio; ci confidence interval the reference group had no exposure to any b2ar agonists or mimics from 30 days before conception through delivery odds ratios were adjusted for maternal education, maternal age, birth type, gestational age, parity, birth hospital, birth year, and sex odds ratios were adjusted for the variables listed above in addition to asthma and preterm labor indications only five case mothers and seven control mothers had exposure to any b2ar agonist during each trimester of pregnancy women with terbutaline exposure for> 2 days (n = 8 (3.2%) cases vs. n = 2 (0.79%) controls) had a fourfold increased risk of having a child subsequently diagnosed with an asd (oradj = 4.1; 95% confidence interval (ci), 0.722.4; table 4). Among women with> 2 days of exposure to terbutaline (range, 548 days), the mean (sd) duration of exposure was 16.4 (12.1) days for cases and 21 (15.6) days for controls . All women with> 2 days of exposure to terbutaline were exposed for this duration during the third trimester . In addition to terbutaline exposure at> 2 days, one case mother had third trimester exposure to albuterol, and two case mothers had second trimester exposure to albuterol . Results were similar for terbutaline exposure of 2 weeks or more (four cases, one control; or = 4.3; 95% ci, 0.538.5). The risk of asd was not elevated among women with> 21 days of exposure to albuterol during pregnancy, (n = 14 cases, n = 11 controls; oradj = 1.3; 95% ci, 0.53.2; table 4). Albuterol exposure of> 21 days duration spanned all three trimesters of pregnancy.table 4adjusted ors and 95% cis for asds associated with duration of exposure to b2ar agonists during pregnancyexposureno . (%) adjusted or (95% ci)adjusted or (95% ci)asd case (n = 291)control (n = 284)terbutaline> 2 days8 (2.75)2 (0.704)4.1 (0.822.4)4.4 (0.824.6) 12 days24 (8.25)19 (6.69)1.0 (0.52.0)1.0 (0.52.0)albuterol> 21 days14 (4.81)11 (3.87)1.3 (0.53.1)1.4 (0.63.5) 21 days13 (4.47)9 (3.17)1.2 (0.53.1)1.3 (0.53.4)b2ar 2-adrenergic receptor; or odds ratio; ci confidence intervalthe reference group had no exposure to any b2ar agonists from 30 days before conception through delivery; cut - points for duration based on distribution of length of exposure among control mothersodds ratios were adjusted for maternal education, maternal age, birth type, gestational age, parity, birth hospital, birth year, and sexodds ratios were adjusted for variables listed above in addition to asthma and preterm labor indicationsall women with> 2 days of exposure were exposed for this duration in the third trimester>21 days of exposure occurred across multiple trimesters adjusted ors and 95% cis for asds associated with duration of exposure to b2ar agonists during pregnancy b2ar 2-adrenergic receptor; or odds ratio; ci confidence interval the reference group had no exposure to any b2ar agonists from 30 days before conception through delivery; cut - points for duration based on distribution of length of exposure among control mothers odds ratios were adjusted for maternal education, maternal age, birth type, gestational age, parity, birth hospital, birth year, and sex odds ratios were adjusted for variables listed above in addition to asthma and preterm labor indications all women with> 2 days of exposure were exposed for this duration in the third trimester> 21 days of exposure occurred across multiple trimesters among women who took any b2ar agonist at some point during pregnancy, 30% of cases and 36% of controls had a diagnosis of asthma during pregnancy and 53% of cases and 43% of controls had a diagnosis of preterm labor . Among women who were exposed to terbutaline during pregnancy, 0 case mothers and 1 (4.5%) control mothers had a diagnosis of asthma only, 28 (84.5%) of case mothers and 18 (81.8%) of control mothers had a diagnosis of preterm labor only, and 3 (9.1%) case mothers and no control mothers had both asthma and preterm labor diagnoses . In order to explore the effect of terbutaline exposure on autism risk independent of the effect of the indicating condition logistic regression models were fit including both the treatment variable and indicating illness as predictor variables . Controlling for indication, the risk of asd associated with terbutaline exposure for> 2 days (oradj = 4.4; 95% ci, 0.824.6) or 2 weeks (oradj = 4.7; 95% ci, 0.453.3) during the third trimester of pregnancy remained elevated . We also estimated the effect of terbutaline exposure in a subsample restricted to those women with an indicating medical condition (asthma or preterm labor). In this subsample, terbutaline exposure for> 2 days (oradj = 5.6, 95% ci 0.934.1) or 2 weeks (or = 4.4, 95% ci 0.539.8) in the third trimester remained associated with autism risk . This estimate is not confounded by the effect of an indicating condition, as all women included had an indication, but it is possible that some of the effect is due to interaction between indication and exposure . All results were similar for the subset of children who were singletons (data not shown). We repeated all analyses in an expanded sample of women who had pharmacy benefits in the third trimester, whether or not they had pharmacy benefits throughout pregnancy (360 cases, 342 controls). In this larger sample, the risk of asd associated with terbutaline exposure longer than 2 days was significantly elevated after adjusting for indication and other covariates (11 cases, 2 controls; oradj = 5.8; 95% ci, 1.228.4). Results were similar for terbutaline exposure for 2 weeks (six cases, one control; or = 5.8; 95% ci, 0.748.6). It is unlikely that a single exposure or insult is responsible for all cases of biologically complex and clinically heterogeneous neurodevelopmental disorders such as those included in the autism spectrum . Yet, it is possible that downstream cellular signaling abnormalities, such as dysregulated camp generation, could act as a common pathway in the development of these disorders in some individuals (connors 2008; witter et al ., we investigated terbutaline and other b2ar agonists since animal studies have shown that these drugs produced functional changes in developing tissues and behaviors comparable to those noted in autism . In this case control study, we did not find evidence of an association between b2ar agonist exposure and autism risk when considering either any b2ar exposure or albuterol exposure specifically . We did find a suggestion of an association between prolonged maternal exposure to terbutaline for more than 2 days in the third trimester and asd risk, although the effect estimate was imprecise due to the small sample size . We did not see an association for second trimester exposure which is where most of the animal studies have focused . This may have been due to the fact that only two women had second trimester exposures to terbutaline in our population . One previous report linked prolonged terbutaline exposure during the second and third trimesters to asd concordance among 36 sets of dizygotic autism twin pairs (connors et al . 2005). Because twin pregnancies are at high risk for exposure to tocolytics, a dizygotic twin series provided an opportunity to explore a potential association in a relatively small sample size . However, twin pregnancies differ in many ways from singleton pregnancies and the twin sample previously studied was a clinical case series and not derived from a population - based frame . In this investigation, we were able to focus on the far larger group of singleton cases drawn from a large kpnc - enrolled sampling frame . Further review of our results also indicates that maternal treatment with b2ar agonists during pregnancy does not explain our previous observation of increased asd risk among asthmatic mothers (croen et al . 2005) although this study employs a larger study population than previous reports investigating b2ar exposure and autism risk in human populations, our analysis still generated statistically imprecise effect estimates . For measures of any exposure to all b2ar agonists, albuterol or terbutaline, odds ratios point estimates were also small in magnitude (below 2.0). For long - duration terbutaline exposure in the third trimester where a large odds ratio point estimate was seen, the proportion exposed was very small (<1% in the controls) and the resulting confidence limits were quite wide . This analysis had the advantage of relying on prospectively collected information on prescribed medications which allowed for examination of risk for specific periods during pregnancy and duration of exposure . However, these records document dispensed prescriptions and therefore exposure could have been misclassified if women did not take the medications as indicated . Although we controlled for many potential confounding factors, including indicators for preterm labor, the possibility that longer duration treatment with terbutaline may in fact represent a marker for an underlying pathogenic process (e.g., maternal stress or inflammation) rather than a risk factor for asd cannot be completely ruled out . A subset of 50 cases was also evaluated with the autism diagnostic interview - revised (lord et al . 94% met criteria for asd on both instruments and 100% met criteria on at least one . In addition, it was reported that at least 90% of children with an asd diagnosis recorded in the kpnc electronic databases had documentation consistent with a diagnosis of autism based on diagnostic and statistical manual of mental disorders (fourth edition) criteria (croen et al ., we were unable to stratify cases into phenotypic subgroups based on severity of asd or other characteristics as the data in the medical records were not sufficient to accurately and completely make these distinctions . Evaluation of genotypic subgroups may also be important in fully understanding the effects of maternal exposure to b2ar agonists . Of note, variants at two polymorphic sites on the b2ar gene whose function is believed to be related to increased responsiveness of the receptor to ligand have been associated preliminarily with autism risk (connors et al . 2005; cheslack - postava et al . 2007). In our study, genetic data were not available and there was no way to investigate whether b2ar exposure effects might have been stronger in subgroups defined by genotype . The possibility of a large - magnitude association between terbutaline exposure of more than 2 days in duration during the third trimester and autism risk does have public health implications . One or 2 days of treatment is consistent with brief exposure as defined in a letter sent by the fda in 1997 alerting practitioners, home healthcare agencies, insurance carriers, and others that the demonstrated value of tocolytics in general is limited to an initial, brief period of treatment, probably no more than 4872 h (us food and drug administration (fda) and nightingale 1997). As indicated above, more than half of the women in this category had exposures for 2 or more weeks duration . There already is a documented lack of evidence for safety and efficacy of maintenance tocolysis (larsen et al . Terbutaline s ability to arrest uterine contractions wanes within 48 h because the beta-2 receptors in the myometrium downregulate and desensitize in response to continuous stimulation (frambach et al . 2005). In addition, other factors that govern uterine contractions are likely to provide a continuing stimulus that is not overcome by b2ar agonists . In contrast to the mature receptor forms found in the uterus, b2ars in the immature brain sensitize in response to sustained stimulation by terbutaline, which leads to persistent activation and abnormal downstream intracellular signaling in animals (slotkin and seidler 2006). In 2003, the american college of obstetrics and gynecology published a practice bulletin for the management of preterm labor, stating that neither maintenance treatment with tocolytic drugs nor repeated acute tocolysis improve perinatal outcome; neither should be undertaken as a general practice (acog practice bulletin 2003). Should prolonged exposure to terbutaline for more than 2 days during pregnancy be associated with increased risk of asd this would be another important factor dissuading the use of maintenance treatment with tocolytic drugs for the management of preterm labor . However, surveys of obstetric providers indicate that terbutaline is still being used as a maintenance tocolytic in other medical communities (fox et al . 2008; morgan et al . Prenatal exposure to any b2ar occurred at a similar rate among children with autism and control children . Asthma treatment with b2ar agonists during pregnancy does not explain our previous observation of increased asd risk among asthmatic mothers . There is a suggestion that maternal exposure to terbutaline for greater than 2 days during the third trimester of pregnancy could be associated with elevated autism risk however, should this association be confirmed, it would provide further contraindication to maintenance tocolysis with b2ars and also raise interesting questions about late pregnancy mechanisms and exposures to be considered in autism risk factor research.
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A 24-year - old lady was referred to our pain clinic with complaint of abdominal pain for last 3 months . She had her second cesarean section 4 months ago and her pain started one month after the surgery . The pain was located around 5 - 7 cm inferior and lateral to umbilicus on the right side and was well localized in a circumferential manner without any radiation . She described her pain as dull aching, stabbing and burning in nature and was severe while getting up from bed, climbing stairs and bending forward . The patient rated her pain as 7/10 on visual analog scale (vas) during above mentioned activities . She had pin - point tenderness just at the lateral border of rectus abdominis with allodynia and hyperalgesia . She had been evaluated by her gynecologist earlier and found no abnormality on ultrasound abdomen . Before coming to us she had used non - steroidal anti - inflammatory drugs, muscle relaxants, tramadol, gabapentin without any relief . After ruling out intra - abdominal pathology by the evaluation of a laparoscopic surgeon, we suspected acnes as there was pin - point tenderness around lateral border of rectus and a positive carnett test (tensing the abdominal muscles by elevating the head and shoulder in prone position lead to increase in pain). After consent and under strict asepsis, we used high frequency linear probe (6 - 13 mhz, m - turbo, sonosite, bothell, wa, usa) to give rectus sheath block . But she didn't allow us to put the probe for rectus sheath block as she had tremendous tenderness . Probe was placed lateral to rectus sheath where all three layers of abdominal muscles were clearly seen . A combination of methyl prednisolone 20 mg and 6 ml of 0.375% ropivacaine was injected between the fascial layer of internal oblique and transversus abdominis using a 22 gauge echogenic needle (sonotap, pajunk, geisingen, germany) utilizing in - plane approach . Patient reported same pain five hours later but she started getting relief 3 - 4 days later possibly because of the depo - steroid . Twelve months after the injection she continues to experience almost complete pain relief and is back to normal life . The second lady was of 30-years and came to us with 7 months history of chronic abdominal pain similar to the previous lady i.e. Pain after 1 month of the second cesarean section . She had pinpoint tenderness at two places viz . At the lateral border of rectus sheath corresponding to t11 and t12 dermatome . She initially responded to gabapentin and tramadol but the pain relief didn't last long and was keen for the injection . We performed tap block lateral to the rectus sheath after which she had very good pain relief (80%). On follow up, after one month, she complained of pain at the lower part closer to surgical scar but there was no pain at the previous painful area . This time we repeated a similar tap block with probe position little down to the previous block location . She had complete relief and without any further injection 6 months down the line till the writing of this report . A 24-year - old lady was referred to our pain clinic with complaint of abdominal pain for last 3 months . She had her second cesarean section 4 months ago and her pain started one month after the surgery . The pain was located around 5 - 7 cm inferior and lateral to umbilicus on the right side and was well localized in a circumferential manner without any radiation . She described her pain as dull aching, stabbing and burning in nature and was severe while getting up from bed, climbing stairs and bending forward . The patient rated her pain as 7/10 on visual analog scale (vas) during above mentioned activities . She had pin - point tenderness just at the lateral border of rectus abdominis with allodynia and hyperalgesia . She had been evaluated by her gynecologist earlier and found no abnormality on ultrasound abdomen . Before coming to us she had used non - steroidal anti - inflammatory drugs, muscle relaxants, tramadol, gabapentin without any relief . After ruling out intra - abdominal pathology by the evaluation of a laparoscopic surgeon, we suspected acnes as there was pin - point tenderness around lateral border of rectus and a positive carnett test (tensing the abdominal muscles by elevating the head and shoulder in prone position lead to increase in pain). After consent and under strict asepsis, we used high frequency linear probe (6 - 13 mhz, m - turbo, sonosite, bothell, wa, usa) to give rectus sheath block . But she didn't allow us to put the probe for rectus sheath block as she had tremendous tenderness . Probe was placed lateral to rectus sheath where all three layers of abdominal muscles were clearly seen . A combination of methyl prednisolone 20 mg and 6 ml of 0.375% ropivacaine was injected between the fascial layer of internal oblique and transversus abdominis using a 22 gauge echogenic needle (sonotap, pajunk, geisingen, germany) utilizing in - plane approach . Patient reported same pain five hours later but she started getting relief 3 - 4 days later possibly because of the depo - steroid . Twelve months after the injection she continues to experience almost complete pain relief and is back to normal life . The second lady was of 30-years and came to us with 7 months history of chronic abdominal pain similar to the previous lady i.e. Pain after 1 month of the second cesarean section . She had pinpoint tenderness at two places viz . At the lateral border of rectus sheath corresponding to t11 and t12 dermatome . She initially responded to gabapentin and tramadol but the pain relief didn't last long and was keen for the injection . We performed tap block lateral to the rectus sheath after which she had very good pain relief (80%). On follow up, after one month, she complained of pain at the lower part closer to surgical scar but there was no pain at the previous painful area . This time we repeated a similar tap block with probe position little down to the previous block location . She had complete relief and without any further injection 6 months down the line till the writing of this report . The purpose of above communication is to have high index of suspicion for acnes as the cause of abdominal wall pain and application of ultrasound guided tap block for the treatment of this pain . History, proper physical examination and a positive carnett test in the absence of any other intra - abdominal pathology should raise the suspicion of acnes . Complete pain relief to a diagnostic nerve block with local anaesthetic usually establishes the diagnosis . In literature, there is description of blind, trigger point injections at neural foraminal level where the nerve enters the rectus abdominis muscle . Though blind injections dominated for a long time, there is a recent report on ultrasound guided rectus sheath block for the same diagnosis . As our patient did not allow us for a rectus sheath block because of severe tenderness, we tried tap block and it was totally successful . We did not go for the classic description of the tap block, in stead injected the anaesthetic in the transversus abdominis plane closer to the rectus muscle, so that the drug is deposited very near to the entrapped site . Despite the increasing use of tap block for perioperative analgesia for hysterectomy, cesarean section, laparoscopic surgeries, there is very sparse literature on chronic pain application . Both of our patients responded to single injections, except the second patient requiring second injection for the lowermost tender point . Tap block under ultrasound guidance may offer a diagnostic possibility for patients with cawp to confirm acnes . This approach may serve as an alternative, safe, effective and reliable method to blind injection or in patients with severe pain and tenderness for probe placement at rectus sheath area.
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Subarachnoid hemorrhage (sah) is a devastating neurological insult that causes significant morbidity and mortality . One of the greatest sources of this morbidity and mortality is cerebral vasospasm (cvs), leading to delayed cerebral ischemia (dci). While angiographic vasospasm is thought to occur in approximately 70% of patients after asah, only 25% develop symptomatic cvs [3, 4]. Morbidity remains high despite years of clinical and basic science research done on the topic, with approximately 50% infarction rates in affected patients [5, 6]. An antecedent inflammatory cascade is one of the many etiologies thought to be responsible for the development of cvs . Experimental studies have shown involvement of cytokines, cell adhesion molecules, and leukocytes, and early clinical studies have attempted to inhibit components of the inflammatory cascade to mitigate cvs [719]. Additionally, endothelin receptor activation, nitric oxide inhibition, thromboxane receptor modification, and many cell signaling cascades are thought to play an integral role in the development of this pathology [2027]. Currently, the primary treatment for this patient population involves hemodynamic augmentation and medically or surgically mediated intra - arterial vasodilation . These treatments, while providing amelioration in the short term, have proved relatively ineffective in staving off neurological decline, in part due to lack of large scale analyses [1, 28]. In our review, we hope to summarize past and current research in the area of inflammation and the development of cvs . An appreciation of these key inflammatory intermediaries will allow for the development of clinically significant interventions in this patient population . This decline was initially attributed to the progression of mass effect secondary to acute sah; however, current evidence suggests that an acute brain injury with subsequent cerebral ischemia plays a primary role . It is thought that the introduction of blood products, specifically hemoglobin, into the extravascular space initiates narrowing of the arterial vasculature within several hours, starting at approximately three days after - injury and lasting up to several weeks . The arterial vasculature demonstrates diffuse or focal stenosis with significant restriction of blood flow visualized via angiography . Cvs presents with focal neurological dysfunction and can precipitate the development of permanent ischemia in approximately 50% of patients with cvs . The gold standard for the diagnosis of cvs is angiography, specifically with digital subtraction angiography (dsa). As an improvement to the technique described by ecker and colleagues in 1951, dsa allows for high quality angiographic images via the digital subtraction of extraneous artifact . In its use as a diagnostic tool, mild vasospasm is defined by less than 25% reduction in vessel patency . A greater than 50% reduction many authors have since espoused the utility of cta and mra as alternatives to the invasive techniques used in dsa [3537]. In addition to being less invasive and severalfold faster than conventional angiography, cta and mra produce images of higher resolution than their predecessor [38, 39]. These techniques however rely heavily on digital reconstruction, and their accuracies vary depending on the vascular territory assessed and operator experience [31, 35, 39, 40]. Transcranial doppler sonography (tcd) is currently the primary tool used in the noninvasive diagnosis of cerebral vasospasm . It is currently the single most important noninvasive measure of cerebral vasospasm [42, 43]. When vasospasm induces a decrease in arterial diameter, cerebral blood flow (cbf) velocity concomitantly increases . Cvs is most times described when cbf velocity exceeds 120 cm / s [44, 45]. An increased ratio of cbf velocity between intracranial vessels, the middle cerebral artery (mca) or anterior cerebral artery (aca), and an extracranial ica can be used to indicate cvs . The diagnostic reliability of tcd has historically been called into question since factors such as the arterial window analyzed, patient movement, and operator experience affect the reliability of the test [31, 43, 47]. Specifically, changes in systemic pressure, cardiac output, and other factors that affect hemodynamic homeostasis can adversely affect reliability of the tool due to its strong dependence on cerebral blood flow dynamics . In a comparative study between tcd and dsa in the assessment of aneurysmal vasospasm, okada and colleagues highlighted the poor sensitivity of tcd for detecting vasospasm and noted that dsa was distinctly superior as a diagnostic tool . Other studies have underscored the incongruences in the diagnostic sensitivity of this tool [49, 50]. Tools such as jugular venous oxygen saturation monitoring, xe - enhanced ct, positron emission tomography (pet), single photon emission ct (spect), and magnetic resonance imaging have also been used to diagnose cvs with varying sensitivities . Walton in 1955 demonstrated that febrile patients who presented with sahs fared worse than their afebrile counterparts . Several decades later, studies by rousseaux et al . Confirmed a definitive relationship between the occurrence of symptomatic cvs and fever in affected patients (table 1). It was in the late 1980's however that spallone and colleagues demonstrated that leukocytosis, the sentinel indicator of systemic inflammation, was significantly higher in patients with asah . These discoveries spurred interest in the role of the inflammatory response as a causative factor in the development of the pathology . Later studies substantiated these earlier findings, and additional inflammatory markers and cytokines were identified to play a pivotal role in the inflammatory process (table 1). As a result of decades of research into the inflammatory mediators of cvs, several theories explaining the pathophysiology have been proposed . One such theory states that the extravasation of erythrocytes into the cerebral parenchyma acts as the nidus for the vasospasms that characterize acute and chronic cvs [11, 18, 55, 56]. Extracorpuscular hemoglobin (hb) is a proinflammatory moiety as opposed to its intravascular homolog [5760]. The presence of hb in the extravascular space liberates reactive oxygen species (ros) that result in the peroxidation of membrane lipids of endothelial cells and proliferation of smooth muscle [61, 62]. It is also thought that these oxidative stressors inhibit bradykinin mediated vascular relaxation after asah . Additionally, it has been hypothesized that the presence of subarachnoid blood is a potent stimulant for nuclear factor - kappa b (nf-b) mediated cytokine release augmenting the inflammatory reaction [11, 64]. Normally, once erythrocytes have entered the extravascular space, exposed hb is rapidly bound to a hepatically produced haptoglobin (hp). Hp exists in two isoforms, hp 1 - 1 and hp 2 - 2 of which hp 2 - 2 is thought to bind with less affinity and as such is relatively pro - inflammatory in comparison to its analog [23, 27, 56, 65]. Macrocytes phagocytize hb bound moieties and remove them from the extravascular space . The initial pro - inflammatory effect elicited by hb and hb bound moieties initiates an inflammatory cascade involving an increase of cytokines, leukocytes, and cell adhesion molecules (cams) characterizing the inflammatory process [7, 13, 18, 56, 66, 67]. The elevation in inflammatory cytokines and cell adhesion markers promotes the initial margination of leukocytes by cams on the endothelial surface . Of these cell adhesion markers, selectins (l-, p-) are most important in the initial capture [9, 20, 6871]. Secondly, leukocyte rolling is facilitated by p- and e - selectins in which captured leukocytes are sequentially bound and released by adjacent cams . The end result is a sequential transmigration of leukocytes down a chemoattractant concentration gradient to the site of inflammation . Finally, firm adhesion to the endothelium effectively halts leukocyte rolling and promotes leukocyte diapedesis [11, 72]. This process is mediated by another class of cams, the integrins of which cam-1, lfa-1 and other moieties play vital roles . Experimental models have successfully demonstrated the temporal relationship between the initial inciting injury and increases in cytokine concentrations [7678]. Of particular interest are il-1, il-6, tl-8, and tnf-. In various studies, increases in the intraventricular concentrations of these moieties have been demonstrated to occur after injury [7981]. Though the csf concentrations of these inflammatory intermediaries increase after - hemorrhage, the change in serum concentrations is not as reliable . In fact, studies have demonstrated both elevations and no relevant changes in the analysis of systemic concentration of these cytokines . It is irrefutable, however, that leukocytosis and changes in c - reactive protein levels (crp) correlate strongly with clinical outcomes after - injury [83, 84]. Transendothelial migration, or diapedesis, is the final step in the recruitment of inflammatory leukocytes and encompasses a multitude of cell adhesion molecules and intracellular cell signaling pathways . Macrophages, monocytes and neutrophils are the primary cells implicated in the initial stages of injury and have been found in postinjury histological analysis to be a highly represented cell type in adjacent tissue [85, 86]. These inflammatory cells mediate macrocytosis of free erythrocytes as part of their natural function; however, after asah there is a disruption of normal cerebral blood flow preventing the re - entry of macrophages and leukocytes into circulation . Ultimately, a release of inflammatory cytokines and other vasoactive compounds, which characterize the chronic vasospasm of cvs, is thought to occur approximately four days after injury . In the final analysis, the upregulation of et-1, destructive free radicals, no dysregulation, and spasmodic cytokines are all thought to play a substantial role in the onset of vasospasm [1, 8789]. Et-1 itself is a potent vasoconstrictor whose upregulation peaks 3 - 4 days after injury . It is thought to be increased due to cerebral ischemia and other stressors with possible augmentation by the aforementioned antecedent inflammatory cascade . Its effect on vasoconstriction is multifaceted [91, 92]. Through a negative feedback loop no plays a significant role in vascular tone through the inhibition of calcium production and subsequent reduction in contractile forces; thus, its inhibition leads to vessel narrowing . Additionally, et-1 is also integral to fibrosis, tissue growth, and inflammation [26, 92, 95]. In summary, through several distinct mechanisms, the inflammatory changes surrounding sah lead to global stenosis of cerebral vasculature [26, 9699]. Several clinical trials have either corroborated or refuted the clinical efficacy of the suggested treatments . Anti - inflammatory agents such as dexamethasone and cyclosporine a (cys - a) have shown favorable improvements in experimental models [100, 101]. However, other known agents, such as tacrolimus, an inhibitor of t - cell maturation similar to cys - a, have not shown similar results . There are also studies implicating the complement system, the 3-hydroxy-3-methyl - glutaryl coenzyme a (hmg - coa) reductase pathway, and multiple other systems; however, results even in the experimental stages have been varied [103106]. There have been experimental reports documenting improvement in cvs using various pharmaceutical agents, however, the variety of these treatments is sparse (table 3). In 2011, velat and colleagues reviewed 44 randomized control trials (rct) and 9 meta - analyses on vasospasm after sah . Their studies suggested that only oral nimodipine was efficacious in stymieing cvs with improvement in neurological decline after sah . In an experimental murine model of cerebral vasospasm, bowman and colleagues demonstrated a statistically significant dose dependent reduction in vasospasm after administration of a polyclonal antibody targeted against il-6 (p <0.05). Such findings indicate that cytokine upregulation is antecedent to radiographic vasospasm, and their attenuation may minimize vessel narrowing . The propensity for magnesium supplementation to mitigate cvs has also been widely studied [108113]. In fact, a small swiss study in 2012 suggested that high dose magnesium administration compared to low dosage suppressed post - sah increases in il-6 (p = 0.021) and il-1 (p <0.001), respectively . Agents such as organic stress reducers including trehalose modulate the effect of the cyclooxygenase-2 (cox-2) catalytic pathway . In experimental murine models, the polysaccharide reduced concentrations of pro - inflammatory markers leading to a reversal of sah induced vasospasm . Of the multiple markers that trehalose modulates is endothelin-1, which modulates vasoconstriction [91, 92], via its effects on nitric oxide, fibrosis, tissue growth, and inflammation . Consequently, it has been the target of major studies on the treatment of vasospasm . These evidences along with other recent experimental findings suggest a multifactorial process that precedes cvs and clinical neurological dysfunction, which may or may not be directly related . Myeloperoxidase (mpo), a marker of inflammation in coronary artery disease, is thought to be implicated in the inflammatory process of cvs . Mpo is a lysosomal enzyme that is a component of the respiratory burst in leukocyte antibacterial function . The enzyme catalyzes the production of hypohalous acids like hypoclorous acid (hocl), which modify cellular membranes . In reports by lim and colleagues, the biomarker demonstrated elevations in csf concentrations that mirror the onset of cbf abnormalities in cvs, supporting mpo as a surrogate predictor of cvs after sah . Because of to its role in inflammation, its upregulation is most likely related to antecedent leukocyte recruitment . The mortality that ensues after cvs is often due to delayed cerebral ischemia (dci). Dci is defined as new focal neurological deficits (symptomatic vasospasm) or ischemic focus in the setting of radiographic vasospasm . While the relationship between inflammation and cvs is irrefutable, it is unlikely to be the single perpetrator behind delayed cerebral ischemia (dci) and subsequent neurologic decline . Results from the conscious trials assessed the effects of the endothelin-1 receptor antagonist clazosentan after sah, and while there was significant improvement in angiographic cvs, neurological dysfunction was only minimally affected . Hansen - schwartz and colleagues concluded that these results point to a more extensive implication of inflammation in the pathophysiology of post - sah neurological dysfunction . Hansen - schwartz also argued that the lack of temporal alignment between angiographic cvs and dci symptoms suggest the need for further studies . Cortical spreading depolarization (csd), intravascular microthrombosis, and early brain injury have all received traction in the field as alternate theories of cvs [120124]. In the immediate posthemorrhage period, rapid increases in cerebral edema, icp [126, 127], and changes in cerebral autoregulation are all thought to disrupt the homeostatic milieu resulting in early brain injury . Transient and sporadic neuroelectrical disruptions also occur in the injured brain after acute injury . Through experimental murine models, researchers have demonstrated that these brief episodes of electrical dampening differentially affect cerebral perfusion resulting in either hyperperfusion or hypoperfusion . This hemodynamic imbalance propagates damage created during early brain injury . Finally, correlations between elevated procoagulant factors and dci occurrence have strongly suggested a role for microthrombosis in the pathophysiology . The detection of microthrombi in postmortem analysis and a 70% prevalence of microthrombi in sah patients also point to the involvement of the coagulation cascade in the development of dci . It is now more likely that cvs is one of many factors that play some part in the neurological decline consistent with dci . Most notably, the earlydrain and lumas trials evaluate early evacuation of csf through a lumbar drain [134, 135]. The lumas trial evaluated 210 patients with asah and randomized 105 to receive standard therapy and a lumbar drain, with the remaining 105 randomized to the control group receiving only standard management . The authors found that lumbar drainage of csf after asah was associated with reduced dind and improved early clinical outcome but had no difference in outcomes at 6 months . Preliminary studies have also shown efficacy using a kinetic treatment, intrathecal thrombolytic therapy in conjunction with head shaking . In this small study, after 14 days of observation, there was decreased dind and vasospasm related infarct in the intrathecal thrombolytic therapy group compared to the control . Lastly, the depolarizations in ischaemia after aneurysmal subarachnoid haemorrhage (discharge-1) trial are an ongoing multicenter diagnostic phase iii study that assesses electrical depression of the cerebral cortex after asah . The aim is to calculate the sensitivity and specificity of the duration of cortical spreading depression as a proxy for dci after asah . As we look to future research, it is hoped that these theories may become incorporated in to standard therapies for the treatment of cvs . From all indications, one of the major limiting factors in the management of cvs is an understanding of the extent of early brain injury secondary to sah . We are currently limited by the difficulty in consistently and accurately diagnosing and treating affected patients . The role of inflammation in cvs represents a possible mechanism behind the development of dci after sah, and targeting the inflammatory process has potential to significantly reduce cvs, though further studies are necessary . Cerebral vasospasm continues to be regarded as an important factor in the progression to dci in patients following sah . It cannot be however regarded as the single factor behind the neurological decline in this patient cohort.
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Age - related macular degeneration (amd) is the main cause of permanent central blindness in the developed countries . It manifests in drusen formation and degeneration / atrophy of the retinal pigmented epithelium (rpe) and neural retina, as well as the formation of abnormal choroidal capillaries [2, 3]. In addition to aging as the principal risk factor, there are others such as smoking, diet, and genetic predisposition [3, 4]. However, it is not yet sufficiently resolved the exact genetic pathways underlying the initiation and progression of amd and the relationship between its genotypes and phenotypes . Although a more recent clinical classification of amd has been published recently, we are using that of newman et al . Since the study specimens were categorized in the public data according to their phenotypes (see table 1 for details), these encompass (1) dry amd, (2) choroidal neovascularization (cnv) or wet amd, (3) geographic atrophy (ga) in macular region of rpe, (4) ga / cnv, (5) pre - amd, and (6) subclinical pre - amd . These phenotypes are typically the progressing manifestations of the disease, and their gene expressions may not harbor the early events responsible for the initiation and progression of the disease . A transcriptomic profiling of these phenotypes will elucidate the affected signaling pathways, reveal their similarities and differences, and clarify whether amd's phenotypes represent a single disease or entities of an assemblage of diseases . In this study, we used systems biology analytical paradigm called parsimony phylogenetics to reveal the various transcriptomic profiles of amd's subtypes . Further specific objectives of this analysis are to find out if gene expression profiling supports the current classification of phenotypes, to identify the shared gene expression aberrations among amd's phenotypes, to find out if the transformations in the neural retina are similar to those in rpe - choroidal region, and to carry out class discovery in order to subtype amd on the basis of gene expression profiles and answer whether it is a single disease or different disease entities . To reach the above stated objectives, we have selected parsimony phylogenetics as the best systems biology tool to analyze microarray gene expression data of amd obtained from public domains . Parsimony is an evolutionary analytical method that has been applied to mass spectrometry data of cancer, gene - expression of various diseases [7, 8], vaccine analysis, and systematics biology of taxa . Parsimony algorithms are capable of utilizing shared derived gene expression aberrations to subtype specimens; they are very suitable for high dimensional heterogeneous data (i.e., with 10,000s of variables). Our analytical strategy can be summarized in the following steps: classify the patient specimens into clades (a cluster of specimens located on the cladogram) onto cladogram through parsimony analysis of their gene - expression data; identify shared genes with abnormal expression (termed synapomorphies in phylogenetic vocabulary) for each clade; and identify genetic pathways affected by abnormal gene expression for all amd specimens and/or for each clade . The gene expression dataset of macular and extramacular encompassed specimens of retinas (55 normal, 13 pre - amd, and 47 amd) and retinal pigment epithelium (rpe-) choroid complexes (96 normal, 21 pre - amd, and 60 amd). The amd specimens encompassed dry amd without geographic atrophy (ga), choroidal neovascularization (cnv), and ga (table 2). Pre - amd and amd gene expression values of retinal and rpe - choroidal specimens were polarized separately against their respective normal specimens (e.g., rpe - choroid data was polarized using normal rpe - choroid specimens data), and the new polarized data matrices were processed separately through mix, a parsimony program of the phylip package (http://evolution.genetics.washington.edu/phylip.html) to produce phylogenetic cladograms for both datasets (for details of this process see [7, 13]). The resulting cladograms were studied for meaningful interpretations and to fulfill the objectives stated in the introduction . Gene lists extracted from the cladograms nodes were processed in genomatix geps (http://www.genomatix.de/) to reveal the affected gene signaling pathway networks . For a more meaningful interpretation of the affected signaling pathways, our analysis focused on sampling different regions of the cladograms to reveal the diversity of the affected signaling pathways within amd lesions . After the extraction of the synapomorphies at several locations of cladograms 1 and 2, we extrapolated from the synapomorphies the affected signaling pathways (tables 3 and 4) by modeling the list of synapomorphies into genomatix geps . The sampled locations represented the basal, the middle, and upper sections of both cladograms . Each dataset analysis with mix produced over 100 cladograms, and only one cladogram was selected (usually the first since the differences between the cladograms were in the upper minor branches) to represent each analysis (figures 1 and 2). Interestingly, the analysis revealed the high heterogeneity of the specimens' gene expression irrespective of their phenotype in both retina and rpe - choroid complex . This was evident by the large number of cladograms produced (over 100) by the two datasets . Usually the fewer the number of cladograms produced the lower the heterogeneity and the higher the confidence in the results . Also supporting this conclusion were several aspects of the cladograms such as the terminal distribution of gene expression aberrations (see below). The specimens of each amd phenotype did not cluster together to form a clade (a clade is a group of specimens sharing one or more abnormal gene expressions) but rather formed mixed clades that encompassed several phenotypes (figures 1 and 2). Therefore, amd phenotypes seemed not to be distinct entities according to their transcriptomic profiles of the retina or rpe - choroid complex suggesting that the clinically recognized phenotypes may not be supported by a classification based on gene expression abnormalities . Macular and temporal extra - macular tissues of the same patient separated in most of the retinal and rpe - choroid complex sets but some clustered together (1215%) indicating similar changes in both locations (macular and extramacular). This could be attributed to the diversity of the disease itself where it is similar in both locations in some patients and different in others, or could be due to sampling from similar locations . The two cladograms (figures 1 and 2) demonstrate that the amd retina and rpe - choroid complex had slightly more transcriptomic subtypes than the currently recognized clinical phenotypes; for example, the number of clades within each cladogram is larger than the number of currently recognized phenotypes . Except for the majority of the retina amd specimens (both macular and extramacular) that shared 113 synapomorphies (shared gene expression aberrations) most of the genetic aberrations were specimen - specific since amd phenotypes did not form their respective clades, there were not any synapomorphies that defined any of the phenotype . While the retina clade was defined by 113 synapomorphies the rpe - choroid complex clade had only two synapomorphies; these are located at the basal section of the cladograms (figures 1 and 2). Tables 3 and 4 summarized the affected signaling pathways of the retina and rpe - choroid complex datasets respectively . Furthermore, the sampled sections of each cladogram had differently affected signaling pathways despite some minor overlap . While the changes in the retina were highlighted in apoptosis, cell cycle, cytoskeleton, and growth signaling pathway, those of the rpe - choroid complex showed affected signaling pathways of oxidative stress, inflammation, cell differentiation, and oncogenecity . The samples of table 4 were selected to represent the various locations of the cladogram of figure 2 in order to explore the affected pathways among various clades . Some of the affected genes included c - x - c motif chemokine 12 (cxcl12) that is a chemokine strongly chemotactic for lymphocytes; glial cell - derived neurotrophic factor (gdnf) that strongly promotes the survival of neurons and prevents apoptosis of motor neurons; secreted frizzled - related protein 1 (sfrp1) that acts as a biphasic modulator of wnt signaling, counteracting wnt - induced effects at high concentrations and promoting them at lower concentrations; which may also affect the differentiation of photo receptors; and superoxide dismutase 1 (sod1) that is associated with macular degeneration when its levels drops below normal . More updates on other genes' functions can be obtained from http://www.ncbi.nlm.nih.gov / gene/. Unfortunately, since the cladograms of figures 1 and 2 show that their clades do not have commonly shared aberrations along the axis of the cladograms, nothing can be said about directionality of gene change in amd from these cladograms . This study is the first transcriptomal analysis of the retina and rpe - choroid complex tissues from amd patients and normal subjects by means of phylogenetic parsimony . The method is a data - based (not specimen - based) analytical paradigm that produces a hierarchical modeling of the specimens into clades (phylogenetic clusters) defined by their shared aberrations, which when identified reveal the affected signaling pathways . The parsimony cladogram is multidimensional tool that exposes the characteristics of its data . In this study, the large number of equally parsimonious cladograms that were produced from the two datasets displayed the massive heterogeneity of the expression pattern within or across the clinical classification of amd . Each dataset produced over 100 cladograms, an unusually high number of cladograms for a dataset of anatomically - related specimens . However, such diversity in advanced degenerative disease could be expected since these diseases are a downhill path toward undifferentiation due to the deregulation of differentiation pathways, and their phenotypes can be reached through several ontogenic pathways . Amd follows the same pattern, and it should not be unexpected that its specimens have shown this considerable heterogeneity . However, it may be surprising to find that the transcriptional profiles of both datasets did not support the current classification of the amds phenotypes and that the neural retina is different from the rpe - choroid complex in their deregulated pathways . The clades produced by the parsimony algorithm did not even come close to the classification of newman et al . As evident in the cladograms of figures 1 and 2 . Further analyses of other data sets, such as metabolomic and proteomic data, are needed to confirm the findings . Pathological aberrations in general are usually divided into driver (clonal) and passenger (nonexpanded). On a cladogram, the driver aberrations are usually modeled at the basal nodes of the cladogram, while the passenger ones are at the terminal level of the clades or randomly distributed on the cladogram . In this study, the vast majority of aberrations are at the terminal level, that is, specimen - specific . This revelation that most of the gene expression aberrations are specimen - specific points out to two conclusions: the first is that the change is mostly patient - specific, and the second is that there are probably multiple etiologies for amd . Our analysis is fundamentally different from that of newman et al . Who mainly used fold change (1.5) as their criteria to identify significantly expressed genes in amd phenotypes . Ours differs in that we used the normal range of gene expression (minimum and maximum values of healthy specimens) as the cutoff for determining the under - and overexpressed genes per specimen . This was followed by a phylogenetic stratification of amd retinal and rpe - choroid specimens to find the natural clusters (clades) and their affected pathways for each of the two groups of specimens . Since these two methods belong to two different schools of thought (specimen - based versus data - based), the congruence of their results was very weak . Therefore, gene lists and pathways of newman et al furthermore, while newman et al . Claimed that their results supported the current phenotypic classification of amd, we think that our unsupervised analysis did not support amd's phenotypes . Maps of significant genes are the best indicators of gene expression heterogeneity within amd's phenotypes and the difficulty in declaring any as global biomarkers; the vast majority of their claimed globally significant genes (newman et al ., figure 2) are actually insignificant except for loc100294179 in retina that is significant in dry amd, ga, and cnv, and c10orf18 in rpe - choroid that is significant in cnv and md . Our analysis indicated that the transcriptomal changes within the neural retina as a group of specimens were different from those in the rpe - choroid specimens, and these two sets of tissues differ from each other in their aberrations; therefore, it is most likely that there are no global biomarkers for amd's phenotypes as defined in table 1 . This conclusion highlights the necessity of stratifying (subtyping) the disease as a priori to declare any aberrations as the global biomarkers of the disease subtypes . As our analysis has shown here, there were different transcriptomal subtypes than the clinical ones . Amd like all degenerative diseases can be bioinformatically modeled on a cladogram as a spectrum that ranges from early stages with initial events to advanced stages with later events . When specimens representing all stages of amd are used to construct a cladogram, the ones harboring early stages of the disease will occupy the basal location of the cladogram while later stages follow . Therefore, revealing early events of amd (i.e., gene expression deregulations that probably are not associated with morphological changes) requires the study of specimens that are less advanced in their pathology . In this study, the identification of early events was not possible; this may be attributed to the lack of specimens with asymptomatic stages or relatively normal pathology of the disease . The presence of drusen in pre - amd and subclinical specimens (see table 1) may also represent part of an advanced stage of the disease rather than a pre - amd or sub - clinical diagnosis since drusen may signify an advanced dysfunction of the mitochondria . Although ophthalmologists rely on morphological criteria that appear to represent advanced events for amd diagnosis, early detection of amd transformations should be carried out on the basis of gene - expression profiling according to our analysis . Additionally, the subtyping of amd may have to be delayed till early gene - expression profiles become available . In spite of some slight overlap, the affected signaling pathways in amd are different in the retina and rpe - choroid complex (tables 3 and 4). In general, the retina specimens shared aberrations within apoptosis, cell cycle, cytoskeleton, and growth signaling pathways, and the rpe - choroid complexes showed aberrations related to inflammation, differentiation, hypoxia, and oncogenecity . It appears from the list of affected signaling pathways that the two tissue types are exposed to different stressors and therefore are responding in a different manner . Tables 3 and 4 detail the affected signaling pathways in the retina and rpe - choroid complex of amd lesions . In conclusion, amd appears to be a diverse disease that involves two major independent but parallel pathological processes, one within the neural retina and the other within the rpe - choroid complex . In both areas, the transcriptomal changes are very heterogeneous and seem to be mostly patient - specific and involve various signaling pathways . Furthermore, the transcriptomal profiles seem to be incongruent with the clinical phenotypes, and the early gene expression events of amd cannot be deciphered from the advanced phenotypes of the disease.
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Based on hcv antibody and rna viral profiles, patients were stratified into two groups: patients who spontaneously cleared hcv and patients with chronic hcv infection who continued to have detectable rna titres . However, the patients with chronic hcv infection and detectable viral rna presented significantly increased alt and ast levels compared with the patients who had spontaneously cleared hcv . Genotype 1a was found in all of the chronically infected patients with detectable rna . Fibrosis staging determined by elastography was conducted in a small number of participants (6) from the chronic hcv group . Patients with distinct stages of liver disease were included in the study and cirrhosis (f4) was found exclusively in one of these patients (table). Tableclinical and demographic characteristics of the patientscharacteristicshcv+ (n = 16)hcv- (n = 17)pgender [female (%)] 68.7576.4-mean age (years sd)53.13 10.349.12 12.830.332mean alt (ui / l sd)64.43 41.1429.8 15.070.005mean ast (ui / l sd)61.86 42.4529.8 16.740.012positive anti - hcv1617-viral genotype1aundetectable - mean viral titre (ui / l sd)1.064 x 10 ^ 7 1.56 x 10 ^ 7undetectable- lipid profile (mg / dl) col170.2 52.67177.3 59.420.724 tg157.3 76.96148.6 63.40.728 hdl38.27 9.7440.29 7.60.514 ldl100.5 50.78119.9 340.210 vldl31.4 15.2830.29 130.826body mass index (kg / m)22.94 1.3822.9 1.260.925grade of fibrosis (n) f11 - -f1-f21 - -f21 - -f2-f32 - -f41 - -alt: alanine aminotransferase; ast: aspartate aminotransferase; col: cholesterol; hcv-: patients who exhibited positive anti - hepatitis c virus test results and undetectable hcv rna titres for more than six months . Averages of quantitative variables are expressed as mean standard deviation (sd); hcv+: chronically hcv - infected patients who exhibited positive anti - hcv test results and detectable hcv genotype 1a viral rna titres for more than six months; hdl: high density lipoprotein; ldl: low density lipoprotein; tg: triglyceride; vldl: very low density lipoprotein . Alt: alanine aminotransferase; ast: aspartate aminotransferase; col: cholesterol; hcv-: patients who exhibited positive anti - hepatitis c virus test results and undetectable hcv rna titres for more than six months . Averages of quantitative variables are expressed as mean standard deviation (sd); hcv+: chronically hcv - infected patients who exhibited positive anti - hcv test results and detectable hcv genotype 1a viral rna titres for more than six months; hdl: high density lipoprotein; ldl: low density lipoprotein; tg: triglyceride; vldl: very low density lipoprotein . In an attempt to rule out the influence of overweight and obesity on the clearance or evolution of chronic hcv infection, this study only included samples from patients with a normal body mass index . No differences in the lipid profiles of the analysed groups were found (table). Representative dot blots showing the relative expression of cytokines in the study groups are shown in a, b in figure . A low detection rate of certain cytokines, including il-2, il-6, il-5, ifn-, il-7, tnf-, mcp-1 (ccl-2), mcp-3 (ccl-7), mig (ccl-9), gro-, gro, il-3, gmcsf and gcsf, was observed in our study groups (a - c in figure). Interestingly, compared with chronic hcv - infected patients with detectable rna, the patients who spontaneously cleared hcv displayed increased levels of il-1, tnf-, tgf-, mcp-2 (ccl-8), il-13 and il-15 (d - f, i, k, l in figure, respectively). In contrast, the chronic hcv - infected patients with detectable rna showed increased levels of il-8 and rantes (ccl-5) compared with patients who spontaneously cleared hcv (h, j in figure, respectively). No significant differences were found in il-10 levels . However, there was a trend towards increased il-10 levels in the chronic hcv - infected patients with detectable rna (g in figure). Viral and host factors are related to the progression of hcv infection . In 2009, it was demonstrated that hcv viral clearance is associated with genetic variation in the il-28b gene (ge et al . Numerous studies have been conducted on this family of cytokines and have led to several inconsistencies and controversies, including the possible correlation between serum protein levels and disease outcomes in chronic viral hepatitis patients (torres et al . Thus, it is accepted that variations in the profile of cytokines involved in the immune response may contribute to the ability to clear hcv . These studies have mainly been conducted in regions where the virus is endemic (shi et al . 2012), thus information on populations in regions of low endemicity is scarce . Herein, we characterised serum cytokine expression profiles in mexican hcv - infected patients and found subtle differences in the cytokine profiles between distinct clinical courses of hcv infection . In our study, persistent infection with detectable genotype 1a viral content resulted in a proinflammatory profile characterised by increased levels of il-8 and rantes (ccl-5) and a trend towards increased il-10 levels . These findings correlate with in vitro studies that have shown that natural killer cells from hcv - infected patients are impaired in their capacity to activate dendritic cells due to the overproduction of il-10 (jinushi et al . Likewise, the increased levels of il-8 found in the chronic hcv group in the present study may be related to the association between il-8 production and ifn- inhibition described in both in vivo and in vitro studies (khabar et al . Moreover, the analysis of cytokines in each patient revealed a trend towards higher serum concentrations of il-10 and il-8 in patients with more severe liver damage (f2-f3 and f4) (data not shown). Altogether, our findings suggest that il-10 and il-8 overproduction may contribute to a lower probability of viral clearance, whereas homeostasis between pro - inflammatory [il-1, tnf-, mcp-2 (ccl-8)], anti - inflammatory (tgf-), fibrogenic (il-13) and immunoregulatory (il-15) profiles allows for viral clearance, as observed in patients with undetectable viral rna levels . However, large - scale studies with adequate statistical power to support this hypothesis are needed . Furthermore, given that our data do not rule out the possibility that cytokine levels may be a consequence and not the cause of hcv clearance, future studies aimed at analysing the possibility of a reverse causation are necessary . The progression of hcv infection is associated with the characteristics of lipid profiles in chronic hcv patients . In fact, it is accepted that lipid components associated with hcv infection are finely modulated in the mexican population (fierro et al . Future studies that include overweight and obese individuals will allow the determination of the exact role of lipids in viral clearance . We previously reported the influence of cytokines on the development of distinct clinical courses in children infected with hepatitis a (fierro et al . 2014) and on the development of occult hepatitis b virus infection in native mexican groups (fierro et al . 2011). Herein, our findings suggest that cytokine expression can influence the extent of hcv development and provide important insights into cytokine - mediated mechanisms underlying the long - term persistence of hcv . In this context, efforts to determine whether biomarkers, including serum cytokines, can accurately predict the outcome of hcv infection are valuable for establishing better hcv control strategies in latin american regions.
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Vascular complications after a total knee arthroplasty (tka) occur in less than 0.5% of cases.1 it is uncommon, with a reported incidence following tka ranging from 0.03 to 0.17%.27 however, when such a complication does occur, it is potentially limb and life threatening . Popliteal artery pseudoaneurysm is one of the sequelae of popliteal artery trauma and also can cause arterial thrombosis, arteriovenous fistulae, or arterial severance.26 we report a case of a false aneurysm of the popliteal artery after a tka with unusual presentation, difficult diagnosis and successful management . Right tka was done using mobile - bearing knee prosthesis (depuy, warsaw, indiana, usa) [figure 1a and b]. Postoperative rehabilitation was started on the 1 postoperative day and consisted of static exercises and continuous passive motion . For postoperative analgesia, iv fentanyl patient controlled analgesia (pca) dose 1.5 ml / h, along with intravenous paracetamol (1 g) thrice daily was used . On the 3 postoperative day, she was disoriented, agitated, and had a fall while trying to get up from bed . The passive range of motion (rom) was not affected following fall and there was no increase in swelling or hematoma formation . Disorientation continued, and on the 4 postoperative day, she had accidental fall again . She gave a significant history of direct blunt injury to the popliteal fossa with the railing of the bed . Hemoglobin dropped from 8.4 gm% (1 postoperative day) to 6.7 gm%, and she was given packed blood cells . (a) postoperative anteroposterior (a) lateral (b) view x - ray of right total knee arthroplasty showing implant in situ on the 6 postoperative day, patient complained of swelling and severe pain in her calf, refusing to move her right lower limb . Swelling extended from popliteal fossa up to midcalf region and was nonpulsatile . Venous doppler examination of the right lower limb was done, which showed no evidence of dvt . While doing the venous doppler, the radiologist suspected an arterial injury . The arterial doppler showed a pseudoaneurysm of the right popliteal artery, of size 3.9 2.6 cm, with hematoma collection extending into calf [figure 2a and b]. Computed tomography (ct) angiography was done for further evaluation and suggested extensive wall calcification of the abdominal aorta and iliac arteries . A 3 2.5 cm pseudoaneurysm in relation to the right popliteal artery just above the knee joint was seen [figure 3a]. (b) color doppler imaging popliteal artery flow pattern across the pseudoaneurysm ct scan with arrow showing location of pseudoaneurysm (posterior) ct angiography of both lower limbs with distal runoffs and good vascularity vascular surgeon opined the need for repair of the rent in the artery causing the aneurysm . A repeat ultrasonographic examination done the next day did not show any increase in the size of the aneurysm . Considering the location of false aneurysm, the vascular surgeon decided to go posterior . On exploration, there were significant clots and the rent was found on the posterior surface of the artery of size 2 2 mm . The rent was not on the anterior aspect, indicating that the injury to the artery was as a result of the impact on the popliteal region due to a fall in the immediate postoperative period and not an intraoperative incident as is seen in most of the reported cases of popliteal pseudoaneurysm following tka . After 12 months, the patient remained asymptomatic without any vascular problems . On followup, she had well felt distal pulsations without any clinical evidence of pulsatile swelling in popliteal area . Trauma to popliteal artery in tka is an intraoperative event . It can present as an acute arterial severance or with arterial thrombosis, pseudoaneurysm, arteriovenous fistula, and occlusion.26 pseudoaneurysm of the popliteal artery is one of the uncommon arterial complications which may present early as a pulsatile mass in the popliteal fossa or remains undetected for a long time.236 it can present as a painful calf swelling as in dvt.5 occasionally, it may present very late.7 the reported mechanisms of injury are due to direct trauma to the vessel, like posterior retractor, oscillating saw or pins used to hold the tibial jig, posterior capsular release.26 the diagnosis of a post tka popliteal artery pseudoaneurysm should be suspected clinically, and diagnostic tools such as doppler ultrasonography and ct angiography should be used to confirm the diagnosis.1516 repeat ultrasonography is also valuable in determining whether the aneurysm is increasing in size . Smaller pseudoaneurysms which are associated with small defects in the arterial wall can be treated with ultrasound guided compression repair (ugcr). This procedure involves two sittings, week apart, by applying compression at the neck of pseudoaneurysm . Compression is performed with the us transducer itself, a procedure that permits direct and continuous visualization of the vessels . Typical protocol includes an initial 10 - 20-min compression of the pseudoaneurysmal neck; if this is not feasible, the pseudoaneurysm itself is compressed . Anticoagulation therapy decreases the success rate; therefore, anticoagulants should be discontinued prior to the procedure if possible . Drawbacks of this procedure are long procedure time, discomfort to the patient, recurrence, and rupture of aneurysm . However, due to the nature of the trauma, only a small proportion of these lesions are amenable to percutaneous therapy which is fairly superficial pseudoaneurysm with less hematoma and commonly used for femoral, axillary, and brachial artery pseudoaneurysms.1416 the established treatment for the remainder is open repair with or without popliteal artery bypass grafting with ligation of the native artery . Open repair has its own set of difficulties as it involves operating from a posterior approach and exposing the recently placed prosthesis to infection . Endovascular treatment involves placement of a covered stent across the defect in the popliteal artery.11316 though no long patient series is available, evidence suggests that endovascular treatment is safe and durable as a treatment modality in the treatment of traumatic popliteal artery pseudoaneurysms and can be performed with an acceptable morbidity rate . All reported pseudoaneurysms of popliteal artery or inferior geniculate artery are on anterior aspect and due to intraoperative direct injury to the vessel with varied presentation.27 though popliteal pseudoaneurysm is rare, it can be a devastating complication post tka . We hereby reported an exceptional case where there was trauma to the popliteal fossa due to a fall in the immediate postoperative period probably due to disorientation caused by the use of iv fentanyl and the location of arterial rent was on posterior aspect . The clinical presentation of this condition can be confused with that of a postoperative dvt or hematoma formation in early postoperative period and the surgeon should be aware of this condition . The treatment options include an open surgical repair or other described noninvasive techniques such as percutaneous embolization or endovascular stenting.
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The stubbed toe is a common innocuous injury, usually sustained barefoot, which can result in a wide range of injuries including nail bed lacerations, fractures of the distal phalanx and, uncommonly, bony or soft tissue mallet injuries . The mainstay of management of simple fractures and nail bed injuries is usually non - operative, with good long - term function and little or no morbidity . Mallet fingers are common injuries with well - established treatments depending on the presence or absence of a fracture or joint subluxation . However, similar injuries to the hallux are rare with only a handful of single case report in the literature [37]. The rarity of these injuries likely relates to the relative sizes of the digits and tendons and the protection offered by footwear . Owing to their relative rarity, there is no consensus as to the optimum treatment which ranged from non - operative treatment in a rigid soled shoe, closed reduction and stabilization with kirschner wires or open repair and reattachment of the tendon with suture anchors . Failure to recognize and treat these injuries can lead to extensor lag of the hallux, which carries some morbidity . A 13-year - old girl injured her left great toe while running downstairs barefoot, incurring a forced plantar flexion injury . There was pain, swelling and bruising to the hallux, but she was able to weight bear . She noted a progressive drooping of the hallux over the following 3 weeks and presented to the trauma clinic via her gp . On examination, she had a swelling overlying the interphalangeal joint (ipj) of the hallux, which was held in 20 of plantar flexion, and she was unable to actively dorsiflex at the ipj . Plain radiographs showed no bony injuries, but the ipj was subluxed in a plantar - flexed position (fig . 1). An urgent ultrasound scan confirmed a full thickness rupture of the extensor hallucis longus (ehl) tendon at its insertion into the distal phalanx and evidence of a healing tear to the dorsal joint capsule (fig . 2). Plain radiograph showing plantar - flexed hallux ipj . Ultrasound image showing ruptured ipj capsule and avulsed ehl tendon . Under general anaesthesia, an l - shaped incision was made to avoid the dorsomedial sensory nerve and avoid injury to the germinal matrix of the nail . The ehl tendon was found to be completely ruptured from the distal phalanx and had retracted back towards the mtpj . The tendon end was debrided back to healthy tissue and was secured back to the distal phalanx using a mitek anchor and a non - absorbable suture in a modified krakow technique . The ipj was stabilized with a 1.2-mm kirschner wire (figs 35). Figure 4:operative photograph showing transfixion of the ipj with a k - wire and insertion of a mitek anchor into the distal phalanx . Operative photograph showing transfixion of the ipj with a k - wire and insertion of a mitek anchor into the distal phalanx . The k - wire was removed 4 weeks postoperatively and she was placed into a thermoplastic extension splint for a further 4 weeks, allowed to weight bear normally in shoes and undertake a passive range of movement exercises with physiotherapy . At this stage, she had good power of dorsiflexion, but limited plantar flexion due to stiffness . At the point of discharge, 6 months following surgery, she had regained a full range of movement compared with the contralateral side, full power of plantar flexion and dorsiflexion, and had returned to normal function . The earliest reports of mallet hallux by rapoff in 1999 and saxby et al in 2001 advocated non - operative treatment with a rigid soled shoe and an extension splint, respectively [3, 7]. These cases were both middle - aged patients who had sustained bony mallet injuries and following treatment had stiff ipjs, but were asymptomatic with good function . More recently, three of these reports describe temporary stabilization with k - wires (either to reduce and stabilize fracture fragments directly or to act as blocking wires) [5, 6, 8]. In all of these cases, all report bony union within 58 weeks and full function at final follow - up . However, only one of these patients (who was 16 years old) regained a full range of movement . Mallet hallux using bio - absorbable suture anchors and non - absorbable sutures, in combination with early mobilization of the joint . They were concerned that popular k - wire transfixion was associated with a high degree of stiffness and the possibility of infection and wire breakage . They showed that with this combination of treatment, both of their patients were able to get back to full function relatively sooner with no stiffness of the joint . Hallux mallet as the ehl tendon is larger than the extensor tendons involved in soft tissue mallet injuries of the fingers, meaning that the repair is of better quality . In our reported case, due to the chronicity of the rupture (nearly 4 weeks at the time of surgery) and degree of retraction of the ehl tendon, it was felt that the patient would benefit from the combined treatment with a suture anchor and k - wire transfixion . Following this combination of treatment, the patient has made a full functional recovery and achieved a full range of movement . We feel that this reflects the strength of the reconstruction and relative protection that the k - wire affords in addition to a suture anchor, with relatively early (4 weeks) passive movement . Additionally, the only other patient to achieve a full range of movement following treatment was also a teenager (despite having ipj transfixion), suggesting that, as with most injuries, the virtues of youth enable better recovery . Unstable bony and soft tissue mallet injuries of the hallux are uncommon . In this more chronic case in a younger patient with significant retraction of the tendon, we advocate open reconstruction with combined suture anchor repair and k - wire transfixion, with a return to normal functional outcomes and a range of movement.
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Auricular defects characterized by absence of the pinna may be congenital (microtia or anotia) or acquired as a result of infection, cancer surgery, or traumatic injury . Congenital defects may be associated with auditory canal, middle ear, and inner ear malformations . The total or partial pinna mutilation may be a consequence of a car accident, war collateral damage, or chemical burn . Recently an increase in the number of patients presenting with pinna lesions due to chemical assault burns, occurring in the domestic or industrial setting, has been reported . Whatever the cause, absence of the pinna is an important aesthetic problem, with a permanent effect on the patient's quality of life, which can often cause severe psychological distress . The modern era of pinna reconstruction surgery has its foundations in the techniques proposed by tanzer, brent, and nagata [46]. The differing techniques for reconstructive plastic surgery of the auricular defect involve usually the insertion of autogenous rib cartilage framework under the skin . Other surgeons have suggested the employment of prostheses made of synthetic material as a good alternative to autogenous costal cartilage [7, 8]. The reconstructive plastic surgery techniques based on the use of autologous cartilage inserts normally require repeated surgery acts at both implant site and donor site . This creates a significant discomfort both for the pain complained about by the patient and for repeated hospital admissions to the detriment of quality of patient life . Initially, these epitheses were held in place by adhesives that, however, provided poor results in terms of stability and were often associated with skin irritations and deterioration of the prosthesis caused by the adhesive substances . Also these defects imply a deterioration of patient quality of life . Currently a superior, innovative surgical technique exists that allows fixation of the ear epitheses by osseointegrated titanium implants (cochlear bone anchored solutions ab, sweden). Basically, this approach reflects an evolution of the implants developed for dental prostheses as proposed by brnemark in 1969, which have been used for over 40 years in the field of odontostomatology and subsequently were proposed for application with osseointegrated craniofacial implants for use with bone - conduction hearing implant solutions [9, 10]. In our study, we describe our experience using these pinna prostheses in 27 patients treated in the last three years . We discuss the indications for auricular epithesis implant, the surgical technique, and outcomes . Our case series comprised 27 patients (24 males and 3 females with a mean age of 33.1 years, range 1687 years) (table 1). The etiology was congenital in 17 patients (figure 1) who were affected by microtia, while eight had posttraumatic mutilation (figure 2) and two surgical amputation due to neoplasia (figure 3). Six of the patients with microtia and three of the patients affected by traumatic mutilation had previously undergone plastic reconstructive surgery with rib cartilage grafting and were not satisfied with the results . These patients requested to have their previously reconstructed ear removed and replaced with an epithesis . One patient (patient 12) had previously undergone surgery, in a different hospital, using bone - anchored titanium implants but one of the abutments had broken because of a further unlucky trauma . After six weeks, all the implants were osseointegrated and a retentive bar was fixed to the abutments . The patients with microtia were offered the option of undergoing treatment with a bone - conduction hearing implant solution (e.g., baha) to concomitantly address their hearing needs in parallel to the auricular rehabilitation with the bone - anchored epitheses . All patients declined the hearing treatment option, preferring to have the option at a later stage instead . Before the surgical field is prepared and with the patient's face still fully and easily visible, the implant sites should be carefully marked, using methylene blue, down to the bone . These are ideally placed approximately 20 mm from the center of the external auditory canal opening or anticipated opening . They are positioned at 8 o'clock and 10:30 on the right side and at 4 o'clock and 1:30 on the left side . In the presence of a complete malformation, the supposed location of the external auditory canal is determined by considering a triangle traced on the contralateral hemiface using the following references: the line between the lateral canthus and the auditory canal, the line between the auditory canal and the labial commissure, and the angle formed by these two lines (figure 1(b)). We usually perform one - stage surgery, removing tags and remnants in cases of microtia and performing the necessary subcutaneous tissue reduction . The one - stage surgical procedure can be used in adults to treat auricular defects involving nonirradiated tissue; the two - stage technique should usually be chosen for paediatric patients and for the treatment of orbital and midface defects and auricular defects in patients with poor bone quality . Drilling begins using the guide drill with the spacer kept on 3 mm . During drilling the bottom of the hole is repeatedly checked for bone at the base of the site . If there is adequate bone thickness, drilling continues to a depth of 4 mm . The next step is to widen the hole to the exact diameter using a 3 or 4 mm drill countersink . The low - speed setting should be used for implant insertion . In compact cortical bone, a torque setting of 40 ncm is recommended, whereas, in soft bone, a lower torque setting of 20 ncm should be used . The self - tapping fixture with the premounted fixture mount it is then picked up with the connection to the hand piece, which is placed into the drill hand piece . The implant is installed without cooling irrigation until the small grooves at the distal end of the implant are well within the canal . When the flange of the implant has seated, the hand piece will automatically stop . The titanium standard abutment is picked up with the abutment holder and placed into the implant . Finally a gauze dressing is applied in a figure eight form (i.e., foam dressing, soft silicone wound contact layer, or antiseptic dressing) around the abutments . All patients were discharged the day after surgery and were revised for the first dressing after seven days . The implants occurs six weeks after implant to permit sufficient healing and stabilization of the implants . Following healing and stabilization of the surgical site, the patient was sent to the anaplastology technician who prepared the epithesis, modeling it with reference to the contralateral ear and carefully matching the individual's skin colour (figures 3(b) and 3(d)). The definitive one has two sides: the inner one is an acrylic plate with clips that allow the attachment to a gold - platinum bar fixed to the abutments and the external one is made of soft silicone . The patients receive two epitheses of different colours: a pale one for winter and a tanned one for summer . When the process of osseointegration is complete, the prosthesis, which has clips, can be easily and securely attached to, or removed from, the gold - platinum cylinder - and - bar system (figures 3(c) and 3(d)). None of the patients we treated experienced problems related to the implants over six months to three years (osseointegration failure or wound healing problems). Only one patient encountered a relatively exuberant scar in the area of surgical remnants removing . The patients were evaluated for their quality of life, during the week before pinna reconstruction with the short form health survey (sf-12) as a baseline assessment (t0). Statistical analysis of changes over time was performed via pearson correlations, with a significant change deemed by p 0.05 . Statistical analysis adopting sf-12 score (short - form health survey, 12 items) suggests a significant role in quality of life of patients who underwent auricular rehabilitation . All our patients expressed their satisfaction regarding the short hospitalization and reduced invasiveness compared to other alternative therapies . They had no adverse psychological reactions; instead, they were able to resume social relations and usual physical activities . Absence of the ear, congenital or resulting from trauma or surgery, is a defect that can be resolved through reconstructive plastic surgery . The surgical reconstruction of pinna defects remains a challenging task typically requiring multiple operations with often compromised aesthetic results . This involves the insertion of either an autogenous rib cartilage framework or a prosthesis made from synthetic material into a subcutaneous pocket behind the ear, created through tissue expansion . In the past, if surgical reconstruction was refused, a patient could undergo an alternative rehabilitative approach using an adhesive - retained prosthesis . The reconstruction using rib cartilage has some inherent disadvantages: it requires more than one surgical procedure, the risk of complications is relatively higher both at the implant site (e.g., infections, bleeding, haematoma, necrosis, and skin graft or cartilage graft exposure) and at the donor site (e.g., infections, haematoma, and scarring). Patients are very often dissatisfied with the final outcome because their new ear looks considerably different from the contralateral one and does not meet their expectations [13, 14]. Wellisz proposed the use of medpore prostheses made from porous polymer material inserted in subcutaneous pockets . This procedure is frequently complicated by partial or total rejection and further build - up of scar tissue . The use of adhesive - retained prostheses is also not without its issues, that is, dermatitis resulting from contact with the adhesives, unpredictable reliability of retention, variability of positioning of the prosthesis, and poorer hygiene directly attributable to the tackiness of the adhesive, as well as a decreased life span of the prosthesis resulting in an increased number of device renewals . Recent studies described the use of osseointegrated auricular prostheses as a good alternative treatment to surgical reconstruction . In this regard, titanium implant systems for bone - anchored implantable hearing solutions have shown us how such prostheses can be attached safely, securely, reproducibly, and without the need for adhesives [11, 1517]. The procedure discussed in this paper is suitable for patients who are unwilling to undergo plastic reconstructive surgery utilizing rib cartilage, which remains a challenging surgical procedure that involves more than one surgical step and is associated with the risk of complications at the donor site and/or the acceptor site . Similarly, in cases of pinna amputation ensuing following damage to the pinna from chemical burns, the usual techniques of plastic surgery employed hold their inherent difficulties as they involve procedures that require the presence of large areas of intact skin around the lesion (necessary for the preparation of sliding skin flaps or the use of tissue expanders prior to the placement of subcutaneous implants). Subsequently, the use of these implants is also the only possible solution in cosmetic treatment of oncology patients who have previously undergone several surgical procedures and/or radiotherapy . Radiotherapy does not constitute a contraindication for this procedure, although implant loss is relatively higher in irradiated cases than in nonirradiated cases at the site of treatment . Granstrom reported that the adjunctive use of hyperbaric oxygen could ultimately reduce the risk of implant loss . It is important to note the reasonably low cost of the implants and epithesis: typically amounting to a total of less than 4000 euros . In all previous reports patients express their satisfaction regarding the short hospitalization and reduced invasiveness compared to other alternative therapies . . Local skin infection around the fixture could occur, as could the formation of granulation tissue and keloids . These are complications that can be avoided or resolved using appropriate medication and topical treatments without loss of the fixture . Absolute contraindications for the use of titanium bone implants in prosthetic reconstruction of the auricle are exceptional and may be local or general conditions (i.e., resp ., contraindications for general anaesthesia need not preclude use of these implants since they can be positioned equally well under local anaesthesia . This surgical technique is contraindicated in patients younger than 14 years of age, whose skull thickness is not sufficient to support the osseointegrated implant . Preoperative evaluation of bone thickness via ct scans should, nevertheless, be a mandatory part of the surgical planning in all the patients [1517]. As mentioned earlier, indications for one - stage surgery are auricular defects, adult patients, and nonirradiated tissue, while two - stage technique should be used in young patients (1417 years old) and to treat orbital and midface defects and auricular defects in patients with poor bone quality . Conductive hearing loss due to malformations of the external and middle ear presents in all subjects affected by microtia and can be corrected by combining the placement of titanium implants for auricular rehabilitation with implantation of the fixture and abutment for a baha . In this way, both the sensory and the aesthetic problems can be resolved in a single operation . However, it should be pointed out that all the patients in our series refused to undergo baha implantation after testing the device prior to surgery; these patients, being well accustomed to hearing on only one side, found the increased auditory perception provided by the baha disorienting and irritating . The pinna epithesis fixed with bone - anchored titanium implants technique is characterized by excellent aesthetic outcome and lasting results . For example, they are able to use also helmet required for some sport activities and can swim without the problems related to adhesive epitheses.
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Benign prostatic hyperplasia (bph) is a progressive and androgen - dependent disease that causes lower urinary tract symptoms (luts). Although bph is not a life - threatening disease, it can have a negative effect on the quality of life . Alpha - adrenergic receptor antagonists (-blockers) and 5-alpha reductase inhibitor (5ari) are the first - line treatment option in patients with bph . 5ari blocks the conversion of the sex steroid hormone testosterone to dihydrotestosterone (dht), which is an essential component of bph pathogenesis . Previous studies reported that the serum prostate - specific antigen (psa) level and prostate volume were decreased by approximately 50% and 20%, respectively, after treatment with 5ari . However, some clinical questions remain as to how to interpret the psa level in men treated with 5ari . Furthermore, the effects on luts and prostate volume after discontinuation of 5ari are not well understood . Therefore, in the present study, we analyzed the changes in luts, prostate volume, and psa level after discontinuation of 5ari . From december 2003 to december 2012, 81 men more than 40 years of age with moderate to severe bph symptoms as determined by the international prostate symptom score (ipss8) and a prostate volume of greater than 25 cm measured by transrectal ultrasonography (trus) were collected retrospectively for the study . Two patients (case 1, 4.91 ng / ml; case 2, 5.43 ng / ml) with a psa level of 4 or more and with a negative pathologic result on the initial prostate biopsy were included in the study . Patients were excluded from the study if they had chronic urinary tract infection; a history of prostate or testicular surgery, prostate cancer, acute urinary retention, or chronically large postvoid residual urine volume; or 5ari treatment history . The patients were classified into group 1 (n=42) and group 2 (n=39) according to 5ari use . A combination of dutasteride 0.5 mg with tamsulosin 0.2 mg was given daily to all patients for 1 year ., group 1 (n=42) received a combination of dutasteride 0.5 mg with tamsulosin 0.2 mg daily, and group 2 (n=39) received -blocker monotherapy only . The ipss, prostate volume, and psa level were measured at baseline and at 12 and 24 months . With trus (aloka, tokyo, japan), prostate volume was measured by use of the prostate ellipsoid formula and was calculated by multiplying the longest anteroposterior (height, h), transverse (width, w), and cephalocaudal (length, l) diameters and 0.524 (hwl/6). Pasw ver . 18.0 (ibm co., armonk, ny, usa) was used for all statistical analyses . Student t - test was used to compare the baseline values of ipss, prostate volume, and serum psa level in each group . The repeated - measures analysis of variance was performed to compare the change in ipss, prostate volume, and psa level from 0 to 12 months and also from 12 months to 24 months . The mean age of the patients (n=81) was 71.70 years (standard deviation [sd], 7.80). The mean prostate volume was 43.96 cm (sd, 17.38) and the mean baseline serum psa level was 3.00 ng / ml (sd, 1.98). The patients were classified into group 1 (n=42) and group 2 (n=39). There were no statistically significant differences between the two groups in mean age, total ipss, baseline prostate volume, or psa level (table 1). The differences in total ipss, prostate volume, and serum psa level according to the absence of dutasteride are shown in table 2 . At 1 year after combination therapy, total ipss had decreased by 17.6% (from 17.88 [sd, 7.77] to 14.95 [sd, 7.27]; p<0.001) in group 1 and by 13.3% (from 15.50 [sd, 7.60] to 13.45 [sd, 2.50]; p<0.001) in group 2 . However, at 1 year after dutasteride had been withdrawn, total ipss had increased by up to 7.6% (from 13.45 [sd, 2.50] to 14.93 [sd, 5.48]; p<0.001) in group 2 (table 2). At 1 year after combination therapy, the prostate volume was reduced by 21.9% (from 41.59 cm [sd, 13.97] to 32.95 cm [sd, 10.10]; p<0.001) in group 1 and by 15.5% (from 45.96 cm [sd, 23.02] to 38.96 cm [sd, 23.91]; p<0.001] in group 2 . At 1 year after dutasteride had been withdrawn, the prostate volume had increased by up to 13.1% [from 38.96 (23.91) cm to 43.82 (23.45) cm; p<0.001]. At 1 year after combination therapy, the transition zone volume had been reduced by 20.0% (from 15.62 cm [sd, 5.76] to 12.23 cm [sd, 4.50]; p<0.001] in group 1 and by 17.6% (from 17.84 cm [sd, 10.35] to 14.61 cm [sd, 9.81]; p<0.001] in group 2 . At 1 year after dutasteride had been withdrawn, the prostate volume had increased by up to 14.2% (from 14.61 cm [sd, 9.81] to 16.46 cm [sd, 10.06]; p<0.001] in group 2 . The psa level had decreased by 48.4% (from 3.30 ng / ml [sd, 2.09] to 1.75 ng / ml [sd, 1.33]; p<0.001) in group 1 and by 49.4% (from 2.67 ng / ml [sd, 1.82] to 1.35 ng / ml [sd, 0.92]; p<0.001) in group 2 . However, 1 year after discontinuation of dutasteride, the psa level had increased up to 108.2% of the baseline level (from 1.35 ng / ml [sd, 0.92] to 2.89 ng / ml [sd, 2.10]; p<0.001) in group 2 (fig . The changes in ipss, prostate volume, and psa over time also did not differ significantly between groups 1 and 2 (table 2). Patients discontinued dutasteride in group 2 owing to adverse events, including loss of libido (n=9) or erectile dysfunction (n=17), lack of therapy efficacy (n=7), abnormal ejaculation (n=5), and gynecomastia (n=1). These symptoms may be associated with bph, which causes extrinsic compression of the prostatic urethra leading to impaired voiding . Bph is a common disease in aging men that leads to deterioration in symptoms, acute urinary retention, bph - related prostatic surgery, gross hematuria, urinary tract infection, bladder stones, and renal insufficiency . Men who have a prostate volume of more than 30 ml are more likely to have moderate - to - severe symptoms (3.5 times), decreased flow rates (2.5 times), and acute urinary retention (3 - 4 times) than are men with a prostate volume of less than 30 ml . Bph can significantly affect quality of life in a negative direction, thus requiring treatment . Bph can be managed medically with alpha - blockers, 5-ari, or a combination of both . Alpha - blockers relax the smooth - muscle tone in the prostate and bladder neck . However, alpha - blockers do not provide long - term reduction in the risk of acute urinary retention or bph - related surgery . The main role of 5ari is to induce glandular epithelial atrophy, which causes volume reduction in the prostate . 5ari is responsible for impeding the conversion of testosterone to dht by inhibiting the nuclear - bound steroid 5-reductase (5ar) isoenzymes . Both normal prostate development and hyperplasia of the prostatic transitional zone are regulated by dht . Men taking 5ari have a 57% reduction in the risk of acute urinary retention compared with those taking placebo and a 34% reduction in the risk of surgery . Dutasteride results in a reduction of 20% or more in the size of the prostate and a reduction of 48% in the risk of bph - related surgery . In our study, total ipss improvement and prostate volume reduction were evident in all men after treatment with dutasteride 0.5 mg . Few reports have discussed the effect of discontinuation of 5ari on luts, prostate volume, and psa level . Stoner demonstrated that the prostate volume returned to near the baseline values at 12 weeks after discontinuation of 5ari . In our study, the mean percentage of prostate regrowth had reached 95.5% of the baseline prostate volume 1 year after discontinuation of dutasteride . Owing to the complex etiologies of prostate enlargement, the mechanism of prostate regrowth after 5ari withdrawal is not fully understood . The hyperplastic changes in bph are an androgen - dependent process, and it is possible that the sensitivity to dht is increased or overly activated as a result of the prolonged pharmacologic inhibition of 5-reductase by the 5ari . Androgen receptors, which become more sensitive or up - regulated during treatment with 5ari, may be considered as an alternative mechanism . Our study showed that the total ipss had increased 1 year after cessation of dutasteride . Therefore, we propose that reuse of 5ari is helpful in cases that have aggravated bph symptoms or complications of bph . The psa level is also commonly used as a screening tool for prostate cancer, and the level of 4.0 ng / ml or greater, or more recently, greater than 2.5 ng / ml, has predictive value for prostate cancer detection . Psa velocity also has some predictive potency for detection of the cancer, especially in men with a psa level of 4.0 ng / ml or less . The epithelial cells of the prostate secrete psa, and it is synthesized by androgen regulation . At 1 year of 5ari treatment, the treatment results in an approximate 50% decrease in the psa level compared with the baseline serum level . The use of 5ari changes the psa level, and this may distract the clinician's attention . The psa level must be multiplied by 2 to maintain its usefulness as a marker . The psa level increases after discontinuation of 5ari because psa is secreted by the epithelial cells of the prostate . In our study, the psa level had decreased 1 year after combination therapy and had increased 1 year after discontinuation of 5ari . In group 1, two patients had continued psa level elevation, and they were finally diagnosed as having prostate cancer . Thus, regular monitoring of the psa level may be helpful for prostate cancer detection . Although it is not mandatory, we suggest measuring prostate volume by using trus in selected patients with aggravated bph symptoms or a high psa level . Trus can provide psa density, which helps clinicians make decisions about further evaluations such as prostate biopsy . The limitations of this study include it being a retrospective review and having a small number of cases . Prospective randomized clinical trials with a larger number of cases are needed to fully determine the effect of discontinuing 5ari . Maximal urinary flow rate is needed to evaluate the changes in urinary symptoms with the use of 5ari . Last, our study investigated changes 1 year after the discontinuation of dutasteride, but longer follow - up is necessary to make therapeutic decisions about the effect of 5ari . Our study showed that withdrawal of 5ari during combination therapy resulted in prostate regrowth and deterioration of luts . The psa level was reduced by the use of 5ari, but discontinuation of the drug resulted in an increase in the psa level . As a result, regular checkup of the ipss and psa level may be helpful for all patients who continue or discontinue use of 5ari . We recommend measuring prostate volume by using trus in selected patients with aggravated bph symptoms or a high psa level.
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Diabetic nephropathy (dn) is an important health problem in the worldwide adult population, and approximately 2040% of dn patients inevitably progress to end stage renal disease (esrd). Albuminuria is an important marker to diagnosis and predicts the progression of dn [2, 3], but some studies have shown that only 3045% of diabetic patients with microalbuminuria have progressed to macroalbuminuria in 10-year follow - up; 30% of them regressed to normoalbuminuria, and 3040% remained at microalbuminuria level . Furthermore, 20% of type 2 diabetic (t2 dm) patients had their glomerular filtration rate (gfr) decline before detecting albuminuria . All the renal cellular elements including the glomerular endothelium, mesangial cells, podocytes, and tubular epithelium are affected in setting of chronic hyperglycemia . Furthermore, renal tubular and interstitial compartments have been increasingly reported to play an integral role in the pathogenesis of dn and they correlate well with progressive renal function decline and represent a final common pathway of chronic kidney disease (ckd). In t2 dm, novel tubular biomarkers that relate to renal tubular injury could improve risk stratification and prediction . Neutrophil gelatinase associated lipocalin (ngal) is expressed in the renal tubular epithelium, and a rise in urinary concentrations may provide a potential biomarker of a chronically injured kidney . Kidney injury molecule-1 (kim-1) is a type 1 membrane protein expressed on the apical membrane of proximal tubule cells . Its ectodomain is cleaved and released in the lumen of the tubule and finally appears in urine, which is stable . Cystatin - c is produced from nucleated cells in the body and easily filtered by the glomeruli and is reabsorbed and catabolized by the proximal tubule . Previous study showed that the urine cystatin - c (ucys - c) level represented an indicator of renal tubular dysfunction . Finally, changes in angiotensinogen could influence ras activity and increases in intrarenal ras components, in parallel with the severity of fibrotic renal damage, have been demonstrated in chronic progressive nephropathy [9, 10]. Thus, all tubular biomarkers serve as a promising biomarker for tubule damage . Starting from renal tubular injury playing a role in the pathogenesis of dn and progressive decline of renal function, we hypothesized that baseline urinary tubular injury biomarkers are an important determinant of lower gfr in a wider cohort of t2 dm patients . However, inconsistent results have been produced with regard to predictors of all biomarkers in type 2 dm with nephropathy . Age, sex, and diabetes duration matched subjects with various stages of normoalbuminuria (urine albumin creatinine ratio (uacr) <30 mg albumin / g creatinine, n = 94), microalbuminuria (uacr 30300 mg albumin / g creatinine, n = 95), and macroalbuminuria t2 dm (uacr> 300 mg albumin / g creatinine and/or persistent proteinuria, n = 114) were recruited in february 2014 and march 2015 and followed up for a least 12 months at the outpatient clinic, department of internal medicine, phramongkutklao hospital . The study was approved by the ethics committee of the institute review board at the royal thai army medical department and all patients gave written informed consent . Exclusion criteria included acute kidney injury (aki) episode, pregnancy, unspecified type of dm, and patient life expectancy <1 year . All patient histories were carefully recorded by interview and confirmed by checking patient records and recording drug prescriptions . Clinical examination, including assessment of body mass index (bmi), systolic and diastolic blood pressure (bp), fasting plasma glucose, and other basic laboratory data, was conducted . Bp was measured three times, and the average value was used to analyze data . Common biochemical parameters including urea, creatinine, hemoglobin a1c, serum lipids and electrolytes, albumin, hemoglobin, and proteinuria were measured at baseline in all patients, according to standard methods in a routine clinical laboratory . Estimated gfr was assessed using the chronic kidney disease epidemiology collaboration (ckd - epi) equation . Urine albumin was measured on a nephelometric analyzer and urine creatinine was measured on a multiple analyzer (modular p chemistry analyzer; roche diagnostics). Urine albumin and creatinine for urine samples collected from participants and albuminuria were reported as albumin creatinine ratio (uacr). Thirty milliliters of fresh urine was centrifuged at 4,000 rpm for 10 minutes and then stored at 80c until assayed . All specimens were diluted often to obtain concentration at the optimal density according to the elisa kit instruction . Coefficients of variation for urine tubular biomarkers assays were <10%, for intra - assay and interassay variation ., usa and canada) levels were expressed as nanograms per gram of creatinine (ungal and ukim-1). Cystatin - c (r&d systems china co., ltd) levels were expressed as micrograms per gram of creatinine (uccr). Urine angiotensinogen (r&d systems china co., ltd) by solid phase elisa technique was expressed as nanograms per gram of creatinine (uang). After the baseline assessments, patients were followed up prospectively until the end of the observation period . The latter was defined by the combined outcomes of percentage changes of gfr decline from baseline and rapid renal progression was defined by decreased estimated gfr 25% from baseline in one year . Patients were personally contacted in case they missed any appointment and at the end of the study, to avoid eventual loss during follow - up . Data were presented as mean sd, median, or percentage frequency, as appropriate . Receiver operating characteristic (roc) analysis was used to calculate the area under the curve (auc) for ungal, ukim-1, uccr, uang, and uacr, and to find the best cut - off values of other tubular biomarkers to identify the progression to renal endpoint . Kaplan - meier curves were generated to assess renal survival in subjects with each cut - off point values of tubular biomarkers above and below the optimal roc - derived cut - off levels . For examination of associations with decline gfr and quartiles of urinary biomarkers, we first examined the unadjusted relationships and then adjusted the models for bmi, systolic bp, anemia, raas blocker, serum creatinine, and uacr by multivariate cox proportional hazard regression analysis . A total of 303 subjects (96%) at the second visit of the study were recruited for analysis . Mean age of patients was 66.4 11.4 years, and more than half were male (55.4%). Onset of t2 dm was 12.2 9.2 years, all patients had follow - up time of 12.3 4 months . Regarding clinical and laboratory data, mean estimated gfr was 50 29.7 ml / min/1.73 m, mean uacr was 887 210 mg / gcr, and hba1c was 7.3 1.5% . During the observational period (median follow - up of 12.3 4 months), 41 patients (13.5%) had gfr decline 25% yearly from baseline as rapid renal progression . The patients with rapid renal progression had a higher prevalence of anemia; raas blocker usage increased systolic bp and decreased baseline estimated gfr and baseline uacr (table 1). Urine tubular biomarker levels of uccr, uang, ukim-1, and ungal were significantly higher in the rapid renal progression group when compared with nonrapid renal progression group, represented by median (interquartile range at 25%75%) value of uccr 3.01 [1.0417.15] versus 7.52 [4.7416.49] mcg / gm (p <0.001), uang 2.41 [0.3110.73] versus 14.26 [3.2924.48] mcg / gm (p <0.001), ukim-1 67.5 [32.1132.2] versus 133.1 [81.9255.6] ng / gm (p <0.001), and ungal 751.3 [413.91350.5] versus 1058.3 [702.11693.3] ng / gm (p = 0.004) as shown in figure 1 . Macroalbuminuric and microalbuminuric t2 dm patients had higher levels of all urine tubular biomarkers as compared to normoalbuminuric patients . However, in subgroup of albuminuria, there were no significant differences in the urine tubular biomarkers between rapid and nonrapid renal progression groups . The relative small sample size and few reported end points were detected in each subgroup (table 2). Roc analysis showed an auc for uccr, uang, ukim-1, ungal, and uacr of 0.72 (95% ci: 0.64 to 0.79), 0.71 (95% ci: 0.63 to 0.79), 0.71 (95% ci: 0.63 to 0.79), 0.64 (95% ci: 0.560.72), and 0.76 (95% ci: 0.680.84), respectively, as shown in figure 2 . For uccr, the best cut - off level was 3.43 mcg / g (sensitivity 80.5%, specificity 56.9%), for uang was 4.52 mcg / g (sensitivity 73.2%, specificity 63.4%), for ukim-1 was 95 ng / g (sensitivity 70.7%, specificity 63.4%), and for ungal was 772 ng / g (sensitivity 68.3%, specificity 51.1%). All tubular biomarkers demonstrated intermediate performance to predict rapid renal progression in t2 dm patients . Kaplan - meier survival curves in patients with uccr, uang, ukim-1, and ungal levels above and below the optimal cut - off are presented in figure 3 . Subjects with uccr, uang, ukim-1, and ungal values above 3.43 mcg / g, 4.52 mcg / g, 95 ng / g, and 772 ng / g, respectively, experienced a significantly faster evolution to endpoint (p <0.05) with a mean follow - up time to progression of 12 months . To identify putative risk factors associated with the progression of ckd, we performed a cox regression analysis in the model for all variables that differed at baseline in patients who reached the endpoint during the whole follow - up period (anemia, systolic bp, raas blocker usage, baseline serum creatinine, and uacr). Univariate analysis showed significantly increased risk for rapid ckd progression in the group of upper quartiles of all urine tubular biomarkers when compared with lower quartiles (table 3). Multiple cox regression analysis after adjusting potential risks for ckd progression revealed that all urine tubular biomarkers including uccr, uang, ukim-1, and ungal were significantly associated with rapid ckd progression at the end of the study . Findings from the present study clearly indicate that all novel tubular biomarkers, that is, uccr, uang, ukim-1, and ungal, represent novel risk markers of dn progression . All novel urinary tubular biomarkers showed a most impressive predictive power in such a contest even after adjusting for conventional risk factors (baseline bmi, systolic bp, anemia, raas blocker used, serum creatinine, and uacr). This suggests that all novel tubular biomarkers would not be simple surrogate indexes of baseline estimated gfr, but markers on their own, predicting dn progression beyond the information provided by serum creatinine and other conventional risk factors . In recent years, however, different studies have underlined the crucial role played by the renal tubule in the genesis of progressive acute and chronic kidney disease and its evolution to terminal stage . Importantly, in diabetic with renal pathology, renal function correlated better with the degree of tubule - interstitial lesions than with that of the glomerular lesions . New biomarkers of the processes that induce these tubulointerstitial changes may ultimately prove to be better predictors of disease progression and long - term prognosis than our current markers . Cystatin - c, angiotensinogen, ngal, and kim-1 are known markers of acute kidney injury . All of these markers are upregulated in renal tubules after renal injury [1518]. Cystatin - c, angiotensinogen, and kim-1 markers located in the proximal tubules following renal injury, whereas ngal was defined in the distal tubules . Recent literature suggests that all of these may be a marker to detect progression of ckd . Prior cross - sectional studies examining the increased urine tubular biomarkers include cystatin, kim-1, ngal, and angiotensinogen in early ckd and early dn [1922]. Similarly, in one - year observational follow - up study in 74 t2 dm patients with nephropathy, high urine ngal levels at baseline correlated with declined levels of estimated gfr and increased serum creatinine . One study in t2 dm demonstrated that kim-1 predicted the decline of gfr in unadjusted analysis and two studies in t1 dm also showed that kim-1 levels were significantly higher in the patients who progressed from nonmacroalbuminuria to ckd stage 3 and macroalbuminuria to late stage of ckd . In addition, urine cystatin - c as reported from a prospective study predicted the ckd progression in the largest study reported to date, with 237 patients with t2 dm . Therefore, our study measured all of promising urinary tubular biomarkers at the same time in t2 dm patients . Our results are consistent with those in additional studies, and we provide more validation for these biomarkers associated with the progressive course of the disease . In this study, we found that t2 dm with rapid renal progression had significantly increased levels of urinary biomarkers when compared with the nonrapid renal progression group . From roc curves (auc 0.640.72) each tubular biomarker presented an intermediate accuracy performance in predicting rapid renal progression and all tubular biomarkers had an auc less than the auc of uacr (0.76) possibly affecting the level of proteinuria in our population . Cox proportional hazard models, representing all novel tubular biomarkers, were independent predictors of ckd progression in t2 dm . Our study integrated novel tubular biomarkers at the same time and compared the performance of each tubular biomarker with the standard urinary biomarker, uacr . Two studies in early ckd and short - term (less than five years) duration of t2 dm showed that only increased urine ngal, but not urine kim-1, was significantly associated with gfr decline [28, 29]. As opposed to previous studies in participants with ckd and early stage of dn, all tubular biomarkers levels in our study were significantly correlated with gfr decline and albuminuria . A significant rise in urinary tubular levels may indicate severe tubular cells damage in setting of established nephropathy in t2 dm population . Study limitations included being a single center study and having a short follow - up period of 12 months . However, in our study, urine biomarkers were measured in a cohort of both early and advanced dn patients who might have a more rapid decline in renal function in limited time of follow - up . Our primary endpoint was also reached by fifteen percent of the patients, and the statistical model was powerful enough to establish independent relationships between biomarkers and gfr decline in t2 dm . Our study did not collect data of main renal outcomes for demonstrating the doubling of serum creatinine and initiating long - term dialysis . The relatively small sample size of our study limits the precision and power to detect associations of moderate strength . Follow - up was based on a single serum creatinine and gfr calculation for detecting a decrease in gfr of> 25% from baseline; it may have some potential bias due to imprecision in estimated gfr . Finally, our data suggest that all the biomarkers measured in the urine are derived mostly or entirely from a renal tubular source . However, the data do not definitively exclude the possibility that some fraction of the excreted biomarkers especially plasma kim-1 may be derived from glomerular filtration . Animal experimental study addressed that plasma kim-1 was highly increased in diabetic animals compared to nondiabetic animals . Another study also indicated that plasma kim-1 level predicted rate of gfr decline and incidence of esrd in a cohort of t1 dm patients with advanced nephropathy . In conclusion, t2 dm patients with high levels of urine tubular biomarkers (urine cystatin - c, angiotensinogen, kim-1, and ngal) presented more rapid decline in renal function . All urine tubular biomarkers were independent predictors of rapid renal progression among t2 dm patients . For future study, in early stage t2 dm in the normo- or microalbuminuria group, novel tubular biomarkers may possibly have a role in early detection compared to the conventional marker to predict ckd progression.
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To report the effective treatment of radiation macular edema following ruthenium-106 plaque brachytherapy for a choroidal melanoma with a dexamethasone 0.7-mg (ozurdex) intravitreal implant . A 65-year - old caucasian woman was suffering from radiation macular edema following ruthenium-106 plaque brachytherapy for a choroidal melanoma on her left eye . She had undergone one intravitreal injection of 0.5 mg bevacizumab (avastin, genentech / roche) in the following months without functional or anatomical improvement . Seven months after the development of radiation macular edema, she received a single intravitreal injection of dexamethasone 0.7 mg (ozurdex). Four weeks following the injection, her best - corrected visual acuity improved from 0.3 to 0.5 . Radiation macular edema resolved with a reduction of central retinal thickness from 498 m before ozurdex injection to 224 m after ozurdex injection, as measured by oct scan . Dexamethasone 0.7 mg (ozurdex) has proven to be an effective treatment option in retinal vein occlusion and noninfectious uveitis . It can also be considered as off - label treatment in radiation macular edema following ruthenium-106 plaque brachytherapy for a choroidal melanoma . Radiation retinopathy is a predictable complication after radioactive scleral plaque application for treatment of intraocular tumors, most commonly choroidal melanoma . Macular edema is a common manifestation of radiation retinopathy that may lead to severe visual loss . According to guyer et al ., it was the most frequent and early finding after proton beam therapy for paramacular choroidal tumors, with 87% of 218 patients who developed macular edema . Other studies suggest rates of radiation maculopathy from plaque radiotherapy of 18%, 23%, and 42.8% . These include laser photocoagulation, photodynamic therapy, pentoxifylline and intravitreal bevacizumab [9, 10]. Corticosteroids are potent anti - inflammatory agents that can counteract many of the pathological processes thought to play a role in the development of macular edema . Recently, a sustained - release dexamethasone implant (ozurdex) proved to be effective for the treatment of macular edema secondary to a variety of underlying diseases with a potentially lower rate of adverse events . There are no cases of radiation macular edema after ruthenium (ru)-106 plaque brachytherapy for choroidal melanoma resolved by an intravitreal dexamethasone 0.7-mg implant described so far in the literature . We report a case of radiation macular edema after ru-106 brachytherapy for a choroidal melanoma, refractory to a previous treatment with intravitreal bevacizumab, and resolved with significant improvement of visual function following an intravitreal injection of dexamethasone 0.7 mg . A 65-year - old caucasian woman developed radiation macular edema 24 months following ru-106 plaque brachytherapy for a choroidal melanoma on her left eye (fig . She underwent one intravitreal injection of 0.5 mg bevacizumab (avastin, genentech / roche) in the following months without functional or anatomical improvement . Seven months after the development of radiation macular edema, she received a single intravitreal injection of dexamethasone 0.7 mg (ozurdex) as off - label treatment . Four weeks following the injection, her best - corrected visual acuity (bcva) improved from 0.3 to 0.5 . 1b) with a reduction of central retinal thickness from 498 m before ozurdex injection (fig . Her condition has been stable for at least 5 months now, as seen on the last examination, with bcva 0.6 and resolved macular edema in the treated eye . Shields et al . Reported the use of intravitreal triamcinolone (4 mg / ml) in a prospective, nonrandomized, single - center case series of 31 patients with visually symptomatic radiation - induced macular edema after plaque radiotherapy . They reported that visual acuity was stable or improved in 91% of patients by one month and 45% by six months; they also found a decrease in the mean central subfield foveal thickness measured by oct . Despite the potential benefits, intravitreal injection of triamcinolone acetate is associated with side effects, including glaucoma, cataracts, retinal detachment, and endophthalmitis [13, 14]. Also intravitreal bevacizumab has been evaluated as a treatment option for radiation maculopathy secondary to plaque radiotherapy [9, 10]. Our patient was initially treated with an intravitreal injection of bevacizumab which has not proved effective . Gupta et al . Stated that intravitreal injection of bevacizumab for treatment of radiation maculopathy caused by plaque radiotherapy was not useful for long - standing macular edema or when direct irradiation was received by the fovea . One of the main mechanisms of the chronic macular edema is the alteration of muller cells functionality; it has been experimentally shown that steroids, by reducing the osmotic swelling of the muller's cells, improve their functionality and reduce the macular edema . This could indicate that dexamethasone implant (ozurdex) might be an effective treatment option not only in retinal vein occlusion and noninfectious uveitis, but can also be considered as off - label treatment in radiation macular edema after ru-106 plaque brachytherapy for choroidal melanoma.
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In the era of prostate - specific antigen (psa) screening, more and more patients are diagnosed with insignificant prostate cancer . Because many of these cancers will not become clinically symptomatic, active surveillance (as) with delayed treatment has been introduced and has been reported to have similar therapeutic effects . The criteria for defining patients who are suitable to enter as protocols are mainly based on psa, clinical stage, and tumor grade . However, many studies have shown that the tumor grade obtained by prostate biopsy does not always correlate with the final pathological grade of the surgical specimen resected in a radical prostatectomy . In many cases, gleason score (gs) upgrading of up to 57% of cases has been reported in some studies . The incorrect assessment of tumor grade may lead to inappropriate estimation of cancer aggressiveness, resulting in under - treatment of these patients . Thus, a critical factor for the success of as is the use of appropriate entry criteria . Although a number of prognostic models have been developed to help to identify men who are appropriate candidates for as, it remains controversial whether to adopt psa density as an appropriate entry criterion . Accordingly, we conducted a prospective data analysis in patients with low - grade prostate cancer and evaluated the potential of several clinical and pathological variables to predict upgrading of the cancer . After we obtained approval from the ethics committee at institutional review board of daegu catholic university hospital, we conducted a prospective analysis of 60 patients with low - risk prostate cancer who were recruited from 4 university hospitals (gs6, psa<10 ng / ml, t1/t2, 2 positive biopsy cores). All patients underwent a retropubic or laparoscopic radical prostatectomy between january 2010 and december 2013 . All clinical, imaging, laboratory, and pathological data were collected and recorded prospectively and were analyzed retrospectively . We excluded patients who had received any preoperative therapy for prostate cancer (hormone therapy, radiation therapy) and patients who underwent less than 10-core transrectal ultrasound (trus)-guided biopsy . Biopsy cores were examined by different university - based pathologists and all radical prostatectomy samples were examined according to the standard protocol of the pathology department at each university hospital . We analyzed age, body mass index (bmi), preoperative psa value, psa density, number of cores, and number of positive cores in biopsy material . We analyzed the association of these factors with upgrading of cancer in the radical prostatectomy specimen . Any increase in gs between core biopsy and radical prostatectomy specimen preoperative psa was measured before the digital rectal exam, trus, or core biopsy . During trus, prostate volume was calculated according to information on the maximum transverse diameter (d1), the maximum antero - posterior diameter (d2), and the maximum longitudinal diameter (d3) and by using the formula based on the prostate ellipse dimension theory: d1d2d3/6 . Psa density was calculated by dividing the preoperative psa value and prostate volume . A histological report concerning tumor grade of the surgical specimen and pathological stage we estimated tumor grade by use of the contemporary criteria of the 2005 international society of urological pathology modified gleason system . Statistical analysis was performed by using pasw statistics ver . 18.0 (spss inc ., the normality condition of the numerical variables was studied by means of the kolmogorov - smirnov test . Psa was the only variable with a normal distribution; consequently, student t - test was used to compare means . To analyze other variables, the mann - whitney u test was used, and the chi - square test was used for categorical variables . The optimal cutoff values and sensitivity and specificity for quantitative variables that were found to be significant predictors were estimated by using receiver operating characteristic (roc) curve analysis . Positive predictive value (true positive/[true positive+false positive]) and negative predictive value (true negative/[true negative+false negative]) were also estimated . In total, 60 patients met our inclusion criteria within the study period and were entered into the analysis . The median patient age was 63 years, the median preoperative psa value was 6.26 ng / ml, and the median psa density was 0.15 ng / ml . As we previously described, every patient underwent trus biopsy with more than 10 cores; 58 patients (96.7%) underwent 12-core biopsy . On the basis of the pathological results from the radical prostatectomy, 2 patients (3.3%) were diagnosed with gs 5 prostate cancer, 40 patients (57.1%) had gs 6 cancer, 27 patients (38.6%) had gs 7 cancer, and 1 patient (1.4%) had gs 8 . Clinical stage on the basis of the digital rectal examination and trus findings was categorized as follows: 61 patients (86.7%) with t1c disease and 9 patients (13.3%) with t2a prostate cancer, respectively . Following radical prostatectomy, a tumor upgrade was noticed in 28 patients (40.0%). Extracapsular extensions were noted in 6 patients (10.0%) and positive surgical margins in 8 patients (17.3%). The demographic, clinical, and pathologic data regarding the presence of pathological tumor upgrading are described in table 1 . A statistically significant correlation between preoperative psa density and postoperative gs upgrading was noted (p=0.030). No statistical correlation with postoperative gs upgrading was found for age, bmi, preoperative psa level, prostate volume, number of biopsy cores, number of positive biopsy cores, the institute where the biopsy was done, or the operation method . In the analysis of pathological outcomes after radical prostatectomy, tumor upgrading was highly associated with extracapsular cancer extension (p=0.000). In the multivariate analysis, psa density was the only statistically significant variable to predict gs upgrading (p=0.030) (table 2). The estimated optimal cutoff value of psa density was 0.13 ng / ml, as obtained by roc analysis (area under the curve=0.66; p=0.020; 95% confidence interval, 0.53 - 0.78), as shown in fig 1 . It is well established that tumor grade is the most reliable and valuable parameter for estimating the prognosis of prostate cancer . By being a surrogate for tumor aggressiveness, the tumor grade allows us to stratify patients as either low, intermediate, or high risk . To date, the calculation of gleason grade has been based on pathological evaluation of the cores obtained by prostate biopsy . For patients who enter an as treatment program with close monitoring until disease progression and radical therapy, biopsy results, in combination with psa levels and clinical stage, are the data that guide treatment decisions . Nevertheless, upgrading of the gs between the needle biopsy and the radical prostatectomy is not rare . Upgrading was found in 30% of patients in one recent meta - analysis, and higher rates have also been published . This observation has important prognostic meaning because a significant percentage of these cases progress outside the prostate capsule . The present data, which showed gs upgrading in 40.0% of samples, also revealed 24.0% extracapsular extension . Given that conservative treatment protocols are mainly applied in low - risk patients, these findings are very important . An underestimation of prostate cancer aggressiveness may lead to under - treatment and inappropriate monitoring of biologically aggressive tumors . Thus, it is clear that many patients classified as having clinically localized disease and being at low risk actually have highly malignant cancer with a risk of clinical progression . Several reports have indicated that the likelihood of gs upgrading will decrease as the number of cores obtained by biopsy increases . King et al . Showed that in patients with a biopsy gs of 6, an extended biopsy strategy reduced gs upgrading from 66.7% to 36.8% . 's study also showed that gs upgrading decreased from 47.9% to 23.5% by taking more than 18 cores in a cohort of patients with low - risk prostate cancer . In coogan . 's study, increasing the number of cores from 6 to10 significantly improved the accuracy of the biopsy gs . The question remains as to what the optimal number of biopsy cores should be . . Showed that at least 10-core biopsies may be needed in patients with low - risk prostate cancer . Adopting this concept, no statistically significant dif ferences were found in the present study when comparing 12-core biopsies with 10-core biopsies . Whereas some groups have shown that an increase in positive cores correlates with an upgrading of gs, other studies could not find any association . In the present study, the association between preoperative psa level and gs upgrading also varies . Whereas the studies of mian et al . And king et al . Showed that psa level was a statistically significant predictor of gs upgrading . In the present study, we were not able to show an association between psa level and gs upgrading . Several reports have shown that an increase in prostate weight reduces the risk of gs upgrading . An explanation for this finding might be that a small prostate size is a surrogate of low in vivo androgenicity, leading to reselection of aggressive cancers in an androgen - depleted hostile environment . However, the results in the literature vary in relation to the weight of the prostate . In the present study however, in the multivariate logistic regression analysis, prostate weight was not an independent predictor of gs upgrading . Even though conf licting results regarding the interobserver reproducibility of gleason scoring in prostate biopsies have been reported, we did not observe a significant difference in gs upgrading between university pathologists in a univariate analysis (table 2). This result could partly be due to the proficiency of the pathologists and also to the number of biopsy cores being more than 10 . Corcoran et al . Examined the predictive characteristics of psa density in patients with low- and intermediate - risk disease on biopsy and subsequently treated with radical prostatectomy . They found that 58.3% of patients with low - grade disease after prostate biopsy were upgraded to higher scores and that psa density was a significant predictor (p<0.001) of upgrading in patients with gs 6 . Similar results were observed by kojima et al . And magheli et al ., with p=0.019 and p=0.037, respectively . Our results add to the above data, revealing a beneficial role of psa density in prediction of upgrading (p=0.030). A significant association of psa density with the pathological outcomes following radical prostatectomy was observed . We tried to produce a threshold level of psa density, over which the possibility of gleason upgrading increased significantly, triggering either a repeat biopsy or definitive therapy . By using the roc analysis, even though the sensitivity and negative predictive value were low, an increased specificity and positive predictive value were found (table 3). Given the complexity of prostate cancer, it seems to be more reasonable to use psa density combined with other predictive factors . First, the sample size was relatively small and might have decreased the strength of the results . Another limitation is that the psa density calculation was based on the estimation of prostate volume during trus made by 4 different operators . Even though a standard calculation protocol was followed, the interobserver differences in the volume measurement may have negatively affected the validity of the results . Last, even though the data collection was made prospectively, the analysis was done retrospectively . However, the strength of our study was that the evaluation of psa density in low - risk prostate cancer was adjusted solely for patients who underwent biopsies with more than 10 cores . Equal distribution of the number of cores obtained by prostate biopsies might have reduced a statistical bias and increased the value of the results for the estimation of tumor grade . With the commonly used criteria for as, gs upgrading in a radical prostatectomy specimen is a challenging problem . Psa density was an independent predictor of gs upgrading and may be included as a criterion for patients eligible for as.
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Peripheral arterial aneurysms are rare, and among these, deep femoral arterial (dfa) aneurysms are extremely rare [13]. Dfa aneurysms are not associated with any characteristic symptoms, and their early diagnosis is difficult . These aneurysms have a high rate of rupture, and surgery plays an important role in their treatment . However, standard methods have not yet been established because of the rarity of dfa aneurysms [36]. We describe a case of dfa aneurysm in a patient who was successfully treated with aneurysmectomy and revascularization with an artificial blood vessel graft . A 58-year - old man presented with paralysis and pain in the left leg, and a mass was found in his thigh . Because of the growth of the mass and the worsening of his symptoms, the patient visited a neighboring hospital . Physical examination revealed a pulsatile mass with a diameter of 8 cm in the left thigh . Multidetector computed tomography performed in our hospital revealed a large dfa aneurysm with an intraluminal thrombus (fig . 1). Surgical intervention was planned because of the large size of the aneurysm, the high risk of perforation and the worsening symptoms . Figure 1:multidetector computed tomography revealed a large dfa aneurysm (arrows show aneurysm). The common femoral artery, superficial femoral artery, superficial femoral vein and deep femoral vein were recognized and preserved (fig . 2a). After clamping the dfa proximal and distal to the aneurysm, we opened the sac and found a massive thrombus . Aneurysmectomy and revascularization of the distal dfa with an artificial blood vessel graft were performed (fig . Pathological examination of the specimen revealed atherosclerosis in the vascular wall and confirmed that it was a true aneurysm . Figure 2:(a) the common femoral artery, superficial femoral artery, superficial femoral vein and deep femoral vein were recognized and preserved . (b) aneurysmectomy and revascularization of the distal dfa with an artificial blood vessel graft were performed . (a) the common femoral artery, superficial femoral artery, superficial femoral vein and deep femoral vein were recognized and preserved . (b) aneurysmectomy and revascularization of the distal dfa with an artificial blood vessel graft were performed . Following surgery, a follow - up multidetector computed tomography scan showed good blood flow in the graft, superficial femoral artery and popliteal artery (fig . Figure 3:a follow - up multidetector computed tomography scan showed good blood flow in the graft, superficial femoral artery and popliteal artery . A follow - up multidetector computed tomography scan showed good blood flow in the graft, superficial femoral artery and popliteal artery . Although many dfa aneurysms have been associated with other peripheral aneurysms or peripheral occlusive diseases, there was no evidence of aneurysms or occlusive lesions in the other arteries of our patient [4, 7, 8]. Therefore, we consider our case of an isolated dfa aneurysm to be extremely rare . There are no characteristic symptoms of dfa aneurysms in the early stage, and their diagnosis is difficult . They are often found accidentally or after their enlargement, when symptoms include pulsating groin swelling, paralysis or pain in the leg, which were the symptoms that our patient experienced . Enlarged dfa aneurysms must be treated because of the high risk of rupture, which may cause severe complications, including hemorrhagic shock, ischemia of the foot, thigh embolization, thrombophlebitis and sciatic or femoral neuropraxia [1, 5, 9, 10]. Several treatment options, including simple ligation, revascularization of the distal dfa and bypass grafting to the popliteal artery, exist for dfa aneurysms; however, standard methods have not been established because of the rarity of dfa aneurysms . In our case, we performed an aneurysmectomy and revascularization to the distal dfa with an artificial blood vessel as the graft . Revascularization was performed to avoid thigh ischemia and preserve the collateral circulation if ipsilateral peripheral occlusive disease occurred . We believe that revascularization to the distal dfa should be done whenever it is technically feasible . In previous reports, if the patient must undergo operations such as coronary artery bypass grafting or other peripheral arterial bypass grafting, the saphenous veins will be required for those surgeries . Therefore, we did not use the saphenous vein as the graft . In this case, we consider the surgery a success because the patient recovered well from the surgery without complications, his symptoms improved and the blood flow in the leg was preserved . In conclusion, dfa aneurysms are extremely rare . Surgery plays an important part in the treatment of this disease, but standard methods have not been established because of its rarity . In our patient, we successfully performed an aneurysmectomy and revascularization to the distal dfa with an artificial blood vessel graft.
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It is characterized by apical migration of the gingival margin from the cement enamel junction, which is caused by the loss of connective tissue fibers and is accompanied by resorption of the alveolar bone crest and necrosis of cementum tissue.1,2 gingival recession results in poor esthetics, particularly while smiling or talking . The primary symptom is root hypersensitivity, and it may also be associated with root caries and cervical abrasion . The primary risk factors for gingival recession include traumatic brushing techniques,3 plaque - induced gingivitis, occlusal trauma, restorations with ill - fitting cervical region, a high buccal or lingual frenum, incorrect orthodontic movements, tooth crowding, and a thin alveolar bone.46 techniques for root coverage in patients with gingival recession vary according to local anatomical features such as the height and depth of the cervical abrasion,7 extension of the affected areas of the crown and root, length of the gingival recession, presence of keratinized tissue, and, most importantly, esthetic requirements of patients.5 only defect restoration will not resolve poor esthetics because it results in an excessively long tooth.2,4,8 similarly, only mucogingival therapy for root coverage cannot completely cover the abrasion, thus resulting in persistent dentin hypersensitivity.2 several surgical procedures for root coverage have been successfully conducted;911 furthermore, several restorative materials are now available for the reconstruction of lost hard tissue before the surgical procedure for root coverage.5,9,10 however, few studies have reported the combination of composite resin restoration for the treatment of cervical radicular abrasions and surgical root coverage.1214 here we present a comparison of the conventional and semilunar coronally positioned flap techniques for root coverage in teeth with cervical abrasion restored with pink resin mimicking the color of the gingiva . A 55-year - old female patient was presented at the department of periodontics at positivo university with chief complaints of unsatisfactory esthetics because of receding gums in her maxillary front teeth and sensitivity to thermal stimuli in the same teeth . Anamnesis and clinical examination revealed good systemic health, with no history of drug abuse or smoking . Radiological examination revealed no interproximal bone loss, while clinical intraoral examination showed good gingival health . The bilateral maxillary canines showed gingival recession, with 3 mm, thick gingival biotype and 4 mm high - keratinized gum (figure 1 a and b). We concluded that traumatic brushing was the possible cause of recession and accordingly provided instructions regarding oral hygiene, brushing technique, use of a soft brush, and regular change of the brush . Next, the restoration was planned only for the cervical abrasion area in the canines, using light - cured composite resin mimicking the color of the gingiva (amaris gingiva, voco, cuxhaven, germany). Following shade selection (figure 2a), initially, the admira bond (voco) adhesive system was used according to the manufacturer s recommendations . This resin was applied to mask the dark background of the stained substrate (figure 2b). Immediately after, the pink resin was applied to mimic the surrounding soft tissue, and thereby it was possible to harmonize the color of the resin with the soft tissues . Finally, the finishing and polishing was performed with sandpaper disks and drills for resin (figure 3). Subsequently, we decided to perform root coverage using the conventional (coronally advanced flap) and semilunar coronally positioned flap technique for the left and right canines, respectively, in order to compare the clinical outcomes of the two techniques . For the left canine, we placed an intrasulcular incision with preservation of the papillae using a scalpel with a no . Then, we placed mesial and distal vertical relaxing incisions and raised a mucoperiosteal flap (figure 4). Using a periosteal molt 7a spatula, we completely shifted the flap apically to provide the tissue with complete mobility, which subsequently facilitated coronal advancement of the soft tissue (figure 5a). To stabilize the flap, each suspension suture was placed in the mesial and distal papillae, while simple sutures were used to close the relaxing incisions (figure 5b). For the right canine, we placed an internal bevel incision using a scalpel with a no . The incision was placed above the mucogingival line in a half - moon shape, preserving the interdental papilla (figure 6a). A vascular pedicle with a minimum of 2 mm thickness was maintained for adequate blood supply . The coronally positioned tissue was stabilized with mesial and distal suspension sutures, leaving the most apical portion of the connective tissue exposed for healing by secondary intention (figure 6b). The patient was prescribed an analgesic (paracetamol, 750 mg every 6 hours) as necessary, an antibiotic (amoxicillin, 500 mg every 8 hours for 7 days), and chemical plaque control measures (0.12% chlorhexidine rinse, every 12 hours for 14 days), and was instructed on oral hygiene and care of the surgical sites . Regular follow - up examinations were conducted at day 21 and 3, 6, and 12 months after the surgeries . Partial root coverage on both sides was observed at 21 days (figures 7 and 8), with no dentin hypersensitivity and satisfactory esthetics . After 12 months, almost complete root coverage was observed on both sides (figures 9 and 10), although esthetics were better for the right canine because of a less visible scar . Written informed consent was obtained from the patient to publish this paper and accompanying images . The initial height of the recession is an important factor that should be taken into consideration because of its influence on the results . Gingival recessions equal to or higher than 3 mm have worse prognosis of root coverage.14 we presented a comparison of the conventional and semilunar coronally positioned flap techniques for root coverage in teeth with cervical abrasion restored with pink resin that mimics the color of the gingiva . We found that both techniques were effective in achieving satisfactory and esthetic root coverage, since gingival recession is less than the remaining height of keratinized gingiva . Patients who complain of gingival recession and root sensitivity or poor esthetics are candidates for root coverage procedures.12 various periodontal surgery techniques that can be used for gingival recession coverage are currently available . 1520 of these, coronally positioned flap techniques show good and predictable root coverage.12,21,22 the coronally positioned flap was classified as a pedicle soft tissue graft because of vertical movement in the coronal direction . This flap provides a great combination of color, texture, contour, technical simplicity, little pain and postoperative discomfort (because scarring is by first intention), and good vascularization of tissue moved through the pedicle.21 this surgery requires only a single surgical site, and it is recommended for the treatment of areas with miller class i recession, in the case of recessions up to 3 mm, where there is sufficient keratinized mucosa apically from the gingival margin and tissue thickness of at least 1 mm as it was observed in this case.20 however, it is not indicated for patients with a shallow vestibule, or thin gingival tissue.23 it is necessary to emphasize the importance of adequate surgical planning, considering multiple factors such as the type of recession, quantity and quality of keratinized tissue, tooth position in the arch, flap type, and degree of bone loss among other factors, in order to gain a favorable clinical outcome . Furthermore, the root surface treatment must also be carried out to produce a positive tissue reaction.8,9 in 1986, tarnow19 modified the apical incision and advocated a half - moon shape parallel to the contour of the recession, naming this technique as the semilunar coronally positioned flap technique . This is indicated for shallow localized or generalized recession that does not extend beyond the mucogingival junction, with no interproximal bone loss (miller class i), as observed in the present case . Possible causes include traumatic brushing in the cervical region, causing gingival recession followed by dental abrasion.3 cervical restorations alone cannot improve esthetics and cause excessive tooth exposure . Moreover, root coverage with soft tissue is contraindicated when the cervical abrasion exceeds 1 mm in depth, because plaque control in the cervical region becomes difficult in this case . Therefore, combined restoration and root coverage techniques are required for such cases.8,9,23 in particular, combined restoration and root coverage are necessary for cases of deep cervical abrasions, because the latter can interfere with the adaptation of soft tissue flaps on the root surface when not restored, while an attempt to plane the surface would require the removal of a large amount of healthy tooth tissue.23 the outcomes observed in our patient were consistent with those reported in other studies.9,10,16,23,24 dragoo13 established the biocompatibility of glass ionomer cement placed under gingival tissue; however, the use of restorative materials such as composite resin was reported to resolve the issue of poor esthetics.8,9,24 histologically, long junctional epithelium formation was reported with both glass ionomer and composite resin, demonstrating the biocompatibility of both materials under gingival tissue.24 this biocompatibility can also be clinically observed, evidenced by the absence of inflammatory signals . We used pink resin for cervical restorations in the present case, the properties of which allow for good finishing and polishing and superior esthetics . The findings of this case suggest that the use of pink resin associated with the conventional and semilunar coronally positioned flap techniques achieves satisfactory esthetics and root coverage, with an excellent combination of color, texture, and gingival contour . Both techniques can be used for covering gingival recession when the teeth show appropriate indications and the surgeon is well trained; however, a conventional technique has advantages over the semilunar technique, as it is more predictable and has a lower surgical morbidity.
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Asymptomatic patients post arterial switch (aso) surgeries are at low risk for myocardial ischemia, and much depends on the type of coronary anomalies and the method of transfer of the coronary arteries during surgery . However certain coronary patterns in transposition of great vessels like a retropulmonary looping of left coronary artery (lca) or an intramural coronary artery predict increased morbidity and mortality . Pediatric myocardial perfusion imaging (mpi) is an established investigation in the evaluation of ischemia and myocardial viability . It is said that aso surgeries are at a low risk for myocardial ischemia, and much depends on the type of coronary anomalies and the method of transfer of thecoronary arteries during surgery . However, certain coronary patterns in transposition of great vessels such as aretropulmonary looping of left coronary artery or an intramural coronary artery predict increased morbidity and mortality . He was diagnosed with taussig - bing anomaly, large subpulmonic ventricular septal defect (vsd), severe coarctation with long - segment hypoplastic arch, retropulmonary looping of lca, and a large patent ductus arteriosus at birth . The patient underwent arterial switch operation, bovine pericardial patch closure of vsd, resection of coarctation and reconstruction with tissue - to - tissue anastamosis posteriorly, and bovine pericardial patch augmentation anteriorly on 5 day of life . Echocardiogram revealed no residual vsd, no regional wall motion abnormalities, and showed no evidence of any left ventricular outflow tract obstruction . Adequate biventricular function was confirmed, normal diaphragmatic movements were seen, and no pericardial effusion was noted . In view of persistent breathlessness (functional classification ii), the patient was referred for mpi with single - photon emission computed tomography (spect) to rule out any myocardial ischemia . Pharmacological same day stress - rest - gated mpi was performed under the supervision of a pediatric cardiologist using intravenous infusion of adenosine at a dose of 140 m / kg per min for 6 min . At 3 min of the infusion 2 mci (millicurie) of technetium sesta mibi (tc - mibi) delayed stress - gated spect images were acquired after 1 h using high - resolution collimators on a dual - head variable angle siemens e cam gamma camera . Six millicuries of tc mibi was injected 3 h later at rest . Delayed rest - gated spect images were acquired . Motionless stress and rest acquisition were performed once the baby was sedated with intravenous midazolam at a dose of 0.1 - 0.2 mg / kg body weight . Spect images of myocardium were acquired at 76 degrees cardiac angle using a 180-degree rotation from the right anterior oblique to the left posterior oblique projection . Images were acquired in tc window centered at 140 kev photopeak with a window of 20% . Images were acquired in a step and shoot mode, 64 64 matrix, for 74 frames at 25 s / frame with a zoom of 2.0 . Images were checked for motion during acquisition and were then processed as per the company provided reconstruction software into short axis, horizontal long axis, and vertical long axis . All images were interpreted visually and quantitatively using emory cardiac toolbox (ectbox, emory university, atlanta, ga, usa). Post - stress - gated spect images [figure 1a] showed a fixed perfusion defect involving entire anterior segment (small arrow) and reversible perfusion defect in lateral segment (bold arrow) indicating a large lad territory infarct with moderate ischemia in lateral segment of left ventricle . (a) post - stress - gated spect images showed a fixed perfusion defect involving entire anterior segment (small arrow) and reversible perfusion defect in lateral segment (bold arrow) indicating a large lad territory infarct with moderate reversible ischemia in lateral segment of left ventricle . (b) coronary angiogram confirmed left main coronary artery ostial occlusion, with retrograde collateral supply from dilated right coronary artery the patient was further taken up for coronary angiogram to confirm the above findings and also to see the coronary artery relationship with right ventricular outflow tract (rvot). Right coronary artery (rca)-guiding catheter and terumo combination was used to enter into the rvot and angiogram was done . A 0.5 fr pigtail / terumo combination was used to enter the aortic root and angiogram was done simultaneously . A 14 4 cordis balloon was inflated across rvot via 8f long sheath over v-18 wire to observe the relationship between the coronary artery and the rvot . Aortis root angiogram showed a dilated rca with delayed and retrograde filling of lca through collaterals [figure 1b]. He had right ventricular (rv) pressures slightly more than half systemic, hence rvot stenting was not done . After balloon dilatation of rvot with 14 4 cordis balloon, there was no significant fall in the rv pressures . He has been advised a close follow - up . Taussig - bing anomaly is a rare congenital heart malformation first described by helen b taussig and richard j bing . It is the transposition of the aorta to the right ventricle and malpositions of the pulmonary artery with subpulmonary vsd . Reports show an increased mortality and morbidity, especially in patients with additional anomalies such as transposition of great vessels such as retropulmonary looping of left coronary artery . Reported that single coronary patterns, both of which loop around the great vessels, were associated with significant mortality [odds ratio (or) 2.9, 95% confidence interval (ci) 1.3 - 6.8], whereas looping patterns that arose from two separate ostia were not (or 1.2, 95% ci 0.8 - 1.9). The current standard procedure includes mobilization and reimplantation of the coronary arteries into the neoaorta . The outcome of such surgeries depends on the variations in coronary anatomy and their successful transfer . There are mixed opinions predicting increased morbidity and mortality in certain types of coronary anomalies such as retropulmonary looping of lca or an intramural coronary artery . Our case highlights the early onset of mi in a patient with retropulmonary looping of lca . It also highlights the relatively asymptomatic presentation of a large lad infarct with reversible ischemia in lcx territory - supplied left ventricular segments within 5 months of surgery . Other causes implicated by wernovsky et al . Were based on postoperative kinking or stretching of these coronary arteries after their transfer . Pasquali et al . Analyzed the relationship between coronary artery variants and mortality after aso in a meta - analysis of 9 case series . Even complete occlusion of a main coronary goes unnoticed as it may remain completely asymptomatic but it has been associated with sudden death . Selective coronary angiography has been recommended after aso; however, there is no consensus as to when it should be performed . Reddy et al . Have recently published their data on adenosine spect mpi in 10 asymptomatic children post - aso . They conclude that postadenosine stress mpi is safe and feasible in patients of aso for transposition of great arteries . This case report highlights importance of identifying coronary artery stenosis in the perioperative period of congenital heart disease patients, which may go unnoticed in the absence of high degree of clinical suspicion . It also reiterates that although post - aso surgery patients are considered as at a low risk for ischemia, there is increased mortality and morbidity, especially in patients with additional anomalies such as transposition of great vessels such as retropulmonary looping of left coronary artery.
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During 2015 in south korea, a total of 10,332,000 pigs were raised, of which 2,338,521 (22.6%) were raised on 1,134 farms in gyeongsang province (6). For this study, we collected 1,030 blood and 97 tissue samples from pigs (645 breeding and 479 fattening pigs) reared on 209 pig farms in gyeongsang province during 20142015 . Sample size was determined using a formula with an expected disease prevalence of 50%, accepted absolute error of 5%, and ci of 99% in a simple random sampling design (technical appendix); a minimum of 664 samples were required . Samples were collected by practicing veterinarians during treatment or regular medical checkups; ethical approval was not required . The number of samplings was based on the number of pigs and farms within each of the province s administrative districts (figure 1). Number of pig farms in the provincial administrative districts and number of farms on which pigs were sampled for the detection and genotyping of coxiella burnetii, gyeongsang province, south korea, 20142015 . The number of samplings was based on the number of pigs and farms within each of the province s administrative districts . Lung, lymph node, liver, spleen, and kidney samples were collected for differential diagnosis of diseases in pigs that aborted or had a stillbirth, respiratory symptoms, or weakness . To detect c. burnetii positive samples, we used 2 different assays and nested pcr . We used an indirect multispecies elisa (i d screen q fever indirect multi - species kit; idvet, montpellier, france) according to the manufacturer s instructions to detect c. burnetii antibodies in samples; a sample optical density to positive - control optical density value of> 50% was considered positive . We also performed an indirect immunofluorescence assay (ifa), using the coxiella burnetii (q fever) fa substrate slide (vmrd, pullman, wa, usa), as recommended by the manufacturer; titers> 64 to phase-1 or phase-2 antigens were considered seropositive . We used the dneasy blood and tissue kit (qiagen, hilden, germany) according to the manufacturer s instructions to extract dna from whole blood and tissue samples . The coxiella 16s rrna gene in extracted dna was then amplified using nested pcr and sequencing primers (technical appendix). We sequenced amplification products with macrogen (seoul, south korea) and analyzed results using sequence alignment programs and statistical methods (technical appendix). Of the 1,030 sampled pigs, 70 (6.8%) were positive for c. burnetii by elisa (table); these pigs were from 32 (15.3%) of the 209 sampled farms . Two of the 32 farms had 8 positive pigs each; the other 30 had 13 positive pigs each . Fifty - three (5.2%) sampled pigs had samples identified as phase-1 or phase-2 antigen seropositive by ifa; these samples were also seropositive by elisa . An additional 17 samples seropositive by elisa were seronegative by ifa . C. burnetii seroprevalence was significantly higher (p<0.0001) in breeding than in fattening pigs by elisa and ifa . Elisa and ifa results were in agreement for 1,013 (98.4%) of the 1,030 samples; 53 (5.2%) samples were positive, and 960 (93.2%) were negative . The cohen coefficient was 0.85 (i.e., very good agreement; 95% ci 0.790.92). Three (0.3%) pigs were positive for c. burnetii by pcr; all were breeding pigs and seronegative for c. burnetii . One positive sample was lung tissue from a pig that appeared to have respiratory signs; other respiratory pathogens were also detected in the sample . However, the infection status of seropositive pigs cannot be determined on the basis of a single titer . C. burnetii seronegative pigs can, however, shed the organism and, thus, might serve as a reservoir for transmission of the bacterium to humans . 16s rrna gene sequences for the 3 c. burnetii pcr - positive samples (genbank accession nos . Kt94501416; figure 2) showed 100% identity with each other; nucleotide sequences showed high (96.6%96.9%) identity with those of other c. burnetii strains . Phylogenetic analysis showed that the 3 isolates belong to clade a, clustering with previously published c. burnetii sequences (figure 2). Phylogenetic tree constructed using the maximum - likelihood method from coxiella burnetii 16s rrna sequences . Arrows indicate c. burnetii sequences from study to detect and genotype c. burnetii in pigs in gyeongsang province, south korea, 20142015 . We found that 6.8%, 5.2%, and 0.3% of tested pig samples in gyeongsang province were positive for c. burnetii by elisa, ifa, and pcr, respectively . These rates of seropositivity are relatively low compared with the rate found in a study in uruguay, in which 18.4% (83/479) of the blood samples were seropositive by layer microagglutination (7). In that study, the innate susceptibility of pigs to c. burnetii was confirmed during a q fever epidemic . Seropositivity in our study was, however, higher than that reported in blood tested by ifa in japan (0/396 samples) (8) and by complement fixation in bulgaria (0.05%; 1/1,809 samples) (9). In c. burnetii we did not test such tissues; however, the positivity rate in our study was similar to that (0/16) in a previous examination of pig placentas by real - time pcr in the netherlands (10). In our study, similar to results from a previous study (11), our results showed that ifa was less sensitive than elisa at detecting c. burnetii in serum . Animals can maintain seropositivity after acute infection has cleared, and they can seroconvert without shedding (12); thus, serologic methods are not useful for determining which animals currently pose a risk for transmission . In our study, in addition, only breeding pigs were positive for c. burnetii by pcr . Because of pregnancy stress, breeding pigs probably experienced a recrudescent infection, making them more likely to shed the organism . A study on the epidemiology of q fever suggested that breeding pigs can cause infection in humans (13). The genus coxiella is divided into 4 highly divergent genetic clades (a d); c. burnetii belongs to clade a (14). Phylogenetic analysis showed that the 3 c. burnetii isolates in our study were closely related to clade a strains from the united states, japan, and greenland, indicating a close epidemiologic link . Although the number of c. burnetii positive pigs was low in our study, a previous study identified contact with pigs as a risk factor for c. burnetii seropositivity in humans (15). Therefore, pigs may serve as potential reservoirs for c. burnetii . However, several questions remain unanswered regarding the epidemiology of c. burnetii infection in pigs and possible transmission to humans . Additional investigations of the infection prevalence in other animals are necessary to understand the epidemiology of c. burnetii.
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Lipomas are the most common soft tissue mesenchymal neoplasms; it involves 15 - 20% in the head and neck region and 1 - 4% in the oral cavity . Lipomas commonly present as slowly enlarging asymptomatic lesions, with a soft, and smooth - surface when it is superficial with a yellow coloration . They most commonly occur in the areas of fat accumulation, especially the cheek, followed by the tongue, floor of the mouth, buccal sulcus and vestibule, lip, palate, and gingiva . Its exact etiology is unknown, possible causes may include trauma, infection, chronic irritation and hormone alterations and 12q, 13q, 6p chromosomes alterations . Its occurrence in the oral cavity is less, but if affected gingiva followed by lip, tongue, and palate are chiefly involved . A 13-year - old boy reported to the department of oral medicine and radiology, with a chief complaint of swelling in his right lower buccal vestibule since 2 years, the swelling was initially smaller in size, when noticed by the parents, which gradually progressed to the present size and not associated with any pain or any functional impairment . On intraoral examination, a solitary, sessile, dome - shaped normal mucosal colored swelling was present in the right lower buccal vestibule, measuring approximately 3 cm 2 cm in diameter, extending anterio - posteriorly from mesial aspect of 44 to the distal aspect of 46, mediolaterally from the attached gingiva of 44, 45, 46 to till 0.5 cm away from lower buccal vestibule and superio - inferiorly 1 cm above the occlusal level of 44, 45, 46 to till the lower buccal vestibule with well - defined borders [figure 1]. On palpation, swelling was measuring about 3.5 cm 2.5 cm in diameter with smooth surface, normothermic, nontender, soft in consistency with few palpable masses, mobile, compressible, nonreducible, fluctuant with no palpable pulsations . Intraoral hard tissue examination showed retained deciduous 55 and yellowish white flecks on all the teeth . Panoramic radiograph was noncontributory [figure 2]. Based on clinical signs and symptoms, a provisional diagnosis of lipoma was established with a differential diagnosis of hemangioma and mucocele . Swelling in the right buccal vestibule panoramic radiograph revealed no loss of bone in relation to the lesion hemogram of the patient was within normal limits . Further investigations such as ultrasonography (usg) with doppler and magnetic resonance imaging (mri) were done, which were noncontributory . Usg revealed 2.6 cm 0.9 cm mass in the right buccal mucosa, which is predominantly hypo - echogenic with no evidence of internal vascularity [figure 3]. Mri study of head and neck showed hypointense area on the right side of the mandible measuring about 26 mm 9 mm 10 mm [figure 4]. The lesion was excised [figure 5] under local anesthesia with all necessary emergency equipment and excised specimen was sent for histopathological examination [figure 6] which revealed overlying epithelium and connective tissue with numerous blood vessels of varying sizes and shapes intermingled with proliferating blood capillaries embedded in fibrous connective tissue . The blood vessels are lined by plump endothelial cells and are engorged by rbc's dense collagen fibers lined by spindle - shaped fibroblasts and fibrocytes are seen, which confirmed the diagnosis of sclerotic capillary hemangioma . The patient was under follow - up since 6-months with a good prognosis [figure 7]. Ultrasonography showing hypoechogenic areas magnetic resonance imaging showing hypointense area on the right side of mandible intraoperative photograph histopathological photomicrograph, 10 and 40 postoperative photograph (after 6-months) hemangiomas are considered to be benign tumors of infancy that are characterized by a rapid growth phase with endothelial cell proliferation followed by gradual involution . Hemangiomas follow a predictable course with three distinct developmental phases: proliferation, quiescence, and final unique phase of involution . Historical reports suggest that involution of 50%, 70%, and 90% of the hemangioma occurs by 5, 7, and 9 years of age with some variability . They are classified on the basis of their histological appearance as capillary, mixed cavernous, or a sclerosing variety that tends to undergo fibrosis . Capillary hemangiomas may be sessile or pedunculated, soft, painless, and may be smooth or irregularly bulbous in outline . Its color varies from reddish blue to deep blue depending on the depth of vascular proliferation within the oral submucosa . They blanch upon application of pressure; conversely, when intraluminal clots form they become palpable and the lesion will usually not blanch as in the present case . The exact etiology of vascular lesions is not known but according to few authors, angiogenesis plays a vital role in the pathogenesis of vascular lesions . Cytokines like basic fibroblast growth factor and vascular endothelial growth factor induce the process of angiogenesis and the factors which conquer angiogenesis (gamma interferon, tumor necrosis factor beta, transforming factor - beta). 60% of the hemangiomas occurs in the head and neck region and its occurrence in the oral cavity is less, but if affected gingiva followed by lip, tongue, and palate are chiefly involved . Hemangiomas occur in the skin of 4 - 10% of caucasian newborn and much more common in females than males at a ratio 3:1 . They show lower incidence in dark - skinned infants, but in our case, the patient was male . According to krishna kripal et al ., nadeem jeddy et al ., alparslan dilsiz et al ., clinically, the hemangiomas were soft, sessile or pedunculated, smooth or lobulated, painless, purple or deep dark blue in color with size varying from small to a larger lesion . The findings in our case were similar to the finding noted by above authors except for the color, which was of normal mucosal color . Clinically and histopathologically, hemangiomas can mimic many other soft tissue tumors like the vascular malformations, pyogenic granuloma, epulis, varicosities, oral squamous cell carcinoma, kaposi sarcomas . The differential diagnosis has a vital role to play in these lesions as it can dedicate the treatment option and the outcome of the disease . Hemangioma classification is based on histological appearance, therefore, histopathological assessment remains the most accurate and satisfactory means of diagnosis . Hemangiomas are histologically classified as capillary, cavernous, mixed and sclerosing type our case is of sclerotic capillary hemangioma . Depending upon anatomic location and clinical manifestation, the treatment of hemangiomas varies . Surgical excision is the treatment of choice and for those lesions which are not amenable to surgery, other treatment modalities such as sclerotherapy, cryosurgery, laser therapy, electrocoagulation, and radiation may be utilized . Sclerotic capillary hemangioma being a very rare lesion, it should be included in the differential diagnosis of benign mucosal lesions with normal mucosal color, which do not blanch on pressure . When a clinician is in a dilemma, not cognizant with the possibility of this lesion in its unusual site, can be solved by histopathological assessment which remains the most accurate and satisfactory.
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Chronic kidney disease (ckd) in the pediatric population has become an important issue . Ckd represents a spectrum of conditions, which result in renal impairment varying from mild renal insufficiency to end stage renal disease (esrd). Fivush et al reported that half of patients with esrd in childhood attain adult's height below the 3 centile . The cause of growth failure in ckd is multifactorial with linear impairment being a final common pathway of various factors including malnutrition, anemia, metabolic acidosis and persistent micro - inflammations ckd can result in impairment in each phase of development from in - utero to adolescence, which can subsequently result in growth retardation, with studies suggesting that the degree of height deficit worsens with duration of disease . The north american pediatric renal transplant cooperative study (naprtcs) reported that more than one third of children with ckd exhibited significant growth failure . Although a correlation between renal function and growth impairment existed, significant short stature was seen at all levels of renal function . Wong et al explored a potential association of serum albumin and mortality in children with esrd . While malnutrition can be seen in pre - dialysis period, it can also be developed by factors such as increased catabolism, loss of nutrients and anti - oxidants, and aggressive dietary restrictions during dialysis . Serum albumin has been identified as a surrogate marker for nutritional status and morbidity / mortality in patients with ckd . Patients <18 years of age initiating dialysis with hypoalbuminemia are at a higher risk for death . The objective of this study was to describe the prevalence of growth failure and malnutrition in pediatric ckd patients according to age group . We also aimed to explore the relationship between growth parameters and treatment modalities in the patients . This was a cross - sectional and observational study that included pediatric ckd patients in stages 1 - 5, followed by peritoneal dialysis (pd), hemodialysis (hd) and pre - dialysis (pre - d) in ege university faculty of medicine, department of pediatric nephrology in turkey from october 2007 to may 2008 . Patients clinical characteristics were retrieved from medical interview, physical examination and careful analysis of patients records . This study included 42 patients (23 males and 19 females) and 29 healthy children matched for age and gender . Patients were grouped according to treatment modalities: 20 patients on pre - d (5 at stage i, 8 at stageii, 2 at stage iii, and 4 at stage iv), 22 patients in stage v ckd (end stage renal disease, esrd); 8 patients in this group were treated with pd, and 14 patients were treated with hd . The duration of renal replacement therapy (rrt) ranged from 6 to 139 (average 50.636) months . Also, we divided the patients into three groups according to the age onset of disease as early childhood (3 - 6 years), pre - pubertal (7 - 10 years), and pubertal (11 - 17 years). Ckd was defined according to schwartz formula as: stage 1 (renal injury) estimated glomerular filtration rate (egfr) of> 90 ml / min per 1.73 m, stage 2 (mild) egfr of 60 - 89 ml / min per 1.73 m, stage 3 (moderate) egfr 30 - 59 ml / min per 1.73 m, stage 4 (severe) egfr of 15 - 29 ml / min per 1.73 m, stage 5 (esrd) egfr of <15 ml / min per 1.73 m. nutritional evaluations were performed according to the kidney disease outcomes quality initiative (kdoqi) guidelines, and we performed adjustments using values from children with the same chronological age as reference . All subjects weights and heights were measured, weight and height standard deviation (sd) scores, and body mass index (bmi) calculated . Sd score (z score) was calculated by subtracting the mean height of children of the same age and sex from the observed height and then dividing by the sd for children of that age and sex . Body mass index (bmi) was determined by dividing weight in kilograms by square of height in meters . For these calculations, we used the epi - info program (version 3.3, october 2004) with centers for disease control (cdc) 2000 references . Growth retardation was described when bmi was below percentile 5 for chronological age, and height / age, weight / age were below -2 z scores for chronological age . Lipid parameters: total cholesterol, high density lipoprotein (hdl), low - density lipoprotein (ldl) and triglycerides, hemoglobin, hematocrit, creatinine clearance (blood plus peritoneal fluid in the dialysis group), serum albumin, calcium, phosphorus, parathyroid hormone, calcium in phosphorus, lipid levels and c - reactive protein were evaluated in patients . The effectivity of dialysis (kt / v) was evaluated in the esrd patients . Smirnov test and values are presented as mean (sd) or as median (interquartile range) unless otherwise specified . Student's t test, analysis of variance (anova), or wilcoxon signed ranked test was used to compare differences between numeric values in different groups . Comparisons of prevalence in different groups were assessed by the chi - square test . Calculations were performed with spss (version 11.0) and a p - value of less than 0.05 was considered as significant . We included 42 (23 males, 19 females) patients and 29 healthy children matched for age and gender . According to their treatment, 22 patients were on rrt (14 on hd, 8 on pd) and 20 were followed in pre - dialysis clinic (5 at stage i, 8 at stage ii, 2 at stage iii, and 4 at stage iv). The mean age at onset of the disease was 11.24.4 years (range 3.3 to 17.5 years). The mean duration of chronic renal failure was 64.556.9 months (range 1 month to 16 years). Nineteen patients (45%) had glomerulo - pathies, 1 (2%) had alport syndrome, 15 (35%) had reflux nephropathy, 4 (9.5%) had neurogenic bladder, 3 had obstructive uropathy and 3 had hereditary conditions . Eight (19%) patients were 3 - 6 years (early childhood), 9 (21%) patients 7 - 10 years (pre - pubertal), and 25 (60%) patients 11 - 17 years old (pubertal). The height was most severely impaired during early childhood (under 6 years old), with mean height sds of -3.362.81 . In this group, height sds in 83% of the patients was below -2 . In pre - pubertal group mean height sds was higher than in early childhood group (-0.991.58 vs -3.36 2.81, p=0.04). This value was lower than that in pre - pubertal period while higher than in early childhood (p=0.4 and p=0.03 respectively). Mean and standard deviation (sd) scores for height by age in all patients according to therapy modalities no significant differences between height sds within the pre - dialysis groups no significant differences between height sds within the peritoneal dialysis groups no significant differences between height sds within the hemodialysis groups significant differences (p<0.05; anova) between height sds within the age groups are marked the mean weight sds was -0.251.46 (range -4 to 1.85), -2.581.78 (range -5.3 to 0.68) and 0.530.95 (range -0.5 to 1.8) in pre - d group, rrt group and controls, respectively (p<0.001). In rrt groups, the mean weight sds was -2.921.71 (range -5.3 to 0.57) and -1.991.87 (range -5.0 to 0.68) in hd and pd groups, respectively (p=0.2) (table2). Demographic and clinical data, and biochemical characteristics in all groups according to the therapy anova between peritoneal dialysis and hemodialysis parameters within the therapy modalities are marked . Sd: standard deviation / bmi: body mass index / crp: c- reactive protein the height sds was -0.771.88 (range -5.7 to 2.3), -2.651.86 (range -6.6 to -0.1) and 1.31.5 (range 0.8 to 2.8) in pre - d, rrt and controls, respectively (p=0.00). In rrt groups, the mean height sds was -2.661.55 (range -6.3 to -0.64) and -2.632.43 (range -6.6 to -0.10) in hd and pd groups, respectively (p=0.96). Seventeen (40%) patients had short stature (height sds below -2 sds). The disease duration was longer in patients with short stature than in other patients (7.13.8 vs 3.84.4 years). There was statistically significant relationship between duration of disease and short stature (p=0.01). In our cohorts growth impairment was related to treatment modalities without age group (p=0.09, p=0.6, p=0.7, pre d, hd and pd, respectively) (table 1). In pre - d group, 2/20 patients had short stature (height sds was below <-2sds), while 12/22 patients had short stature in rrt group (p=0.03). Significant difference was seen between bmi sds in pre - d, rrt and controls groups (p=0.03), but in rrt groups, the mean bmi sds doesn't have significant difference in hd and pd groups (p=0.4). In all groups, 19/42 (45%) (4 on pre - d, 5 on pd, 10 on hd) patients had malnutrition which was attributed to sga . Mean albumin was 3.820.97 which was below the normal reference values (3.5 mg / dl) in only 14% of patients . In pre - d group, the mean serum albumin was 3.990.4 mg / dl (range 3.4 to 4.8), while it was 3.870.45 (range 3 to 4.5) and 3.271.29 (range 0.3 to 4.5) in hd and pd group, respectively (p=0.04) (table2). Protein catabolic rate was higher in hd groups than in other patients (0.820.10 g / kg / per day) (p=0.03). The growth and nutritional status were better in pre - d group than in rrt group . Most patients were receiving the usual medications, such as phosphate binders, antihypertensive agents, vitamins, and nutritional supplements . For patients in hd, mean kt / v was 1.75, whereas for patients in pd, it was 4.2 . Mean crp concentration was 1.071.86 mg / dl (range 0.18.41 mg / dl). Sixteen (45.7%) patients presented crp levels above 0.3 mg / dl and therefore were considered inflamed growth failure is a major obstacle for full rehabilitation and may result in severely diminished adult height in childhood with ckd . Chronic kidney disease can result in impairment in each phase of development from in - utero to adolescence, which can subsequently result in growth retardation, with studies suggesting that the degree of height deficit worsens with duration of disease . In this study we found that a relation exists between disease duration and short stature . Previous studies have described aspects of growth pattern, either focusing on infancy, pre - puberty or puberty [1416]. These studies showed that infancy and early childhood represent the most affected growth periods in children with ckd . In this study, our patients under 6 years of age showed the most severe growth deficit in height (mean height sds was -2.45). In the age cohort, zivicnak et al have shown that growth kinetics differed during early and late puberty, beginning with a slowing - down of growth during early puberty . The mean height z score decreased in early childhood and pubertal groups than in prepubertal group (table 1). The degree of kidney failure and treatment modalities did significantly influence the severity of growth impairment without age dependence . Despite good progress with regard to both conservative treatment and rrt, 30% to 60% of children with esrd still grow up to become stunted adults [1820]. Growth failure is associated with mortality, morbidity and especially psychosocial problems in children with ckd [5, 7]. Reduced adult heights have been reported in about 30% to 50% of patients with ckd [15, 16, 21]. The age at onset of esrd, the duration of chronic renal failure, gender and primary disease were related to final height . In our cohort, longer duration of chronic renal failure was found to be associated with short stature while gender, age at onset of crf and primary disease were not related to final height . At the end of the follow up time 17 (40%) patients had short stature (9 patients on hd, 4 on pd and 4 on pre - d). The pre - dialysis patients were found better than rrt patients . In hd group, there was lower mean height sds, which was not statistically significant . In our cohorts, the body mass index can be prognostic, because extremes are associated with increased mortality and morbidity . Wong et al demonstrated that the adjusted relative mortality risk of children with esrd is 60% higher at bmi standard deviations of -2.5 and + 2.5 as compared with an ideal bmi standard deviation of 0.5 . In our study, 10 patients bmi sds was under -2.5 sds (1 on pre - d, 5 on hd and 3 on pd). The pre - dialysis patients were found to be better than renal replacement therapy patients . Malnutrition is an important risk factor for mortality and it is too prevalent in adults and children with ckd[10, 23, 24]. The prevalence of loss of energy and protein in pediatric hd patients is reported to be 56% . However, measurement of nutritional parameters is complicated in ckd because of salt and water imbalance . Anthropometric and nutritional measures are usefully expressed as a score of the number of standard deviations from the mean for a normal population of the same age (e.g. Height, weight or body mass index sd scores). This allows comparison with the normal population and helps follow progress in the individual patient . However, patients on peritoneal dialysis have significant protein and amino acid losses in the dialysate . In our patients, the mean serum albumin level was higher in pre - d group than hd and pd patients . Previous studies have shown that hypo - albuminemia is related to inflammation [28, 29]. We did not find a correlation between hypo - albuminemia and elevated crp . In summary, we investigated growth parameters in children with ckd according to both age groups and therapy . Growth impairment in our patients was age dependent with the most vulnerable period of longitudinal growth being early childhood . Secondly we observed that growth impairment was found more frequently in patients under hd therapy . Growth failure and malnutrition remain a significant age dependent clinical problem in children with ckd.
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The mhc is a genomic region located on the short arm of chromosome 6 (6p), encoding hla molecules . Loss or down - regulation of mhc class i expression in cancer cells is a major immune escape route used by a large variety of human tumors to evade anti - tumor immune responses mediated by cytotoxic t lymphocytes [1 - 3]. Multiple mechanisms are responsible for such hla class i alterations [4 - 11]. Among them, loss of heterozygosity (loh) of chromosome 6p has been reported in a number of solid tumors and some hematologic malignancies, mainly in all [12, 13]. In patients with leukemia, loh at the mhc region can give false homozygosity results in hla typing when the typing is performed using peripheral blood containing a high proportion of leukemic cells . However, such a problem has rarely been reported because hla typing is not usually carried out on a blood specimen containing a high proportion of neoplastic cells . A case of a cll patient has been reported involving loh at the mhc region at the initial hla typing, which was then detected at confirmatory typing for hematopoietic stem cell transplantation . Here we report false homozygosity results in hla genotyping due to loh associated with the loss of whole chromosome 6 in an adult korean patient with all . A 33-yr - old korean female was admitted to the hemato - oncology ward of seoul national university hospital on october 4, 2010 for the evaluation of leukocytosis found during a workup for headache . A complete blood cell count showed a hemoglobin level of 7.8 g / dl, a platelet count of 1510/l, and a white blood cell count of 336.910/l, of which 84% were blasts . The blasts were homogeneously small sized with high nuclear / cytoplasmic ratio and inconspicuous nucleoli . Flow cytometric analysis revealed that the neoplastic cells were positive for tdt, cd19, cd34, and cytoplasmic cd79a, and a diagnosis of precursor - b all was made . On october 7, 2010 (the third day of hospitalization), before initiation of induction chemotherapy, high - resolution hla dna typing of the patient was performed using the peripheral blood sample . For future hematopoietic stem cell transplantation, hla class i (a, b, and c) serological typing of her 2 siblings high - resolution hla typing was performed by sequence - based typing (sbt) using the alleleseqr hla sequencing kit (atria genetics, south san francisco, ca, usa). Hla genotyping of the patient revealed homozygosity at class i and ii loci, except for the drb1 locus: a02:01, b54:01, c08:01, drb104:05, drb111:01, dqb104:01 (table 1, fig . 1). One of the deduced hla haplotypes (a02:01-b54:01-c08:01-drb111:01-dqb104:01) of the patient was a fairly unusual one for korean ethnicity . Moreover, homozygosity at the hla - b locus (b54:01) in the patient could not be explained, considering the hla - b types of her 2 siblings (b51, b54 in one and b60, b62 in the other). Retyping of the patient's dna for hla - a, -b, and -dr loci by the sequence specific oligonucleotide (sso) method using dynal reli sso hla - b, -drb, and -dqb1 kits (invitrogen, wirral, uk) also revealed homozygosity at the following loci: hla - b54:01 group, drb104:05 group (drb401:01 group), and dqb104:01 group . A retrospective review revealed that the patient's hla genotyping had been requested when she had a high blast count in the peripheral blood, with a white blood cell count of 91.710/l, of which 87% were blasts . Conventional karyotyping performed on a bone marrow aspirate sample obtained on october 11, 2010 (the seventh day of hospitalization) revealed chromosomal abnormalities with loss of multiple chromosomes, including chromosome 6, and reduplication of the remaining chromosomes: 29,x,+x,+8,inv(9)(p11q13),+10,+14,+18,+21/58,idemx2/46,xx, inv(9) (fig . 2). Retrospective analysis of short tandem repeat (str) markers revealed loh in the str markers on chromosomes 3, 4, 5, 7, 12, and 13 (table 2), which was in accordance with conventional karyotyping results . Minor alleles due to loss of pertinent chromosomes in neoplastic cells comprised on average 4.1% (range 2.9 - 6.9%). Hla genotyping was repeated on december 13, 2010, with the peripheral blood in a remission state, 67 days after the initiation of induction chemotherapy . A complete blood cell count showed a hemoglobin level of 10.0 g / dl, a platelet count of 16010/l, and a white blood cell count of 2.310/l, of which 0% were blasts . All of the hla - a, -b, -c, -drb1, and -dqb1 loci were typed as heterozygous: a02:01, a02:06, b40:01, b54:01, c03:04, c08:01, drb104:05, drb111:01, dqb103:01, dqb104:01 (table 1, fig . 1). Karyotyping performed on a peripheral blood sample obtained on the same date (in a remission state) revealed a normal karyotype: 46,xx, inv(9)(p11q13). Str analysis performed on the same blood sample also revealed heterozygosity results in all 9 markers . Thus the heterozygosity results of hla - a, -b, -c, -dr, and -dq loci were confirmed and finally used to search for an unrelated donor for bone marrow transplantation . Hla class i antigens are vital in the recognition of tumor cells by tumor - specific cytotoxic t lymphocytes . Loss or down - regulation of hla class i antigens, therefore, represents a way by which tumors can escape t - cell surveillance, adversely affecting the course of disease and the outcome of t - cell - based immunotherapy [1 - 3]. This can have several causes: absence of 2-microglobulin or transporter associated with antigen processing (tap) expression [4 - 6]; loss of heterozygosity, large deletions, or mitotic recombination in chromosome 6 [4, 8, 9]; transcriptional down - regulation; or point mutation, partial deletion, or somatic recombination [4, 7, 11]. Loh in chromosome 6p has been detected in about 10 - 55% of solid tumors such as those of the colon or larynx, melanoma, and breast cancer [12, 15]. Previously reported frequencies of loh in 6p in hematologic malignancies are variable according to the detection method and definition of loh . Masuda et al . Reported loss or down - regulation of hla class i expression in only 4 out of 43 freshly isolated adult hematologic malignancy samples and no haplotype loss in the hla genome . In childhood all, the frequency of loh in 6p has been reported to be 7 - 8% [17, 18]. In contrast, mcevoy et al . Reported a high frequency of mhc loh in all patients (19/56), which was usually associated with whole chromosome 6 loss (13/19). Loh in chromosome 6p in hematologic malignancies can cause false homozygosity results in hla genotyping when the typing is performed with leukemic blood samples . This can give rise to a serious problem in hematopoietic stem cell transplantation in that the patient would be at a risk of being transplanted from a falsely matched donor if the false typing results are not properly recognized . Serologic typing of hla antigens with blood samples containing high blast counts is difficult, and therefore, typing is usually not attempted . Nowadays dna typing is increasingly used for hla typing, and such problems may occur if clinicians inappropriately order hla genotyping with leukemic blood samples, and the hla typing laboratory is not aware of the problem in the blood sample requested for typing . False homozygosity results for hla class i due to loh at the mhc region has been reported in a cll patient, which was detected at confirmatory typing for hematopoietic stem cell transplantation . In this patient, loh in chromosome 6p was confirmed by microarray - based comparative genomic hybridization of tumor cells . In our case, whole chromosome 6 loss in leukemic cells caused false homozygosity results in hla - a, -b, -c, and -dqb1 sbt and hla - b, -drb, and -dqb1 sso typing . Retesting with a peripheral blood sample in a remission state using sbt revealed heterozygosity results in all of the 5 hla loci tested . In hla - drb1 typing in the initial sample, a sequencing electropherogram showed amplification peaks of the minor allele (drb111:01, present only in a small fraction of normal leukocytes) which were much lower than those of the major allele (drb104:05, present in both leukemic blasts and normal leukocytes) (fig . The discrepancy in hla - drb1 typing results between the 2 typing methods, differences in the primers used for these methods might be the cause . For hla - drb1-specific amplification, different hla - drb1 alleles cannot be amplified by a single pair of primers and thus several different primer pairs amplifying different groups of drb1 alleles are used in hla - drb1 sbt . The primer pairs that can amplify each of the 2 drb1 alleles (drb104:05, 11:01) in this patient are expected to be different and the minor allele could thus be amplified in sbt . In hla - drb sso typing, using a dynal reli sso kit, single primer pairs amplifying drb1/drb3/drb4/drb5 genes are used, and the minor allele in this patient seems not to have been effectively amplified to be detected . Likewise, minor alleles in hla - a, -b, -c, and -dqb1 loci in this patient seem not to have been effectively amplified to be detected by sbt . Thus, when hla typing is performed on blood samples containing a high proportion of blasts with loh in chromosome 6p, typing results may differ between different typing methods according to differences in their detection sensitivity . In the pcr - sequence specific primer (ssp) method, different primer pairs amplify different hla alleles, and this method is expected to detect minor alleles with higher sensitivity than the sso or sbt methods . We later performed pcr - ssp typing (allset gold ssp hla typing kit, invitrogen, brown deer, wi, usa) for hla - b (48 primer pairs) and -dr loci (24 primer pairs) using the dna sample from our patient at the time of diagnosis . As expected, the pcr - ssp method detected heterozygosity for both hla - b and -dr loci, although the minor alleles showed much weaker amplification bands on the electropherogram than the major alleles . To avoid false hla typing results in patients with hematologic malignancies, clinicians as well as laboratory personnel need to be aware of such problems and take appropriate precautions . In leukemia patients, hla typing is usually performed at the time of diagnosis or during the first complete remission . Clinicians should not request hla typing using blood samples at the time of diagnosis if patients have blasts in the peripheral blood . There is no data available on what percentage of blasts or neoplastic cells in the white cell differential count of a peripheral blood sample can be present without affecting the hla molecular typing results in patients with loh in 6p . Because blasts generally have considerably larger nuclei than normal leukocytes, the dna fraction from blasts would comprise a much larger portion of total dna extracted from leukocytes than that simply calculated from the percentage of blasts in the blood sample this is evidenced in our case; whereas the blood sample used for hla dna typing contained 87% blasts, the minor alleles detected by str analysis comprised a much lower percentage (average 4.1%) than expected (above 10%). If hla typing is required in such patients before remission is obtained, alternative samples, such as a buccal swab or hair follicles, should be considered for hla typing . Hla typing laboratories involved in the typing of patients with hematologic malignancies should be aware of such problems and should check patients' hematologic data whenever they have unusual hla typing results or discrepancies between different typing methods . In addition, the patients' hla typing results should be reconfirmed by confirmatory typing . For hematopoietic stem cell transplantation, confirmatory hla typing using newly collected samples should be performed not only for donors but also for patients [20, 21]. However, confirmatory hla typing of patients is not reimbursed by the national medical insurance system and is not routinely carried out as a standard clinical practice in this country . We have described a case of false homozygosity results in hla genotyping in a patient with precursor - b all due to the loss of whole chromosome 6 in the neoplastic cells . To avoid false hla typing results in patients with hematologic malignancies, clinicians and hla typing laboratory personnel need to be aware of the problem and take appropriate precautions . Hla typing should not be performed with blood samples containing a high proportion of neoplastic cells, and alternative samples of buccal swab or hair follicles should be used if needed . In addition, confirmatory hla typing should be routinely performed for hematopoietic stem cell transplantation.
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Additional reports were also extracted from promed mail (8) and healthmap (9). We performed the analysis by using 3 dates: 1) date of case registration in the surveillance system of the brazilian ministry of health (model 1); 2) earliest of either date of symptom onset (if available) or registration date (model 2); and 3) earliest of either case registration date, date of symptom onset, or date of case report by other sources (model 3). Surface trend analysis was used to interpolated a continuous estimate of disease spread speed in magnitude and direction (10) by using available spatial and temporal information . Time of dispersal was calculated from the start of the epidemic for each model (technical appendix). Data provided by the brazilian ministry of health on may 31, 2016, indicated that zika had been confirmed in 316 of 5,564 municipalities in 26 states; 6 additional municipalities were identified from other reporting sources . Contour maps of interpolated temporal trends (figure 1) indicate a trend of spread into southern and western brazil, and initial outbreak reports originated from municipalities along the northeastern coast . On the basis of confirmed cases, the earliest location of spread was the northeastern coastal area between the states of paraba, cear, baha, alagoas, and rio grande do norte . There were also earlier dates of self - reported symptom onset in the northwestern state of amazonas (january 1, 2015), the west - central state of matto grosso (january 4, 2015), and the southeastern coastal state of rio de janeiro (january 1, 2015). Contour surface trends and directional vectors for reconstructing zika introduction in brazil . A) date of case registration (model 1); b) earliest date between date of symptom onset (if available) and date of registration (model 2); c) earliest date between date of case registration, date of symptom onset, and date of case reporting by other sources (model 3). Each contour line represents a 1-day period, and contour lines farther apart show that the disease spread rapidly through an area, whereas lines close together show slower progression in an area . . Magnitude of speed and direction should be interpreted cautiously near the edges of the study area . Estimates of speed are subject to edge effects, which indicates that estimates are less stable because they are based on fewer data (not as many neighboring values). Contour maps (figure 1) indicate slight differences in patterns of dispersion between the models . Model 1 indicates the strongest trend of a southward spread from the northeastern coast toward the populous southeastern coastal states of rio de janeiro, esprito santo, and so paulo; the estimated time of dispersal was 22 weeks (figure 1, panel a). In addition to west to east spread of zika in southern brazil, there was a pattern of movement west toward bolivia . The dispersal trend for model 2 was more varied but also indicated spread to the southeastern coastal states of rio de janeiro, esprito santo, and so paulo (figure 1, panel b). This model also suggests an initial spread north from the earliest reports in the northeastern region and a spread west toward bolivia . Model 3 suggests a strong southward spread originating from the northeastern coast toward the southeastern coastal states (approximate dispersal time of 29 weeks) and toward the western border and northwestern state of amazonas (figure 1, panel c). Overall, the average speed of diffusion was 42.1 km / day or 15,367 km / year . The minimum speed across all 3 models was 6.9 km / day, and the maximum speed was 634.1 km / day (figure 2). Municipalities in northeastern and northern regions had the slowest speeds, and municipalities in the west - central and southeastern regions had the highest speeds . More cases occurred closer in time and over larger areas in southern, southeastern, and west - central regions, which resulted in faster rates of case introduction . Speed or log speed (km / d) of zika introduction into municipalities in brazil . A) june 2015may 2016; b) january 2015may 2016; c) january 2015may 2016 . Gray circles indicate central western region, green circles indicate northern region, light blue circles indicate northeastern region, black circles indicate southern region, and dark blue circles indicate southeastern region . All models were consistent in agreement that zika dispersal in brazil followed a general pattern of southward spread toward the populous coastal states (average speed of introduction of 42 km / day), which could be explained by multiple introductory cases into different areas probably caused by movement of viremic persons . We estimate that it took 56 months for zika to spread from the northeastern coast to the southeastern coast and western border of brazil . These findings are supported by the first report of local transmission of zika virus in paraguay in late november 2015 (11) and in bolivia in january 2016 (12), 7 months after the first registered case in brazil . Limitations of this analysis include quality and timeliness of surveillance data that provided the basis for this study . Symptom onset date is subject to error because it is based on self - report, and earlier introductions of zika in some municipalities might not have been captured by the ministry of health surveillance system and supplementary data sources, given the mild and generic nature of zika symptoms and the high proportion of asymptomatic persons (3). The northern region of brazil had a major dengue outbreak in early 2015, and given symptom similarities between dengue and zika, it is probable that some suspected dengue cases were in fact early cases of zika . Sporadic geographically disparate cases were recorded in various parts of brazil, which increased the uncertainty associated with speed analysis . These cases, such as those in northwestern brazil, increased uncertainty in direction and speed estimates, which are also related to edge effects . Edge effects occurred along the boundary of the study area, which in this study were constructed by using fewer data points and are therefore less stable . This effect is shown with directional arrows pointing toward earlier areas of spread versus toward later areas of spread (figure 1, panels b, c). The arrival and rapid spread of zika virus in the americas resembles that of chikungunya virus, which was introduced into saint martin in the caribbean in 2013 (13,14). Increased knowledge of the speed of spread and direction of zika spread can help in understanding its possible future directions and pace at which it travels, which would be essential for targeted mosquito control interventions, public health messages, and travel advisories . Future work will investigate underlying causes for the southward and westward spread in brazil by incorporating mobility data and seasonal events, such as movement of persons between northeastern and southeastern regions for vacations, which could have driven the spatial transmission pattern . Furthermore, multicountry analysis is needed to understand continental spatial and temporal patterns of dispersion of zika virus and co - circulating viruses, such as chikungunya virus.
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The car is a convenient means of transport and enables people to expand their range of activity . In particular, for people with disability of the lower extremities who must use a wheelchair have limited use of public transportation, it is very important to be able to drive a car . The ability to drive is associated with employment, community participation, and overall satisfaction in individuals with spinal cord injury1,2,3,4 . To be able to drive independently, these individuals need to transfer to the driver s seat (i.e., car transfer [ct]), operate the steering wheel, and load their wheelchairs in their car by themselves . Among these activities, the ct movement to the driver s seat is particularly important because it is related to other transfer movements in daily living . Kinematic analysis of the ct movement in individuals with c6 tetraplegia showed that these individuals prefer the rotatory movement pattern in which their head moves in the opposite direction to their buttocks5 . In an analysis of the ct movement in individuals with tetraplegia, the subjects confirmed that they could move their buttocks by inclining their trunk forward inclination by via neck control, because of trunk muscle paralysis5 . However, this previous study only analyzed the head, trunk, and buttocks during ct movement in individuals with tetraplegia, and it did not examine the position of the lower limbs whereby individuals with c6 tetraplegia could control their paralyzed trunks . Several studies have been conducted on transfer movement in individuals with spinal cord injury . These earlier studies indicated the importance of the eccentric control of trunk forward flexion and the coordinated flexion movement between the head and trunk to assist the buttocks to lift off the seat during transfer movement6,7,8,9 . Individuals with tetraplegia used a variety of methods to facilitate the ct movement . For example, they perform ct after placing one or both legs in the car or they fix their head against the door during the movement . For individuals with tetraplegia to participate socially, it is very important them to achieve more efficient ct movement . However, few studies have kinematically examined ct movement in individuals with tetraplegia . The purpose of the present study was to examine the kinematic characteristics and explore the most efficient methods of ct movement in individuals with tetraplegia, focusing on trunk and hip joint movement . Eleven adult males with c6 tetraplegia, with grade a impairment according to the american spinal injury association (asia) guidelines, participated in this study . Their asia motor scores ranged from 18 to 24, while the time since injury was 1131 years (age: 39.6 8.1yerars, height: 174.7 5.6 cm, weight: 55.3 6.8 kg). The inclusion criteria were as follows: no limitation in range of motion or pain in the upper extremities and inability to lift themselves off the buttocks . Subject characteristics are presented in table 1table 1.subject characteristicssubjectsage (years)height (cm)weight (kg)bmi (kg / m)time since injury (years)asia motor score of upper extremities (/50)height of wheelchair seat (cm)height of driver s seat (cm)a41168.560.021.121195053b26175.055.018.012205053c39182.066.520.122225354d51168.056.019.831205358e51174.055.018.227185354f28167.045.016.110205252g39180.065.020.119245360h33172.047.015.911215452i42183.050.014.922215060j43178.051.616.327205554k42174.057.018.818205253mean sd39.6 8.1174.7 5.655.3 6.818.1 2.120.0 6.920.5 1.652.3 1.754.8 3.0 . This study was approved by the ethics committee of the school of comprehensive rehabilitation, osaka prefecture university (2013 - 108). The purpose of the study was explained to the subjects orally and in writing, and written informed consent was obtained from all participants . Subjects were instructed to perform ct movement as they would usually from their own wheelchairs to their own cars . The difference between the height of the driver s seat and the seat surface of the wheelchairs was less than 10 cm (table 1). The wheelchair was positioned on the correct side of the car, and the subjects attempted to transfer themselves from the wheelchair into the driver s seat . The passenger - side door was held open maximally, and a digital video camera (30 hz) was placed on the passenger side (fig . The passenger - side door was held open maximally, and a digital video camera was placed on the passenger side . ). The passenger - side door was held open maximally, and a digital video camera was placed on the passenger side . The duration of the movement, defined as the total time of buttocks movement from the wheelchair to the driver s seat was determined from videos recorded on the camera . The starting point was the moment that the buttocks movement was visible, when the left great trochanter was confirmed to have moved forward . The end point was the moment that the buttocks movement was completed when the participant was in the driver s seat . On the basis of the videos, the duration and the trunk inclination and hip angles the trunk inclination and hip angles were calculated using image - j (nih) from static images . The trunk inclination angle was defined as the angle from the left acromion process to the great trochanter and the vertical line . The hip angle was defined as the angle between the left acromion process to the great trochanter and the left great trochanter to the knee (fig . The trunk inclination angle was defined as the angle from the left acromion process to the great trochanter and the vertical line . The hip angle was defined as the angle between the left acromion process to the great trochanter and the left great trochanter to the knee . ). In other words, a small hip angle indicates a high hip flexion angle . In the case of multiple buttocks movements, these angles were calculated for each movement, and the maximum angle was used in the data analysis . After the buttocks movement, the angles of the moment their acromion and knee position closing were measured as the maximum angle . The trunk inclination angle was defined as the angle from the left acromion process to the great trochanter and the vertical line . The hip angle was defined as the angle between the left acromion process to the great trochanter and the left great trochanter to the knee . The relationship of the trunk inclination and hip angles with duration were analyzed using spearman s rank - correlation coefficient . The duration of the ct movement ranged from 9 to 39 s (table 2table 2.duration of the ct movement, trunk inclination angle, and hip angle recorded for all subjectssubjectasia motor score (/50)duration of the ct movement (s)maximum trunk inclination angle (degrees)maximum hip angle (degrees)a193245.461.9b203635.064.9c22937.746.4d202234.058.3e181842.564.1f201336.257.7g243932.066.9h212631.561.5i211940.581.8j202041.058.0k201737.654.8mean sd20.5 1.622.8 9.537.6 4.461.5 8.8). No correlation was found between the asia motor score and duration (=0.07, p>0.05). The maximum trunk inclination angle ranged from 31.5 to 45.4 degrees, while the maximum hip angle ranged from 46.4 to 81.8 degrees (table 2). No correlation was observed between the trunk inclination angle and the duration of ct movement . However, a significant correlation was observed between the hip angle and duration (=0.67, p<0.05). In other words, transfer movements are very common in daily life, and the ability to perform these movements is one of the most important and meaningful skills for individuals with tetraplegia . Independence in transfer movement would encourage individuals with tetraplegia to participate more fully in the community by allowing them to use a car . Thus, understanding the kinematic characteristics of patients with c6 tetraplegia is important in order to promote their participation in the community . The purpose of this study was to examine the kinematic characteristics of individuals with tetraplegia and identify the most efficient method of ct movement for them, with a focus on trunk and hip joint movement . Previous studies showed that coordinated flexion movement between the head and trunk assist the buttocks to lift off the seat6,7,8 . Additionally, the strategy of neck flexion and forward inclination of the trunk was found to differ among subjects with c6 tetraplegia during ct movement5 . In the present study, no correlation was found between the asia motor score and the duration of the ct movement . A reason for this could be that all subjects with c6 tetraplegia have paralyzed triceps brachii muscles, and the asia motor score of the upper limbs does not significantly affect the ct movement . In addition, it has been reported that the pectoralis major, latissimus dorsi, and serratus anterior muscles are important for the ct movement in individuals with spinal cord injury8 and that in order to analyze the ct movement in individuals with c6 tetraplegia, these muscles should be tested in addition to those tested to determine the asia motor score . Further, the present study showed no correlation between the trunk inclination angle and the duration of the ct movement . This could be because of the limited space in which the movement was performed and because the subjects had paralyzed trunk muscles . Cars have several obstacles, such as the door, handle, and dashboard, and the subjects had to incline their trunks forward in a limited space . However, these obstacles might actually assist the subjects in inclining their paralyzed trunks forward and control them . On the other hand, a significant correlation was observed between the hip angle and duration of the ct movement . This could be because c6 tetraplegia enabled smooth ct by allowing for control of trunk inclination, such as placing the lower extremities in the car in order to tilt pelvis backward . Although the hip flexion angle was high, the trunk inclination angle was low, despite which the individuals with tetraplegia could perform the dynamic ct movement . As mentioned above, it has been reported that for transfer movement among individuals with tetraplegia, head and trunk flexion may facilitate efficient buttocks movement . However, with regard to transfer movement in confined spaces such as ct movement, it has been suggested that the magnitude of the hip flexion angle affects smooth movement of the buttocks . Compare the changes in the trunk and shoulder angles and reaction forces under the two hands elicited by different hand base - of - support positions during sitting pivot transfer10 . They reported that the lower hand position should be recommended as an effective and safe method for sitting pivot transfer for patients with spinal cord injury, because this position was associated with a significantly high trunk angle of forward and lateral flexion, even though the angle of rotation while transferring to the 20-cm lower support position was reduced . In the present study, the difference between the height of the driver s seat and the seat surface of the subjects wheelchair was less than 10 cm . However, since even a small difference in these levels is considered to influence the ct movement in individuals with c6 tetraplegia who have paralyzed upper limbs, this factor needs further investigation . The trunk inclination angle probably showed no effect on the duration of the ct movement because the movement was performed in a limited space and because the trunk muscles of the subjects were paralyzed . In contrast, c6 tetraplegia enabled smooth ct by allowing for control of trunk inclination, such as placing the lower extremities in the car, whereby the pelvis backward tilt angle increased . A drawback of the present study was that the ct movement was examined in a limited space, and the differences in the cars and wheelchairs used by the subjects were not taken into consideration . Third, the experimental conditions were not fixed, and the subjects were allowed to select their own strategy for ct movement . Further, the motions of the ct movement, including rotational motion, were examined by two - dimensional analysis . The angles in the sagittal plane were determined, and the numerical values of these angles are not precise . Thus, although the trends of the trunk inclination angles and hip angles required for the ct movement have been understood, further studies are needed for a more accurate analysis with a greater number of subjects.
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Mouth opening is a clinical parameter, which we encounter routinely in our daily practice . A known normal range of mouth opening is necessary to enable the clinician conduct a thorough oral examination conveniently . A reduced mouth opening capacity (moc) may be one of the first clinical signs of pathological changes and traumatic conditions in the masticatory system . Early recognition of decreased or limited mouth opening is necessary for a prompt and efficient approach to diagnosis and to plan out the treatment options judiciously . To make a diagnosis of decreased mmo, it is essential to establish what constitutes the normal mmo for children of different age . Research has shown that the measurement of mouth opening varies significantly with age, gender, and race . Numerous studies across the world have characterized the mmo in their adult as well as pediatric population . However, very little data exist on normal mmo in the indian pediatric population . In a growing population, furthermore, most existing datasets are of limited value because they do not cover the entire age range . Despite these limitations, the existing normal values indicate a significance of growth on the mmo and a wide range of normalcy within a certain age category . In india, it is fairly common to report the mouth opening in terms of finger breath in our daily practice . Understandably, this is semi - quantitative measurement subject to many variations . Hence, establishing numerical value will be of significant value . The present study aims to establish age- and height - related percentiles for the mmo of healthy children and adolescents which may serve as a basis for various studies and will have the clinical utility in treatment and diagnosis of diseases directly or indirectly affecting the mouth opening . The present study was carried out in the pediatric outpatient clinic in a tertiary care center in western india . A total of 985 children, 560 males and 425 females, in the age range of 518 years were studied . The children were asked to open their mouth maximally unassisted till no further opening was possible . Mmo was measured when the children rested their heads against a firm wall surface in an upright position . The distance from the incisal edge of the upper incisor teeth to the incisal edge of the lower incisor teeth was measured using a calibrated fiber ruler, and the findings were recorded in millimeters [figure 1]. Three readings were recorded for each individual, and their average was recorded as the final reading . To control for inter - examiner and intra - examiner reliability, the age, sex, height, and weight of all the children were recorded . Method of measuring interincisal distance with fiber ruler the study included all the children between the ages of 518 years of age attending pediatric outpatient clinic, having all the incisors and able to understand and cooperate with the investigators . Exclusion criteria for the study were children with (1) habit of smoking, tobacco, or betel nut chewing, (2) no natural front teeth, (3) previous maxillofacial trauma, (4) oral malignancies, (5) angular cheilosis or oral ulcers affecting mouth opening, (6) chronic systemic disease, and (7) pain while opening mouth . This study was approved by the research ethics committee of the institute . As children involved in the study were minors, weight and height for each child were obtained using a standard calibrated anthropometric scale, with precision of 100 g and 1 mm, respectively . Mean mmo values were analyzed according to age, height, and weight ranges, which were established appropriately for statistical analysis . Hence, the sample was divided according to age and height: 13 age groups (between 5 and 18 years) and 10 height groups of 10 cm each (8695 cm; 96105 cm; 106115 cm; 116125 cm; 126135 cm; 136145 cm; 146155 cm; 156165 cm; 166175 cm; 176185 cm) were assigned . Continuous variables are reported as mean standard deviation . To determine correlation of mouth opening with age and height, a linear regression analysis was performed and statistical significance was tested by the application of t - test and f - test . The statistical analysis has been performed using open source software r version 3.0.1 . Of r foundation, vienna, austria . Mean mmo values were analyzed according to age, height, and weight ranges, which were established appropriately for statistical analysis . Hence, the sample was divided according to age and height: 13 age groups (between 5 and 18 years) and 10 height groups of 10 cm each (8695 cm; 96105 cm; 106115 cm; 116125 cm; 126135 cm; 136145 cm; 146155 cm; 156165 cm; 166175 cm; 176185 cm) were assigned . Continuous variables are reported as mean standard deviation . To determine correlation of mouth opening with age and height, a linear regression analysis was performed and statistical significance was tested by the application of t - test and f - test . The statistical analysis has been performed using open source software r version 3.0.1 . Of r foundation, vienna, austria . Our study included a total of 985 children with 560 males and 425 females among them . The median age for the entire sample is 10.5 years, and median height was 130 cm for girls and 130139.99 cm for boys . The mean mmo was 43.5 5.19 for girls and 44.24 5.84 cm for boys . Table 1 presents the number of children in each age group with mean moc values (mm) for indian children . Table 2 presents the mean moc values (mm) for the indian children in each height group . The scattered diagram relation of moc with age and height was plotted [figure 2a and b]. Number of individuals in each age group for boys and girls with mouth opening capacity values (mm) variation in mouth opening with height (a and b) the scattered diagram showing relation of mouth opening capacity with age and height diagrammatic representation of mean mouth opening capacity value according to the height there was a significant difference between the moc of male and female in all age groups . A linear relationship was observed between the mouth opening and age but more significantly with height in entire sample, with correlation coefficient of 0.63, which showed that moc increases as the age and height increases, and this relation was found to be statistically significant (p <0.0001). As the height reflects the skeletal growth, we also found that the mmo is more significantly associated with height than age . The dataset was entered into the r program for the creation of age - related percentiles . Best results were achieved using the following settings: l = 1, i.e., l constant, set to l = 0.65 by the program, m = 5, s = 2 for girls, and l = 1, set to 0.5 by the program, m = 7, s = 2 for boys . Table 3 depicts four linear regression models evaluating impact of age (model 1), height (model 2), age and height (model 3), and age, height, and gender (model 4) on moc . In isolation, age and height significantly impact mouth opening . However, impact of height on mouth opening is more significant than that of age . Further, when age and height were considered together, it was only height that had influenced moc and not age . Norms could be designated for age with gender and height with gender [tables 47]. Age - related percentiles for moc in boys and girls are given in figure 4a and b. height - related percentiles for moc in boys and girls are given in figure 5a and 5b . Linear regression models for mouth opening capacity age - related percentiles of mouth opening capacity for males (in mm) age - related percentiles of mouth opening capacity for females (in mm) height - related percentiles of mouth opening capacity for males (in mm) height - related percentiles of mouth opening capacity for females (in mm) (a and b) age - related percentiles for mouth opening capacity in boys and girls (a and b) height - related percentiles for mouth opening capacity in boys and girls the dataset was entered into the r program for the creation of age - related percentiles . Best results were achieved using the following settings: l = 1, i.e., l constant, set to l = 0.65 by the program, m = 5, s = 2 for girls, and l = 1, set to 0.5 by the program, m = 7, s = 2 for boys . Table 3 depicts four linear regression models evaluating impact of age (model 1), height (model 2), age and height (model 3), and age, height, and gender (model 4) on moc . In isolation, age and height significantly impact mouth opening . However, impact of height on mouth opening is more significant than that of age . Further, when age and height were considered together, it was only height that had influenced moc and not age . Norms could be designated for age with gender and height with gender [tables 47]. Age - related percentiles for moc in boys and girls are given in figure 4a and b. height - related percentiles for moc in boys and girls are given in figure 5a and 5b . Linear regression models for mouth opening capacity age - related percentiles of mouth opening capacity for males (in mm) age - related percentiles of mouth opening capacity for females (in mm) height - related percentiles of mouth opening capacity for males (in mm) height - related percentiles of mouth opening capacity for females (in mm) (a and b) age - related percentiles for mouth opening capacity in boys and girls (a and b) height - related percentiles for mouth opening capacity in boys and girls patients with temporomandibular joint diseases, craniofacial syndromes, maxillofacial trauma, oral malignancies and those who have been treated for these conditions often have complaint of restricted mouth opening . As with any other disease or condition, the aim of treatment of disorders affecting mouth opening is to restore the mouth opening to its normal value, hence the relevance of establishing normal values . A large number of methods have been described in the literature to measure the mouth opening . The measurement most often used to assess mouth opening is the interincisal distance attained during active opening by the subject . This method underestimates the movement of the mandible as it does not include the overbite . However, as pointed by mezitis et al ., the functional opening of the mouth is more important . Sufficient mouth opening to allow normal social function is clinically important for the patients and adequate access to the oral cavity for clinicians, which in effect is the interincisal distance without consideration for the overbite . Hence, the interincisal distance has been used as a measurement of mmo in this study . An advantage of the incisal edge distance is that the measuring point is relatively more consistent, permanent, and more easily determined . Wood and branco examined three methods of measuring interincisal distance and concluded that direct measurement using a ruler was the most accurate . Described how the mmo decreases in order of forward, natural, and retracted head positions . In the present study, the mmo was measured when the subjects rested their heads against a firm wall surface in an upright position to eliminate the possible influence of different head positions . Mouth opening is influenced by a number of factors in children as they are in the period of skeletal growth . The age, weight, height, gender, and race affect the mouth opening . Rothenberg noted a significant relationship between the maximum vertical opening of the mouth and age, among 189 caucasian children, aged 414 years . The present study also reported a gradual increase in mmo in different age groups of children . A few studies have observed a gender difference in mmo among children . In the present study, a statistically significant difference was observed in between boys and girls in various age groups . The study also revealed a definitive correlation between mmo and height as well as age . Rothenberg also observed a positive correlation between mmo values in relation to weight and height in children aged 414 years of age group . The observed gradual increase in mmo with increasing age, height, and body weight, as found in the present study, is due to changes in the temporomandibular joint apparatus, facial morphology, muscle development, and growth of cranial base and mandible, particularly in length . Although age has the significant influence on the mmo values, a definite and stronger correlation exists between mmo and height . Children with poor nutrition often have stunted growth, and their skeletal growth is hampered . In such cases, measurement of mmo will not correspond to the established norm for the age since the height is unaccounted for, in these malnourished children from lower socioeconomic group . In such cases, to measure height by age can give a false - positive value of reduced mouth opening . This study is therefore providing the normal percentile for different height groups for the first time . These data are important because a high number of complex surgical procedures are routinely performed in this part of the world without a proper reference value for established norms . Assessment of mouth opening is an important part of clinical examination for the physician, surgeon, or clinician involved in the treatment of head and neck disorders and dental disorders . To diagnose an abnormality, knowledge of normal values is very important . Reference to international data is obviously not justified as the mouth opening is clearly varied among different ethnicities . To the best of our knowledge, this data sample of 985 unselected children covers the entire age range of indian children where unassisted mouth opening can be measured . We believe that these percentiles will therefore be of great importance for future studies as well as clinical assessment of indian children with affections of the masticatory system.
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Nephrotic syndrome in the first year of life in two thirds of cases is caused by mutations in four genes (nphs1, nphs2, wt1, and lamb2). The wilms tumor suppressor gene 1 (wt1) encodes a transcription factor involved in kidney and gonadal development . Drash syndrome (dds) and frasier syndrome (fs), two syndromes characterized by nephrotic syndrome (ns) with either diffuse mesangial sclerosis (dms) in dds or focal segmental glomerulosclerosis (fsgs) in fs, genitourinary defects and a higher risk of developing wilms tumor in dds, or gonadal dysgerminoma in both dds and fs . . Recently published three children with fsgs on renal biopsy and wt1 mutations, who responded to intensified therapy consisting of oral cyclosporine a (csa) in combination with oral or intravenous glucocorticoids . The mechanism of csa action in minimal change disease was the inhibition of nuclear factor of activated t cell (nfat) signaling in t lymphocytes . However, the therapeutic effect of csa in genetic nephrotic syndrome is probably related to a direct influence of csa on podocytes . In this report, we present a patient with denys drash syndrome, in whom the calcineurin inhibitors were found to induce total remission . Our patient is the first child of non - consanguineous young parents (mother 32, father 34 years) without any past medical history of kidney diseases . The infant was born at 41 weeks gestation (birth weight 3,450 g, length 55 cm). The delivery and neonatal period were normal . At the age of 7 months, she was hospitalized for the first time in the pediatric nephrology department because of proteinuria and hematuria, which was found in routine urinalysis performed before the vaccination . On admission, physical examination revealed no edema, normal female external genitalia, and normal body weight and height for chronological age . Initial laboratory investigations revealed variable non - selective proteinuria 200960 mg / dl in morning sample, protein / creatinine ratio 27 g / g, hypoproteinemia (4.79 g / dl), hypoalbuminemia (3.0 g / dl), hypercholesterolemia (299 mg / dl), thrombocytosis (567 10/l), and reduced levels of igg and iga . The serum creatinine level was 0.23 mg / dl, urea was 8 mg / dl, and the glomerular filtration rate was 133 ml / min/1.73 m by the schwartz formula . Antinuclear antibodies were negative, and the level of c3 and c4 were within normal limits . Chromosomal analysis demonstrated karyotype 46, xx, and the biopsy of the kidney demonstrated diffuse mesangial proliferation and diffuse fibrillar increase, what may represent early stage of diffuse mesangial sclerosis . The glomerular lesions contained high amount of fibrils in the mesangial matrix with diffuse mesangial proliferation . Immunofluorescence on frozen sections using rabbit polyclonal antisera against human igg, iga, igm, and c3 revealed only weak segmental igm staining . The molecular genetic analysis was performed in the laboratory at the university of michigan in the usa . Mutation analysis of exons 8 to 9 of wt1 was performed, and the mutation r394w (c. 1180 c> t) was found in our patient . The treatment with enalapril (1.25 mg / day), which was started after the admission and was continued for 11 months, did not change the proteinuria significantly . There was no reduction in proteinuria after 4-week oral prednisone treatment (60 mg / m / day). Seven months later, we started the therapy with csa (6 mg / kg/24 h in two divided doses). The pre - dose / trough (c0) csa level was 104.4 ng / ml . The serum total protein was (6.47 mg / dl), albumin (4.26 mg / dl), cholesterol 197 mg / dl, and platelet count 385 10/ql . Creatinine level was 0.32 mg / dl and the glomerular filtration rate was 147 ml / min/1.73 m. the pre - dose / trough (c0) csa level was 95.03 ng / ml . At the 6 month of the treatment, we decided for csa dose reduction to 4.5 mg / kg, but a month later, slight proteinuria was observed . Now csa treatment is continued with the higher dose (6 mg / kg/24 h). The ultrasonography examination, performed every 3 months, has not revealed any signs of tumor . We report the benefits of csa treatment of the patient with glomerulopathy associated with wt1 mutation as seen in denys drash syndrome . The girl in this report had early - onset srns secondary to dms, what was confirmed in kidney biopsy . Genetic studies showed mutations in intron 9 of wt1 gene c. 1180 c> t. this is the most common mutation found in dds . Mutations in exons 8 and 9 of the wt1 gene have been found in patients with isolated srns and in srns associated with wilms tumor or urogenital malformations . . Indicated that screening of wt1 exons 8 and 9 in patients with sporadic srns is sufficient to detect pathogenic wt1 mutations . It is possible that our patients have an incomplete form of dds; however, it corresponds to the observation of ismaili et al ., who reported the presence of wilms tumor in only one of two patients with dds . Chernin et al . In the recent publication suggested that missense mutations can occur with and without wilms tumor . In our patient, in this situation, csa therapy was started with a significant reduction of proteinuria and remission of nephrotic syndrome . Three months later, complete remission of proteinuria was found . Whereas genetic forms of nephrotic syndrome do not respond to therapy with immunosuppressive drugs, partial remission has been reported following therapy with calcineurin inhibitors in patients with nphs2, frasier syndrome, mutations of the phospholipase c epsilon gene (plce1), and alport syndrome [3, 4]. Reported three children with fsgs associated with wt1 mutation treated with combined csa and prednisone therapy given for 6 and 12 months . In long - term observation, the authors observed remission of ns and significant reduction of proteinuria . The therapy does not seem to be accompanied by a significant loss of renal function on the short term . We plan to continue the treatment with the lowest possible dose which controls the proteinuria . It was considered that the main effect of csa treatment in nephrotic syndrome caused by t cell dysfunction was the inhibition of nfat signaling in t cells . However, it was found that csa can also reduce proteinuria in non - immunological diseases, raising doubts about the above hypothesis . In genetic diseases, mechanisms similarly, the afferent arteriole vasoconstriction, which is induced by csa, does not seem to play the main role in the reduction of proteinuria . This fact was suggested by zietse et al ., who studied the influence of csa on proteinuria in different glomerulopathies and found the reduction of protein excretion in minimal change disease, but not in membranoproliferative glomerulonephritis or fsgs . The possible mechanism in genetic ns may be that proposed by faul et al . . The authors suggested the influence of csa on synaptopodin, an actin binding protein that was highly expressed in podocytes . It was shown that calcineurin caused the dephosphorylation of synaptopodin and caused proteinuria via its degradation . This mechanism seems to be possible because significant reduction (30%) of csa dose caused the slight proteinuria in our patient, so we increased the dose to initial one . Our observation seems to confirm the theory of mundel and reiser, who suggested that proteinuria is due to an enzymatic disease of the podocytes . It is likely that podocytes are a direct target of csa, which preserves the phosphorylation - dependent synaptopodin-14 - 3 - 3beta interaction, protects synaptopodin from degradation, and preserves a stable filtration barrier [7, 17]. The fact that the antiproteinuric effect of csa results from a direct effect on podocytes explains not only its beneficial role in most steroid - resistant and steroid - dependent nephrotic syndromes but also the fact of cyclosporine dependency . Our observation and observations of other authors [3, 4, 9, 12, 15] confirm the beneficial effect of csa treatment in genetic ns; however, the potential nephrotoxicity of this drug will probably not allow the long - term use.
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Males and females of c57bl/6-tg(ubc - gfp)30scha / j, c57bl/6j, b6.129p2(c)mecp2/j, b6.129p2-lyz2/j (lysm), b6.129p2-mecp2/j, mecp2, and c57bl/6j mice were purchased from jackson laboratories (bar harbor, me); b6.cg-mecp2 mice were a generous gift from dr . Andrew pieper, (southwestern medical school, dallas, tx) and were maintained in our lab on c57bl/6j background . All procedures complied with regulations of the institutional animal care and use committee (acuc) at the university of virginia . Four week - old mice were subjected to lethal split - dose -irradiation (300 rad followed 48 hours later by 950 rad). Four hours after the second irradiation, mice were injected with 510 bone marrow cells . After irradiation, mice were kept on drinking water fortified with sulfamethoxazole for two weeks in order to limit infection by opportunistic pathogens . Full methods and any associated references are available in the online version of the paper at www.nature.com/nature . Males and females of c57bl/6-tg(ubc - gfp)30scha / j, c57bl/6j, b6.129p2(c)mecp2/j, b6.129p2-lyz2/j (lysm), b6.129p2-mecp2/j, mecp2, and c57bl/6j mice were purchased from jackson laboratories (bar harbor, me); b6.cg-mecp2 mice were a generous gift from dr . Andrew pieper, (southwestern medical school, dallas, tx) and were maintained in our lab on c57bl/6j background . All procedures complied with regulations of the institutional animal care and use committee (acuc) at the university of virginia . Four week - old mice were subjected to lethal split - dose -irradiation (300 rad followed 48 hours later by 950 rad). Four hours after the second irradiation, mice were injected with 510 bone marrow cells . After irradiation, mice were kept on drinking water fortified with sulfamethoxazole for two weeks in order to limit infection by opportunistic pathogens . Full methods and any associated references are available in the online version of the paper at www.nature.com/nature.
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The advent of multislice ct technology with ecg - gating along with innovations in x - ray tube and ct detector technology have resulted in revolutionary progress in cardiac imaging . Ecg - gated ct angiography with 64 + slice systems has demonstrated efficacy for the diagnosis of coronary disease, cardiac morphology, and valvular assessment . Recent studies suggest additional applications of ct for assessment of myocardial perfusion and cardiac event risk . Among the clinical applications of cardiac ct, coronary ct angiography (ccta) numerous studies suggest that ccta is a cost - effective alternative to the nuclear stress test and diagnostic cardiac catheterization . Although ct angiography has replaced conventional arteriography for most visceral applications, issues related to radiation exposure and functional versus anatomical information have been raised as objections to widespread adoption of ccta . Recent advances in ct imaging methodology and ct image reconstruction algorithms address many of these concerns . The primary focus is on ccta with a review diagnostic accuracy, appropriate clinical indications and limitations for evaluation of the coronary arteries . Although the principle indication for cardiac ct is most often for evaluation the coronary arteries, ccta is often requested for the dual purposes of non - coronary and coronary evaluation . Several of the more common non - coronary applications are reviewed, although a complete review of these applications is beyond the scope of this pictoral essay . Despite clinical advances in diagnosis and treatment, heart disease remains the leading cause of death for both men and women, and is responsible for 26% of all deaths in the united states . Coronary computed tomographic angiography (ccta) provides a non - invasive alternative to cardiac catheterization for direct visualization of coronary anatomy (fig . 1). Recent meta - analyses of ccta studies have demonstrated high sensitivity (9699%) and specificity (8891%) for the anatomic presence of cad [25]. Although functional information about wall motion can be obtained during ccta, the primary utility of ccta is for imaging of the coronary arteries to define the presence of cad (figs . 2, 3, 4). The high sensitivity of ccta for cad results in an extremely high negative predictive value, especially in patients with low to intermediate risk of coronary disease . Although the specificity of ccta is limited by arterial calcification, post - processing techniques using vessel tracking and curved multiplanar reconstructions are often useful for evaluation of calcified vessels (fig . 5). Nonetheless, blooming artifact associated with densely calcified plaque may preclude visualization of the underlying vascular lumen in the absence of significant stenosis (fig . A left anterior oblique view demonstrates the left anterior descending artery coursing down the anterior interventricular groove (black arrowhead) and the circumflex artery coursing in the left atrioventricular groove (white arrowhead). B view of the base of the heart from the left side demonstrates the continuation of the circumflex artery in the posterior left atrioventricular groove (arrowhead), extending to the crux of the heart where it continues as the posterior descending artery (white arrow)fig . 2severe stenosis of the proximal left anterior descending artery (lad) in a patient presenting with chest pain . A curved mip demonstrates greater than 70% narrowing in the proximal lad (arrow). B straightened lumen mip again demonstrates severe stenosis of the proximal lad (arrow). 3severe stenosis of the proximal circumflex artery (lcx) in a patient complaining of chest pain . A curved mip demonstrates greater than 70% narrowing in the proximal lcx (arrow). B curved mip in an orthogonal projection again demonstrates greater than 70% narrowing in the proximal lcx (arrow). C straightened lumen mip confirms the severe stenosis just proximal to the first obtuse marginal branch (arrow)fig . B straightened lumen mip demonstrates the presence of non - calcified plaque at this level with diameter reduction in the range of 5070%fig . 5minimal calcified plaque in the proximal left anterior descending artery (arrowheads) with no significant stenosis . Although the calcified plaque overlies the vessel in the globe mip view (bottom right), the curved mip and straightened lumen views are rotated to project the calcium off the vessel lumen and demonstrate that there is no significant stenosisfig . Because of the large amount of calcified plaque, the vessel lumen is not visualized on curved mip projections . Ccta was not able to clear this vessel, even though conventional cardiac catheterization did not demonstrate an area of high - grade stenosis volumetric rendering of a left dominant coronary circulation from ccta . A left anterior oblique view demonstrates the left anterior descending artery coursing down the anterior interventricular groove (black arrowhead) and the circumflex artery coursing in the left atrioventricular groove (white arrowhead). B view of the base of the heart from the left side demonstrates the continuation of the circumflex artery in the posterior left atrioventricular groove (arrowhead), extending to the crux of the heart where it continues as the posterior descending artery (white arrow) severe stenosis of the proximal left anterior descending artery (lad) in a patient presenting with chest pain . A curved mip demonstrates greater than 70% narrowing in the proximal lad (arrow) b straightened lumen mip again demonstrates severe stenosis of the proximal lad (arrow). The lumen disappears in the middle image at the site of maximum narrowing severe stenosis of the proximal circumflex artery (lcx) in a patient complaining of chest pain . A curved mip demonstrates greater than 70% narrowing in the proximal lcx (arrow). B curved mip in an orthogonal projection again demonstrates greater than 70% narrowing in the proximal lcx (arrow). C straightened lumen mip confirms the severe stenosis just proximal to the first obtuse marginal branch (arrow) moderate stenosis of the proximal left anterior descending artery (lad). B straightened lumen mip demonstrates the presence of non - calcified plaque at this level with diameter reduction in the range of 5070% minimal calcified plaque in the proximal left anterior descending artery (arrowheads) with no significant stenosis . Although the calcified plaque overlies the vessel in the globe mip view (bottom right), the curved mip and straightened lumen views are rotated to project the calcium off the vessel lumen and demonstrate that there is no significant stenosis heavily calcified plaque in the right coronary artery . A and b orthogonal curved mip views of the right coronary artery . Because of the large amount of calcified plaque, the vessel lumen is not visualized on curved mip projections . Ccta was not able to clear this vessel, even though conventional cardiac catheterization did not demonstrate an area of high - grade stenosis whereas ccta provides an anatomic depiction of coronary stenosis, stress testing provides additional information to predict the functional relevance of coronary stenosis [6, 7]. When ccta demonstrates no more than minimal disease of the coronary arteries or when ccta demonstrates the presence of high - grade coronary stenosis, the clinical implication is clear . When ccta demonstrates coronary stenosis of uncertain hemodynamic significance, a stress test may be useful to evaluate the functional / clinical significance of cad found by ccta . Conventional arteriography, which demonstrates the degree of luminal narrowing and can provide measurements of pressure gradients but does truly evaluate the impact of coronary stenosis on myocardial perfusion . In the setting of moderate cad, ccta and stress testing may provide complementary information for the evaluation of cad . A recently published decision analytic model for evaluation of the symptomatic patient with chest pain demonstrates how ccta may be used to improve diagnostic accuracy, reduce the effective radiation dose and limit overall imaging costs for the evaluation of cad . A workup strategy that employs stress ecg or stress echocardiography prior to ccta minimizes effective radiation exposure compared to all other strategies that use ccta and/or nuclear perfusion scanning . When the pre - test probability of cad is below 10%, a strategy that employs stress echocardiography followed by ccta before proceeding to cardiac catheterization will result in an effective radiation dose that is lower than a strategy that uses stress echocardiography followed by conventional cardiac catheterization . Imaging costs are minimized by a strategy that employs stress ecg or stress echocardiography with follow - up by ccta for positive stress tests, as the ccta can identify false - positive stress tests and eliminate many negative diagnostic cardiac catheterization procedures . The most appropriate testing strategy for the workup of symptomatic cad depends upon the pre - test probability of disease, which should be estimated on the basis of demographics and clinical presentation . The symptomatic patient with a high pre - test probability of cad (> 50%) may benefit from direct triage to cardiac catheterization . For the patient with a low pre - test probability of disease (20%), the combinations of stress ecg + ccta or stress echocardiography + ccta provides optimal options as judged by cost and radiation dose, while stress echocardiography + ccta will result in the lowest rate of negative cardiac catheterization . As the pre - test probability of cad is increased above 20%, ccta alone may be a more appropriate option for the diagnostic workup in order to avoid a large number of false - negative test results that are associated with stress testing . Based upon the high negative predictive value, ready availability and rapid diagnostic time of ccta for the exclusion of coronary disease, ccta has been proved to be a cost - effective examination for evaluation of low to intermediate risk patients with suspected acute coronary syndrome presenting to the emergency department [11, 12]. Ccta examinations are usually performed as a dedicated study of the coronary arteries, tailored to image the coronary arteries with a minimum dose of iodinated contrast and radiation exposure . However, in the setting of a patient who presents with acute chest pain, the differential diagnosis often includes other potentially life - threatening processes such as pulmonary embolism and aortic dissection . Furthermore, contrast injection for a dedicated ccta is usually timed with a saline bolus to wash out the right heart during imaging . In order to optimize imaging of the coronary arteries, thoracic aorta and pulmonary arteries, a triple rule - out protocol may be applied . When performed with proper attention to technique, the triple rule - out study provides excellent opacification of the aorta and pulmonary arteries without loss of coronary artery image quality, although there is an increased radiation dose proportional to the increased scan length (fig . 7triple rule - out study with opacification of the aorta, coronary arteries and pulmonary arteries . A oblique sagittal mip demonstrates the thoracic aorta (ao) as well as the bifurcation of the pulmonary arteries (pa). This scan was acquired in four steps using prospective ecg - gating to reduce patient dose . B left anterior oblique coronal mip demonstrates the origins of the right coronary artery (arrowhead) and left coronary artery (arrow). Shape of the right coronary artery triple rule - out study with opacification of the aorta, coronary arteries and pulmonary arteries . A oblique sagittal mip demonstrates the thoracic aorta (ao) as well as the bifurcation of the pulmonary arteries (pa). This scan was acquired in four steps using prospective ecg - gating to reduce patient dose . B left anterior oblique coronal mip demonstrates the origins of the right coronary artery (arrowhead) and left coronary artery (arrow). Shape of the right coronary artery ccta is currently not recommended for evaluation of asymptomatic patients . Atherosclerotic cad is highly prevalent in the population of developed countries, and screening for cad by ccta is likely to identify many patients with cad who might not benefit from interventional therapy . Several recent studies have suggested that ccta may be useful for risk assessment in the asymptomatic population (see below), but this suggestion must be tempered by the potential for overtreatment of anatomical disease, which is found during such screening ccta procedures . A similar problematic situation arises when low - risk asymptomatic patients are referred for cardiac catheterization after a positive stress test in the course of a preoperative or routine cardiac evaluation . Ccta, which may obviate the performance of an unnecessary invasive conventional diagnostic cardiac catheterization . An overwhelming majority of patients presenting with acute coronary syndrome have calcification in the coronary circulation . Several large retrospective studies of asymptomatic patients have demonstrated that the presence of coronary calcium is a predictor of cardiac events and risk of death [18, 19]. Prospective studies have confirmed these results and demonstrated that coronary calcium scoring provides additional prognostic information beyond standard risk assessment based upon clinical and laboratory tests [20, 21]. Nonetheless, calcium scores are not recommended to evaluate the patient who presents with acute symptoms . Coronary calcium imaging does not detect the presence of non - calcified plaque, and a minority of patients experience coronary events in the absence of coronary calcification (fig . A recent meta - analysis of 64-slice mdct studies included 10 studies with 5,675 patients . After a mean follow - up of 21 months, major adverse cardiac events occurred in 0.5% of patients with normal ccta, 3.5% of patients with non - obstructive disease on ccta and 16% of patients with obstructive ccta (p = 0.0001). Individual studies have demonstrated an increasing frequency of cardiac events based upon both increasing plaque burden and more proximal location . Although ccta is not currently recommended for risk stratification, continued advances in plaque characterization by cta may make risk stratification by ccta a reality in the future . A major limitation of traditional ccta is the lack of a functional imaging component to gauge whether the coronary stenosis results in myocardial ischemia . The extent of inducible ischemia associated with coronary disease is predictive of the risk of a coronary event as well as the expected benefit of revascularization compared with medical therapy [27, 28]. In order for ccta to serve as a comprehensive evaluation for coronary disease several groups have used ct to perform perfusion imaging either as a stand - alone examination or as part of a ccta study . A first - pass dynamic evaluation of myocardial perfusion might be the most accurate technique to quantify myocardial perfusion, but such an approach requires repeated scanning of the heart with a relatively high radiation exposure . Several groups have used a single arterial phase to estimate myocardial perfusion based upon myocardial blood pool enhancement during ccta and a delayed phase to image late enhancement that may be associated with infarcted myocardium . Low - dose scans for late enhancement can provide valuable diagnostic information on the functional significance of coronary artery disease . Given the relatively small changes in ct density provided by enhancement of the myocardium with iodinated contrast material, various techniques have been proposed to improve the sensitivity of ct for perfusion imaging . One group has suggested that ischemic myocardium may be recognized by a pattern of subendocardial hypoperfusion during systole with apparent normal perfusion during diastole . Using this technique, they have demonstrated a sensitivity of 90% and specificity of 83% for ct detection of myocardial perfusion defects identified by nuclear perfusion imaging . A more conventional approach given the limited temporal resolution of ct, the tachycardia associated with a dobutamine stress study can result is substantial degradation of image quality . One group has measured a transmural perfusion ratio during adenosine stress ct (subendocardial attenuation density / subepicardial attenuation density) to detect myocardial perfusion abnormalities with a sensitivity / specificity of 86%/92% on a per - patient analysis . Another group has combined adenosine with first pass stress imaging followed by rest images, and has demonstrated good correlation with spect myocardial perfusion imaging for both stress and rest imaging [34, 35]. The combination of ccta stress imaging with subsequent rest imaging as well as a third scan for delayed enhancement can be performed with an effective radiation exposure similar to that of spect imaging (mean 12.7 msv). In order to improve the detection of perfusion defects with ccta a relatively higher kvp is needed for optimal tissue penetration to optimize coronary imaging, while a lower kvp is useful to improve sensitivity for the detection of small differences in myocardial enhancement . A dual - source scanner may be used to simultaneously provide adequate penetration for coronary ccta as well as low kvp imaging to improve detection of myocardial perfusion . A low energy iodine perfusion map is displayed as a color overlay on the high - resolution coronary cta image to provide excellent diagnostic correlation with spect imaging (sensitivity: 91%, specificity: 92%). They have suggested that such defects may be visible without stress imaging because of the superior spatial resolution and dynamic range of ct as compared with nuclear spect or secondary to the intrinsic vasodilatory effects of iodinated contrast . Ccta provides clear definition of the anatomic course of the coronary arteries, even when image quality is limited with respect to assessment of coronary stenosis . Although the coronary arteries are clearly visualized by conventional cardiac catheterization, ccta more clearly demonstrates the relationship between coronary arteries and adjacent anatomical structures such as the pulmonary artery . For this reason, ccta is an excellent study to demonstrate various coronary anomalies (figs . 8, 9). Course between the aorta (ao) and the right ventricular outflow tract (rvot). A globe mip demonstrates the course of the left coronary artery (arrowhead), which originates from the right sinus of valsalva and travels between the aorta (ao) and right ventricular outflow tract (rvot). The left coronary artery is seen to branch into the left anterior descending (lad) and circumflex (lcx) arteries . B sagittal mip demonstrates compression of the left coronary artery (arrowhead) into an ovoid shape as it courses between the aorta (ao) and right ventricular outflow tract (rvot)fig . A volumetric - rendering demonstrates the right coronary artery (white arrowhead) as it courses in the right atrioventricular groove . The circumflex artery (black arrowhead) b volumetric rendering from a more superior projection again demonstrates the right coronary artery (white arrowhead) as it courses in the right atrioventricular groove . The circumflex artery (black arrowhead) c globe mip again demonstrates the circumflex artery (black arrowhead), which courses between the aorta (ao) and left atrium (la) anomalous left coronary artery with a malignant course between the aorta (ao) and the right ventricular outflow tract (rvot). A globe mip demonstrates the course of the left coronary artery (arrowhead), which originates from the right sinus of valsalva and travels between the aorta (ao) and right ventricular outflow tract (rvot). The left coronary artery is seen to branch into the left anterior descending (lad) and circumflex (lcx) arteries . B sagittal mip demonstrates compression of the left coronary artery (arrowhead) into an ovoid shape as it courses between the aorta (ao) and right ventricular outflow tract (rvot) anomalous origin of the circumflex artery from the right coronary artery with a benign a volumetric - rendering demonstrates the right coronary artery (white arrowhead) as it courses in the right atrioventricular groove . The circumflex artery (black arrowhead) branches off the right coronary artery and courses posterior to the aorta . B volumetric rendering from a more superior projection again demonstrates the right coronary artery (white arrowhead) as it courses in the right atrioventricular groove . The circumflex artery (black arrowhead) c globe mip again demonstrates the circumflex artery (black arrowhead), which courses between the aorta (ao) and left atrium (la) ccta provides a useful roadmap in preoperative planning for bypass surgery as well as in post - surgical assessment . In addition to localizing the site of coronary stenosis, ccta can be used to assess whether a more distal segment of a coronary vessel is epicardial in location and is accessible for bypass grafting . In the postoperative patient, ccta can be used to assess the patency of bypass grafts that may be difficult to cannulate by conventional arteriography (fig . Ccta clearly demonstrates the position of bypass grafts relative to other structures and is therefore useful for planning of reoperation bypass procedures . 10patent bypass grafts with narrowing at the origin of the vein graft to the circumflex distribution . A volumetric - rendering in the right anterior oblique demonstrates a vein graft (black arrowhead) coursing from the aorta to the distal right coronary artery . The superior portion of the left internal mammary artery (lima) bypass is also visible (white arrowhead). B volumetric rendering in the left anterior oblique again demonstrates the vein graft (black arrowhead) from the aorta to the distal right coronary artery . The left internal mammary artery (lima) bypass (white arrowhead) is seen to course into the anterior interventricular groove where is anastamoses with the left anterior descending artery . A second vein graft (arrow) extends from the aorta to the circumflex territory . C volumetric rendering in the left lateral projection viewed from a more inferior approach again demonstrates the left internal mammary artery (lima) bypass (white arrowhead) to its anastamosis . The anastamosis of a vein graft (arrow) to an obtuse marginal branch of the circumflex artery is visualized . There is greater than 50% narrowing at the origin of this vein graft (arrow) patent bypass grafts with narrowing at the origin of the vein graft to the circumflex distribution . A volumetric - rendering in the right anterior oblique demonstrates a vein graft (black arrowhead) coursing from the aorta to the distal right coronary artery . The superior portion of the left internal mammary artery (lima) bypass is also visible (white arrowhead). B volumetric rendering in the left anterior oblique again demonstrates the vein graft (black arrowhead) from the aorta to the distal right coronary artery . The left internal mammary artery (lima) bypass (white arrowhead) is seen to course into the anterior interventricular groove where is anastamoses with the left anterior descending artery . A second vein graft (arrow) extends from the aorta to the circumflex territory . C volumetric rendering in the left lateral projection viewed from a more inferior approach again demonstrates the left internal mammary artery (lima) bypass (white arrowhead) to its anastamosis . The anastamosis of a vein graft (arrow) to an obtuse marginal branch of the circumflex artery is visualized . There is greater than 50% narrowing at the origin of this vein graft (arrow) the stable patient with a suspected thoracic aortic injury, dissection or aneurysm is often evaluated with a ct for preoperative planning as well as a cardiac catheterization to assess patency of the coronary arteries . Ccta can provide simultaneous evaluation of pathology in the aorta as well as assessment of the coronary arteries for preoperative planning (fig ., ccta can provide clear anatomic definition of prosthetic valves, aortic grafts and residual dissection flaps (fig . Ccta is also useful for evaluation of complex aortic anomalies such as coarctation, interruption of the aortic arch and collateral vascular supply (fig . A oblique mip demonstrates a dissection flap in the ascending aorta (black arrowheads). A metallic stent is present in the proximal right coronary artery, obscuring the underlying vessel . B oblique mip again demonstrates a dissection flap in the ascending aorta (black arrowheads). The communication between the true lumen and false lumen is visualized as a defect in the dissection flap (black arrow). 12a and b status post repair of type a dissection with a mechanical aortic valve (white arrowhead) at the aortic annulus and a tube graft (white arrow) of the ascending aorta above the native aortic root . A dissection flap is present beyond the tube graft (black arrowheads) with visible communication between the true lumen and false lumenfig . A sagittal mip demonstrates the interruption (arrow) with a large left common carotid exiting the aortic arch just proximal to the interruption . An aorto - femoral graft extends caudally from the ascending aorta and is visualized in the retrosternal space (arrowhead). B coronal mip demonstrates a large, tortuous collateral branch (arrow) extending down from the left external carotid artery to the aortic arch, just beyond the interruption . C four - chamber view of the heart demonstrates the aorto - femoral graft anteriorly (arrowhead). There is an unusual contour to the right ventricle, which appears to be narrowed in its mid - portion (arrow). D curved mip view of the lad demonstrates non - calcified plaque in the proximal portion of this vessel with mild - moderate approximately 50%narrowing (arrow). E curved mip view of the lcx demonstrates eccentric calcified plaque in the mid portion of this vessel without narrowing (arrow) type a dissection of the ascending aorta with extension into the aortic root . A oblique mip demonstrates a dissection flap in the ascending aorta (black arrowheads). A metallic stent is present in the proximal right coronary artery, obscuring the underlying vessel . B oblique mip again demonstrates a dissection flap in the ascending aorta (black arrowheads). The communication between the true lumen and false lumen is visualized as a defect in the dissection flap (black arrow). The proximal right coronary artery is again visualized (white arrow) a and b status post repair of type a dissection with a mechanical aortic valve (white arrowhead) at the aortic annulus and a tube graft (white arrow) of the ascending aorta above the native aortic root . A dissection flap is present beyond the tube graft (black arrowheads) with visible communication between the true lumen and false lumen interruption of the aortic arch with collateral circulation and graft . A sagittal mip demonstrates the interruption (arrow) with a large left common carotid exiting the aortic arch just proximal to the interruption . An aorto - femoral graft extends caudally from the ascending aorta and is visualized in the retrosternal space (arrowhead). B coronal mip demonstrates a large, tortuous collateral branch (arrow) extending down from the left external carotid artery to the aortic arch, just beyond the interruption . C four - chamber view of the heart demonstrates the aorto - femoral graft anteriorly (arrowhead). There is an unusual contour to the right ventricle, which appears to be narrowed in its mid - portion (arrow). D curved mip view of the lad demonstrates non - calcified plaque in the proximal portion of this vessel with mild - moderate approximately 50%narrowing (arrow). E curved mip view of the lcx demonstrates eccentric calcified plaque in the mid portion of this vessel without narrowing (arrow) preoperative planning for aortic valve replacement surgery requires assessment of the ascending aorta for morphology and calcification, assessment of the aortic valve annulus for appropriate sizing of the replacement valve and evaluation of the coronary arteries . Ccta is an excellent modality to assess simultaneously the thoracic aorta, the aortic valve and the coronary arteries (figs . Ccta can provide accurate imaging and measurements of the aortic annulus and valve for preoperative planning of aortic valve replacement . Ccta provides essential information prior to percutaneous valve replacement, especially with respect to the location and extent of aortic root and valve calcifications and the location of the coronary ostia relative to the valve plane . 16), and the presence of aortic valve thickening and insufficiency may also be visible (fig . A closed aortic valve demonstrates normal coaptation of the leaflets in diastole at 70% of the r - r interval . Three raphe lines are visible within the valve (arrowheads). B aortic valve opens with a triangular shape, demonstrating three independent leaflets at 30% of the r - r interval . The presence of a trileaflet valve can only be confirmed by demonstrating this normal systolic openingfig . A closed aortic valve demonstrates normal coaptation of the leaflets at the three raphe lines (arrowheads). B systolic image demonstrates a normal - size opening with mild myxomatous thickening and redundancy of the leafletsfig . A diastolic image in short axis of the aortic valve demonstrates a linear closure of the aortic valve with only two leaflets (arrowheads). A small regurgitant orifice is visible between the mildly thick leaflets the more anterior leaflet is in the position that would typically be occupied by the right and left coronary leaflets . B systolic image in short axis of the aortic valve demonstrates a fish - mouth-shaped opening of the two leaflets (arrowheads) that is typical for a bicuspid valve . The overall aortic valve area is minimally restricted (2 cm by planimetry)fig . 17bicurpid aortic valve with moderate aortic insufficiency and focal thickening, likely related to endocarditis . A diastolic image in short axis of the aortic valve demonstrates incomplete coaptation of the valve leaflets with a regurgitant orifice (*). Just anterior to this regurgitant orifice, there is a focal thickening of the valve, representing a vegetation . B systolic image in short axis demonstrates a fish - mouth opening of the aortic valve with a visible raphe (arrowhead) within the anterior leaflet . Pseudoraphe as it does not open as a normal raphe, but can mimic the appearance of a trileaflet aortic valve on diastolic imaging . The regurgitant orifice in diastole appears to be related to the free edge of this pseudoraphae . C three - chamber view in diastole demonstrates incomplete coaptation of the aortic valve (black arrowhead), with focal thickening (a vegetation) associated with the free edge of the anterior leaflet normal aortic valve in short axis . A closed aortic valve demonstrates normal coaptation of the leaflets in diastole at 70% of the r - r interval . Three raphe lines are visible within the valve (arrowheads). B aortic valve opens with a triangular shape, demonstrating three independent leaflets at 30% of the r - r interval . The presence of a trileaflet valve can only be confirmed by demonstrating this normal systolic opening mildly thickened aortic valve with normal motion pattern . A closed aortic valve demonstrates normal coaptation of the leaflets at the three raphe lines (arrowheads). B systolic image demonstrates a normal - size opening with mild myxomatous thickening and redundancy of the leaflets bicuspid aortic valve with regurgitation and minimal restriction of motion . A diastolic image in short axis of the aortic valve demonstrates a linear closure of the aortic valve with only two leaflets (arrowheads). The more anterior leaflet is in the position that would typically be occupied by the right and left coronary leaflets . B systolic image in short axis of the aortic valve demonstrates a fish - mouth-shaped opening of the two leaflets (arrowheads) that is typical for a bicuspid valve . The overall aortic valve area is minimally restricted (2 cm by planimetry) bicurpid aortic valve with moderate aortic insufficiency and focal thickening, likely related to endocarditis . A diastolic image in short axis of the aortic valve demonstrates incomplete coaptation of the valve leaflets with a regurgitant orifice (*). Just anterior to this regurgitant orifice, there is a focal thickening of the valve, representing a vegetation . B systolic image in short axis demonstrates a fish - mouth opening of the aortic valve with a visible raphe (arrowhead) within the anterior leaflet . Pseudoraphe as it does not open as a normal raphe, but can mimic the appearance of a trileaflet aortic valve on diastolic imaging . The regurgitant orifice in diastole appears to be related to the free edge of this pseudoraphae . C three - chamber view in diastole demonstrates incomplete coaptation of the aortic valve (black arrowhead), with focal thickening (a vegetation) associated with the free edge of the anterior leaflet when reoperation is required for a prosthetic aortic valve that has failed, coronary angiography is often contraindicated, especially in the setting of endocarditis when catheter angiography carries an increased risk of embolization . Ccta can provide high - resolution functional imaging of prosthetic valves with simultaneous evaluation of the coronary arteries for preoperative planning . Prosthetic endocarditis may also be associated with additional complications including valve malfunction and pseudoaneurysms, which may be clearly defined by ccta (fig . A pocket of contrast just anterior to the aortic prosthesis (arrowheads) represents a pseudoaneurysm of the left ventricular outflow tract . The pseudoaneurysm is visualized to extend as a tubular structure into the aorto - pulmonary window (arrowheads) pseudoaneurysm of the left ventricular outflow tract following prosthetic valve endocarditis . A pocket of contrast just anterior to the aortic prosthesis (arrowheads) represents a pseudoaneurysm of the left ventricular outflow tract . The pseudoaneurysm is visualized to extend as a tubular structure into the aorto - pulmonary window (arrowheads) mri is generally considered the test of choice for evaluation of cardiac morphology since this technique does not require ionizing radiation . However, ccta has been used extensively in preoperative planning for ablation procedures in the left atrium . The pulmonary veins and the intricate details of the left atrial anatomy are more clearly visualized by ccta, which provides superior spatial resolution as compared to mri, and their location can be mapped into a three - dimensional volume that is used during the ablation procedure (fig . Integration of cta image data during ablation in the left atrium is often useful to shorten procedure times and improve patient outcome . Although echocardiography and mri are generally preferred for evaluation of cardiac morphology, changes in cardiac morphology (fig . A volumetric surface rendering of the left atrium from a posterior vantage point demonstrates four pulmonary veins . The right inferior vein consists of several small branches that unite at the level of the left atrium (arrow). This information is important when performing an ablation procedure, as these branches have small orifices that might be damaged with resulting pulmonary vein stenosis . B endoluminal rendering demonstrates multiple small branches entering the left atrium in the location of the right inferior pulmonary vein (arrow). The orifice of the left atrial appendage (*) is separated from the two left - sided pulmonary veins by the warfarin ridgefig . A four - chamber view of the heart demonstrates a mass along the left heart border (arrowheads) adjacent to the left atrium (la) and left ventricle (lv). B coronal view of the left atrium (la) demonstrates that this mass (arrowheads) corresponds to the typical location of the left atrial applendage . Stagnant flow within the mass results in slow filling, but no thrombus was visible on delayed views . C four - chamber view in a delayed phase demonstrates contrast filling of the mass . D coronal view in a delayed phase also demonstrates homogeneous enhancement of the mass that represents a herniated and dilated left atrial appendage . F lateral view of the chest again demonstrates the mass corresponding to the herniated and enlarged left atrial appendagefig . 21left atrial myxoma in a patient with a recent transient ischemic episode . A axial image demonstrates a small focal mass within the left atrium (arrowhead). The more common location for a left atrial appendage would be along the interatrial septum . B sagittal image demonstrates that this mass (arrowhead) extends from the roof of the left atrium adjacent to the orifice of the left atrial appendage pulmonary venous mapping of the left atrium demonstrates a compound right inferior pulmonary vein . A volumetric surface rendering of the left atrium from a posterior vantage point demonstrates four pulmonary veins . The right inferior vein consists of several small branches that unite at the level of the left atrium (arrow). This information is important when performing an ablation procedure, as these branches have small orifices that might be damaged with resulting pulmonary vein stenosis . B endoluminal rendering demonstrates multiple small branches entering the left atrium in the location of the right inferior pulmonary vein (arrow). The orifice of the left atrial appendage (*) is separated from the two left - sided pulmonary veins by the warfarin ridge partial absence of the pericardium with herniation of the left atrial appendage . A four - chamber view of the heart demonstrates a mass along the left heart border (arrowheads) adjacent to the left atrium (la) and left ventricle (lv). B coronal view of the left atrium (la) demonstrates that this mass (arrowheads) corresponds to the typical location of the left atrial applendage . Stagnant flow within the mass results in slow filling, but no thrombus was visible on delayed views . C four - chamber view in a delayed phase demonstrates contrast filling of the mass . D coronal view in a delayed phase also demonstrates homogeneous enhancement of the mass that represents a herniated and dilated left atrial appendage . F lateral view of the chest again demonstrates the mass corresponding to the herniated and enlarged left atrial appendage left atrial myxoma in a patient with a recent transient ischemic episode . A axial image demonstrates a small focal mass within the left atrium (arrowhead). The more common location for a left atrial appendage would be along the interatrial septum . B sagittal image demonstrates that this mass (arrowhead) extends from the roof of the left atrium adjacent to the orifice of the left atrial appendage enlargement of the right heart is often related to intracardiac shunts . Ccta is an excellent test for identification of anomalous pulmonary veins that are not visualized by echocardiography . Furthermore, we have found ccta to be a useful study in the pre - procedure planning for percutaneous closure of atrial septal defects (fig . The superb three - dimensional anatomical definition provided by ct imaging allows assessment of the size and location of the defect relative to adjacent structures such as the aortic root . A sagittal mip demonstrates a posterior left atrium (la), with a more anterior right atrium (ra). The superior vena cava (svc) is identified as it enters the right atrium . A jet of contrast material (arrowhead) extends from the left atrium through an asd into the right atrium, just below the orifice of the svc b axial mip demonstrates the asd with left to right shunting of contrast (arrowhead). The proximity of the asd to the aortic root (ao) is clearly demonstrated . The location of the asd and the presence of a rim of tissue must be ascertained prior to attempted percutaneous closure . If the asd is too close to the aortic root, the closure device may clamp the root with a possible long - term complication of erosion into the aortic rootfig . A four - chamber view demonstrates a defect in the distal portion of the interventricular septum (arrowhead). B short axis view demonstrates that the defect is larger than appreciated on the four - chamber view and extends through the inferior half of the distal septum (arrowhead) atrial septal defect (asd) with left to right shunt . A sagittal mip demonstrates a posterior left atrium (la), with a more anterior right atrium (ra). The superior vena cava (svc) is identified as it enters the right atrium . A jet of contrast material (arrowhead) extends from the left atrium through an asd into the right atrium, just below the orifice of the svc . B axial mip demonstrates the asd with left to right shunting of contrast (arrowhead). The proximity of the asd to the aortic root (ao) is clearly demonstrated . The location of the asd and the presence of a rim of tissue must be ascertained prior to attempted percutaneous closure . If the asd is too close to the aortic root, the closure device may clamp the root with a possible long - term complication of erosion into the aortic root post - infarct ventricular septal defect . A four - chamber view demonstrates a defect in the distal portion of the interventricular septum (arrowhead). B short axis view demonstrates that the defect is larger than appreciated on the four - chamber view and extends through the inferior half of the distal septum (arrowhead) evaluation of the right heart by ccta is limited by the mixing of opacified blood from the svc with unopacified blood from the ivc . Nonetheless, with appropriate modifications of the contrast injection protocol, ccta may be used successfully to evaluate right ventricular morphology, as well as for evaluation of the tricuspid valve (fig . 24ebstein s anomaly with apical displacement of the septal leaflet of the tricuspid valve (arrowhead), a large, redundant sail - like anterior tricuspid leaflet (arrow) and marked enlargement of the right heart . A four - chamber view . B short axis view ebstein s anomaly with apical displacement of the septal leaflet of the tricuspid valve (arrowhead), a large, redundant sail - like anterior tricuspid leaflet (arrow) and marked enlargement of the right heart . A four - chamber view . B short axis view given the development of new low - dose cta protocols (see below) and the more rapid acquisition time of cta compared to mri, it is likely that ccta will play an increasing role in the evaluation of cardiac morphology and congenital heart disease . As a full discussion of these topics is beyond the scope of this pictoral review, the reader is referred to a recent textbook for more in - depth discussion of these topics . Ccta is a powerful diagnostic tool, but necessitates injection of intravenous contrast material as well as exposure to ionizing radiation . Ccta is often limited by image quality in obese patients as well as patients with elevated or irregular heart rates . The presence of blooming artifact from dense arterial calcification limits the quality of ccta and results in overestimation of the degree of stenosis (fig . Evaluation of metallic stents is also limited by blooming artifacts from the stent struts, though ccta has been increasingly successful for evaluation of coronary stents, especially with stent diameters above 3 mm (figs . 25, 26). Although a normal ccta effectively excludes the presence of cad, the finding of moderate disease by ccta is often of uncertain clinical significance as it may not be obvious whether the patient s symptoms are related to the visualized cad . Because of these limitations, competing technologies for evaluation of suspected cad including ecg - stress testing, stress testing with echocardiography, stress testing with nuclear perfusion imaging and mri may be more appropriate in specific clinical scenarios . Functional stress testing may be appropriate to evaluate the clinical significance of cad detected by ccta . 26slab mip of the proximal to mid - right coronary artery demonstrates an ulcerated plaque at the origin of the rca (arrowhead) as well as an occluded stent in the mid - portion of the rca (arrow) curved mip of the right coronary artery and posterior descending artery . There is no significant narrowing of the rca slab mip of the proximal to mid - right coronary artery demonstrates an ulcerated plaque at the origin of the rca (arrowhead) as well as an occluded stent in the mid - portion of the rca (arrow) a meta analysis of 147 consecutively published reports of exercise treadmill studies reported a sensitivity of 68% with a specificity of 77% for the detection of cad . Diagnostic accuracy is somewhat higher for stress echocardiography and stress perfusion imaging, with both of these tests demonstrating superior diagnostic accuracy as compared to the basic ecg stress treadmill test . A pooled analysis of stress tests for the diagnosis of significant cad demonstrated a sensitivity / specificity of 80%/86% for stress echocardiography and 84%/77% for stress perfusion imaging . Although most studies suggest a relatively similar diagnostic accuracy for stress echocardiography and stress perfusion imaging, a recent meta - analysis suggests that stress echocardiography is superior to stress perfusion imaging for evaluation of high risk (left main disease and/or triple vessel) disease because of the greater risk of missing severe cad with perfusion imaging (sensitivity 75% versus 94%). Although echocardiography does not provide direct visualization of the coronary arteries, stress ecg combined with echocardiography is a well - established non - invasive examination for stratifying the risk of cad and influencing the decision for subsequent coronary angiography and revascularization . Based upon the decision analysis cited above, initial evaluation of suspected cad with stress echocardiography (with ccta reserved for follow - up of positive stress tests) may be more cost - effective as compared to an initial ccta for the low risk patient . A minority of cardiac imaging procedures are performed for structural or functional evaluation, unrelated to cad . For these examinations, echocardiography is a less expensive and less invasive technique compared to ccta . Transthoracic echocardiography can be performed portably and does not generally require administration of intravenous contrast material . Transesophageal echocardiography can be performed when there is a need for higher spatial resolution or to visualize parts of the heart such as the left atrial appendage that may be difficult to image from the transthoracic approach . Echocardiography is superior to ct when the primary objective of diagnostic imaging is for assessment of ventricular wall motion, valve morphology and function, as well as for quantification of flow velocities and pressure gradients within the heart . Doppler evaluation provides a simple method to grade the severity of valvular stenosis or insufficiency, as well as other functional abnormalities such as tamponade and restrictive physiology . Based upon the superior temporal resolution of echocardiography, echocardiography is often superior to ccta and mri for detection of mobile masses and vegetations within the heart . Mri is limited by spatial resolution for evaluation of coronary artery branches, but is an excellent modality for evaluation of myocardial function, perfusion and viability [52, 53]. As compared with ccta, there is no ionizing radiation and no need for a contrast injection to evaluate function with steady - state free - precession sequences . The excellent contrast between the blood pool and the endocardial surface allows clear depiction of cardiac morphology and reproducible measurements of chamber size . When gadolinium is administered, different patterns of myocardial perfusion and delayed enhancement may be used to characterize various types of cardiomypathy and to document the extent of viable tissue . The major clinical applications for cardiac mri at this time include imaging of structural morphology and myocardial evaluation . A major limitation of ccta compared with stress echocardiography and mri is the exposure to ionizing radiation . Various strategies have been implemented in order to reduce this exposure . At the level of ct hardware, there is a constant effort to produce more efficient detectors . At the level of image acquisition, newer techniques have been developed that limit exposure to smaller portions of the cardiac cycle . Finally, at the level of image reconstruction, newer technology is being developed to improve signal to noise in ct images with fewer photons in the raw data . Conventional ccta is performed with an ecg - gated helical image acquisition to reconstruct an image during the quiescent phase of the cardiac cycle, usually in mid - diastole . In order to obtain sufficient data for reconstruction, this low pitch provides sufficient overlap between successive helical rotations of the detector array such that adequate data are obtained in each portion of the cardiac cycle for image reconstruction . The data obtained from the quiescent portion of diastole are used to reconstruct high - resolution images of the coronary arteries, while data obtained from the remainder of the cardiac cycle are used to reconstruct lower resolution functional images that may be used to demonstrate valve motion and ventricular contraction . However, much of the data acquired during the non - quiescent portions of the cardiac cycle is not needed . A technique known as tube - current modulation has been developed to adjust the current to the x - ray tube such that the full dose is delivered during the quiescent phase of the cardiac cycle for high - resolution imaging, while a much reduced dose is delivered in other phases of the cycle to allow reconstruction of lower resolution functional images . At a heart rate of 60 beats per minute, tube current modulation may result in a dose reduction of greater than 50% without loss of image quality . Prospective ecg - gating with an axial step and shoot technique has been introduced to further reduce radiation dose . This technique acquires images in axial mode during a single, short (usually mid - diastolic) interval . The prospective gating technique relies on a stable heart rate to predict the time for mid - diastole and to trigger the x - ray tube only during that interval for imaging . Evaluation of cardiac motion is not possible with standard step - and - shoot acquisition since image data are not obtained during the systolic portion of the cardiac cycle . Furthermore, image quality may be adversely impacted if a variable heart rhythm results in image acquisition during a suboptimal phase of the cardiac cycle . Nonetheless, a recent multicenter international study included 685 patients, with 99 patients evaluated using prospective ecg - gating and 586 patients evaluated with a standard helical approach . Mean radiation dose was reduced by 68%, from 11.2 msv for the helical mode to 3.6 msv for the step - and - shoot technique, without loss of image quality . High pitch spiral acquisition is a new variation on helical scan technology that can be used to acquire ccta of the entire heart in a single spiral acquisition for patients with a sufficiently low heart rate . Rapid table motion is timed so that the entire heart is imaged during the quiescent phase of a single cardiac cycle . A recent study of 100 patients comparing prospective ecg - gating with step - and - shoot technology to prospective ecg - gating with a high pitch spiral mode (pitch 3.4) demonstrated diagnostic image quality in over 98% of coronary artery segments with no significant difference in image quality between the two methods . Mean effective radiation dose was 1.4 0.4 msv for prospective ecg gating with step - and - shoot technology and 0.9 0.1 msv for prospective ecg gating with high - pitch spiral mode . Most contemporary ct scanners utilize a mathematical technique called filtered back projection to reconstruct ct images . Iterative reconstruction is another category of techniques that has recently gained popularity for reconstruction of ct images with decreased image noise . Iterative reconstruction techniques employ an initial estimate of voxel attenuation to predict projection data; estimates of voxel attenuation are iteratively adjusted to minimize the difference between the predicted projection data and the measured projection data . These techniques are more computationally intensive as compared to filtered back projection, but can result in reduced image noise; alternatively, iterative techniques may be used to maintain an acceptable signal - to - noise ratio and image quality with reduced radiation (fig . A recent study suggests that iterative techniques may allow a reduction in effective radiation exposure by approximately 50% . Based upon the doses recorded from studies of prospective ecg - gating and high pitch helical technique, new acquisition techniques combined with iterative reconstruction should routinely provide ccta imaging with effective radiation exposures of 1 msv or less . 27iterative reconstruction illustrated for a curved mip of a normal rca scanned with very low dose technique . A standard reconstruction with filtered back projection demonstrates a noisey image related to inadequate signal - to - noise characteristics . B iterative reconstruction using idose (philips medical systems) with the same raw data demonstrates improved signal - to - noise characteristics with improved definition of the smaller distal rca branches iterative reconstruction illustrated for a curved mip of a normal rca scanned with very low dose technique . A standard reconstruction with filtered back projection demonstrates a noisey image related to inadequate signal - to - noise characteristics . B iterative reconstruction using idose (philips medical systems) with the same raw data demonstrates improved signal - to - noise characteristics with improved definition of the smaller distal rca branches advances in ct technology over the past decade have resulted in marked improvements in ccta image quality along with reduction in effective radiation dose . We have reviewed the application of ecg - gated cta of the heart to various clinical applications, including evaluation of the thoracic aorta, aortic valve, bypass grafts, left atrium and various structural cardiac abnormalities . However, the major indication for cardiac ct is ccta for evaluation of cad in symptomatic patients with low to intermediate risk of cad . High - risk symptomatic patients should be triaged directly to cardiac catheterization . In patients with a risk of cad below 20%, ccta is useful in these patients to follow - up on positive stress tests in order to avoid catheterization of patients with false - positive stress test results . As the pre - test probability of cad in a symptomatic patient rises above 20%, ccta provides a cost - effective initial evaluation . Stress perfusion imaging may be useful as a secondary study in these patients when moderate stenosis (5075% diameter reduction) is identified on ccta . New developments that allow myocardial perfusion imaging with ccta are likely to result in a single comprehensive ct examination for cad in the future.
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Experimental birds: seven - day - old white leghorn male chickens were used in this study . The birds were fed a commercial balanced diet and water ad libitum and maintained under controlled light conditions (12-hr light/12-hr dark). At the end of the experimental period, the birds were euthanized, and their tissues were used as described below . The birds were treated in accordance with the guideline for regulation of animal experimentation (1997) of the faculty of agriculture, shinshu university . Tissue samples: the experimental methods were divided into three kinds as follows: a pre - embedding technique for immunoelectron microscopy reprocessed from paraffin sections stained by the linked streptavidin - biotin method (prlsb) that had been coverslipped with entellan (merck, darmstadt, germany) and completed light microscopic observation 1 year or more before; a pre - embedding technique for immunoelectron microscopy reprocessed from paraffin sections that had been newly sliced at a thickness of 5 m from previously embedded paraffin blocks and immunohistochemically stained by colloidal - gold immunolabeling (prcg); and a post - embedding technique for immunoelectron microscopy from sections newly sampled and stained by colloidal - gold immunolabeling (pocg). Paraffin sections in both prlsb and prcg were mounted on mas - gp coated glass microscope slides (matsunami glass industries ltd ., the birds (n=4) used for prlsb and prcg were perfused with physiological salt solution, followed by 4% paraformaldehyde in 0.1 m phosphate buffer (ph 7.6). The distal ileum was immediately dissected out after the perfusion and cut into small blocks with razor blades . The tissue blocks were cut open along the mesenterium, stretched on a cork plate, immersed in the same perfusate at 4c overnight, washed several times with phosphate buffer and embedded in paraffin wax according to standard procedures . The birds (n=4) used for pocg were perfused with 4% paraformaldehyde and 0.01% glutaraldehyde in phosphate - buffered saline (pbs). The distal ileum was sliced under a dissecting microscope into 1 1 2-mm blocks, immersed in the same perfusate at 4c overnight, dehydrated through a graded series of ethanol and embedded in quetol 812 (nisshin em, tokyo, japan). The linked streptavidin - biotin staining was visualized with 0.05% 3,3-diaminobenzidine (dab) as described in our previous paper . Briefly, the following antisera were used: rabbit antiserum against synthetic human glp-1 (amino acids 119) (1:2,000 dilution; ga1176; affiniti research products, devon, u.k . ), which does not cross - react with other proglucagon - derived peptides; and biotinylated goat anti - rabbit igg serum (diluted 1:300; ap132b; millipore, billerica, ma, u.s.a . ). Sections on glass microscope slides were soaked in xylene for 12 hr to remove the coverslips and then rehydrated through a decreasing graded series of ethanol for 5 min each . After washing with pbs, the sections were examined under a light microscope, and the intestinal mucosa containing glp-1-positive cells was dissected into 1 1-mm pieces . The sections were immersed in 2.5% glutaraldehyde in 0.1 m phosphate buffer (ph 7.4) for 15 min, postfixed in 1% osmium tetroxide (oso4) for 15 min, dehydrated through a graded series of ethanol and embedded in quetol 812 . After polymerization of the resin, the sections were detached from the glass slides by warming . Ultra - thin sections (80 nm) were obtained using an ultramicrotome (super nova; reichert - jung, vienna, austria), mounted on 200-mesh nickel grids (nisshin em), stained with 10% (v / v) ti blue (nisshin em) and 2% (w / v) lead citrate and observed under a transmission electron microscope (jem-1400; jeol, tokyo, japan). Paraffin sections were newly cut at 5 m thickness from blocks embedded 1 year before . After routine dewaxing and rehydration, the sections were pretreated with 2.5% normal donkey serum (ihr-8135; immunobioscience, mukilteo, wa, u.s.a .) For 20 min and incubated with the above - described rabbit antiserum against synthetic glp-1 (1:2,000 dilution) for 12 hr . After three washes with pbs, the sections were incubated with colloidal gold (6-nm diameter)-labeled donkey anti - rabbit igg serum (1:100 dilution; 711195 - 152; jackson immunoresearch, west grove, pa, u.s.a .) For 3 hr . The sections were then immersed in 2.5% glutaraldehyde for 15 min and serially processed for the silver - enhanced colloidal gold method by immersion in 0.1 m acetic acid buffer (ph 7.0) for 10 min to yield detailed electron - immunoreactive structures and enhancement using a silver enhancement kit (hq silver; nanoprobes, yaphank, ny, u.s.a .) In a dark room for 8 min . After thorough washing with 0.1 m phosphate buffer, dissection, postfixation with oso4, embedding in quetol 812 and electron microscopic observation were performed as described for prlsb . Ultra - thin sections from blocks embedded in quetol 812 were obtained as described above . The ultra - thin sections were pretreated with 1% (w / v) sodium periodate solution for semi - deresination to readily allow reactions between the antibody and antigen and incubated with 1% normal donkey serum for 15 min . After three washes with pbs, a mouse monoclonal primary antibody against synthetic human glp-1 (amino acids 736) (1:8,000 dilution; a6104.1; immundiagnostik, bensheim, germany) was applied to the sections for 24 hr . After three washes with pbs, the sections were treated with colloidal gold (12-nm diameter)-labeled donkey anti - mouse igg serum (1:200 dilution; 715 - 205 - 150; jackson immunoresearch) for 2 hr, stained with 10% (v / v) ti blue and 2% (w / v) lead citrate and observed under a transmission electron microscope . The specificity of the primary antibody used in this study has been documented by the manufacturer and in previous reports [4, 12, 21]. Morphometry of glp-1 granules: morphometric analyses were carried out to characterize the secretory granules of l - cells . L - cells that contained a nucleus and more than 10 granules were selected for morphometric analyses . A total of 471 secretory granules from 15 cells in prlsb, 195 secretory granules from 10 cells in prcg and 207 granules from 12 cells in pocg located at the basal or perinuclear region of the cytoplasm were randomly selected . The shortest and longest diameters of the secretory granules were measured using an image analyzer (ks 400; carl zeiss, oberkochen, germany). Differences between the mean diameters of granules from each method were tested by anova and with a selected tukey immunoreactive cells for the glp-1 antiserum, so - called l - cells, were observed in the full length of the distal ileum in prlsb and prcg under a light microscope . The cells were distributed in the epithelium of the crypt and the lower part of the intestinal villi and had comma- or flask - like shapes (fig . (a) a section stained with the prlsb procedure and counterstained with mayer s hematoxylin . The immunoreactive cells are distributed in the crypts and lower part of the villous epithelium (arrows). (a) distal ileum embedded in paraffin and paraffin sections mounted on a glass slide . (b) a section stained with the prcg procedure examined under a light microscope without a coverslip . The arrow indicates an immunoreactive cell observed under the electron microscope in panel d. (c) dissected sections including immunoreactive cells embedded in quetol 812 . The cell surrounded by a dashed line is the same cell indicated by the arrow in panel b. secretory granules accumulate in the basal cytoplasm of l - cells (asterisk). . Shows results of the prcg procedure from a paraffin block to electron microscopic observation . (a) a section stained with the prlsb procedure and counterstained with mayer s hematoxylin . The immunoreactive cells are distributed in the crypts and lower part of the villous epithelium (arrows). Bars=200 m . Summary of the prcg procedure from a paraffin block to electron microscopic observation . (a) distal ileum embedded in paraffin and paraffin sections mounted on a glass slide . (b) a section stained with the prcg procedure examined under a light microscope without a coverslip . The arrow indicates an immunoreactive cell observed under the electron microscope in panel d. (c) dissected sections including immunoreactive cells embedded in quetol 812 . The cell surrounded by a dashed line is the same cell indicated by the arrow in panel b. secretory granules accumulate in the basal cytoplasm of l - cells (asterisk). The glp-1-containing cells were easily identified as precipitated dark products resulting from dab in prlsb and gold particle enhanced by silver reactions in prcg (fig . 3.low-magnification electron micrographs of the chicken distal ileum stained with the prlsb (a) and prcg (b) procedures . Glp-1-positive cells are easily identified at low magnification by the dark dab reaction products and gold particles enhanced by silver reactions (arrows). An enterochromaffin cell is observed adjacent to the glp-1-positive cell in panel (b). Bars=2 m . ). On the other hand, it was difficult to identify the granules labeled with the 12-nm - diameter gold particles under low magnification in pocg . Although degradation of the nucleoplasm and cytoplasm was observed, secretory granules could be identified in prlsb and prcg (fig . These cells contained round to oval secretory granules without a halo that mainly accumulated in the basal cytoplasm or perinuclear region of l - cells (fig . 4.electron micrographs of glp-1-positive cells in the chicken distal ileum stained with the prlsb (a), prcg (b) and pocg (c) procedures . The electron densities of the secretory granules observed with the three different experimental methods are medium and almost the same (arrows). The secretory granules of an enterochromaffin cell adjacent to the glp-1-positive cell show polymorphous shapes and high - electron density (asterisk) (b). The post - embedding procedure shows fine preservation of the nucleoplasm and cytoplasm (c). (d) high - magnification view of the secretory granules in panel (a). The dab reaction products precipitate around the secretory granules (arrows) and in the cytoplasm (arrowheads). (e) high - magnification view of the secretory granules in panel (b). The particles of enhanced colloidal gold mainly precipitate around the secretory granules (arrows), and a few of them are located in the cytoplasm (arrowheads). The profile of the secretory granules is clearly observed . (f) high - magnification view of the secretory granules in panel (c). Almost all of the particles of colloidal gold are diffusely arranged on the secretory granules (arrows), and very few of them are located in the cytoplasm (arrowheads). The electron densities of the secretory granules observed among the three different experimental methods were medium and almost the same . 4b, the immunopositive granules for glp-1 had lower electron density than the secretory granules of adjacent enterochromaffin cells, which were widely distributed in the gastrointestinal tract and had polymorphous secretory granules . Low - magnification electron micrographs of the chicken distal ileum stained with the prlsb (a) and prcg (b) procedures . Glp-1-positive cells are easily identified at low magnification by the dark dab reaction products and gold particles enhanced by silver reactions (arrows). An enterochromaffin cell is observed adjacent to the glp-1-positive cell in panel (b). Bars=2 m . Electron micrographs of glp-1-positive cells in the chicken distal ileum stained with the prlsb (a), prcg (b) and pocg (c) procedures . The electron densities of the secretory granules observed with the three different experimental methods are medium and almost the same (arrows). The secretory granules of an enterochromaffin cell adjacent to the glp-1-positive cell show polymorphous shapes and high - electron density (asterisk) (b). The post - embedding procedure shows fine preservation of the nucleoplasm and cytoplasm (c). (d) high - magnification view of the secretory granules in panel (a). The dab reaction products precipitate around the secretory granules (arrows) and in the cytoplasm (arrowheads). (e) high - magnification view of the secretory granules in panel (b). The particles of enhanced colloidal gold mainly precipitate around the secretory granules (arrows), and a few of them are located in the cytoplasm (arrowheads). The profile of the secretory granules is clearly observed . (f) high - magnification view of the secretory granules in panel (c). Almost all of the particles of colloidal gold are diffusely arranged on the secretory granules (arrows), and very few of them are located in the cytoplasm (arrowheads). Bars=200 nm . Examination under higher magnification clarified the differences in the immunoreactivities among the three different experimental methods . In prlsb, the dab reaction products precipitated around secretory granules and within the cytoplasm (fig ., the gold particles enhanced by silver products mainly precipitated around secretory granules, and a few were located in the cytoplasm (fig . The gold particles were mostly found on secretory granules, and very few of them were located in the cytoplasm (fig . The profile of the secretory granules was clearly observed in prcg and pocg, but was difficult to identify in prlsb (fig . 4d4f). Because the electron density of the dab reaction product was similar to that of the secretory granules, the deposition of dab reaction products around the whole profile of the granules tended to make the boundary between the granules and cytoplasm ambiguous . The electron density of the dab reaction product was quite low compared with that of the gold particles (fig . 4e and 4f). The results of the morphometric analyses of the secretory granules are shown in table 1table 1.shortest and longest diameters of the secretory granules in the different proceduresprocedureshortest diameter (nm)longest diameter (nm)prlsb311 70 * 378 80*prcg288 58355 69pocg291 56343 * p<0.01, significant difference from the other procedures .. the longest and shortest diameters of the secretory granules in prlsb were significantly longer than those in both prcg and pocg . It is mainly classified into three methods, pre - embedding, post - embedding and non - embedding (ultra - thin frozen section) methods . These three methods each have advantages and disadvantages, and researchers are required to select the most effective method appropriate to the tissues under observation . Pre - embedding immunocytochemical staining has provided successful observations by light microscopy and electron microscopy in the same specimen [9, 16]. However, permeabilization treatments are sometimes necessary to obtain optimal labeling of intracellular antigenic sites, particularly with thick sections, such as vibratome sections [7, 9, 14]. The use of penetration agents, such as enzymes, or detergents, such as triton x-100, often leads to destruction or disorganization of more labile cytoplasmic structures and frequently gives rise to false - positive staining . In contrast, post - embedding immunocytochemical staining of ultra - thin sections at the electron microscopic level produces high - resolution localization and good morphological preservation . However, epoxy resin blocks a number of antigenic epitopes through interactions with tissue components and forms a hydrophobic polymeric network that prevents the diffusion of water - soluble substances, such as antibodies [2, 6]. To expose antigenic sites to the immunoreagents, etching of epoxy resin ultra - thin tissue sections with reagents, such as periodic acid or hydrogen peroxide, is necessary [2, 12, 23]. Some researchers have taken advantage of hydrophilic embedding media, such as lr white / gold embedding resin, to avoid this problem . In either case, post - embedding procedures tend to prevent antibody binding to a sufficient number of antigenic sites, which frequently gives rise to false - negative staining . Indeed, the primary antibody used in prlsb and prcg did not react in pocg in the present study, although the specificity of the primary antibody has been proven . A non - embedding approach, which uses ultra - thin frozen sections and colloidal gold, can overcome the penetration problem and also preserve the tissue antigenicity [9, 19]. Paraffin - embedded tissues have been used for immunohistochemical staining for a long time . The medium of paraffin allows tissues to be thinly sliced, typically in the range of 515 m . The specimen is rehydrated through a decreasing graded series of ethanol, which facilitates the penetration of antibodies . In this study, we attempted to observe 5-m sections that were cut from paraffin blocks and immunostained for observation under an electron microscope . It has been thought that ultrastructural examination of paraffin - embedded tissues is associated with disruption of membranes, loss of clarity and varying degrees of tissue degradation for reasons, such as series of hydration / dehydration processes . However, some studies have documented that electron microscopic observation produced the opposite result [8, 17]. Reported that the ultrastructural features of paraffin - embedded tissues that were reprocessed for electron microscopic observation were quite variable and also pointed out that the fixation, rather than the paraffin embedding, seemed to be most important step that affected the quality of the ultrastructural features . Moreover, they documented that optimal initial fixation in formalin at an appropriate concentration and ph allowed ultrastructural preservation and that ultrastructural examination of paraffin - embedded tissues could be optimized by avoiding paraffin blocks prepared from poorly fixed tissues and tissues adjacent to areas of necrosis . Some studies have clearly documented that organelles, such as nuclei, desmosomes, sarcomeres, intermediate filaments, tonofilaments and electron - dense granules, can be recognized from reprocessed paraffin - embedded tissues under electron microscopic observation, although mitochondria, polyribosomes, microtubules, lipids and glycogen can exhibit various degrees of degradation [8, 22, 24, 26]. The above - mentioned studies on reprocessed paraffin - embedded tissues were performed on newly cut sections from paraffin - embedded blocks, but not on already sliced paraffin sections attached to the glass . To our knowledge, a study about the possibility of observing hormone - containing endocrine cells reprocessed from paraffin - embedded tissues has not been documented . In the present study, we investigated the possibility not only of endocrine cells in the conventional paraffin section with pre - embedding immunocytochemical electron microscopic observation but also from newly sliced paraffin sections that were subjected to immunostaining . We observed secretory granules containing chicken glp-1 using prlsb and prcg techniques . At the electron microscopic level, the immunopositive secretory granules in prlsb and prcg could be recognized more easily than those in pocg under low magnification, owing to the precipitated dark products resulting from dab and gold particles enhanced by silver reactions . Incubations with antisera and other reagents were performed before the ultra - thin sectioning in prlsb and prcg . In addition, the incubations were performed after the ultra - thin sectioning in pocg . Immunoreaction sites were identified around the secretory granules in prlsb and prcg and on the content of the granules in pocg . Although the granules had the same shape among the three procedures, both the longest and shortest diameters of the secretory granules were significantly longer in prlsb than in both prcg and pocg . The electron density of the dab reaction product was similar to that of the secretory granules in prlsb, which made the boundary of the granules ambiguous . Baskin et al . Suggested that osmium - dab interactions resulted in background problems . Because oso4 is well known as a post - fixative solution, it was used in prlsb and prcg . We presumed that this use led to the result that the diameter of the measured granules tended to be longer in prlsb . Conversely, the gold particles enhanced by silver products mainly precipitated around secretory granules, and a few of them were located in the cytoplasm in prcg . Since the electron density of the silver - enhanced gold particles was much higher than that of the granules, the boundary between the granules and the immunogold labeling was clearly recognized under both low and high magnifications . It was presumed that this clarity provided similar evaluation of the granule diameters between prcg and pocg . Because of the difficulty in obtaining sagittally sectioned electron micrographs of l - cells that contained many granules, the measured numbers of granules differed among the three groups . This study is the first to identify the ultrastructure of secretory granules containing an amino acid peptide (glp-1) obtained from paraffin - embedded and sliced sections with similar evaluation results to pocg, which is a commonly used method for post - embedding immunocytochemical studies . The reprocessing method from thinly sliced paraffin sections in the present study has limitations compared with reprocessing from paraffin blocks . The reprocessing method from paraffin blocks, in which paraffin - embedded tissues are dewaxed in xylene, hydrated, postfixed, dehydrated and embedded in epoxy resin, shows fine preservation of nuclei and many cytoplasmic organelles [8, 13, 22, 24, 26]. We presumed that this resulted from removal of the coverslip and repeated hydration / dehydration procedures . However, prcg provided satisfactory information for the secretory granules containing the amino acid peptide hormone . The fixative solution used in pocg contained glutaraldehyde, which is commonly used for electron microscopic studies and preserves the fine structure of nuclei and many cytoplasmic organelles . We presumed that the fine preservation in pocg was also caused by glutaraldehyde . To the best of our knowledge, the present study is the first to report the advantages of the reprocessing method with sliced paraffin sections, which not only provides information on secretory granules containing an amino acid peptide, but is also inexpensive, does not require special devices and can effectively use precious samples that are already paraffin - embedded and unable to be obtained twice, such as the case for endangered animals and rare pathological tissues . In addition, this method may contribute to ethical ideas that have led to a required reduction in the number of laboratory animals used by researchers . 2, is a useful procedure from the following viewpoints: 1) the thinly sliced and hydrated sections allow penetration of antibodies; 2) the gold enhanced by silver provides fine information of immunoreactive cells at the light microscopic level; and 3) the investigator can observe the same cell under a light microscope and the electron microscope.
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Desmoplastic small round cell tumor (dsrct) is a highly aggressive, rare tumor of mesenchymal origin whose oncogenic effect is presumed to originate from the unique chromosomal translocation t(11;22)(p13:q12) [1, 2], leading to the fusion of the n - terminal domain of ewing's sarcoma gene ews, to the c - terminal domain of wilms' tumor suppressor gene, wt1, which is found in most but not all dsrcts . It tends to grow along a serosal lining, most commonly the peritoneal surface, but other primary sites have been described [47]. The typical histology includes variably sized clusters of small round or spindled cells lying within a desmoplastic, collagenous stroma . No large population data exists regarding the epidemiology and best treatment of this tumor due to its rarity . Studies have shown that aggressive and complete surgical resection is a major determinant in patient survival [4, 812]; however, since dsrct most commonly presents as a multicentric abdominal mass, complete upfront resection is not often possible . Dsrcts are chemosensitive, but often recur, necessitating multimodality therapy with radiotherapy, surgery, and/or high dose chemotherapy (hdct) with stem cell rescue . Seer registry of the national cancer institute (nci) is a well - reputed source of incidence, prevalence, and cancer survival statistics currently covering 28% of the united states population . Seer database, seer 17 regs research data + hurricane katrina impacted louisiana cases, was queried for all dsrct cases from 1973 to 2007 using the icd - o-3 = 8806/3 code . Cases of dsrct were selected based on the hist / behav (malignant) code 8806/3 . The frequency session was used to ascertain the primary site and treatment by radiation and/or surgical treatment . Radiation (external beam or method nos) was dichotomized as absent or present (at any time during therapy). Surgery was recorded in seer as no cancer directed surgery or surgery in combination with radiation . No data were available on outcome of patients with surgery alone and the extent of surgical resection ., stata se 13.0 (college station, tx), and graphpad prism software version 5.04 (la jolla, ca). The rate session of seerstat program 7.0.5 generated the overall gender- and ethnicity - based age - adjusted incidence rates standardized to the 2000 united states census population count . Age - adjusted incidence rates were calculated and compared between gender and ethnicity groups . Among cases with a known survival time, gender-, ethnicity-, and treatment - based survival curves were generated using the kaplan - meier method in graphpad prism software . Cox regression models were constructed to estimate the hazard ratio for the association of race and radiation therapy with survival at 5 years . For radiation therapy, two models were constructed and adjusted for age at diagnosis: radiation at any time and radiation following surgical resection . Hazard ratios (hr) are presented with a 95% confidence interval (ci). Statistical significance was defined as alpha less than 0.5 with two - sided alternative hypotheses . From the seer database, 192 cases (age 060 years) of dsrct were identified . The common primary site(s) of disease were the peritoneum or soft tissue of abdomen and pelvis . Less common primary sites included the ovary / fallopian tube (6 cases), orbit (1 case), cerebellum (1 case), and cerebral ventricle (1 case) (table 1). The age - adjusted incidence rate of dsrct was 0.3 cases / million, with a peak incidence of 0.74 cases / million in persons 2024 years of age (figure 1(a)). In blacks, the peak incidence was seen in the age range of 2529 years (figure 1(c)). Age - adjusted incidence rates for males and females were 0.4 and 0.1 cases / million, respectively (p <0.001) (figure 1(b)). Age - adjusted incidence rates for caucasians, african - americans, american indians, and asian pacific islanders were 0.2, 0.5, 0.3, and 0.1 cases / million, respectively (data not shown), with a statistically significant difference between caucasians and african - americans (p = 0.037) (figure 1(c)). There was no difference in 5-year survival between patients of 18 years and younger (30.9%) and over 18 years (34%). Compared to caucasian cases, death was 30% more likely among african - american cases (hr: 1.30; 95% ci: 0.86, 1.95) (figure 2(a)). After adjusting age at diagnosis, radiation therapy at any time was not associated with improved survival (hradj = 0.73; 95% ci 0.49, 1.11) (figure 2(b)). Radiation administered following surgical resection improved survival at 5 years (hrunadj: 0.49; 95% ci 0.30, 0.79) (figure 2(c)). The statistically significant association remained after adjusting for age at diagnosis (hradj 0.53; 95% ci 0.32, 0.87). This is the first seer database analysis evaluating both incidence and survival data among patients with dsrct . The majority of published data on dsrct are single institution studies analyzing small numbers of cases given heterogeneous treatment . The diagnostic process is done locally and information including immunohistochemistry or molecular confirmation of the diagnosis is not available . The analysis of a rare event such as dscrt must be analyzed from data that is gathered over a long time period and is subject to variability from different suppliers of data . One such limitation is the lack of information on dose or format of radiation therapy given . Also data on specific chemotherapy agents, doses, and schedules are not available in this data set . Summarization of end results is imperfect given these limitations; however, the data collected can be useful to provide insight and direct future investigations on patient outcomes . This study is consistent with previous reports in that drsct was found to be more prevalent in males [4, 6, 1012] and african - americans . Male to female ratio is 4: 1 and similar to previously reported [3, 15]. Although the survival differences between blacks and whites suggest that blacks are 33% more likely to die of disease progression at 5 years than whites, this trend did not reach statistical significance likely secondary to small sample size . Disparities in socioeconomic status and availability of medical care are risk factors in the ethnic minority populations which are known to result in worse outcomes in several disease types including pain management, depression, and cancer . Whether such differences influence outcome in dsrct or whether there are differences in biology of this tumor between various ethnic groups as previously described, patients who received radiation therapy did not show a statistically significant survival advantage compared to patients who had not received radiation therapy . There was, however, a statistically significant difference in survival for patients who received radiation after surgery compared to patients who received no radiation . The patients described in the literature with the longest survival tend to have received multimodality therapy including aggressive surgery, multiagent chemotherapy including hyperthermic peritoneal perfusion, and radiation therapy . This review of seer data supports inclusion of multimodality therapy in treatment for dsrct, with data revealing improvement in survival by 32% compared to those without radiation treatment . Overall survival however remains poor and the need for additional novel targeted therapies for this rare tumor is paramount.
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Literacy involves a complex set of abilities to understand and use symbolic systems of a culture for personal and social improvement, which are viewed as necessary for any grown up person in order to work and behave in the society . One of these abilities is health literacy, which involves a series of reading, listening, analysis, and decision - making skills and the ability to use them in health situations . This type of literacy is a result of cooperation between personal and social factors and deals with aspects and concerns of health domain . The capacity of an individual (including the inherent potential and interpersonal skills), is an essential part of health literacy, which can be adjusted with suitable training and its quality is affected but culture, language, and characteristics of health - related situations . This type of literacy is an essential skill for all individuals and since the lack of health literacy can have severe economic repercussions and has a close relation with the economic health . The world health organization (who) considers the health literacy as one of the most important factors in determining the health status of a sociality and urges the countries around the world to create a community for monitoring and coordination of strategic activities involving the promotion of health education . Since health literacy involves both learning the information regarding health issues and the ability to understand and use these information, there is a close relation between health literacy and quality of life . People with low health literacy will have trouble understanding and following the instructions provided by health experts, incur additional medical costs, have poorer health, higher rates of hospitalization, and use of emergency service and use less preventive care . Typically, lower health literacy causes repeated and unnecessary referrals to doctors and longer hospital stays, which in turn increases the medical costs and wastes a part of health budget . Therefore health literacy is an important factor in the outcomes and cost of healthcare and its lack of improvement causes longer use of medical facilities . Patients with lower health literacy have more problems in the use of medicines . In other words improvement of health literacy is a way to tackle the inequalities in healthcare . Given the importance of this issue, many national and international quantities studies have been conducted . Some of these studies are as follows: kohan et al . In a study called the relation between mother's health literacy and prenatal and delivery care claimed that 18% of women had good, 48% of them had mediocre, and 34% of them had poor health literacy . The results of a study by reisi et al . Titled the relationship between health literacy, health status and healthy behaviors among elderly in isfahan showed that the level of health literacy in the elderly was inadequate . The results of a study called evaluation of health literacy of pregnant women in urban health centers of shahid beheshti medical university by ghanbari et al . Showed that poor health literacy is a common problem in pregnant women that can cause misunderstanding in interpretation of health advices . Health literacy and use of health service in the city of kerman, 1390 claimed that the level of health literacy in city of kerman is poor . The results provided in a study by tol et al . Called determination of knowledge and health literacy among women with type 2 diabetes in teaching hospitals of tums also tol et al . In a study titled assessing the effect of educational program based on small group on promoting knowledge and health literacy among women with type 2 diabetes referring to selected hospitals affiliated to tehran university of medical sciences showed that in the intervention group small group teaching strategies are more effective compared with conventional diabetes education . Williams et al . In their study titled the role of health literacy in patients - physician communication showed that poor health literacy is especially common in elderly and sick people . He also claimed that the complex communication problems caused by this can affect the healthcare of these patients . The results provided by chew et al . In a study called brief questions to identify patients with inadequate health literacy showed that 4.5% of the participants had poor and 7.5% of them had marginal health literacy . The results of a study titled health literacy and anticoagulation - related outcomes among patients taking warfarin by fang et al . Showed that health literacy is related to lack of knowledge concerning warfarin . Health literacy, health status, and healthcare utilization of taiwanese adults: results from a national survey showed that almost 30% of the participants had poor health literacy . In a study titled health literacy in a population of primary healthcare patients in belgrade, serbia, jovic - veranes et al ., showed that the patients do not have the necessary skills to act in healthcare environments . Edwards et al . In a study called the development of health literacy in patients with long - term health condition: the health literacy pathway model the patients with long - term problems can improve their health literacy through training and consulting . The current study aims to investigate the health literacy level of patients admitted to the hospitals under the supervision of isfahan university of medical science in two domains of reading comprehension and numeracy . The data gathering tool is test of functional health literacy in adults (tofhla). This questionnaire was previously translated by the institution of researchers without border in tehran and has proven credibility . Cronbach's alpha was used to test the stability of the questionnaire and the number of participants was 60 . The stability was calculated to be 0.89% and the justifiability of the questionnaire was confirmed by related experts . The first section involves the demographic information and the second part is the numeracy part . This part contains 10 health explanation or advice about prescribed medicine, timing of visiting doctors, the stages of using financial assistances, and an example of the results of a medical examination . This section measures the participant's ability to understand and act according to the advice given by doctors and health educators that require numeracy . The third section includes three texts and measures the reading comprehension of participants of texts titled preparing for imaging of the upper gastrointestinal tract, rights and responsibilities of patients in insurance forms, and standard hospital consent form . Each of these sections has a mark from 0 to 50 and the total of these marks gives the total health literacy marks of the participant in a 0 - 100 scale . At the end the health literacy, marks of the participants is divided into three categories: poor (0 - 59), marginal (borderline, 60 - 74), and satisfactory (75 - 100). The studied society is the patients admitted into teaching hospitals under the supervision of isfahan university of medical science from september 2012 to march 2013 who are capable and willing to participate in this study and aged 20 - 65 years . These hospitals include ayatollah kashani hospital, al - zahra hospital, amin hospital, farabi hospital, feiz hospital, imam mousa kazem hospital, isabn - e - maryam hospital, noor and hazrat - e - ali asghar hospitals, shahid beheshti hospital, seyed - al - shohada hospital, shahid chamran hospital . For the sake of this study al - zahra hospital from south, feiz hospital from north, shahid chamran hospital from east, shahid beheshti hospital from west, and ayatollah kashani hospital from central part of isfahan were selected . Since during this study, some patients are repeatedly admitted and discharged, the statistical population is large and somewhat uncertain . Therefore the patient sampling was carried out based on large, unlimited population and the number of samples was calculated to be 384 using the following equation n = z*s / d . The sampling was done using convenient (accessible) method . Before the beginning of data gathering, the necessary correspondence and coordination were carried out with deputy director of research in the faculty of the researcher, directors of each hospital, the head of each department in the hospital, and hospital security staff and the necessary permits were obtained . Then the data in the two sections of numeracy and reading comprehension was gathered from each patient by personally visiting them and after gaining their consent . During the data gathering procedure, the patients could refuse further cooperation at any given time; in this case another patient would replace the previous one . Therefore all ethical guild lines concerning dealing with patients were followed in this study . For the descriptive data analysis, mean, standard deviation, and frequency distribution was used and statistical tests of independent t - test, anova, pearson's correlation, and spearman were used for deductive statistical analysis . Demographic dataamong the investigated patients, 44.5% were from al - zahra hospital, 13.5% from feiz hospital, 13% from shahid chamran hospital, 13.3% from shahid beheshti hospital, and 15.6% were from ayatollah kashani hospital.among the investigated patients, 45.6% of the investigated patients were female and 54.4% of them were male; 47.7% of the patients were in the age group of 20 - 30, 24.2% in the 31 - 40, 19% in the 41 - 50, and 9.1 of them were aged over 50 years . Also 26.3% of the patients had elementary school level education, 32.3% of them had high school diploma, 4.4% of them had associate degree, 10.4% of them had bachelor degree, and 1.2% of them had master's degree or higher . A total of 25.3% of the investigated patients were single and others were married.a total of 31.5% of the patients were self - employed, 10.4% of them were office workers, 4.7% of them were laborers, 9.4% of them were unemployed, 36.5% of them were housewives, 3.4% of them were retired, and 4.2% of them were students . Also 50.3% of them were without income, 46.1% of them had a monthly income of less than 10,000,000 rials, and 3.6% of them had a monthly income greater than 10,000,000 rials . Finally 47.7% of the patients were from the city of isfahan, 38.5% of them were from surrounding cities, and 13.8% of them were from other towns and provenances.patients health literacy markthe average health literacy mark of the patients was 31.35 in numeracy and 31.94 in reading comprehension section from a range of 0 - 50 for each section . The total health literacy mark of the patient is in range of 0 - 100 and in this study the average total health literacy mark of the patients were 63.29 . As mentioned earlier, health literacy is divided into three categories of poor, marginal (borderline), and satisfactory . According to this, 41.1% of the patients (158 patients) had poor health literacy, 25% of them (92 patients) had marginal and only 33.9% of them (130 patients) had satisfactory health literacy.the relation between patient health literacy mark and demographic factorsas seen in table 1 and independent t - test, there were no meaningful relation between the health literacy mark of the patients and their gender (p> 0.05). Among the investigated patients, 44.5% were from al - zahra hospital, 13.5% from feiz hospital, 13% from shahid chamran hospital, 13.3% from shahid beheshti hospital, and 15.6% were from ayatollah kashani hospital . Among the investigated patients, 45.6% of the investigated patients were female and 54.4% of them were male; 47.7% of the patients were in the age group of 20 - 30, 24.2% in the 31 - 40, 19% in the 41 - 50, and 9.1 of them were aged over 50 years . Also 26.3% of the patients had elementary school level education, 32.3% of them had high school diploma, 4.4% of them had associate degree, 10.4% of them had bachelor degree, and 1.2% of them had master's degree or higher . A total of 31.5% of the patients were self - employed, 10.4% of them were office workers, 4.7% of them were laborers, 9.4% of them were unemployed, 36.5% of them were housewives, 3.4% of them were retired, and 4.2% of them were students . Also 50.3% of them were without income, 46.1% of them had a monthly income of less than 10,000,000 rials, and 3.6% of them had a monthly income greater than 10,000,000 rials . Finally 47.7% of the patients were from the city of isfahan, 38.5% of them were from surrounding cities, and 13.8% of them were from other towns and provenances . Patients health literacy mark the average health literacy mark of the patients was 31.35 in numeracy and 31.94 in reading comprehension section from a range of 0 - 50 for each section . The total health literacy mark of the patient is in range of 0 - 100 and in this study the average total health literacy mark of the patients were 63.29 . As mentioned earlier, health literacy is divided into three categories of poor, marginal (borderline), and satisfactory . According to this, 41.1% of the patients (158 patients) had poor health literacy, 25% of them (92 patients) had marginal and only 33.9% of them (130 patients) had satisfactory health literacy . The relation between patient health literacy mark and demographic factors as seen in table 1 and independent t - test, there were no meaningful relation between the health literacy mark of the patients and their gender (p> 0.05). The average health literacy mark of the patients admitted in hospitals supervised by isfahan university of medical science divided based on gender and marital status also t - test showed that the average health literacy mark and the marital status of the patients had a meaningful relation (p <0.05) and that married patients had lower health literacy marks . Table 2 shows the relation between the health literacy mark of the patients with their age and monthly income . Pearson's correlation showed that the average health literacy mark and the marks of the different sections are reversely proportional to the age of the patients . However, no relation existed between average health literacy mark and monthly income of patients . Pearson's correlation between age and monthly income with average health literacy mark of patients admitted in hospitals supervised by isfahan university of medical science table 3 shows the relation between average health literacy mark of the patients and their education level . It can be seen that there is a direct relation between the health literacy mark of the patients and their education level (p <0.001) and the higher their education, the higher their health literacy mark will be . Spearman correlation between education level and health literacy mark of the patients admitted in hospitals supervised by isfahan university of medical science table 4 shows the relation between health literacy mark and employment and the place of residence of the patients . Anova test showed that there is a meaningful relation between the patients health literacy mark and their employment (p <0.001) and that the highest health literacy mark belonged to the students and the lowest marks belonged to retired patients and laborers . In contrast, no meaningful relation was observed between the place of residence and the health literacy mark of the patients (p <0.05). The average health literacy mark of the patients admitted in hospitals supervised by isfahan university of medical science divided based on employment and place of residence the findings of this study show that among the investigated patients, most had poor or marginal health literacy while only a few had satisfactory levels of health literacy . This is in agreement with most of the previous studies such as kohan et al ., tehrani banihashemi et al ., reisi et al ., ghanbari et al ., nekouei - moghadam et al . Inside iran and international studies conducted by williams et al ., chew et al ., shoou - yih lee et al ., and jovic - veranes . This is the case even though enough health literacy is necessary in order for the patient to participate in healthcare systems and for making proper health - related decisions and increases the ability of the populace in using health advices and information . The results also show that there is a meaningful relation between the education of the patient and the average health literacy ., nekouei - moghadam et al ., lee et al ., fang et al ., sun et al ., and the research findings of center for health care strategies for america . Patients with higher education have better health literacy and better understand and use health information and advices . However, patients with lower education also have lower health literacy and have problems in understanding and using medical information, application, and methods of using medicines and understanding the orders of doctors and therefore need special attention . Findings showed that health literacy is reversely proportional with the age of the patients and younger patients have higher health literacy ., tol et al ., lee et al ., fang et al ., sun et al ., and findings of center for health care strategies for america . Younger patients have fewer problems in understanding and comprehension of medical and health information and as was mentioned previously, have more concentration compared to the elderly . It is necessary to note that age, education and marital status affect each other . Therefore healthcare officials must endeavor to develop ways of provide the elderly with easy to understand health and medical information . One of the other findings of this study is a meaningful relation between health literacy and marital status and occupation of the patients, which is similar to the findings by tol et al . This difference can be due to the effect of age and education of the patients . In other words, most single individuals are young and have higher education compared with married people . Also usually younger people have better concentration and attention compared with older ones, which can help them to answer the questionnaire more accurately . Therefore, according to the current study, in health - related situations, single individuals act better and more effectively compared with married people . Another reason behind this difference can be the fact that married people have more responsibilities in taking care of their family and children and the problems of everyday life and therefore spend less time learning about correct information and actions and this lack of attention is also true for health - related situations . As a result married people have less attention in filling medical forms, following medical instructions and in using other information regarding healthcare situations . About the difference in health literacy level of different occupations, one can say that the difference originates from the difference in age and education of people . Therefore, the results show that the best health literacy level belonged to office workers and students while laborers and retired people had the lowest health literacy level . In other words, compared with younger, single people of higher occupation levels, married and elderly people, laborers, and people of lower education need more training, information provided in simple and easily understandable terms and spending more time for establishing health - related communications and also require more aid about medical systems and information such as how to use drugs . Furthermore, the results show that there is no meaningful relation between health literacy and gender and place of residence of the patients . Shows that women and rural people have lower health literacy, which points to a relation between place of residence and health literacy and is the opposite of the findings in this study . The reason can be that in this study, the place of residence was not clearly divided between rural and urban areas but instead were divided by the city of isfahan, its suburban area, and other cities or rural areas . Therefore the investigated patients were from different cities and not rural areas and had no marked difference in their health literacy . In the previous studies like the one by tehrani banihashemi et al ., the place of residence differentiated between urban and rural areas and it is expected for the health literacy of rural people to be lower than that of urban residents . Also points out that the reason behind the lack of health literacy in women and in rural people is their lower education level . The results also show that the monthly income and education of the participants are not meaningfully related, which is in agreement with the findings of nekouei - moghadam et al . And is the opposite of the results reported by lee et al . And sun et al . And center for health care strategies for america . For the reason behind these results can be due to the fact that the income is divided into three categories of no income, lower than 10,000,000 rials, and higher than 10,000,000 rials . However, students that have the highest education and health literacy, belong to the no income category but the laborers that have lower education and health literacy have some in come mostly in the lower than 10,000,000 category . The no income category in this study represents house wives, students, jobless young people, and the people that are unable to work for whatever reason . The results of the current study showed that most of the patients have poor or marginal health literacy and therefore need more explanation from medical staff in order to understand and use the health and medical instructions . They also need to spend more time to communicate with their doctor or nurse and other medical staff, and learn the needed information in a simpler and easier to understand language . The officials and medical staff also need to understand this fact and spend more time communicating and explaining medical and health instructions to these patients . Since based on the results of this study, patients education directly affects their health literacy, it is necessary to pay great attention to education, specially the health education of the sociality as a whole . Also since based on the results, the health literacy of the patients had no relation to their gender, officials must tend to the education of both genders equally . Creating useful health programs, producing simple and easy to understand educational materials and encouraging doctors and medical staff to spending more time and talk slower when communicating with patients are some of the ways of helping patients with lower health literacy and increasing their knowledge . The results of this study also highlight the necessity of increasing the medical knowledge of people in general and admitted patients specially . Suggesting ways of increasing the health literacy of patients to the related officials, identifying the patients with lower health literacy, and conducting this type of research among admitted patients, which had not been attempted before, are some of the strong points of this study . There have been a total of seven studies regarding health literacy, three of which were conducted at the same time as this study . It is necessary to note that due to the importance of health literacy, there are various studies in this area internationally but health literacy still needs a lot of work in iran . It is necessary for the officials of healthcare areas to provide facilities in order to increase the health literacy of people that have unsatisfactory health literacy levelsit is necessary to devise suitable learning programs for married and elderly people, laborers, and those of lower educational backgroundsit is advised for those responsible for healthcare (doctors, nurses, medical librarians, etc .) To distribute leaflets containing elementary medical and healthcare information to people and patientsit is necessary to train healthcare experts and medical staff about the importance of communication with admitted patients and to teach them to spend more time and effort explaining medical procedures to the patients with lower educational backgrounds . It is necessary for the officials of healthcare areas to provide facilities in order to increase the health literacy of people that have unsatisfactory health literacy levels it is necessary to devise suitable learning programs for married and elderly people, laborers, and those of lower educational backgrounds it is advised for those responsible for healthcare (doctors, nurses, medical librarians, etc .) To distribute leaflets containing elementary medical and healthcare information to people and patients it is necessary to train healthcare experts and medical staff about the importance of communication with admitted patients and to teach them to spend more time and effort explaining medical procedures to the patients with lower educational backgrounds . It is necessary for the officials of healthcare areas to provide facilities in order to increase the health literacy of people that have unsatisfactory health literacy levelsit is necessary to devise suitable learning programs for married and elderly people, laborers, and those of lower educational backgroundsit is advised for those responsible for healthcare (doctors, nurses, medical librarians, etc .) To distribute leaflets containing elementary medical and healthcare information to people and patientsit is necessary to train healthcare experts and medical staff about the importance of communication with admitted patients and to teach them to spend more time and effort explaining medical procedures to the patients with lower educational backgrounds . It is necessary for the officials of healthcare areas to provide facilities in order to increase the health literacy of people that have unsatisfactory health literacy levels it is necessary to devise suitable learning programs for married and elderly people, laborers, and those of lower educational backgrounds it is advised for those responsible for healthcare (doctors, nurses, medical librarians, etc .) To distribute leaflets containing elementary medical and healthcare information to people and patients it is necessary to train healthcare experts and medical staff about the importance of communication with admitted patients and to teach them to spend more time and effort explaining medical procedures to the patients with lower educational backgrounds.
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Alternative rna splicing, export and translational regulation all contribute to cancer formation and progression (venables, 2006). These are dependent on the complex secondary structure of the 3 utr of the mrna recognised by binding proteins such as t - cell intracellular antigen-1 (tia-1) (eberhardt et al ., tia-1 contains three domains with rna recognition motifs (rrm1, rrm2 and rrm3), the latter two of which have been shown to be important for rna binding and selectivity (bauer et al ., 2012) (cruz - gallardo et al ., 2014). It has been shown to be alternatively spliced in exon 5 to form two isoforms (a and b, supplementary figure 1a) with b being the most active in its splicing activity, but tia-1 also acts as a stress induced translational inhibitor, localising to stress granules with polya rna (izquierdo and valcarcel, 2007). Tia-1 alters both co - transcriptional and post - transcriptional rna processing and binds vegf - a rna (suswam et al ., 2005), the alternative splicing of which influences the angiogenic capability of colorectal carcinomas (crc) (ferrara et al ., 1991). Vegf - a exists as multiple alternatively spliced isoforms . Exons 6 and 7 endow heparin binding (houck et al ., 1991), whereas exon 8 determines angiogenicity (harper and bates, 2008). Exon 8 has two splice acceptor sites, a proximal one, and a distal one, 66 bases downstream . The stop codon for the two isoforms lies the same number of bases downstream from the respective splice sites, resulting in two families of isoforms of the same amino acid number but with differing six amino acid sequence at the end, which alter the angiogenicity of the protein . Proximal splice site (pss) selection in exon 8 results in the angiogenic family (vegf - axxx where xxx is the number of amino acid e.g. Vegf - a165), but distal splicing (dss) results in vegf - axxxb, anti - angiogenic proteins of the same size (e.g. Vegf - a165b) (bates et al ., 2002). A variable but specific upregulation of the angiogenic isoforms is seen in colorectal cancer (varey et al ., 2008), but although the mechanisms underlying this splice specificity are being elucidated in epithelial cells (amin et al ., 2011; nowak et al ., 2008) and lung cancer (merdzhanova et al ., 2010), isoform specific control of expression, particularly in colon cancer is still not known . Hek293, colonic carcinoma cell lines ht29, hct116, hac7, sw480, osteosarcoma saos-2 and u2os, the cervical cancer cell line, hela, prostate cancer cells, pc3 and lncap (atcc; rockville, md) ls174 t (ecacc, salisbury, uk), adenoma cell lines aa / c1 and rg / c2, the in vitro transformed adenoma cell line aa / c1/sb/10, referred to as 10c cells) and breast cancer mda - mb231 (caliper, perkinelmer, usa), human haemangioma endothelial cells (esc and stem cells (hsc, a kind gift from joyce bischoff at harvard university), myeloma rpmi-8226, and cardiac myocytes (hcm, from promocell) were maintained as described previously (williams et al ., 1990) or according to manufacturer's instructions cancer cell lines were validated by str profiling (identicell, aarhus, denmark). An initial study was undertaken on six frozen paired colon samples (normal and cancerous) from partial colon resection . Additional rna was extracted from 40 formalin fixed, paraffin embedded (ffpe) samples of normal and tumour tissues . These were taken from 22 male, 18 female patients, mean age 69 years, 28 with colon carcinoma, 18 with rectal carcinoma and 1 with carcinoma of the rectosigmoid junction . An antibody to stia-1 was generated by abgent europe using a klh - conjugated vslkngq ncpg peptide . For access cell experiments used at least 3 independent biological replicates (independent experiments), with each western blot, elisa and rt - pcr carried out on independently replicated samples . For western blot, and rt - pcr where gels are shown, each is indicative of a representative of 3 experiments, and quantitated as per the figures . Protein samples from homogenised tissue (100 g) and whole cell extract (50 g) were prepared using standard protocols . Protein lysates were resolved on sds page, and probed with a mouse monoclonal anti - cox-2 antibody (sc-19999; santa cruz), a / b - tubulin (sigma) anti - vegf - a165b igg (a56/1; r&d systems), rabbit polyclonal anti - tia-1 antibody (sc-28237; santa cruz, detects full length and stia-1 isoforms), or sc-166246 c10 mouse monoclonal n - terminal; santa cruz and rabbit anti - vegf - a igg (a-20, sc-120; santa cruz), goat polyclonal laminin (santa cruz) and mouse monoclonal lamin (cell signalling) antibodies . Proteins were detected using chemiluminescence and analysed by nih image, or by fluorescently labelled secondary antibody imaging and imaged using a licor odyssey, and quantified using licor imaging software . Elisas were carried out as described previously (varey et al ., 2008). The vegf - a165b elisa uses an antibody that can detect all vegf - axxxb isoforms but no vegf - axxx isoforms, but its affinity to all of the different vegf - axxxb isoforms is not known . We have assumed that it is the same as vegf - a165b and therefore calculated the levels making this assumption . For immunoprecipitation, 50 l of protein a or protein g (depending on capture antibody) magnetic beads (millipore) was washed with 500 l pbs containing 0.1% tween 20 . After removing the buffer, the beads were resuspended in 100 l of pbs/0.1% tween, capture antibody added to the beads and incubated at room temperature for 10 min before adding the protein samples . The protein samples and antibody were incubated at 28 c overnight with continuous mixing, washed with pbs / tween0.1% before adding sample buffer . For spectrometry, after running the samples on page, the gel was fixed in methanol: acetic acid (50%:7%) for 30 min and stained with sypro ruby (molecular probes) overnight . The gel was de - stained in methanol: acetic acid (10%:7%), washed and imaged . The band was excised and subjected to in - gel tryptic digestion using a progest automated digestion unit (digilab uk). The resulting peptides were fractionated using a dionex ultimate 3000 nanohplc system in line with an ltq - orbitrap velos mass spectrometer (thermo scientific) as described in the supplementary material . Raw data files were processed and quantified using proteome discoverer software v1.2 (thermo scientific) and searched against the swissprot human database using the mascot search engine (matrix science) with parameters as described in the supplementary material . Total rnas from tissue (200 mg) and cells (10) was extracted using the rneasy kit (qiagen) and quantified . Rna was extracted from 25 m thick sections of the ffpe samples by rneasy ffpe kit (qiagen) according to the manufacturer's protocol . Standard rt - pcr protocols were used . For cdna synthesis form ffpe samples random primer or gene specific primers were used . Extracted dna was cloned into the p - gem t - easy plasmid (promega) and sequenced using standard synthetic primers complementary to the vector dna at the junction of the cloning site (t7, sp6) and a ptarget sequencing primer 5-ttacgccaagttatttaggtgaca-3 by the dundee sequencing service (dundee university). 2002). In brief, 5 10 cells were harvested, cross linked with 1%formaldehyde at room temperature for 10 min and quenched with 0.25 m glycine . Protein was extracted, and 100 g of pre - cleared protein incubated with 3 g tia-1 antibody immobilized using seize primary immunoprecipitation kit (pierce biotechnology) and immunoprecipitates isolated by centrifugation . Total rna was extracted from immunoprecipitates and subjected to rt - pcr as described . The vegf - a sequence from 35 bp upstream of exon 8a to 35 bp downstream of exon 8b was inserted into the adml - ms2 plasmid as previously described (nowak et al ., 2008). Nuclear protein (75 g) from hek 293 or aa / c1/sb/10 (10c) cells were prepared and added to the ms2-mbp fusion protein / vegf - rna mix in 0.5 mm atp, 6.4 mm mgcl2, 20 mm creatine phosphate for 1 h at 30 c . Proteins bound to the ms2-mbp / vegf - ms2 rna complex were affinity selected on amylose beads and immunoblot analysis was performed on 30 g of protein samples using tia-1 antibody and -tubulin antibodies as previously described (nowak et al ., 2008). The cdna for fltia-1was generated by rt - pcr amplification using pfu turbo dna polymerase (stratagene) and primer tia-1-forward and tia-1-reverse from rna from the rg / c2 adenoma cell line . The 1239 bp fragment was cloned in p - gem t - easy vector and sequenced in both directions . The cdna for both variants (13-exon tia-1ncbi nm_022173.2 and 12-exon tia-1ncbi nm_022037.2) were obtained using these primers . For in vitro expression, the cdna were subcloned into mammalian expression vectors, ptarget (promega) and pcdna3 according to the manufacturer's instructions . Two custom sirnas targeting stia-1 (sirna1: cagaattgccctggctaacta and sirna2: gccctggctaa ctacaagcta) and control sirna (lopez de silanes et al ., 2005) were assessed . Two colon cancer cell lines were transfected with mixture of the 2 sirnas (50 nm) using hiperfect transfection reagents (qiagen). Transfection: ls174 t and 10c cells were electroporated with 2 g cdna of ptarget - fltia-1 cdna in ptarget and pmaxgfp vectors ptarget vector or pmaxgfp with an amaxa nucleofector system using a basic epithelial cell transfection kit (vpi-1005, amaxa gmbh). Transfectants were analysed 24 h post nucleofection by trypan blue staining and fluorescence microscopy for gfp control plasmid . Transfected cells were selected using g418 (500 g / ml) for 4 weeks . The cells were 4% formaldehyde fixed (15 min) and incubated in 1%bsa/10% normal goat serum in tbs - tween (0.1%) for 1 h. the cells were then incubated with the antibody (ab40693 rabbit polyclonal c - terminal, or sc-166246 c10 mouse monoclonal n - terminal, 5 g / ml) overnight at + 4 c . The secondary antibody (red) was alexa fluor donkey anti - rabbit and (green) alexa fluor 488, used at a 1/500 dilution for 1 h. vector shield hoescht was used to stain the cell nuclei (blue). For confocal imaging cells were imaged using a 60 oil immersion objective with a numerical aperture (na) of 1.45 cell clumps were selected at random and a z stack of 25 images taken for each clump of cells . The central slice of each of these stacks was selected and used for the analysis of the ratio of staining between the nucleus (hoescht) and cytoplasmic regions of the cell . In nih image, the tia-1 positive area and the hoescht positive area were determined by tracing and nuclear area subtracted from the tia-1 positive area and expressed as a proportion of the tia-1 positive area . The vegf - a full length 3utr was cloned downstream of the thymidine kinase promoter and the firefly luciferase orf (construct luc - vegf165), (from jean - jacques feige and nadia cherradi, cea grenoble). This was modified using the quick - change ii xl site - directed mutagenesis kit (stratagene) to delete the first 66 nucleotides (luc - vegf - a165b) of exon 8 . The plasmid prl - tk (promega) encoding renilla luciferase was used as an internal control . Luciferase activities were measured with the dual - luciferase reporter assay system (promega) on a victor 3 1420 multilabel counter luminometer (perkinelmer). Vegf - a mrna decay was measured in 10c cells after transfection with tia-1 and treatment with 10 mol / l actinomycin d. cells were harvested every hour over a 4-h period, and vegf - a isoform mrna levels analysed by rt - pcr . Experiments were undertaken under home office license after review by the university of bristol ethical review group . 2 10 untransfected, fltia-1, vegf - a165, doubly or vector transfected cells in 0.2 ml pbs were injected subcutaneously in the dorsum of 6 balb / c nude mice (from university of bristol colony) for each group . Mice were treated bi weekly with mouse igg (sigma), or an anti - mouse vegf - a neutralising antibody (liang et al . Sample size was calculated a priori using g - power on the basis of being able to have 80% power to detect a mean sd 2 0.5 fold change in size and vascular density . 5 m thick de - waxed, re - hydrated sections were microwaved in 0.01 m sodium citrate, ph 6.0 for 15 min at 800 w. sections were incubated with vegf - a receptor 2 (rabbit mab 2479 cell signalling) or cd31 antibodies at 1:100 overnight at 4 c in a humidity chamber and after application of secondary antibodies and appropriate enzymatic process (vector dab kit), vessel number counted in 10 randomly selected areas for each tumour (n = 6). Results were analysed using prism software, given as mean sem unless otherwise stated and compared using one - way anova or t tests for comparison of means . P <0.05 was considered significant . Power analysis on non - significant results were carried out to ensure power of at least 80% (using g - power). In five carcinoma (ls174 t, hct116, sw480, ht29, hca7) and an adenocarcinoma line (10c) tia-1 rna, but not two other rna binding proteins, ttp and hur, was lower than in adenoma lines rg / c2, and aa / c1 (figure s1a, b), amplified using primers to detect full length (fltia-1). Expression of the 4243 kda tia-1 protein was similar across cell lines, but in all carcinoma and the 10c cells, an additional band at 16 kda was seen (figure 1a, figure s1c) suggesting the presence of a truncated tia-1 protein . Amplification of tia-1 mrna sequence from the 10c cells using primers that flank exons 6 and 7 revealed an additional product only in carcinoma and 10c cells (figure 1b, figure s1d), due to the insertion of a 66bp sequence (figure 1c) from the intron between exons 6 and 7 of the tia-1 gene (figure 1e). This was also present in braf v600e + ve a375 melanoma cells (figure s1e). The 66base sequence is flanked by a classical consensus splice sequence (ag gu) and has a 73% cu sequence immediately upstream, suggesting a cassette exon undergoing alternative splicing . This sequence results in a unique 11 amino acid sequence and a premature stop codon, which should generate a 144 amino acid truncated tia-1 peptide of 15.8 kda (figure 1d), with only rna recognition motif (rrm)-1 and part of the rrm 2, consistent with the short band (16 kda) detected by the anti - tia-1 antibody in figure 1a . Sequencing of tia-1 genomic dna from 10c cells showed no mutations in the region around either the 5 or 3 acceptor or donor sites . This mrna is identical to a splicing event described as exon 6a when tia-1 itself was over - expressed in a cancer cell line (le guiner et al ., 2001), but its endogenous expression has not previously been demonstrated . A blast search of this sequence showed identity in 66 ests, all consistent with it being this tia-1 isoform . A rabbit antibody raised to the unique peptide sequence of stia-1 detected the protein in 10c cells (figure s1f). Immunoprecipitation of 10c protein (figure s1 g) and immunoblotting for tia-1 showed both short and full length tia-1 . Ip with a fltia-1 antibody and ib with stia-1 demonstrated that the protein detected with this antibody was tia-1 in ls174 t cells as well . (figure s1h). Both protein (figure 1e top image) and rna expression (figure 1e bottom image) of stia-1 was seen in sarcoma (saos), hela, prostate cancer (pc3 and lncap), and mda - mb-231 breast cancer cells, but not osteosarcoma (u2os), haemangioma endothelial (a differentiated phenotype), haemangioma stem (hsc), human embryonic kidney (hek), or rpmi-8226 leukaemia cells or human cardiac myocytes (hcm). Of the 18 cell lines in which stia-1 was investigated, only 2 of the 8 that lacked stia-1 have ras mutations or over - expression, whereas 8 of the 10 that did express stia-1 had ras over - expression or activating mutations, and the other two are braf mutants, (ht29 and a375 cells). In both ls174 t and 10c cells the ras inhibitor farnesyl thiosalysilic acid (fta) significantly inhibited splicing to stia-1 in a dose dependent manner (figure 1f). Downstream signalling through mek and pi3k was required for this splicing in both cell lines (figure s2a). Ras is active in aac1 and rgc2 cell lines (stia-1 negative) indicating that it is necessary, but not sufficient to induce splicing of tia-1 . Ras inhibition (figure 2a, figure s2b d) and mek and pi3k inhibition (figure s2e) also switched splice form expression from vegf - a165a to vegf - a165b in both 10c and ls174 t cell types . Vegf - a165b protein expression was confirmed by immunoprecipitation and peptide sequencing (figure s3a and b). These results suggest that hyperactivated ras signalling also acts to repress vegf - axxxb splicing, so we investigated a possible link between vegf and tia-1 . Isoform specific sirna knockdown of stia-1 (figure s3c g), reduced vegf - a165a mrna in both 10c and ls174 t and increased vegf - a165b mrna in 10c cells but made no difference in aa / c1 cells (figure 2b). Vegf - a165b but not vegf - a165 protein was significantly increased by sirna to stia-1 in ls174 t cells (p = 0.013), whereas only a significant reduction in the vegf - a165a isoform expression was seen in 10c cells (p = 0.03, figure 2c). Thus there was a significant switch in the ratio of isoforms in both cell types towards a less angiogenic phenotype after knockdown of stia-1 (figure 2d, p <0.01 anova). Conversely, vegf - a165b expression was rescued by fltia-1 expression in 10c and ls174 t cells measured by rt - pcr (figure 3e), immunoblot (figure 2f) and elisa (figure 2 g), in 10c cells to 90% vegf - a165b (p <0.001 compared with ctrl), similar to aa / c1 cells and in ls174 t from 5 1.8% to 12 1.5% of total vegf - a (p <0.01). The reverse experiment (over - expression of stia-1) resulted in lethality of unknown cause . Fltia-1 also reduced cyclooxygenase-2 protein expression (figure s3h), a known tia-1 target . Rna immunoprecipitation (rip) of tia-1 showed that, whereas 10c, aa / c1 and rg / c1 all expressed both vegf - a165 and vegf - a165b, immunoprecipitation with an anti - tia-1 antibody pulled down only vegf - a165 in aa / c1 and rg / c1 (cells expressing only fltia-1), but no vegf - a mrna in stia-1 expressing cell lines (10c, hca7 and ls174 t, figure 2h). In 10c and ls174 t cells, transfection with fltia-1 restored tia-1 binding to vegf - a165a mrna, as evidenced by pull down of vegf - a165 mrna, compared with untransfected cells (ctrl, figure 2i). The binding site of tia-1 to vegf - a mrna was demonstrated, in hek cell nuclear extract using an ms2-mbp binding assay (nowak et al ., 2008) to reside in the vegf - a exon 8a sequence and 35 bases of intron 7 (figure 2j). Nuclear extract from 10c cells, while containing tia-1 (as seen in the flow through unbound eluate in the lower panel), was unable to bind to vegf - a mrna sequences (figure 2j). Fltia-1 has been shown to function both as a translational repressor (piecyk et al ., 2000) and an alternative splicing factor (barron and lou, 2012; del gatto - konczak et al ., 2000). Expression of fltia-1 did not affect the half - lives of vegf - a165b and vegf - a165 mrna in 10c cells, determined by investigating mrna decay rates in cells treated with the transcriptional inhibitor actinomycin d (actd) (figure 3a and b). Although it did increase the amount of vegf - a165b at the start of the experiment, figure 3c shows that there was no difference in the half - life . There was no significant difference in expression of a luciferase reporter containing the 3utrs of vegf - a165a or vegf - a165b (figure 3d) in either 10c or ls174 t cells (figure 3e), but transfection with fltia-1 resulted in significantly increased luciferase activity with the vegf - a165b 3utr compared with the vegf - a165a 3utr (figure 3f) indicating preferential translation of vegf - a165b by tia-1 as well as inhibiting splicing of vegf - a165 . Rrm 2 and 3 are required for 3 translational repression (bauer et al ., 2012), and this is lacking in stia-1, which only contains the full length rrm1, which cannot by itself bind to rna (dember et al ., 1996) but is required for fltia-1 binding to specific sequences (e.g. U rich sequences) (bauer et al ., 2012), thus conferring specificity . Thus, while stia-1 prevents fltia-1 mediated repression of splicing of the pro - angiogenic isoforms of vegf - a, the increased level of vegf - a165b mrna and protein induced by over - expression of fltia-1 in colon cells suggest that it also acts to selectively represses translation of the exon 8a containing isoforms . This may explain why the effects of stia-1 sirna knockdown on rna and protein were subtly different in the two cell types (in 10c it increased vegf - a165b rna but not protein, and vice versa in ls174 t cells). Nuclear / cytoplasmic separation of proteins (figure s4a) showed that in aac1 cells, tia-1 is found as the full length form but only in the nucleus, whereas in ls174 t and 10c cells a substantial proportion of all tia-1 isoforms and products were expressed in the cytoplasm, including stia-1 (figure 3 g). This was confirmed by transfection of cells with full length tia-1 fused to gfp (figure 3h), and by immunofluorescent staining with antibodies to the n terminus of tia-1 (detects all isoforms, figure s4b). Stia-1 knockdown, surprisingly, resulted in enhanced cytoplasmic staining for tia-1 (figure 3i). This was confirmed in fltia-1-gfp over - expressing 10c cells, with stia-1 knockdown (figure s4c). N - terminal staining (which would detect stia-1) was reduced, consistent with a reduction in only the stia-1 component (figure s4c). Nuclear localisation of gfp - fltia-1 was reduced by stia-1 knockdown (figure 3j). Stia-1 therefore alters localisation of the fltia-1 within the cell, thereby preventing it from binding vegf - a mrna . The increased not decreased cytoplasmic tia-1 resulting from reduced of stia-1 in k - ras mutant cells suggests that the gfp - fltia-1 fusion protein localisation is stia-1 dependent . Thus stia-1 expression in k - ras mutant cells may act by preventing fltia-1 nuclear trafficking (to and from the nucleus), preventing it from interacting with other potential regulators of splicing and/or translation . This in turn alters the expression of vegf - a isoforms and results in a more angiogenic phenotype of the tumours . Co - immunoprecipitation indicated that it is possible that the two isoforms could complex together (figure s1 the localisation experiments indicate that in carcinoma cells fltia-1 excess either by over - expression with gfp, by knockdown of stia-1 or both results in cytoplasmic fltia-1 and/or vegf - a165b expression, whereas in adenoma cells, where k - ras is not over - expressed, fltia-1 is nuclear . Stia-1 and vegf - a165b rna were detected in freshly isolated human colorectal cancer samples (figure 4a) confirmed by sequencing . Quantification showed both a significant increase in stia-1 expression in cancer (figure 4b) and a significantly lower vegf - a165b: vegf - a165 ratio in the tumours than the controls (figure 4c). In histological samples, fltia-1 rna (figure 4d) was found only in 13 of 31 tumours (42%) but 22 of 30 normal tissues (73%, p = 0.02, fisher's exact test) samples (figure 4e). The proportion of tumours that were k - ras mutant was significantly higher (p <0.01) in those that had no fltia-1, and k - ras mutant tumours rarely (2/13 cases) had fltia-1 detected (figure 4f). Quantification showed that stia-1 exceeded fltia-1 in 20 of 31 tumours (65%) but in only 9 of 30 normal tissues (30%, p = 0.01, figure 4 g). Moreover, stia-1 was present in 14 of the 15 dukes' c (93%), but only 9 of the 16 dukes' b (56%, p <0.01, figure 4h). There was a significantly higher microvascular density (figure 4i) in samples in which stia-1 but not fltia-1 was found (figure 4j). Over - expression of fltia-1 inhibited tumour growth in nude mice (figure 5a), resulting in significantly smaller tumours at 18 and 21 days after injection (figure 5b). This reduction was rescued by vegf - a165 cdna expression i.e. Vegf - a that was not under the control of alternative splicing (figure 5b). Tumours from fltia-1 transfected cells had reduced tumour vessel density (figure 5c and d, p <0.01). Mice with ls174 t tumours expressing fltia-1 treated with an anti - vegf - a antibody (liang et al ., 2006) grew significantly faster compared with igg (figure 5e), at the same rate as wild type tumours treated with anti - vegf - a treatment (figure 5f), but still more slowly than untreated wild type tumours (figure 5 g). Here we identify a novel k - ras dependent endogenous splice variant of tia-1, short tia-1 (stia-1) in crc patients and cancer cells, which controls vegf - a isoform expression . This stia-1 had been identified as being capable of being induced by over - expression of recombinant mouse tia-1 in human hek cells (le guiner et al ., 2001). This is the first demonstration that this splice variant may be present in human cancers, and that it may regulate vegf splice form expression . Stia-1, containing only one rna recognition motif (rrm), prevented binding of full length tia-1 to vegf - a165 mrna, blocking both its angiogenic vegf splice site repression and its promotion of translation of anti - angiogenic vegf - a165b . It has previously been shown that rrm2 and 3 are required for binding of tia-1 to rna (bauer et al ., 2012), and interestingly, rrm3 is required for binding to c rich stretches (cruz - gallardo et al ., 2014). Moreover, inclusion of exon 5 in tia-1 results in a higher splice activity, indicating that the activity of the protein can be altered by alternative splicing (izquierdo and valcarcel, 2007). As both of these domains are disrupted in stia, it is likely that stia-1 does not act by directly binding to the vegf rna and preventing fltia-1 from exerting its effects, and the direct mechanism of repression of distal splicing is not known . As stia-1 did co - immunoprecipitate with fltia-1 it is possible that stia-1 acts by heterodimerising with fltia-1 and preventing it from repressing splicing and/or translation of the pro - angiogenic isoform, vegf - a165a . Cancer cells over - expressing fltia-1 formed smaller, less vascular tumours than those expressing stia-1, and were more resistant to anti - vegf antibodies such as bevacizumab . These results indicate that k - ras mediated splicing of an rna binding protein may regulate isoform specific expression of vegf, providing an added layer of complexity to the angiogenic profile of colorectal cancer and their resistance to anti - angiogenic therapy . This opens up the question as to what is the downstream mechanism through which k - ras regulates tia-1 splicing . There is no clear evidence as to how k - ras activation can induce splicing switches . Splicing is regulated by the complex interaction of splicing factor proteins that can be phosphorylated, localise to the nucleus and take part in a complicated interaction with other rna binding proteins and auxillary factors that control splice site location . The mechanisms through which tia-1 is spliced is not known, and none of the known splicing factors have yet been identified as controlling exon 6a splicing . Interestingly, over - expression of human fltia-1 did not induce stia-1 splicing in cancer cells, in contrast to that shown by mouse flyia-1 in human hek cells (le guiner et al ., 2001). The finding that over - expression of vegf - a165 can reverse the effect of fltia-1 expression on tumour growth inhibition suggests that the effect was vegf splicing dependent, through anti - angiogenic activity, or through direct effects on the cells low vegf - a165b levels predict bevacizumab response in crc (bates et al ., 2012), but, bevacizumab provides significant clinical benefit in patients irrespective of k - ras mutations (hurwitz et al ., 2009). Although patients with k - ras mutations did do better on bevacizumab compared with controls (9.3 compared with 5.5 months, an increase of 3.8 months), they did not perform as well as patients with wild type k - ras (13.5 versus 7.4, an increase of 6.1 months). It would therefore be interesting to know how patients with k - ras mutations, in which stia-1 is expressed, responded . It is not yet clear whether stia-1 predicts survival, or can drive angiogenesis by reducing the anti - angiogenic vegf - a165b splice variants, or by increasing the vegf - a165 variant . These vegf - a165b variants have been shown to be down regulated so far in melanoma (pritchard - jones et al ., 2007), colon (varey et al ., 2008), prostate (woolard et al ., 2004), and kidney (bates et al ., 2002) cancers, but the mechanism of regulation of these variants in cancer is still poorly described (amin et al ., 2011; nowak et al ., 2010, 2008). The loose relationship between stia-1 and vegf - a165b down regulation in patients suggests a complex interaction, just as for alternative splicing of p73 and vegf - a165b isoforms, also demonstrated in colon cancer (diaz et al ., 2008). This is supported by the findings that ras inhibition in 10c cells was more effective at lower doses than in ls174 t cells, and that the rna and protein levels of vegf - a165b in the presence or absence of shtia-1 are not directly correlated . The effect of tia-1 on rna stability, as shown in figure 3 indicates that tia-1 is likely to act in a pleiotropic manner on the amount of vegf isoforms present acting by altering translation specifically for one set of isoforms, by regulating splicing at the proximal splice site, or, most likely, by changing both splicing and translation . Binding of tia-1 to the vegf rna was shown, but it is also possible that other vascular growth factors are regulated by tia-1, although these have not yet been published . In summary, we show that alternative splicing of tia-1 in human colon cancer may regulate the balance of the pro- and anti - angiogenic vegf - a isoforms, and control tumour growth and angiogenesis in animal models . This suggests that tia-1 can act as a splicing and translational regulator of vegf - a and alternative splicing of tia-1 may lead to enhanced angiogenesis and tumour growth in colorectal cancer, and resistance to anti - vegf - a therapy . Mhz, ema, cha designed and carried out experimental procedures, ed, ks, xy, kh, as, od's, kbb carried out experimental procedures, acw, djk, ahjs, rsm, mrl, and sjh contributed to the design and secured funding for the experimental work, ahrv contributed to design and interpretation of the experiments, dob designed the experiments, secured funding, and wrote the paper.
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Advanced acquisition strategies and image reconstruction have become major research topics in cardiovascular magnetic resonance (cmr). New techniques are commonly validated using numerical phantoms . However, computer simulations are often based on oversimplified and unrealistic models . Furthermore, the lack of standardized reference models has resulted in a large diversity in simulation methods hampering appropriate cross - validation . Comparability is, however, of particular relevance in cmr, where cardiac and respiratory motions add to simulation complexity . Considerable efforts have been invested into the development of non - cartesian and accelerated data acquisition strategies, as they permit reduced scan time, increased spatial and/or temporal resolution or a combination of these benefits . Despite the growth of the research field, it remains difficult to benchmark the various image reconstruction approaches . Among others, one reason for this is the lack of comparability between simulation setups . The first analytical phantom to be used widely for imaging simulations was the shepp - logan head phantom . Further development led to computationally more complex cerebral phantoms specifically designed for mri, using elliptical shapes or combinations of different geometrical contours . Voxel based phantoms, on the other hand, are usually derived from acquired images of a single volunteer or patient, with a few segmented tissue types only and adapted to the purpose of the study . Voxel based phantoms are realistic and easily accessible, but limited to the acquisition parameters they are based on, such as spatial and temporal resolution . Furthermore, for voxel based phantoms the image to k - space relation is approximated by the discrete fourier transform, which ignores k - space truncation errors . These effects are omitted with analytical phantoms, where the continuous fourier transform is well - defined . In contrast, analytical phantoms usually do not incorporate motion as required in cmr . A practical approach to numerical phantoms is the integration of advantages of both analytical and voxel - based methods into hybrid phantoms . Hybrid phantoms address the key limitations of analytical and voxel - based approaches, leading to a versatile, realistic and reproducible simulation framework . The mathematical cardiac - torso (mcat) and the extended cardiac - torso (xcat) phantoms present such approaches by combining voxel - based in - vivo data with non - uniform rational b - splines (nurbs) or polygon meshes to define anatomical objects . The anatomical information in xcat is based on the visible human project of the national library of medicine . Several numerical phantoms for cmr applications have been proposed so far, including analytical, voxel based and hybrid models . These in part allow for reproducible figures of merit based on known simulation parameters such as spatial resolution . Despite the attempt to generate versatile and realistic phantoms, none of the above - mentioned studies have made their phantoms available to the community . Therefore, reproducibility is limited and reimplementation of the phantom is cumbersome . In this paper, mrxcat is a simulation framework designed for research on cmr sampling and reconstruction strategies in the presence of motion . In order to promote comparability and reproducibility in cmr simulation research, mrxcat cine and myocardial perfusion mrxcat implementations are presented alongside with reconstruction and post - processing examples as showcase applications . The xcat software allows generation of realistic human anatomical masks including cardiac contraction and respiratory motion . Spatial resolution, matrix size, temporal resolution, coverage, geometry and non - rigid object motion are adjustable . For the mrxcat implementation, each tissue mask is assigned mr properties as illustrated in figures 1 and 2 . Tissue masks in desired orientation with optional cardiac and respiratory motion are generated using xcat . Optional gridding to non - cartesian trajectories and resampling, followed by image reconstruction and post - processing complete the simulation procedure . The tissue and sequence operators depend on tissue properties, sequence parameters and the signal model m. the coil operator and noise object use coil geometry and snr as input . The trajectory governs the effect of the gridding and sampling operators . Mathematically, the mrxcat phantom dk?,t in k - t space is expressed as(1)dk?,t=?i=1nsri?g?f?snc, rc, x?c?cmte, tr,?,nr?tt1,t2,?,ct?oix?,t+nix?,t, where oix?,t is the xcat object, which is a function of space and time, and nix?,t denotes object noise . The circle denotes application of the operator on the left hand side of? Onto the object on the right hand side . T is the tissue operator, which depends on relaxation times t1, t2, the proton density? And the contrast agent concentration c(t). The sequence operator cm implements the signal model m with various parameters including echo time te, repetition time tr, flip angle? And number of repetitions nr . Coil operator s applies coil sensitivities of nc receive coils with radius rc and location x?c as input . After fourier transform f, the gridding operator g and the sampling operator ri convert the raw k - space phantom into simulated mri data, as detailed below . The full xcat and noise objects are created for each segment i and the sampling operator ri extracts the segment i from the full k - space data . A number of physiological parameters such as heart beat and respiratory cycle duration can be adjusted in xcat . Several parameters are defined at the xcat input including spatial and temporal resolution, matrix size, slice offcentres and angulation and the number of simulated heart beats . The timing information allows setting the temporal resolution during the heart phase and the number of segments needed to acquire the full k - space . The blood pool and myocardial tissue of the atria and ventricles in the left and right heart are divided into eight individual partitions . Arteries, veins and pericardium form separate compartments . Using the tissue and sequence operators t and cm the organ masks (xcat object oi) are assigned realistic mr signal intensities based on tissue properties t1, t2, proton density? And contrast agent concentration c(t). Sequence timing parameters such as repetition time tr, echo time te, number of repetitions nr and the flip angle? Examples of available signal models are balanced ssfp, and saturation - recovery gradient echo sequences, . Dynamic contrast enhancement during first - pass myocardial perfusion is simulated based on a population average arterial input function (aif) from six healthy volunteers . A contrast dose of 0.025 mmol / kg b.w . And a short saturation delay (30 ms) mid - ventricular signal intensity vs. time curves were extracted from the left ventricle and manually shifted in time to the same bolus arrival time before averaging, which mitigates temporal broadening of the aif peak by averaging . The population average aif was fitted by a 3-parameter gamma - variate function to obtain a first - pass aif of the contrast bolus . The myocardial concentration - time curve is obtained by convolution with a fermi function . The blood pool and myocardial concentrations are then converted to signal intensities using the relationship between t1 and concentration c(t),(2)r1=1t1,0+ct?r, where t1,0 is the baseline tissue t1 in the absence of contrast agent and r the relaxivity of the contrast agent . Three - dimensional coil sensitivities are simulated using the biot - savart law,(3)b?r?=?04??coilidl?r??r?'r??r? '3, by discretising the integral and calculating the effective magnetic field at the voxel positions of the xcat masks . Adjustable parameters in this module are the number of receive coils nc, the radius rc and position x?c of the individual coil elements . The coil elements are arranged on one or more circles around the mrxcat phantom to mimic typical cardiac coil arrays around the chest . Note that the biot - savart law is only valid in the near field approximation, i.e. For field strengths?<?4 t. since most cmr protocols are designed for 3 t or lower field strength the approximation is considered a valid assumption . A spatial low - pass filter option is implemented at this point with variable filter strength to obtain realistic signal intensities at tissue interfaces . White gaussian noise is added to obtain the desired signal - to - noise ratio (snr). In case of dynamic contrast enhancement, the noise level is defined by the contrast - to - noise ratio (cnr). After this step, the image - domain phantom is transformed into k - space via discrete fourier transform f. the gridding operator g allows interpolation of the data onto non - cartesian trajectories . To apply these trajectories, the non - uniform fast fourier transform (nufft) implementation by j. fessler and the nufft wrapper by m. lustig the sampling operator r defines the sampled k - space profiles and enables application of undersampling schemes by omitting certain k - space lines . If the simulated acquisition consists of multiple k - space segments, the required segment is extracted from the full k - space . The mrxcat phantom production is repeated for each segment to yield the final mrxcat phantom, which is saved as matlab output . Other output formats such as the ismrmrd data structure are possible . To allow comparison of the numerical phantoms with in - vivo cmr, cine and first - pass myocardial perfusion cmr was performed on two philips achieva scanners (philips healthcare, best, the netherlands) at 1.5 t and 3 t field strengths . Cine in - vivo parameters were: spatial resolution: 1.561.56 mm, 10 mm slice thickness, field - of - view: 400400 mm, tr?=?3.4 ms, te?=?1.7 ms, flip angle?=?50, 20 heart phases . Parameters for in - vivo myocardial perfusion imaging were: spatial resolution: 2.142.255 mm, field - of - view: 36036080 mm, tr?=?1.97 ms, te?=?0.78 ms, flip angle?=?15. Perfusion image acquisition was performed using a 10-fold accelerated saturation - recovery gradient echo sequence with a saturation delay of 150 ms followed by k - t pca reconstruction . A gadolinium bolus (gadovist, bayer healthcare, zurich, switzerland) with a contrast agent dose of 0.025 mmol / kg b.w . Was applied during a breathhold acquisition of 30 time frames . Example cine and myocardial perfusion mrxcat phantoms were generated with t1 and t2 values according to textbook literature . Relative proton densities were extracted from a proton - density weighted in - vivo measurement . Two - dimensional (2d) cine mrxcat parameters were equal to the in - vivo measurement, except for the number of heart phases, which was set to 24 to enable 8-fold undersampling and k - t reconstruction without zero - filling (cf . Parameters for simulated first - pass myocardial perfusion imaging were as in - vivo except for the in - plane resolution, which needed to be isotropic in xcat and was set to 2.142.14 mm . Further parameters were: cnr of 30, 40 profiles from acquisition start to k - space centre, contrast agent dose: 0.075 mmol / kg b.w ., contrast agent relaxivity: r?=?5.6 rest and stress myocardial blood flow were set to 1.0 and 3.5 ml / g / min, respectively . Both the 2d cine and the 3d myocardial perfusion fully sampled cartesian phantoms were retrospectively undersampled and reconstructed using k - t pca and k - t sparse . For both cases, an acceleration factor of r?=?8 was chosen . To visualise local variations in reconstruction accuracy, error maps were calculated by subtracting the reconstructed images from the fully sampled reference phantom reconstruction . All error maps were normalized by the average signal intensity over all time frames of the reference image to yield percentage errors . Root mean square error (rmse) values were calculated for each case to estimate the total error . Quantification of myocardial blood flow (mbf) was performed on the myocardial perfusion reconstruction results . Using the xcat masks, signal intensity - time curves were extracted from left ventricle and myocardium and converted back to contrast agent concentration using equation (2) and the signal model . The signal intensity - time curves allow display of temporal filtering effects introduced by image reconstruction from undersampled data . Analysis of the mbf from fully sampled versus undersampled data enabled estimation of the additional uncertainty introduced by undersampling compared to measurement noise . The mrxcat extension for xcat was implemented in matlab (mathworks, natick, ma, usa). All computations were run on a laptop pc equipped with an intel i7 m620 2.67 ghz cpu with 2 cores and 8 gb memory . A number of physiological parameters such as heart beat and respiratory cycle duration can be adjusted in xcat . Several parameters are defined at the xcat input including spatial and temporal resolution, matrix size, slice offcentres and angulation and the number of simulated heart beats . The timing information allows setting the temporal resolution during the heart phase and the number of segments needed to acquire the full k - space . The blood pool and myocardial tissue of the atria and ventricles in the left and right heart are divided into eight individual partitions . Using the tissue and sequence operators t and cm the organ masks (xcat object oi) are assigned realistic mr signal intensities based on tissue properties t1, t2, proton density? And contrast agent concentration c(t). The concentration is used to simulate dynamic contrast enhancement by shortening t1 . Sequence timing parameters such as repetition time tr, echo time te, number of repetitions nr and the flip angle? Examples of available signal models are balanced ssfp, and saturation - recovery gradient echo sequences, . Dynamic contrast enhancement during first - pass myocardial perfusion is simulated based on a population average arterial input function (aif) from six healthy volunteers . A contrast dose of 0.025 mmol / kg b.w . And a short saturation delay (30 ms) mid - ventricular signal intensity vs. time curves were extracted from the left ventricle and manually shifted in time to the same bolus arrival time before averaging, which mitigates temporal broadening of the aif peak by averaging . The population average aif was fitted by a 3-parameter gamma - variate function to obtain a first - pass aif of the contrast bolus . The blood pool and myocardial concentrations are then converted to signal intensities using the relationship between t1 and concentration c(t),(2)r1=1t1,0+ct?r, where t1,0 is the baseline tissue t1 in the absence of contrast agent and r the relaxivity of the contrast agent . Three - dimensional coil sensitivities are simulated using the biot - savart law,(3)b?r?=?04??coilidl?r??r?'r??r? '3, by discretising the integral and calculating the effective magnetic field at the voxel positions of the xcat masks . Adjustable parameters in this module are the number of receive coils nc, the radius rc and position x?c of the individual coil elements . The coil elements are arranged on one or more circles around the mrxcat phantom to mimic typical cardiac coil arrays around the chest . Note that the biot - savart law is only valid in the near field approximation, i.e. For field strengths?<?4 t. since most cmr protocols are designed for 3 t or lower field strength the approximation is considered a valid assumption . A spatial low - pass filter option is implemented at this point with variable filter strength to obtain realistic signal intensities at tissue interfaces . White gaussian noise is added to obtain the desired signal - to - noise ratio (snr). In case of dynamic contrast enhancement, the noise level is defined by the contrast - to - noise ratio (cnr). After this step the image - domain phantom is transformed into k - space via discrete fourier transform f. the gridding operator g allows interpolation of the data onto non - cartesian trajectories . To apply these trajectories, the non - uniform fast fourier transform (nufft) implementation by j. fessler and the nufft wrapper by m. lustig the sampling operator r defines the sampled k - space profiles and enables application of undersampling schemes by omitting certain k - space lines . If the simulated acquisition consists of multiple k - space segments, the required segment is extracted from the full k - space . The mrxcat phantom production is repeated for each segment to yield the final mrxcat phantom, which is saved as matlab output . To allow comparison of the numerical phantoms with in - vivo cmr, cine and first - pass myocardial perfusion in - vivo data were acquired in two healthy volunteers . Cmr was performed on two philips achieva scanners (philips healthcare, best, the netherlands) at 1.5 t and 3 t field strengths . Cine in - vivo parameters were: spatial resolution: 1.561.56 mm, 10 mm slice thickness, field - of - view: 400400 mm, tr?=?3.4 ms, te?=?1.7 ms, flip angle?=?50, 20 heart phases . Parameters for in - vivo myocardial perfusion imaging were: spatial resolution: 2.142.255 mm, field - of - view: 36036080 mm, tr?=?1.97 ms, te?=?0.78 ms, flip angle?=?15. Perfusion image acquisition was performed using a 10-fold accelerated saturation - recovery gradient echo sequence with a saturation delay of 150 ms followed by k - t pca reconstruction . A gadolinium bolus (gadovist, bayer healthcare, zurich, switzerland) with a contrast agent dose of 0.025 mmol / kg b.w . Example cine and myocardial perfusion mrxcat phantoms were generated with t1 and t2 values according to textbook literature . Relative proton densities were extracted from a proton - density weighted in - vivo measurement . Two - dimensional (2d) cine mrxcat parameters were equal to the in - vivo measurement, except for the number of heart phases, which was set to 24 to enable 8-fold undersampling and k - t reconstruction without zero - filling (cf . Parameters for simulated first - pass myocardial perfusion imaging were as in - vivo except for the in - plane resolution, which needed to be isotropic in xcat and was set to 2.142.14 mm . Further parameters were: cnr of 30, 40 profiles from acquisition start to k - space centre, contrast agent dose: 0.075 mmol / kg b.w ., contrast agent relaxivity: r?=?5.6 rest and stress myocardial blood flow were set to 1.0 and 3.5 ml / g / min, respectively . Both the 2d cine and the 3d myocardial perfusion fully sampled cartesian phantoms were retrospectively undersampled and reconstructed using k - t pca and k - t sparse . For both cases, an acceleration factor of r?=?8 was chosen . To visualise local variations in reconstruction accuracy, error maps were calculated by subtracting the reconstructed images from the fully sampled reference phantom reconstruction . All error maps were normalized by the average signal intensity over all time frames of the reference image to yield percentage errors . Root mean square error (rmse) values were calculated for each case to estimate the total error . Quantification of myocardial blood flow (mbf) was performed on the myocardial perfusion reconstruction results . Using the xcat masks, signal intensity - time curves were extracted from left ventricle and myocardium and converted back to contrast agent concentration using equation (2) and the signal model . The signal intensity - time curves allow display of temporal filtering effects introduced by image reconstruction from undersampled data . Analysis of the mbf from fully sampled versus undersampled data enabled estimation of the additional uncertainty introduced by undersampling compared to measurement noise . The mrxcat extension for xcat was implemented in matlab (mathworks, natick, ma, usa). All computations were run on a laptop pc equipped with an intel i7 m620 2.67 ghz cpu with 2 cores and 8 gb memory . Temporal profile plots of breathhold mrxcat cine, free - breathing mrxcat cine and in - vivo breathhold cine data are demonstrated (figure 3c). Individual coil maps of a simulated 8-element cardiac coil array are displayed in figure 3f . Full field - of - view at systole and expiration (a) and at diastole and inspiration breathhold (b). (c) profiles along dashed line in (b) for 24 heart phases for breathhold and free - breathing mrxcat with 15 segments and the breathhold in - vivo case for comparison . (d - e) cardiac region - of - interest at different cardiac phases for breathhold mrxcat (d) and in - vivo scan (e). (f) single coil images from 8 individual coils . In figure 4 the whole - heart myocardial perfusion mrxcat phantom is displayed . Apical, mid - ventricular and basal slices at the time points of bolus arrival in the right and left ventricle as well as in the myocardium are shown in figure 4a . In - plane profiles as a function of time extracted from the breathhold, free - breathing phantom and in - vivo acquisition are demonstrated in figure 4b . A cardiac region - of - interest (roi) in addition, the effect of timing of the saturation pulse with respect to the image acquisition is shown for a long saturation to acquisition delay of 150 ms relative to a short saturation delay of 10 ms to demonstrate the non - linearity between mr signal and contrast agent concentration . (a) three slices from apex to base at maximum contrast agent enhancement in the right ventricle, left ventricle and myocardium . (b) anterior - posterior profiles along dashed line in (a) for all heart beats for breathhold mrxcat, free - breathing mrxcat and in - vivo breathhold myocardial perfusion images (left - right). (c) 16 slices of the mrxcat perfusion phantom, covering the whole left ventricle at a time frame during contrast enhancement . (d - e) signal intensity curves extracted from the left ventricle (aif) and the myocardium of stress and rest mrxcat perfusion phantoms generated with saturation delays of 150 ms (d) and 10 ms (e). Figure 5 shows the results of image reconstruction of the breathhold 2d cine mrxcat phantom with 8-fold data undersampling . A cardiac roi and a profile through the slice as a function of heart phase are shown . Absolute error maps reveal that both k - t pca and k - t sparse yield reconstructed images of good quality . Reconstruction errors are found at the borders of the myocardium, where cardiac contraction causes most motion . The rmse in the cardiac roi was 10.0% and 10.2% for k - t pca and k - t sparse, respectively . Diastolic and systolic heart phases and profile along the dashed line in (a) for all heart phases . Reference images (a), reconstruction results and difference maps using 8x k - t pca (b) and 8x k - t sparse (c). Figure 6 illustrates reconstruction results for the 8x undersampled myocardial perfusion phantom at time points of bolus enhancement in the left ventricle and myocardium . Error maps show pronounced edge effects for k - t sparse, whereas the error distribution in k - t pca is more homogeneous . The rmse was 16.1% and 14.4% for k - t pca and k - t sparse, respectively . Fermi model based mbf quantification in eight mid - ventricular slices of the simulated stress perfusion image is shown for reference, k - t pca and k - t sparse reconstructions in six angular sectors per slice . Mean and standard deviation of mbf were 3.48? ?0.13 ml / g / min in the reference image, 3.48? ?0.36 ml / g / min for 8 k - t pca and 3.55? ?0.33 ml / g / min for 8x k - t sparse . Fully sampled reference (a), 8x k - t pca (b) and 8x k - t sparse (c) reconstruction results and error maps . A mid - ventricular slice at peak left - ventricular (lv) and myocardial enhancement and profile along the dashed red line for 32 time frames are shown . (d - f) signal intensity - time curves for reference and reconstructed data from 4 voxels (inset). (g - i) fermi mbf quantification of stress perfusion simulation in 8 slices with 6 angular sectors per slice (inset). Total computation times for mrxcat generation were 38 and 124 minutes for the breathhold and free - breathing cine phantom, respectively . For the perfusion phantom, calculations took 13.6 and 10.7 minutes for breathhold and free - breathing simulations . In all cases the large difference between cine and perfusion computation times mainly stems from the fact that 360 xcat time frames were created for cine (24 heart phases, 15 segments), while only 32 time frames were needed for perfusion . By executing only the matlab part of mrxcat, calculation times spanned from 54 s (breathhold cine) to 166 s (free - breathing perfusion) in this work a cmr extension to the xcat phantom has been proposed to enable realistic simulation to aid and benchmark image reconstruction and quantitative post - processing approaches . The mrxcat framework is composed of several sequential software modules, which enable user interaction via parameters at each stage of the simulation . The xcat hybrid phantom, which is used to simulate anatomy and motion, delivers realistic and generalized tissue masks . Although the xcat object is rasterised, the drawbacks of voxel based phantoms can be addressed by increasing the spatial resolution in xcat . Thus, the 0.330.331 mm voxel size of the visible human segmentation is sufficient for most cmr applications . All subsequent software parts act on that rasterised xcat object, which simplifies implementation and data handling in array - based numerical software . The individual operators or software modules allow setting parameters specific for tissue and sequence, receiver coils, measurement noise and k - space characteristics . The independency between modules potentially enables parallel computing and division of large data portions into chunks on computers with less available memory . The use of mrxcat phantoms for research on accelerated imaging was exemplified using k - t pca and k - t sparse . The effect of cardiac contraction in conjunction with image acceleration was showcased with the cine phantom, where the biggest residual errors after reconstruction remain at the inner and outer border of the myocardium . Using the myocardial perfusion showcase, the effect of signal changes due to contrast agent administration was demonstrated . In this example, residual reconstruction errors mainly stem from temporal filtering of the signal - time curves . Moreover, mrxcat phantoms can be used for the development of post - processing methods such as myocardial blood flow quantification . A key advantage of quantification using mrxcat is that noiseless ground truth is always at hand and available for assessment of errors this issue was accounted for by adding a low - pass filter module before noise addition . A similar problem is the missing organ texture in xcat, which has recently been addressed . However, mrxcat assumes instantaneous acquisition of the individual segments . Extending the framework to enable individual timing of single k - space lines would require full xcat masks for each k - space line . Since the xcat part of mrxcat is the computationally most time - consuming step, this would yield unacceptably long simulation times . As such, it is suitable for the simulation of sampling strategies, trajectory optimization and post - processing methods . However, a number of error sources are excluded, such as magnetic field inhomogeneity, magnetization evolution across time frames or signal alteration due to motion during sampling . For those applications, other simulation frameworks based on solving bloch equations may be more suitable . Extending the framework to enable individual timing of single k - space lines would require full xcat masks for each k - space line . Since the xcat part of mrxcat is the computationally most time - consuming step, this would yield unacceptably long simulation times ., it is suitable for the simulation of sampling strategies, trajectory optimization and post - processing methods . However, a number of error sources are excluded, such as magnetic field inhomogeneity, magnetization evolution across time frames or signal alteration due to motion during sampling . For those applications, other simulation frameworks based on solving bloch equations may be more suitable . Extensions to other cmr modalities, such as diffusion or flow encoded mri, are possible . Aif: arterial input function cmr: cardiovascular magnetic resonance cnr: contrast - to - noise ratio mbf: myocardial blood flow mcat: mathematical cardiac - torso mrxcat: magnetic resonance extended cardiac - torso nufft: non - uniform fast fourier transform nurbs: non - uniform rational b - splines rmse: root mean square error roi: region - of - interest snr: signal - to - noise ratio xcat: extended cardiac - torso lw, cs, sk devised mrxcat in matlab, created phantom examples, performed image reconstruction and post - processing.
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About 96% are symptomatic at presentation with a history of stricture disease in more than half and sexually transmitted disease in one fourth of all patients . Rarely, it can manifest as a periurethral abscess, fistula, or fungating perineal tumor . While urethral carcinoma is known to aggressively infiltrate periurethral tissue; concomitant involvement of suprapubic catheter tract has not been reported in the literature . In this paper, we reiterate the importance of high clinical suspicion in making this rare diagnosis, the consequences of delayed diagnosis and the role of biopsy of a suprapubic tract with clinical features of malignancy . A 65-year - old diabetic male with a long history of urethral stricture developed rapid worsening of his lower urinary tract symptoms and noticed a painful swelling in his upper scrotum for 2 weeks . He was diagnosed with a periurethral abscess and underwent incision and drainage at another center . His perineal wound did not heal and over the next 3 months it developed into a fungating ulcer . He consulted his surgeon, who performed a biopsy revealing squamous cell carcinoma (scc). At presentation to us, he had a 10 6 cm, fungating, malodourous, ulceroproliferative scrotal growth fixed to the region of the bulbar urethra . The abdominal entry site of the suprapubic catheter was ulcerated with rolled out edges and induration [figure 1a]. Ct scan showed an enhancing ill - defined soft tissue density in the region of bulbar urethra infiltrating corpora cavernosa, adjacent skin and subcutaneous tissue [figure 1b]. Transverse section [figure 1c] at the level of bladder and suprapubic cystostomy (spc) tract showed induration surrounding the tract . He underwent a total penile amputation, wide local excision scrotum, radical urethrocystoprostatectomy, ileal conduit diversion, and pelvic lymphadenectomy [figure 2a]. We included wide local excision of the spc site, en bloc with the cystoprostatectomy specimen . Histopathological examination showed moderately differentiated scc of the bulbar urethra [figure 2b] with 2 right external iliac lymph nodes positive for metastasis . Postoperative staging was t3n1; he was given a course of adjuvant chemotherapy (cisplatin and gemcitabine) and has completed follow - up of 6 months . (a) ulceroproliferative growth at the penoscrotal junction and, suprapubic cystostomy (spc) site with ulcerated, everted mucosal edges; (b) computed tomography scan showing an ill - defined, enhancing mass in the region of posterior urethra infiltrating cavernosae (arrow); (c) transverse section showing bladder and spc tract (arrow head) (a) postoperative picture following radical urethrocystoprostatectomy with en - bloc excision of suprapubic cystostomy (spc) tract and wide local excision of scrotum; (b) h and e, 10 showing primary squamous cell carcinoma (scc) of urethra with keratin pearls; (c) h and e, 20 showing foci of scc in the spc tract our patient had a history of long - standing urethral stricture complicated by a periurethral abscess . Others are chronic irritation after intermittent catheterization, following urethroplasty, external - beam radiation therapy, radioactive seed implantation, and chronic urethral inflammation or urethritis following sexually transmitted diseases . Male urethral carcinoma most commonly occurs at the bulbomembranous urethra (60%), while the penile and the prostatic urethra are afflicted in 30% and 10% of cases respectively . Histologically, scc accounts for 80% of urethral malignancy, 15% are transitional cell carcinoma and less than 5% are adenocarcinoma . Symptoms of this malignancy are neither pathognomonic nor diagnostic . The often insidious onset of the disease and non - diagnosis at initial presentation might result in substantial delay between first symptoms and treatment . Common presentations include bleeding per urethra, discharge, urinary obstruction, penile or perineal pain, hematospermia, and a palpable mass . In rare cases such as this, a periurethral abscess or urethral fistula in the background of stricture disease may be the sequel of advanced infiltrative malignancy . The early obstructive symptoms can mimic stricture disease and indeed, progressively difficult dilatation of urethral stricture may presage the diagnosis in the presence of adequate clinical suspicion . Similarly, urethral bleeding in the absence of prior trauma or disease may reveal urethral malignancy on evaluation . Regional lymph nodes are to be examined carefully in all cases; palpable lymphadenopathy present in 20% of cases most often represents metastatic disease rather than inflammatory process as seen in penile cancer . Local spread occurs by direct infiltration of adjacent tissue through the spongiosum and periurethral tissue into the urogenital diaphragm, perineum and scrotal skin . Spread via lymphatic embolization into regional lymph node basins generally occurs late in the process . Lymphatics from the anterior urethra generally drain into the superficial and deep inguinal nodes and less often directly into the external iliac group . Faced with advanced clinical stage of disease, we undertook radical resection of the lower urinary tract with urinary diversion . In this case, malignancy at the suprapubic site was unlikely to have been caused by chronic irritation from the catheter given the relatively short duration of the diversion . Previous reports of scc at the suprapubic tract involved period of catheterization from 5 to 35 years . This is the first report of suprapubic tract scc in the presence of primary urinary tract malignancy . We presume that the seeding of the tract occurred either from extension of tumor via transdermal lymphatic spread or exposure to malignant cells in urine . Urine cytology in this case was however negative and the bladder was also free of tumor . Perhaps the distal obstruction in combination with the breach of tissue planes by both tumor and intervention allowed viable malignant cells in urine access to the suprapubic site around the time of its creation . Our anecdotal experience leads us to recommend biopsy of the suprapubic tract whenever examination arouses suspicion, especially when urinary tract malignancy is present . Locally advanced urethral scc must be excised with a wide margin along with flap reconstruction of the defect when indicated, to allow optimal local oncologic clearance.
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Chemotherapy - induced neutropenia is characterized by a marked decrease in the peripheral blood neutrophil count . Since neutrophils are an integral part of the innate immune system, this may result in severe complications such as life - threatening infections (1, 2). As a result, neutropenia is considered to be the most serious chemotherapy - related hematological adverse event, frequently leading to dose delays or reductions which may compromise treatment outcomes (3). Neutropenia dampens most of the signs and symptoms of infection, and patients typically present with only fever, which is why this neutropenia - associated complication is referred to as febrile neutropenia (fn) (4). Depending on the number of comorbidities, chemotherapy - induced fn is thought to be responsible for the deaths of up to 50% of affected patients (5). Therefore, prevention and treatment of fn is considered a primary goal of supportive care in cancer patients at high risk undergoing cytotoxic chemotherapy (6). Antibiotics, additional interventional care like use of granulocyte colony - stimulating factor (gcsf) and further treatment in the outpatient setting (7). This may result in chemotherapy dose delays or dose reductions, and interferes with delivery of optimal treatment, adversely affecting patient outcomes, including survival . Beside medical consequences of fn, there is a significant economic impact of this condition in terms of its treatment . One of the main drivers of costs related to antineoplastic chemotherapy is hospitalization which often includes other direct and indirect medical costs such as antibacterial treatment and inability to work (8, 9). In the united states (us), the direct cost of neutropenia reported in the literature ranged from $us 2.893 to $us 38.583 (2006 values $us 4.842-$us 49.917) per episode for inpatients (10). For outpatients, the cost was $us 1893 (2006 value $us 2632) per episode . Outside of the us, the cost per episode ranged from $us 300 (2006 value) for non - febrile cases to $us 32.395 for elderly breast cancer patients with neutropenic complications . In general, the cost of neutropenia appeared to be lower in other countries compared with the us, with most estimates being <$us 7.000 (2006 value) per episode . Hospitalization was the largest driver of the cost of neutropenia, comprising as much as 82% of the total direct medical costs for neutropenia (11). Major components of the hospital costs included the room (36 - 38% of hospital costs), pharmacy (27 - 33%), supplies (4 - 17%) and blood bank (3 - 14%) (12). Indirect costs for neutropenia, such as costs associated with patient work loss, caregiver work loss, paid caregiver and/or non - revenue - generating support centers, were estimated to be $us 3.834-$us 5.738 per episode (2006 values $us 4.741-$us 8.781), thus representing approximately 34 - 44% of the total cost of managing neutropenia (12). Study published in 2009 reported that one of the most common reasons for cancer patient hospitalization was fn resulting in average hospitalization of 12,9 days and inquiring significant costs (14). Treatment of fn is mainly based on different gcsfs (15), and antibiotics in order to prevent or treat serious infections since this patients are immune compromised (16). As authors best knowledge, there is no any publication describing this issue in bosnia and herzegovina . In this observational, retrospective cohort study, data from a database that included costs from one hospital / oncology department were used to calculate the hospitalization costs and overall treatment costs associated with neutropenic complications . The aim of this study was to analyze cost of fn treatment from one hospital / oncology department in bosnia and herzegovina . Retrospective cohort studies of patients treated at oncology department of cantonal hospital zenica have been analyzed . Only patients who were admitted and treated for chemotherapy induced febrile neutropenia have been considered . Data on age, cancer type, fn grade, chemotherapy protocol and duration of hospitalization as well as medical treatment have been extracted and taken into the consideration . Only direct medical cost of drugs, laboratory tests, hospitalization and drug administration have been included . Data on cost and prices have been taken from the official price list applied in this hospital . No discount rate on cost has been applied due to short time frame of the study . All results have been summarized and analyzed in ms excel, and presented in form of tables and figures . In total we have included 27 patients with different cancer types diagnosed and treatment protocols applied . All patients have had grade iv of fn occurred at different stage of therapy / cycle . Therapeutic regimens (protocols) used in treatment of different cancer types are presented in figure 1 . Febrile neutropenia episode has occurred at different stages of treatment or cycle and detail overview is given in table 2 . Majority of fn episodes appeared after second and third cycle (22% and 26% respectively). Five patients have been treated in outpatient conditions, one has combined treatment (hospitalization and outpatient care) while majority of them have been hospitalized (81%). Total hospital days consumed was 180, and on average patients have been hospitalized for 6.7 days . For all patients routine laboratory tests have been performed meaning complete blood count (cbc) and differential blood count (dbc), and for some of them, depending on health status and diagnosis additional test like chest x - ray (4 patients; 15%), creatinine, the blood glucose and blood urea nitrogen test - bun (5 patients; 19%). All patients have been treated with granulocyte colony - stimulating factor (gcsf) and majority of patients required introduction of antibiotic therapy (59%). Filgrastim or lenograstime as gcsfs were applied one vial per dose, and meropenem and imipenem / cilastatin intravenous in doses 1 g every 8 hours and 500 mg/500 mg every 6 hours, respectively, were antibiotics of choice . Totally 191 vials of gcsfs have been consumed, and on average antibiotics treatment duration was 3,5 days or 96 days in total . Detailed overview of services provided and unit doses used with unit and total cost in euro are presented in table 3 . In total costs, cost of hospitalization is the highest and amount in 11.044,80 eur (40%) followed by cost of gcsf amount in 10.023,68 eur (36%). Significant part in cost structure is allocated to preparation and application drugs and amount 2.954,24 eur or 11% . Total cost of treating febrile neutropenia induced by chemotherapy in five months period is 27.945,56 eur, or 1.035,02 eur per patient or episode per average . Clinical and demographic characteristics of the subjects therapeutic regimens used in treatment occurence of fn at different stage of treatment overview of resources utilized and costs for treating fn this observational cohort study have included just direct medical cost and showed relatively high average cost per patient or episode of fn . A study by caggiano et al, using 1999 data from a longitudinal hospital discharge database that contained data from across seven us states, reported average neutropenia hospitalization costs (sd) of $13.400 ($21.000) across 13 cancer types (17). A study by kuderer et al, using a longitudinal hospital discharge database with data from 115 us academic medical centers collected over 6 years (1995 to 2000), reported average febrile neutropenia hospitalization costs of $19.110 . Study by weycker et al, using 2001 to 2003 data, reported average neutropenia related hospitalization costs of $7,813 (95% ci 6,537 - 9,379) (19). Regarding cost structure, one study from singapore reported highest cost allocated to drugs (20), while most of the studies from western countries reports hospitalization as highest cost part . Even our findings showed similar portion among cost allocation between major cost types, we have noticed high difference in absolute value . This due to very low cost of hospital days and low prices for medical services overall, and due to different time frames when studies are conducted . It is reported in previous studies that hospital costs in general differ from country to country (21). Time frame of this study included year 2015 when lot of generic drugs penetrated market, cgsfs and antibiotics, which impacted on relatively low cost of drugs in comparison to the results of studies published in western countries . This is also interesting to take into the consideration by health care policy decision makers in terms of evaluating introduction of new medicines into the reimbursement system in regards to transferability of data from studies published abroad (22). Recently, pharmacoeconomic criteria has been introduced into legislation as part of reimbursement submission dossier (23), but still not fully implemented, mainly because of lack of experts in this field employed in decision making institutions and lack of education in this field (24, 25). Significant costs are incurred when fn develops in a patient treated with chemotherapy, as mentioned above . These costs include both direct medical costs and indirect costs that are borne by the patient and his or her family . Economic analyses estimated the different types of aggregate costs that are incurred in hospitalization for fn, and these can be weighed against the costs of the use of prophylactic gcsfs . Primary prophylaxis with gcsfs significantly reduces fn incidence in adults undergoing chemotherapy for solid tumors or lymphoma (26). An early cost - minimization analysis calculated that, when the risk of fn was about 40%, the cost of universal prophylactic gcsf was equaled by the reduction in the costs of hospitalization for fn (27). In bosnia and herzegovina, due to restricted budgets and in order to make savings (28), decision on prophylactic use of gcsf sometimes is not approved by the payer; even published evidences clearly show benefits of prophylactic use in terms of avoiding complications and cost savings (29). In general, health insurance fund (hif) cover all costs for oncology patients and all drugs used in treatment are procured by central tender, meaning that medication prices are unified in all hospitals and clinical centers . In our study cost estimates include direct medical costs but not nonmedical costs that patients incur, such as time lost from work and transportation costs . Focusing on only part of the costs, such as the direct medical expenses, may lead to cost shifting and fails to consider the actual financial impact on all affected parties . Main limitation of our study is small sample of patients taken into analysis, and short time frame counting just first five months . Based on this, it is possible to estimate that cost of treating chemotherapy induced neutropenia on a national level is much higher especially taking into the consideration total number of treated patients at more than ten oncology clinics and departments of different sizes . Our study showed that approximately cost of treating febrile neutropenia is significant and count 1.035,02 eur per patient or episode . Results are based on analysis of small patient population and experiences from middle - sized oncology department . Proportion of costs of hospitalization, drugs and services are in line with other studies published but with significant differences in absolute values due to low prices for these services in bosnia and herzegovina and generic drugs . Proper treatment and prophylaxis, especially in case of high risk patients and chemotherapy protocol, could result in significant cost savings and better resources allocation . It is also important to take into the consideration pharmacoeconomic analysis results at national level as useful tool for decision making and resource allocation, but also at micro level (hospital). In order to implement current legislation on reimbursement criteria, it is necessary to popularize and educate health care professionals and stakeholders in field of pharmacoeconomic . Further research on this issue is recommended through inclusion of other oncology clinics and departments in bosnia and herzegovina, and also taking into the account societal perspective in order to estimate total burden of fn treatment.
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Human beings walk upright and maintain their postures with a narrow base of support and with the center of gravity of the upper trunk . Even if muscle tension slightly loosens, low back pain occurs because of stress from the mechanical posture of the muscle involved1 . This causative factor of low back pain can lead to problems in sense and timing of muscle contraction awareness, sense of heaviness, acting force, and acting load force2 . Of the cases of low back pain syndrome, 80% are related to the lumbar disk; and herniation of disk material is known as a secondary inflammatory response to stimulation of the dorsal root ganglion and nerve root is known as the cause of low back pain . The remaining 20% of cases are directly related to posterior elements such as lumbar facet joint, ligament, lamina of the vertebral arch, and fascia . In addition, the action of the local muscles attached directly to the spine plays an important role in maintaining spinal segmental stability3 and providing trunk stability generally4 . In particular, lumbar disk herniation causes herniation of part or all of the nucleus pulposus by rupture of the mid, internal, or lateral fiber of the annulus fibrosus through an external force or lumbar disk degeneration . It is a disease that induces neurological signs such as hypoesthesia, muscle weakness, and radiating pain in the constricted innervation area, with back pain in the local part by pressing the dura mater or nerve root . Generally, thermotherapy and electrotherapy have been used in physical therapy as interventions for low back pain syndrome . In addition, application of surface heat such as a hot pack and infrared light, and deep heat such as ultrasonography, short waves, and microwaves has been used in thermotherapy6 . Spinal stabilization exercise is another widely used intervention method that improves muscle strength and trunk endurance, and stabilizes nervous system control of muscles and muscle coordination . Therapeutic methods for herniated intervertebral disks are divided into conservative and operational therapies . Excluding 5% of patients, most undergo conservative therapy but anti - inflammatory drugs, joint mobilization, acupuncture, traction, and so on are being used as interventions to recover degenerative disks and improve neurological symptoms8 . Among these interventions, traction therapy is essential in clinical therapy, but its effect is not clear and differences in viewpoints at home and abroad are evident9 . Recently, spinal decompression therapy is being performed to improve the limitations of traction therapy, as it does not have to set a direction and traction force unlike traction therapy . Adjusting the angle and direction of the traction force by adjusting the position of the disk by using a computer program therefore, this study measured disk height after decompression therapy and measured the activities of muscles that provide overall trunk stability by overcoming the gravity applied to the body . In addition, we aimed to clarify the difference between the therapeutic effect of traction therapy and that of decompression therapy, and their clinical therapeutic significance by comparing low back pain disability index between the two intervention groups . This study was approved by the bioethics committee (institutional review board) of sehan university center (approval no . The subjects were 31 patients aged 35 to 50 years who had unilateral or bilateral lumbar and radicular leg pains, who attended a medical institution located in jeollanamdo, south korea, between february 4, 2016, and april 3, 2016, for 2 months . They showed more than one bulging of the intervertebral disks on magnetic resonance imaging mri, had had the onset of illness for less than 6 months, and did not receive spinal depression therapy . In addition, their complaint of pain matched the neurological symptoms, and their mean score in the oswestry disability index (odi)korean version questionnaire was> 30 points (table 1table 1.characteristics of the study subjectsitemsexperimental group (n=15)control group (n=16)age (years)42.5 6.345.2 7.3height (cm)167.1 4.7168.3 3.9weight (kg)61.7 6.865.4 7.1odi (point)34.4 3.935.3 4.7gender (male / female)8/77/9data are presented as mean sd, obtained by using the shapiro - wilk test . Data are presented as mean sd, obtained by using the shapiro - wilk test . Odi: oswestry disability index in this study, an intervention program was conducted in 31 patients with lumbar herniated intervertebral disks . Fifteen subjects were randomly selected for the experimental group and received decompression therapy and trunk stabilization exercise . Sixteen subjects were randomly selected for the control group and received traction therapy and trunk stabilization exercise . These intervention programs were conducted for 3 weeks, and the disk heights at l4 and l5 were measured by using mri . Moreover, trunk muscle activities were measured by using surface electromyography (semg), and the lumbar pain disability index was calculated by using the odi and then analyzed and compared between the groups . The participants understood the purpose of this study very well and agreed to participate in this study voluntarily by providing written informed consent . Vertebral height and disk herniation index were determined by using mri (signa hde 1.5 t, ge corporate, usa). Disk height was analyzed by using t2-weighted axial - section mri, and the images were analyzed by using the pacs (picture archiving and communication system). The subject lay supine, and then the researcher put a cushion under the subject s knee and had the subject bend the knee at a 25 angle . In this position, 1.measurement of lumber disk height . A: anterior disk height, b: middle disk height, c: posterior disk height . A: anterior disk height, b: middle disk height, c: posterior disk height . Disk height: (a + b + c)/3 (mm) disk herniation index measurement: the disk and intervertebral foramen were measured at intercept of the axial direction . The sagittal distance of the herniated disk was recorded as the maximum anteroposterior disk height (ab), and that of the vertebral foramen was recorded as the maximum anteroposterior canal length (ef). The width of the herniated material was measured by drawing a line at the mid level of the maximum anteroposterior disk length (cd). The width of the spinal canal was calculated at the same level (gh). The formula used for calculating the disk herniation index was ([ab cd]/[ef gh]) 1,000 (fig . Ab: maximum anteroposterior disk height, cd: width of the herniated material at the level of the mid ab distance, ef: maximum anteroposterior canal length, gh: width of the spinal canal at the level of the mid ab distance . The herniation index is calculated as ([ab cd]/[ef gh]) 1,000)10, 11 . Ab: maximum anteroposterior disk height, cd: width of the herniated material at the level of the mid ab distance, ef: maximum anteroposterior canal length, gh: width of the spinal canal at the level of the mid ab distance . The herniation index is calculated as ([ab cd]/[ef gh]) 1,000 the four - channel semg system mp 100 (biopac, usa) was used to measure the activities of the trunk muscles, and the sampling rate for collecting emg signals was set at 1,000 hz . The subjects skin was cleaned by removing hair, and dead skin cells were removed by using thin sandpaper to minimize skin resistance to the emg signals . Two ag / agcl surface electrodes were attached to the belly of each muscle in a direction parallel to the muscular fiber at 2-cm intervals . Standard electrodes were attached to the spinous process of the 12th thoracic vertebrae and scapula spine, which are the protruding parts . The emg signals from the right rectus abdominis, transverse abdominis, external oblique abdominis, and erector spine muscles were collected . The sampling rate for emg signal collection was set at 1,000 hz, and the notch filter was set at 60 hz . The band pass filter signals from the measured muscles were converted to root mean square (rms), and emg signal analysis was performed by using the acqknowledge 3.9.1 software program (biopac). Emg data of the rectus abdominis, transverse abdominis, and external oblique abdominis muscles were collected while the subject was touching the knees with both hands in supine position for 5 seconds . For the measurement at the erector spine muscle, the subjects lay in a prone position and flexed both arms maximally and then extended their trunks maximally for 5 seconds . The emg signal produced while performing this movement was set as a particular action, and the emg signal while standing motionless for 5 seconds was set as the standard action . It was regarded as the reference voluntary contraction (rvc), and the% rvc method standardized based on rvc was used . Patients who complained of pain and difficulty in performing movement were excluded from this experiment . The odi was measured by using the korean version of the odi questionnaire to quantify physical difficulty in daily life due to lumbar pain . It is composed of 10 items, and each item was evaluated on a scale of 05 points . In this questionnaire, the higher the score, the more severe the disability . In the korean version, in consideration of the korean culture, the item about sexual life is excluded . Thus, it consists of 9 items; and each item was evaluated with a score from 0 to 5, and the highest possible total score was 45 points . The total score was divided by the number of items 5 and was converted into a percentage12 . The trunk stability exercise described by lee et al.13, 14 was used as spinal and pelvic stabilization exercise . It is composed of step 1 (awareness stage), step 2 (associative stage), and step 3 (exercise task stage) based on the research of ok stage)15 . The exercise method was performed for 30 minutes per session, four times a week, for 3 weeks and was applied step by step in subjects considering their exercise ability (table 2table 2.intervention program for trunk muscle stabilizationstagetimecontentsprogramwarm - up exercise5 minwhole - body flexibility exerciseupper and lower extremity stretching and joint moving exercisesmain exercise20 mintrunk muscle stabilization exercise1 . Pelvic posterior tilting in the supine position with flexion of both kneesstage 12 . Abdominal draw - in in the supine position with flexion of both knees3 . Pelvic anterior tilting in the supine position with extension of both knees4 . Abdominal draw - in in the supine position with extension of both kneesstage 25 . Unstable bridging: bridging with extension of the left / right legcooling - down exercise5 minbreathing and aerobic exercisesbreathing with the abdominalis muscle). The automatic electric traction equipment rehatrac rc-100 (sakai, japan) was used for traction therapy . The duration of traction was 15 minutes, and the ratios of hold and rest times were 2:1 and 60:30 sec, respectively . The traction force for resting time was set to half of the traction force used in hold time . Traction force was started at one fourth of each weight and gradually increased in each session until the subjects did not feel uncomfortable . In the last week, the standard of the maximum traction force was half of the subjectsthweights . The pelvis was fixed with a pelvic stabilization belt, and the thorax was fixed with a thorax stabilization belt . Spinal decompression therapy was performed by using a lumbar decompression device max - d (medirex, korea), and the subjects lay on a traction table in the supine position . Their knees were naturally positioned in a 90 angle on the knee support fixture installed in the device . The ankle was fixed with a hydraulic device according to the subject s body type . Traction force and movement of the low extremity were supposed to be delivered through the fixed ankle . The upper extremity was fixed by using a trunk stabilization belt and an air pumping apparatus to maintain lumbar lordosis . A pelvic tilt of 15 was applied for patients with a lumbar herniated intervertebral disk at the l4 and l5 levels . The subjects were comfortably positioned in the device; then, their lower limbs were pulled within range so as not to make them feel uncomfortable during steps 1 to 5 of delivering traction force through the hydraulic device . The subject s upper extremities were pulled within range so as not to make them feel uncomfortable during steps 1 to 5, using a trunk stabilization belt . First, the multi - mode of an automatic program of a decompression therapy device was used for steps 1 to 5 . This multi - mode was programmed to make the lower part move in multiple directions . Traction force was started at one fourth of the subjects weight and increased by 2 kg at a steady rate daily . When pain occurred according to the increase in traction force, the traction force was lowered or maintained . The traction force for rest time was set to half of the traction force used in hold time . Both the traction and decompression therapy groups received their respective therapies once a day and five times a week for the first week and then once a day, four times a week (total 8 times) for the next 2 weeks . The normality of the general characteristics of the subjects, and the respiratory muscle and physical activities of both groups were tested by using the shapiro - wilk test . No special variables related to the general characteristics of the subjects were found between the groups, thus indicating normality (table 1). The activities of the rectus abdominis, transverse abdominis, and external oblique muscles increased significantly in both the experimental and control groups (p<0.05). The activity of the erector spinal muscle decreased significantly in comparison with the changes in both groups (p<0.05 and p<0.01, respectively; table 3table 3.comparison of changes in muscle activity and disk height within and between the experimental and control groupsgroupprepost%rvcraexperimental group50.8 4.754.1 3.2*control group51.2 5.153.8 5.2*taexperimental group 47.1 3.649.7 4.1*control group45.9 3.748.7 4.9*eaoexperimental group 45.3 2.247.5 2.4*control group45.7 3.248.1 2.1*esexperimental group 59.4 5.253 5.4***control group55.1 4.852.7 5.2*disk height (mm)experimental group 7.1 2.77.3 1.9control group6.8 2.36.8 1.7disk herniation indexexperimental group284 12.3198 22.6**control group264 9.5248 16.3**p<0.05, * * p<0.01 . Ra: rectus abdominis muscle, ta: transverse abdominis muscle, eao: external abdominal oblique muscle, es: erector spine muscle). This was the only significant difference between the groups (p<0.05; table 3). The disk herniation index in the experimental group decreased significantly (p<0.01) in comparison with the changes in both groups . The difference in the changes in disk herniation index between the groups was significant (p<0.05; table 3). Ra: rectus abdominis muscle, ta: transverse abdominis muscle, eao: external abdominal oblique muscle, es: erector spine muscle in this study, 31 patients with lumbar herniated intervertebral disk were randomly assigned to receive either trunk stabilization exercise and decompression therapy or traction therapy and trunk stabilization exercise . The disk heights of the l4 and l5 segments were measured before and after the intervention by using mri . In addition, trunk muscle activities were measured by using semg, and lumbar pain disability index was calculated by using the odi questionnaire and then analyzed and compared between the groups . Guehring et al.16 reported that while decompression by distraction in an animal model with degenerative disk was conducted for 28 days, the detection rate of degenerative change on mri decreased, gene expression levels of disk matrix components increased, and the nucleus pulposus was regenerated . Formation of the structural material of the matrix increases with the increase in hydrostatic pressure through decompression, moisture content increases through the improvement in the combining ability with moisture, and nutrition supply can be expected to increase owing to the improvement of degenerative change . Apfel et al.17 insisted that 30 patients with discogenic low back pain or disk herniation received treatment by using decompression therapy equipment for 6 weeks and conducted a cohort study that compared disk heights by using ct . Kwon et al.11 performed acupuncture, cupping therapy, medicine treatment, and decompression therapy in six patients with disk disease . They reported significant differences in disk height and disk herniation index, and positive changes as results of their research . Moreover, park and kim18 compared disk herniation index and disk height by using mri after applying traction and decompression therapies in 60 patients . As a result, they observed a significant difference in disk herniation index but no significant difference in disk height . This study supported the results of advanced studies because no significant difference in disk height was found in both groups, but a significant difference in disk herniation index was found in the decompression therapy group . Various factors cause the discrepant results between the present study and the advanced study, such as disk disease duration and activities of daily living . In particular, the reasons for having no difference in disk height could be that the patients were exposed to gravity in daily living and that treatment periods differed between the two studies however, a significant difference in disk herniation index was found between the two groups . Only disk separation was discussed as the mechanism that explains the effectiveness of conventional traction therapy, and the effectiveness of automatic traction therapy for patients with chronic discogenic back pain in relation to spine decompression remains unclear . By comparison, kwon et al.11 suggested that decompression therapy supplemented the limitation of traction therapy and had a great advantage of inducing physical change to the disk . It is known that it can measure load applied to the spine by using an electronic control system and allows delivery of traction force to the spine because based on the result, muscle tone did not occur by controlling the speed of traction therapy . In terms of muscle activity, the activity of the erector spine muscle in the decompression therapy group decreased significantly . Inflammation, pain caused by the inflammation, and neurological manifestations were observed in the paraspinal muscles as symptoms of disk disease increased intervertebral foramen diameter by decompression can induce an increase in blood flow in the blood vessel within the foramen and intervertebral foramen, and increased blood flow can lead to decreased muscle tension and pain by removing inflammatory exudation20 . Based on these results, the paraspinal muscles were verified to have a positive response to trunk stabilization exercise after decompression therapy, it is considered more effective clinically than conventional traction therapy as intervention method for patients with disk disease . Normalizing the general characteristics of the study subjects was difficult because the study period was short and the subjects daily lives were not properly controlled . However, decompression therapy can be considered a more effective intervention method for patients with disk disease in the light of disk height and disk herniation index, change in muscle activity, and many advanced research studies . Prospective research on this intervention with adjustment of the study and disease periods will be needed in the future.
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Werner syndrome (ws) is a rare autosomal recessive disease associated with premature age - related phenotypes such as cancer, osteoporosis, diabetes mellitus and early graying of the hair (review). The gene responsible for ws (wrn) is one of a five human recq helicases including blm, recql1, recql4, and recq5 . Like ws, the absence of blm and recql4 gives rise to the clinically distinct diseases, bloom's syndrome (bs) and rothmund - thomson syndrome, respectively . Although a variety of different wrn mutations have been discovered, many result in a truncated nonfunctional wrn (summarized in). Cells from ws patients depict an aging phenotype including reduced proliferation associated with an increase in s - phase and early passage senescence [3, 4]. Furthermore, ws cells show increased levels of genomic instability thought be caused from increased levels of illegitimate recombination . These observations lead us to investigate the role of wrn in vivo . For this study we used a wrn mouse model with a deleted helicase domain in combination with the well - established murine pink - eyed unstable (p) mouse model that can be used to determine changes in the spontaneous frequency of somatic homologous recombination (hr) events [68]. Though rare, this particular wrn mutation has been found in a small population of ws patients [9, 10] and is therefore relevant to the human disease . The p assay is based on an hr - mediated deletion of one copy of a 70 kb dna duplication that encompasses exons 618 of the p gene . The exact deletion of one copy of the repeated region will restore the function of this pigmentation gene, and this can be observed as somatic events in pigmented tissues such as the fur and the retinal pigment epithelium (rpe) [8, 12]. The further development of the p eye spot assay which identifies p reversion events on a monolayer of clear rpe cells has proven to be significantly more sensitive and informative than the fur spot assay . Studies in yeast using a similar duplication / deletion assay to the p reversion assay have identified several possible mechanisms of hr that may mediate this type of deletion event . These include intrachromatid exchange, one - sided strand invasion, unequal sister chromatid exchange (sce), sister chromatid conversion, and single - strand annealing (ssa). Excluding ssa, each of these hr mechanisms is considered rad51-dependent . Rad51-dependent hr is considered a high fidelity dna repair mechanism that is frequently associated with dna replication (review). For example, replication forks can stall or collapse when the replication machinery encounters dna damage like a single strand break, and hr is capable of repairing the damaged dna template and restoring the replication fork . Recently reported that in the absence of parp1, somatic hr events, measured by the p system, are highly elevated and that a majority of these events were large clonally expanded cells . These authors went on to suggest that these clonally expanded populations of cells are the result of an hr - mediated deletion that occurred during replication, probably in a rad51-dependent mechanism (as compared to ssa). Wrn helicase mutant (wrn) mice on an fvb strain background were obtained from dr . P. leder, and c57bl/6j and c57bl/6j p/ mice were obtained from the jackson laboratory (bar harbor, me). In order to obtain congenic c57bl/6j p / wrn mice (hereafter called wrn), wrn mice were backcrossed 5 times to c57bl/6j followed by two additional crosses to c57bl/6j p mice . Control (wrn) and experimental (wrn) animals result from crossing wrnmice together . Genotypes for the wrn allele were determined by a pcr amplification protocol obtained from aya leder, harvard medical school, ma consisting of the following 3 primers: (1) 5-gtttcctctatcatctgtaacagg-3, (2) 5-gcgaaggagcaaagctgctat-3 and (3) 5-agtgagacatgtatgactacc-3 and the thermo profile: 1 cycle of 94c for 5 min; 30 cycles of 94c for 30 s, 60c for 30 s, and 72c for 1 min; 1 cycle of 72c for 3 min . Amplicon size for the wt pcr product is 350 bp and wrn mutant 450 bp . When necessary, genomic dna was isolated from fixed rpe using the qiagen dneasy blood and tissue kit according to manufacture's recommendations . Harvesting of the eye and dissection of the rpe were carried out as previously described in . Rpe whole mounts were visualized and imaged using a zeiss lumar v.12 stereomicroscope, zeiss axiovision mrm camera, and zeiss axiovision 4.6 software (thornwood, ny). P reversion events were identified on the transparent monolayer of the rpe as pigmented cells or eye spots . Total number of eye spots and number of cells making up that eye spot were recorded for each rpe according to the criteria set forth by bishop et al . In . Additionally, the relative distance from the optic nerve of each eye spot was recorded . This was done by using the measurement tool in adobe photoshop, by first measuring from the center of the optic nerve to the proximal edge of the eye spot and then from the center of the optic nerve to the edge of the rpe . These include tests for normality (shapiro - wilk test), equal variances (fmax test), two group comparisons (mann - whitney test), and contingency tables (fisher's exact test). The frequency of spontaneous hr for mice with helicase domain - deficient wrn protein was previously reported as being increased 2-fold using the p fur spot assay . Though the p fur spot assay can be considered a faithful assay for measuring hr frequency in vivo, the peye spot assay affords many advantages, including being more sensitive to changes in hr frequency and can reveal information about the timing of events during development, developmental patterning, and even information about whether the hr events are associated with replication . Therefore, we set out to recapitulate the fur spot study and to determine whether we might be able to reveal any additional phenotypes associated with the wrn hr events . Surprisingly, when we compared the number of eye spots per rpe in wrn versus wrn (table 1 and figure 2(a)), we were unable to detect a significant increase in the overall frequency of hr events (p = .35, mann - whitney test) (figure 2(b)). The nonparametric mann whitney test was used because our data was found to be not normal (data not shown) with unequal variances using a fmax test (p <.0001). Of interest, the variance within the wrn rpe was larger than expected due to three rpes with higher than usual numbers of reversion events . To determine whether the lack of difference in hr frequency between wrnand wrn was due to the wild - type rpe with elevated hr frequency, we compared the frequency of eye spots of our wrn wild - type rpes with an independent wild - type data set that was recently reported by our laboratory . No statistical difference in p reversion frequency was observed between these two groups of wild - type rpe (data not shown). We therefore combined these wild - type datasets, compared their combined p reversion eyespot frequency with wrn, and still did not observe any statistical difference between genotypes (figure 2(b)). Therefore, it appears that wrn helicase activity is not required for hr, and no additional hr events are instigated by the wrn mutation . We classify eye spots as having either single (1 cell) or multi - cell (2 cells) events . Due to the edge - biased proliferation of the rpe and the apparent position shift between single - cell and multi - cell eye spots, we speculate that multi - cell (clonally expanded) p reversion events are associated with dna replication (discussed below). Approximately 60% of eye spots are normally single cell events . Even though we did not observe an overall difference in hr frequency, we wanted to see if wrn helicase activity affected the clonal expansion of p reversion events . Here we found no significant difference between single versus multi - cell eye spots when comparing wrn versus wrn rpe (p = .39, fisher's exact test figure (3)). These data indicate that the helicase activity of wrn does not affect clonal expansion of mouse rpe cells following hr . The mouse rpe develops radially outward from the optic nerve with an edge - biased pattern of proliferation . The rpe begins to form in the developing eye cup at ~8.5 dpc and continues through the first week of postnatal development [20, 21]. Much like the age of a tree that can be determined using its concentric rings, the retrospective mapping of an eye spot onto an rpe suggests when during development a p reversion event occurred . Previously we have reported mutant genotypes that affected either the timing of p reversion events during rpe development or the pattern of rpe development by examining eye spot patterns [6, 18]. In order to determine if wrn helicase function has a role in hr at a specific point during murine development, each rpe was divided into 10 concentric rings where the inner most ring contains the optic nerve depicting the beginning of rpe development (0.0 - 0.1) to the outer most ring at the edge of the rpe (0.9 - 1.0). At each interval, the pattern of positional distribution was similar for both wrn and wrn for all eye spots (p = .22, chi - square test, figure 4). Of note, the positional analysis of the eye spots measures the distance from the center of the optic nerve to the most proximal cell of an eye spot, irrespective of the number of cells that constitutes the eye spot . These results suggest that the effect of the wrn helicase mutation on hr does not alter the timing or distribution of p reversion events during mouse rpe . In summary, mice expressing a helicase - deficient wrn allele did not have an increase in the frequency of spontaneous hr . Our results differ substantially from earlier work done using this same mouse model with the less sensitive p fur spot assay which observed at least a 2-fold increase in p reversion events . In our experience we have never observed a discrepancy in p reversion frequency between the neural crest - derived melanocyte - dependent fur spot assay and the neural epithelium - derived rpe - based eye spot assay . However, it should be noted that we have not examined the frequency of p fur spots in our c57bl6/j congenic animals, and it is formally possible that the difference may be due to difference in strain background . Our result also differs from a more recent report using the p eye spot assay with the expression of a transgenic dominant negative human wrn allele . This latter report observed a 30% increase in eye spots (from an average of 6 to 8 eye spots per rpe in wild - type and wrn impaired, resp . ), a relatively slight, though significant increase for the p eye spot assay, that could be simply explained by sample size; yamamoto et al . Alternately, the difference could be due to a difference in using a mouse helicase deficient wrn mutant mouse and a mouse model that expressed a transgenic dominant negative allele of human wrn . The p fur spot assay is reliant upon detecting large clonal expansions of melanocytes following p reversion that are visible in the fur (at least 1 mm). In contrast, the p eye spot assay can detect both single cell and clonally expanded (multi - cell) reversion events . Additionally, the p eye spot assay can be used to determine any differences in the timing of hr events during embryonic development . In this study we found that the helicase activity of mouse wrn does not affect the frequency of single versus multi - cell events and nor does it affect the timing of spontaneous hr events during mouse embryonic development . Although ws cells are described as having genomic instability, there is some discrepancy as to the role wrn plays in hr . In support of wrn as a suppressor of illegitimate hr, ws patients exhibit variegated chromosomal translocations, elevated recombination levels between microhomology plasmids, approximate 2-fold increase of rad51 focus formation, and sensitivity to agents that lead to replication stress [5, 2628]. Additionally, wrn is known to associate with proteins tied to replication machinery like rpa [2932], pcna [3335], pol [36, 37], and rad52 . In contrast though, ws cells do not have elevated amounts of sce which are the hallmark of bs . More recent studies have begun to show that recq helicases (e.g., wrn) promote hr via mechanisms like dna resection . Following double - strand breaks (dsbs), the helicase domain of sgs1 (yeast recq orthologue of wrn) is required for resection of dna ends to produce single strand dna substrates for rad51 [40, 41]. Additionally, the helicase function of sgs1 is required for normal kinetics of hr at the mat locus, and yeast mutants for sgs1 and exo1 nuclease exhibit sensitivity to dna damaging at similar levels to rad52 mutants (which are diminished for hr repair). These same authors subsequently went on to demonstrate that the recq helicase blm also has some dna resection functions following camptothecin - induced dsbs and that blm function of dsb resection is in parallel with an exo1-dependent pathway . A separate study investigated the role of wrn following exposure to chromium(vi), an agent known to induce dsbs, and found that chromium(vi) treated human cells depleted of wrn and ws cells had delayed or absent rad51 focus formation . This study again suggests that wrn is important for promoting hr, potentially in early steps of this process (e.g., initiation via resection) following dna damage . Unlike the other human recq helicase members, wrn also has exonuclease activity, so understanding which enzymatic function of wrn is involved in promoting hr is valuable to our knowledge of this protein, as well as insightful to the syndrome . As it pertains to this study, we found that the helicase function of wrn is neither necessary for, nor suppresses spontaneous hr . With regard to other recq helicases, we recently found hr to be significantly elevated in the absence of blm using the p eye spot assay (data not shown). Considering the lack of effect observed in our study, and only a mild suppressive effect in the yamamoto et al . Study compared to a deficiency in blm (data not shown), it would appear that wrn only plays a minor role in suppressing hr, possibly the result of redundancy amongst the different recq family members . Together, these studies give insight into the potential differences between two of the known five human recq helicases and suggest that future studies are warranted to better understanding the functions of wrn (and blm) in hr.
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For the past several decades, the mainstream scientific opinion on healthy diets and the recommendations for dietary intake have favored high - carbohydrate and low - fat (hclf) diets . This position has led most countries in the world to issue dietary guidelines in favor of lowering dietary fat and increasing starch and fiber intake (myers et al ., 2013). These guidelines have largely been followed as dietary carbohydrate content has gradually increased at the expense of fat (johnston et al ., 2014). Despite the scientific and dietary progress in the recent decades, overweight and obesity have been rising among adults worldwide in parallel with type 2 diabetes (shaw et al ., 2010) and cardiovascular disease (naghavi et al ., while the mainstream science has advocated hclf diets, in recent decades a group of scientists, practitioners, and the general public have explored the efficacy of low - carbohydrate - high - fat (lchf) diets such as the atkins diet as a means of weight loss (gudzune et al ., 2015). In addition, a number of clinical studies revealed the beneficial effects of lchf diets on a wide range of metabolic risk factors exceeding, or at least matching, those of hclf diets . A less well explored question is whether lchf diets may exert beneficial effects on exercise and athletic performance . Despite the concept that eating a lchf diet goes counter the traditional view that athletes require high - carbohydrate intake to maintain sufficient muscle glycogen for high - intensity and endurance performance there is emerging scientific evidence that lchf diets at least maintain, if not enhance, endurance performance while at the same time improving body composition compared to hclf diets . These results have raised the awareness that a re - examination of lchf diets for sport performance is necessary (burke, 2015). The aims of this review were to examine the evidence for the efficacy of lchf diets in body composition, performance in endurance and high - intensity exercise, central fatigue, and perceptual - motor ability in athletes . Lchf diets, or ketogenic diets, usually contain less than 20% of energy from carbohydrate, more than 50% of energy from fat, and variable amounts of protein (hession et al.,2009; hu et al . Several studies used extreme lchf diets that contained less than 5% of carbohydrate (langfort et al ., 2004; martin et al ., 2011; paoli et al ., 2012) lchf diets usually lead to ketosis when the liver oxidizes high concentrations of non - esterified fatty acids (nefa) into ketone bodies, including 3-hydroxybutyrate, acetoacetate, and acetone (owen et al ., 1967; paoli et al ., 2015). The physiological ketosis from lchf diets results in blood ketone body concentration of around 7 - 8 mm and blood ph of 7.4 (paoli et al ., 2015). Although the brain prefers glucose as the main energy source, it can metabolize ketone bodies as fuel for long periods of time during starvation and hypoglycemia (owen et al ., 1967). Muscles and other organs also are able to oxidize ketone bodies as an alternative source of energy when carbohydrate supply is limited . Oxaloacetate becomes a limiting factor for fat oxidation after several days of lchf diets because of inadequate glucose availability . In order to maintain citric acid cycle function, oxaloacetate has to be provided by deamination of glucogenic amino acids such as aspartate and asparagine . Therefore, daily consumption of between 1.3 to 2.5 g / kg of protein is necessary to ensure the maintenance of muscle mass, gluconeogenesis and fat oxidation when consuming lchf diets (paoli et al . Many studies have shown that consuming a lchf diet over months or years does not lead to metabolic imbalances or serious adverse effects provided that it supplies sufficient energy and adequate amounts of protein (bueno et al ., 2013; hession et al ., 2009; hu et al ., 2012; paoli et al ., 2015). Contrary to the popular concept that diets high in fat would increase the risk for obesity, cardiovascular disease, and diabetes, several meta - analysis and systematic reviews document that long - term lchf diets actually reduce these metabolic risk factors (bueno et al . Hession et al ., 2009; hu et al ., 2012; sharman et al ., 2004; volek et al ., several systematic reviews and meta - analyses have shown that long - term lchf diets combined with reduced energy consumption are more effective than, or at least as effective as, hclf diets in reducing body weight and fat mass in overweight and obese populations . (hession et al ., 2009) and - 1.05 kg (bueno et al ., 2013) compared to hclf diets after 12 months or longer . Although the advantage of lchf diets over hclf diets in reducing body weight may seem small, it is crucial in weight - sensitive sports . Deliberate pre - match weight loss is practiced to a great extent by athletes in such sports (brito et al ., 2012; franchini et al ., 2012 . However, extreme energy restriction and dehydration practices implemented by these athletes to achieve weight loss produce acute negative energy balance, carry serious health risks, and thus may be detrimental to athletic performance (khodaee et al ., 2015). Even though some athletes may maintain physiological performance after rapid weight loss, the detrimental effects on psychological functions and mood states are still present (marttinen et al ., 2011). The potential of lchf diets in reducing body weight and fat mass has drawn little attention from the scientific and athletic community . A cross - over study revealed that after 4 weeks of a lchf diet, body weight and fat mass were significantly decreased in well - trained offroad cyclists (zajac et al ., 2014). Consuming a hypocaloric lchf or hclf diet for 3 weeks resulted in similar decreases in body weight and fat mass in high school taekwondo athletes (rhyu and cho, 2014). In addition to fat mass, the effect of lchf diets on fat - free mass is important to athletic success . In studies that used hypocaloric diets with the aim to induce weight loss in overweight or obese subjects, lchf and hclf diets resulted in similar degrees of loss in fat - free mass (brinkworth et al . In a study that encouraged normal - weight subjects to consume adequate energy to maintain body weight, a lchf diet led to a significant increase in fat - free mass, while a western diet did not change body composition (volek et al ., 2002). Few studies investigated the effects of the combination of resistance training and lchf diets on body composition . A study in overweight women suggested that a lchf diet in combination with resistance training reduced body weight and fat mass while maintaining lean body mass, whereas resistance training in combination with a regular higher - carbohydrate diet increased fat - free mass but maintained fat mass (jabekk et al ., 2010). These changes are at least partially due to reduced anti - lipolytic insulin action and protein - protective action of increased total and free thyroxine index after a lchf diet (volek et al ., 2002). Furthermore, a lchf diet led to a similar magnitude increase in the muscle protein synthesis rate, activation of amp - activated protein kinase and 4e - binding protein-1 compared to a hclf diet after resistance exercise in rats (roberts et al ., 2016) therefore, it is possible that lchf diets, in combination with resistance training, can maintain fat - free mass while receiving the benefit of loss in fat mass and body weight . It is well - known that the maximal fat oxidation rate is reached at moderate - intensity exercise corresponding to 59 - 64% vo2max in endurance - trained individuals, and 47 - 52% vo2max in the general population . The fat oxidation rate drops significantly above this exercise intensity and is almost zero above 90% vo2max (achten and jeukendrup, 2004). Nefa availability is one of the limiting factors for fat oxidation during exercise . At 80% of maximal effort, nefa utilization is 30% lower than at 65% of relative effort, while muscle glycogen utilization is increased (romijn et al ., 1993). Elevating plasma nefa by infusion of medium - chain or long - chain fatty acids significantly increases the fat oxidation rate during exercise at 40 to 80% vo2max . However, endurance exercise capacity is similar despite these metabolic changes (hawley, 2002). Excess availability of plasma nefa by infusion of intralipid and heparin during exercise at 85% vo2max restores only about a quarter of the decline in the fat oxidation rate from the maximal 43 mol / kg / min at 65% vo2max, indicating that other factors are involved in regulating fat oxidation during high - intensity exercise (romijn et al ., these other factors may include enzymes responsible for fatty acid transport across mitochondrial membrane and the free carnitine pool (jeukendrup, 2002; stephens et al ., 2007). Sport nutritionists have long advocated high - carbohydrate diets for athletes to ensure sufficient muscle glycogen during exercise . The inference that hclf diets are better than lchf diets for endurance performance was generated in studies contrasting the short - term (less than 2 weeks) effects of exposure to hclf and lchf diets . Indeed, in the absence of long - term adaptation, reduced muscle glycogen content after lchf diets leads to hypoglycemia, impaired endurance performance (karlsson and saltin, 1971; walker et al ., 2000), and an increased feeling of fatigue (white et al ., 2007). However, endurance performance, measured by time trials after fixed - intensity prolonged exercise, was maintained in elite cyclists when muscle glycogen was restored with a 1-day high - carbohydrate diet after 5 - 6 days of a lchf diet (burke and hawley, 2002). Even such short - term consumption of a lchf diet resulted in a significantly higher fat oxidation rate of 0.7 to 0.8 g / min and a lower carbohydrate oxidation rate of 2 to 2.3 g / min during exercise at 70% of vo2max in these highly - trained endurance athletes, compared to those who consumed a hclf diet during the study period (burke and hawley, 2002). The glycogenolysis rate and pyruvate dehydrogenase activity were also lower compared to a hclf diet, during prolonged and sprint cycling after such dietary intervention (stellingwerff et al ., 2006). Long - term adaptation to lchf diets produces even greater metabolic benefits, including a higher rate of fat oxidation and lower rates of carbohydrate oxidation and glycogenolysis . Endurance athletes who adapted to lchf diets for 9 - 36 months could reach the maximal fat oxidation rate of approximately 1.5 g / min at about 70% vo2max (volek et al ., 2016). This value is higher than what carbohydrate - adapted endurance athletes ever reported (~1.0 g / min) (venables et al ., 2005). Several researchers have suggested that long - term lchf diets may be as effective, or even beneficial in many aspects of endurance performance as hclf diets while providing several metabolic advantages in athletes (brukner, 2013; burke, 2015; noakes et al ., 2014; paoli et al ., 2015; volek et al ., 2015). Large decreases in the carbohydrate oxidation rate (from 15.1 to 5.1 mg / kg / min) and muscle glycogen utilization rate (from 0.61 to 0.13 mmol / kg / min) without compromising the time to exhaustion at moderate intensity were found after consuming a lchf diet for 4 weeks in well - trained cyclists (phinney et al ., 1983). Even though these athletes had lower resting muscle glycogen levels after consuming a lchf diet, the post - exercise level of muscle glycogen was similar to that before the dietary intervention . Surprisingly, a recent study (volek et al ., 2016) revealed that ultra - endurance athletes who had consumed lchf diets (<20% energy from cho,> 60% from fat) for at least 6 months achieved higher peak exercise intensity supported by fat oxidation than athletes on hclf diet (lchf: 70.3 6.3; hclf: 54.9 7.8% vo2max). These fat - adapted athletes also showed a higher fat oxidation rate (lchf: 1.54 0.18; hclf: 0.67 0.14 g / min) and a lower carbohydrate oxidation rate during prolonged exercise at 64% vo2max . They also had similar muscle glycogen contents at rest (approximately 140 mol / g wet tissue) and immediately after a 3-hr run at 65% vo2max (approximately 50 mol / g wet tissue) compared to their counterparts consuming normal or hclf diet . It is noteworthy that the glycogen resynthesis during the 2-hr post - exercise recovery remained the same in both groups (lchf: 44.8 7.5; hclf: 34.6 23.9 mol / g wet tissue) despite the fact that the lchf group consumed only 5% carbohydrate, while the hclf group consumed 50% carbohydrate during that period . This study indicated that endurance athletes could maintain normal muscle glycogen content, utilization and recovery after long - term adaptation to lchf diets . Thus, it has been hypothesized that long - term lchf diet may enhance performance in ultra - endurance events such as the ultra - marathon and ironman triathlon by supporting a higher fat oxidation rate at higher relative exercise intensity and by having a glycogen sparing effect (langfort et al ., 1996). In these events, the lchf - adapted athletes may be able to maintain higher relative exercise intensity during most of the distance, while preserving muscle glycogen for sprints at the later stage of competitions . Future studies may evaluate endurance performance in lchf - adapted athletes using a race - like design, rather than constant workload protocols . Subcellular changes that support skeletal muscle adaptation to lchf diets include upregulation of enzymes involved in fatty acid oxidation such as -hydroxyacyl coa dehydrogenase (cameron - smith et al ., 2003), fatty acid translocase / cd36 (cameron - smith et al ., 2003), and carnitine palmitoyl transferase-1 (goedecke et al ., 1999), and downregulation of enzymes supporting carbohydrate oxidation such as pyruvate dehydrogenase (chokkalingam et al ., 2007). These enzymatic changes after lchf diets may account for the increase in the fat oxidation rate . The potential effects of long - term adaptation to lchf diets on endurance performance are presented in figure 1 . Potential mechanisms to improve endurance and repeated high - intensity exercise after long - term adaptation to low - carbohydrate - high - fat diets exercise above 70% vo2max requires significant energy from the anaerobic metabolism (brooks and mercier, 1994). A single bout of short - term high - intensity exercise mostly utilizes energy from creatine phosphate and glycolysis (gaitanos et al ., 1993b). Therefore, an elevated fat oxidation rate after adaptation to a lchf diet is unlikely to increase performance in this mode of exercise . In many field - based sports such as soccer, rugby, basketball, and hockey, the ability to perform multiple sprints at the highest speed after short rest is crucial for game performance (rampinini et al ., 2007; ross et al ., 2015; spencer et al ., 2005). Although a single sprint relies mostly on the anaerobic metabolism, repeated sprints significantly increase the demand for the aerobic metabolism during the later stages of exercise . In 10 6-s sprints separated by 30 s rest periods, the anaerobic metabolism provided most energy in the first sprint, but it fell to approximately 60% in the last sprint (gaitanos et al ., the proportion of energy from the aerobic metabolism increased from almost zero in the first sprint to 40% in the last one (girard et al ., 2011). A 4-fold increase in plasma glycerol in combination with a much smaller increase in plasma nefa during intermittent sprints also indicated a significant contribution of fat utilization and the aerobic metabolism (chang et al . In addition, subjects with greater aerobic capacity are more able to maintain power output during later stages of repeated sprints (bishop and edge, 2006; brown et al ., 2007). Thus, the increased fat oxidation rate and muscle - glycogen sparing effect after long - term adaptation to lchf diets may be helpful to maintain and/or improve performance in latter stages in these sports . Only a few studies have examined the impact of lchf diets on high - intensity exercise . Several of them failed to account for the changes in body weight and/or body composition associated with lchf diets when evaluating exercise performance . Maximal repetitions of exercises that support body weight, such as push - ups and chin - ups, are widely used for regular monitoring of strength performance . However, performance of these exercises depends on strength as well as endurance factors and can be affected by body weight and composition . The maximal height in squat jumps can also be affected by body weight and composition . Thus, caution should be taken in interpreting the results that are not normalized to body weight . It has been shown that elite male gymnasts maintained maximal repetitions of push - ups, reverse grip chins, and parallel bar dips after 30 days of a lchf diet while experiencing significantly reduced body weight and fat (paoli et al ., 2012). In another study, combining a lchf diet for 3 weeks with a 25% energy restriction resulted in the loss in lean body mass and impairment in anaerobic performance measured by the wingate test in high school taekwondo athletes (rhyu and cho, 2014). It is noteworthy that performance requiring greater endurance capacity such as a 2000 m sprint, and a fatigue index in the wingate test were actually increased in the lchf group . Zajac et al . (2014) reported that maximal power output was significantly decreased after consuming a lchf diet for 4 weeks in well - trained off - road cyclists . However, the lchf diet also resulted in lower body mass and body fat, and increases in vo2max and vo2 at lactate thresholds . The decrease in maximal power output, expressed as w, was not normalized to body weight to account for its change after the diet manipulation (zajac et al ., 2014). Future studies should normalize the results to body weight or fat - free mass in power and strength performance, as in high - intensity repetitive exercise, such as w / kg in squat jumps, in order to clarify the changes . The potential effects of long - term adaptation to lchf diets on repeated high - intensity exercise performance are shown in figure 1 . More research is needed to elucidate whether long - term adaptation to a lchf diet would affect repeated sprint performance in field - based sports, especially in later stages of the competition . In addition to the aforementioned physiological changes in the muscle, the metabolic changes induced by long - term lchf diets may also affect the central nervous system during exercise (figure 2). The role of the central nervous system in the development of physical fatigue has long been recognized (asmussen, 1993; gandevia, 2001). Alterations in the metabolic fuel use during exercise after adaptation to a lchf diet can affect cerebral amino - acid uptake, the energy metabolism, and neurotransmission . The increased rate of fat oxidation during exercise after adaptation to a lchf diet is likely to increase brain uptake of free tryptophan . This is the consequence of increased competition for binding to albumin by rising concentrations of nefa . Free tryptophan is the precursor of serotonin (5-hydroxytryptamine), a brain neurotransmitter associated with the feeling of lethargy and tiredness that may contribute to the loss of central drive and motivation (davis and bailey, 1997). Increased brain uptake of free tryptophan has been reported to favor cerebral serotonin synthesis and contribute to central fatigue (pardridge, 1998). Potential effects of long - term adaptation to low - carbohydrate - high - fat diets on central fatigue and perceptual - motor performance . High protein content of lchf diets also leads to elevated ammonia production during exercise (maclean et al ., 1996; ammonia is another factor that could induce central fatigue by altering the cerebral energy metabolism and neurotransmission, and affect signaling pathways within the neural circuits (mutch and banister, 1983; wilkinson et al ., subjects adapted to lchf diets experienced higher plasma concentrations of nefa and ammonia, two agents contributing to central fatigue, during exercise at various intensities (langfort et al . A pilot study with untrained adults consuming a hypocaloric lchf diet for 2 weeks suggested that increased blood concentration of ketone bodies was associated with the feeling of fatigue and disturbance of the mood during submaximal exercise, indicators of central fatigue (white et al ., 2007). There is currently a knowledge gap regarding the possible effect of long - term adaptation to lchf diets on central fatigue in various types of exercise that is in need of additional research . Perceptual - motor performance, i.e., the ability to better anticipate opponent s movements, plan appropriate strategies and activate the musculoskeletal system to execute necessary actions, is a major factor that separates experts from non - experts in many sports (north et al ., 2009). Consuming lchf diets for 1 - 2 years was reported to slightly improve certain cognitive functions and mood states in middle - aged overweight subjects, compared to hclf diets (brinkworth et al . A recent animal study also suggested that ketone bodies may protect against obesity - induced cognitive impairment (davidson et al ., 2013). However, these studies used simple cognitive tests that are not related to athletic competitions . There is evidence that central fatigue may contribute to the decline in perceptual - motor performance during exhausting exercise (chen et al ., 2016; yang et al ., 2016) whether a long - term lchf diet would increase central fatigue and subsequently impair perceptual - motor performance in athletes is still unclear . Long - term lchf diets appear to be safe and may even improve several metabolic risk factors for chronic diseases in the general population . Lchf diets provide a promising way to help control body weight and fat mass while maintaining lean body mass in athletes engaged in weight - sensitive sports . There is emerging evidence that lchf diets could be beneficial, particularly for performance in ultra - endurance sports . Their effect on field - based sports that require repeated high - intensity activities is also promising . It appears that at least several months of adaptation to a lchf diet are required for the metabolic changes and restoration of muscle glycogen to occur . However, some aspects regarding the effects of long - term lchf diets in athletes are still unexplored and in need of investigation, including: strength, power, psychological status, and perceptual - motor performance after weight loss, especially in weight - categorized sports such as wrestling, judo and taekwondo.performance in repeated high - intensity exercise in field - based sports such as soccer and basketball.the development of central fatigue during endurance events.perceptual-motor performance during prolonged intermittent sports such as tennis and soccer.the ideal composition of saturated, monounsaturated and polyunsaturated fatty acids in lchf diets . Strength, power, psychological status, and perceptual - motor performance after weight loss, especially in weight - categorized sports such as wrestling, judo and taekwondo . Performance in repeated high - intensity exercise in field - based sports such as soccer and basketball.
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Obesity is a condition that causes damage to the various body functions, such as cardiovascular, musculoskeletal, and metabolic functions amongst others . The respiratory function is also affected by obesity, as excess fat deposited on the chest wall and the abdominal cavity affects the chest mechanics . This results in increased work of breathing, reduced lung volumes, dysfunction of the respiratory muscle, impairment in gas exchange, and reduced exercise tolerance [29]. A few studies have demonstrated that weight loss due to bariatric surgery has resulted in a huge improvement in some functions, such as decrease in hemoglobin and hematocrit, decreased heart rate and oxygen consumption, and reduction of insulin resistance . In addition, especially improved lung function with increased forced vital capacity (fvc) [3, 12, 13] and forced expiratory volume in one second (fev1), improved alveolar - capillary diffusion capacity and improvement in gas exchange [12, 13] have also been observed . There is strong evidence supporting the increase in fvc and erv (expiratory reserve volume) after weight loss [3, 12, 13]. However, controversy still persists regarding the behavior of the respiratory muscle strength and irv (inspiratory reserve volume) after weight loss caused by the roux - en - y gastric bypass surgery . The objective of the present study was to evaluate the effect of weight loss, after 1 year of the roux - en - y gastric bypass surgery (rygb), on the lung volumes and the respiratory muscle strength in obese women . Were recruited 24 obese women candidates for rygb at the meridional hospital . Patients with body mass index (bmi) 3550 kg / m were included if they met the minimal criteria for bariatric surgery proposed by the world health organization (who) report of 2000 . The following were not included in the study: patients suffering from pulmonary diseases or those unable to carry out the pulmonary function tests adequately, smokers, patients who did not attend the reevaluation 1 year after surgery, and patients refusing to sign the informed consent term . The present study was approved by the meridional hospital ethics committee (protocol number 01/07). The evaluation of the pulmonary function was carried out by conventional spirometry using a personal computer version of the ndd easyone spirometer model 2001 (medizintechnik ag, zurich, switzerland). Parameters, such as volume, capacity, and flow of the lungs, were directly evaluated by using the slow vital capacity (svc), the forced vital capacity (fvc), and the maximum voluntary ventilation (mvv) tests, with volunteers in a sitting position and a minimum of three repetitions, as recommended by the american thoracic society (ats) and the european respiratory society (ers). The obtained results were expressed in absolute values and as percentages of the predicted reference values for the brazilian population . The svc test produced the following variables: vital capacity (vc), tidal volume (vt), inspiratory reserve volume (irv), and expiratory reserve volume (erv). The fvc test allowed the determination of the forced expiratory volume in 1 s (fev1) and the fev1/fvc ratio . The mvv, a variable that evaluates the respiratory endurance, was expressed in liters per minute and as a percentage of the predicted reference value for the brazilian population . The respiratory muscle strength was determined through the maximal static respiratory pressures measured during forced inspiration and expiration maximal inspiratory pressure (mip) and maximal expiratory pressure (mep). The measurement was carried out using an aneroid manometer (wika, iper, sp, brazil), calibrated in centimeter h2o (300 cm h2o), and equipped with a 2 mm hole to relieve the oral pressure . Mip and mep were determined using the residual volume and the total lung capacity, respectively, with the subjects in a sitting position . Patients carried out at least three acceptable inspirations / expirations wearing a nose clip for determining the two reproducible inspirations / expirations . The mip and mep values were also expressed as percentages of the predicted values, according to the equation proposed by neder et al . . The patients were evaluated in the preoperative period, and one year after surgery, they were asked to return for a reevaluation of the pulmonary function tests, by the spirometry and respiratory muscle strength . The data collected were expressed as mean standard deviation and analyzed by the shapiro - wilk test . After verifying the normal distribution of the variables, the paired t - test was used to compare the preoperative and 1-year postoperative results . The pearson correlation was used to correlate these variables: w / h ratio and erv; irv and mip . The sample size had an 80% power at the 5% level of significance with mep as the main variable . The characteristics of the patients, such as age, bmi, weight, and w / h ratio, are shown in table 1 . There was a significant reduction in the values of weight, bmi, and w / h ratio 1 year after the surgery . The bmi value returned to normal in 11 patients; in the other 11 patients, it lowered to the range of overweight (2530 kg / m), and only 2 patients remained obese, despite significant reduction in the bmi one year after surgery . Before surgery, 12 patients had hypertension, 7 had dyslipidemia, and 8 had diabetes . One year after the surgery, 6 patients continued with hypertension and 2 with dyslipidemia . On analyzing the variables that measure lung volumes, it was observed that there was a significant increase in the vc, fvc, and fev1 . However, when examining the components of the vc separately, an increase in erv and reduction of irv, keeping the vt unchanged 1 year after surgery, was observed . Furthermore, respiratory endurance assessed by mvv also increased after weight loss (table 2). Moreover, in assessing respiratory muscle strength, a reduction statistically significant in the values of mip and mep was recorded (table 3). There was a significant negative correlation between w / h ratio and erv (r = 0.37; p = 0.01) (figure 1) and a significant positive correlation between mip and irv (r = 0.41; p = 0.004) (figure 2). Based on the obtained results, it was established that 1 year after the rygb surgery, the patients showed a significant reduction in the measures of weight, bmi, and w / h ratio, especially changes in the lung function tests, such as spirometry and respiratory muscle strength . Studies by some authors [9, 12, 18] have shown an improved lung function in patients evaluated after 1 year following weight loss induced by bariatric surgery, and others have attributed this improvement mainly to the reduction in the w / h ratio . (2005) found that the patients showed an improvement in dyspnea and a reduction in the respiratory drive after weight loss induced by bariatric surgery . In the present study, one year after surgery, the patients showed increased lung volumes and decreased the respiratory muscle strength . With respect to the volumes, an increase in vc, fvc, and fev1 could be observed after weight loss . Other authors have also found similar results in previous studies [9, 12, 18, 2022]. However, in this study, a finding still not published and discussed in the literature was noted: the irv reducing associated with an increase of erv after weight loss . The reduction of erv is a major known change in the respiratory function caused by obesity . According to koenig (2001), this is because the diaphragm is pressed upwards due to the expanded abdominal volume and w / h ratio of the obese individuals, which is a mechanical disadvantage for this muscle . Besides these detrimental mechanics aspects to the pulmonary function of obese individuals, young et al . 2003 also suggested that the reduction of the erv could lead to an increase in areas of atelectasis . As a result, the ventilation / perfusion mismatch could be harmed, thereby leading to arterial hypoxemia in those individuals . The reduction of w / h ratio after weight loss may have contributed to improvement in chest mechanics and consequent increase in erv, as demonstrated by the significant correlation between these variables (r = 0.37; p = 0.01) (figure 1). (2008) compared the pulmonary function in the obese and nonobese subjects, and the authors found a higher irv and lower erv in the first and the opposite in the other one, with no significant changes in the vc values between the groups observed . According to the authors, this is due to the problems in the chest mechanics of obese individuals, which could have resulted in a compensatory increase in irv by reducing the erv caused by obesity, while retaining an unchanged cv . Thus, in the present study there was a tendency to return to the patterns of distribution of the lung volumes of the non - obese individuals in studied patients . Therefore, the obtained results suggested that the weight loss induced by bariatric surgery altered the chest mechanics, by a rearrangement of the volumetric lung compartments inside the rib cage, especially for abdominal decompression after weight loss of these obese patients . El - gamal et al . (2005) evaluated obese patients in the preoperative and 1 year after bariatric surgery and found that the low value of erv is related to increased of respiratory drive and dyspnea, with improvement in these parameters after weight loss . These results help to consolidate the hypothesis that obesity, by reducing erv, leads to a respiratory overload (inspiratory mainly), verified by the increase in dyspnea and respiratory drive, leading to increased of irv . And finally, after weight loss these changes are reversed . Besides these changes of the chest mechanics due to weight loss, the reduction of inspiratory muscle strength (mip) may also have contributed to the reduction of irv, since the inspiratory muscles are responsible for expanding the rib cage and promote entry of air into the lungs . This finding can be confirmed by the significant and positive correlation between these variables mip and irv (r = 0.41; p = 0.004) (figure 2). The respiratory muscle strength is also an important variable that influences the lung function, and the available data in the literature are still few and controversial about this variable after weight loss . (1998) also measured mip and mep after bariatric surgery and found an increase in these variables . (1991) found a reduction in the respiratory muscle strength after weight loss induced by bariatric surgery, in agreement with the results of the present study . One explanation for this finding could be the loss of lean body mass after bariatric surgery, as described by some authors [2528]. The average weight loss with rygb is 30%, which is similar to the results of the present study . However, this loss is not only fat of mass but also lean body mass . Stegen et al . Found a reduction in the lean body mass associated with a reduction of static and dynamic muscle strength, whereby the authors suggested that physical activity prevents the reduction of muscle strength after bariatric surgery . However, one limitation of the present study was that it did not evaluate the lean body mass of the patients . Another hypothesis is that the reduced work of breathing in the obese individuals achieved with weight loss, no longer exerted training on the respiratory muscles of these individuals, thereby reducing the values of the respiratory muscle strength . Despite a trend to decrease the maximal static respiratory pressures mip and mep, there was an increase in mvv (a variable that evaluates the respiratory endurance). This fact can be justified as the weight loss promotes an improvement of the chest mechanics, increases the lung volumes [9, 22], and reduces the work of breathing . Based on the obtained findings, it was concluded that weight loss induced by bariatric surgery provides an improvement in the ventilatory mechanics, as evidenced by the increase in lung volumes (erv, vc, fvc, and fev1) and respiratory endurance (mvv) of obese women . Furthermore, the reduction in irv appears to show a trend distribution of static lung volumes in the pattern as seen in the nonobese patients . Additionally, there was also a reduction in the respiratory muscle strength, which could be caused by a loss of lean body mass and a reduction in the work of breathing after weight loss.
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Although there is no direct link between exposure to radio waves and the genotoxic mechanisms that lead to cancer, studies to address the association between use of cellular telephones and cancer risk have been performed . Case - control study of uveal melanoma patients in germany using three groups of control subjects: population - based, siblings of patients, and ophthalmology control subjects . No association between cellular telephone use and uveal melanoma risk was observed . Using cellular telephones subjects who agreed to participate in the study were more likely to regularly use cellular telephones than subjects who did not agree to participate . Although there is no direct link between exposure to radio waves and the genotoxic mechanisms that lead to cancer, studies to address the association between use of cellular telephones and cancer risk have been performed . Case - control study of uveal melanoma patients in germany using three groups of control subjects: population - based, siblings of patients, and ophthalmology control subjects . Subjects who agreed to participate in the study were more likely to regularly use cellular telephones than subjects who did not agree to participate . German science foundation (dfg) (grants kfo 109/1 - 1 and 444 usa 111/7/06 to as); federal radiation protection agency (bfs) (grant m 8811 to as).
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Biofilm growth on the surface of biomaterial implants is generally recognized as a cause of biomaterial - associated infection (bai). Regardless of the high sterile conditions and improved techniques in the operating theatre, both perioperative and postoperative contamination by microorganisms suspended in the air and from the skin flora continue to be the most common pathway for the contamination of biomaterial implants and medical devices [1, 2]. The biofilm mode of growth protects the organisms against the host defense system and antibiotics . Therefore complete removal of an infected implant or device is often the final result of bai . Bai starts with the initial adhesion of microorganisms and then subsequently grows to form a biofilm . . Showed that bacterial adhesion was on materials with different wettabilities . A hydrophilic polymer brush coating is included, since these have been shown to discourage microbial adhesion . Several attempts have been made to develop nonadhesive coatings, such as polymer brush coatings, in order to prevent bacterial adhesion and subsequent biofilm growth [8, 9]. Polymer brushes are end tethered polymer chains, having high density of chains per unit surface area due to which it stretches away from a surface into the adjacent solution . Polyethylene oxide (peo) brush coating forms a highly hydrated layer of chains that is compressed upon bacterial approach, leading to a repulsive osmotic force and weak repulsive forces and reduced mobility of the polymer chains . Though most types of brush coatings show significant reductions in microbial adhesion [1113], bacteria adhere more weakly to the surface, being capable of growing into a mature biofilm . Moreover these brush coatings only prevent adhesion and are incapable of killing the bacteria present . Nanoparticles are less than 100 nm in diameter and as a result properties such as surface area, chemical reactivity, and biological activity alter dramatically . The antibacterial efficacy of metal nanoparticles has been suggested to be due to their high surface - to - volume ratio rather than to the sole effect of metal - ion release . A high surface - to - volume ratio is generally accompanied by increased production of reactive oxygen species, including free radicals [17, 18]. These characteristics allow nanoparticles to interact closely with microbial membranes, damaging their structure and inactivate bacteria . Metal oxide nanoparticles are of particular interest as antibacterial agents, as they can be prepared with extremely high surface areas and unusual crystalline morphologies with a high number of edges and corners and other potentially reactive sites . Iron - oxide nanoparticles are a special class of metal oxide nanoparticles with unique magnetic properties and superior biocompatibility . Therefore, the aim of the study was to evaluate the effect of iron - oxide nanoparticles over biofilm formation on different biomaterial and polymer brush coated surfaces . Poly(methyl methacrylate) (pmma) (industrial insulation, chennai, india), polystyrene (ps) (industrial insulation, chennai, india), tissue culture polystyrene well plates (tcps) (nest biotech co. ltd ., china), glass slide (gs, control), and surfaces (pmma and tcps) coated with a hydrophilic polyethylene oxide (peo) layer were used . All samples except hydrophilic peo coating and tcps hydrophilic peo - coated surface (polymer brush coating) was prepared by first cleaning the surfaces in sterile water, ethanol, and water again and finally washing with sterile water . Exposure to a solution of 1 g / l pluronic f-68 solution (himedia laboratories pvt . Mumbai, india) in phosphate - buffered saline (pbs: 10 mm potassium phosphate, 0.15 m nacl, ph 7.0) for 20 min created a hydrophilic polymer brush coating over the surface . The wettability of the surfaces was determined by water contact angle measurements at room temperature with an image analyzing system, using sessile drop technique . 4 ml of ferrous chloride and 1 ml of ferric chloride were added to a flask . Initially formed brown precipitate with time should be changed into a black precipitate, indicating the formation of iron - oxide nanoparticles . The size of the synthesized particles was determined using transmission electron microscopy (tem). The optical measurement of the nanoparticles was studied by uv - visible spectrophotometer (unico) over the spectral range of 2001000 nm . Bacterial strains used in this study were obtained from the culture collection of the centre for drug discovery and development, sathyabama university, chennai, india . Bacteria were first grown aerobically overnight at 37c on blood agar from a frozen stock . One colony was inoculated in 10 ml of tryptone soy broth (tsb; hi media, mumbai, india) and cultured for 16 h. bacteria were harvested by centrifugation at 3000 rpm for 5 min . Bacterial adhesion was performed on six different surfaces (gs, ps, pmma, polymer brush coated pmma, tcps, and polymer brush coated tcps). Each well was filled with 1 ml of bacterial suspension and allowed to adhere and grow aerobically at 37c for 30 min . Subsequently, wells were washed with sterile phosphate buffer saline (10 mm potassium phosphate, 0.15 m nacl, ph 7.0) to remove unbound bacteria and images were taken using phase contrast microscopy and the number of adherent bacteria per cm was determined using imagej software . Bacterial cultures were inoculated to the agar plates and incubated at 37c for 30 min . Holes were filled with 100 l of iron - oxide nanoparticles (0.01 mg / ml, 0.05 mg / ml, 0.10 mg / ml, and 0.15 mg / ml) and incubated at 37c for 24 h. the antibacterial activity was assessed by measuring the zone of inhibition . In this study, . 1 ml of bacterial suspension was added to each well and allowed to adhere and grow aerobically at 37c for 30 min . Then, iron - oxide nanoparticles were introduced in different concentrations (0.01 mg / ml, 0.05 mg / ml, 0.10 mg / ml, and 0.15 mg / ml). Thereafter, biofilms were allowed to grow for 24 h. subsequently, wells were washed with sterile water to remove unbound bacteria and biofilm development was assessed by measuring the optical density using spectrophotometer . To this end, 500 l of 0.1% crystal violet staining was added to each well . The wells were washed with sterile water and 33% acetic acid was added to each well . The optical density (absorbance at 590 nm) was performed followed by a tukey's hsd post hoc test and a p value <0.05 was considered to be significant . The water contact angles of biomaterial and polymer brush coated surfaces are shown in figure 1 . The biomaterial surfaces extend over a wettability range from 52 to 73. the polymer brush coating on pmma and tcps has an average wettability of 36 and 41, respectively . The tem images of synthesized iron - oxide nanoparticles are shown in figure 2(a). The uv - visible spectrum of iron - oxide nanoparticles was shown in figure 2(b) where the absorbance of nanoparticles steadily decreases with time which confirms the formation of oleic acid coated iron - oxide nanoparticles . Initial adhesion of bacteria after 30 min of incubation was significantly (p <0.05) reduced on polymer brush coated surfaces compared to bare surfaces (figure 3). This trend holds good for all the three bacteria (staphylococcus aureus, escherichia coli, and pseudomonas aeruginosa) on both pmma and tcps surfaces . The zone of inhibition of iron - oxide nanoparticles was directly proportional to the increase in concentration of iron - oxide nanoparticles (table 1). At 0.15 mg / ml of iron - oxide nanoparticles, the highest inhibition (29 mm) was observed in s. aureus compared to e. coli and p. aeruginosa . Influence of iron - oxide nanoparticles at different concentrations against biofilm growth on polymer brush coated surface was shown in figure 4 . Significant reduction (p <0.05) in biofilm growth on all the three bacteria was observed in the presence of iron - oxide nanoparticles compared to control (absence of iron - oxide nanoparticles). The highest reduction (p <0.05) was observed in the presence of iron - oxide nanoparticles at 0.15 mg / ml compared to other concentrations (0.01 mg / ml, 0.05 mg / ml, and 0.1 mg / ml) and control . This paper presents the experimental study on the bacterial adhesion and biofilm growth on various biomaterials including polymer brush coated surfaces and the strategy of using iron - oxide nanoparticles in eradication of biofilms . Staphylococcus aureus, escherichia coli, and pseudomonas aeruginosa are the frequently isolated pathogens from infections related to biomaterials implant surfaces . Therefore, these pathogens were considered in our experiments . Amongst other material properties, studies showed that staphylococci adhesion to different biomaterials showed no differences irrespective of differences in wettability, whereas in our study a significant reduction in bacterial adhesion after 30 min was observed in gs compared to other surfaces (pmma, tcps, and ps). And polymer brush coated pmma and tcps surfaces showed significant reduction (p <0.05) in bacterial adhesion (s. aureus, e. coli, and p. aeruginosa) compared to bare pmma and tcps surfaces . Similarly, nejadnik et al . Showed that the polymer brush coatings reduced adhesion of staphylococci considerably but the few adhered bacteria still formed a biofilm when allowed to grow . Metals have been used as antibacterial agent for centuries and their efficacy has been surpassed by the use of modern antibiotics . Taylor and webster showed that iron - oxide nanoparticles in a concentration range of 0.01 to 2 mg / ml were able to kill up to 25% of s. epidermidis in a 48 h old biofilm . And, similar results were observed in our previous and current studies with iron - oxide nanoparticles on s. aureus biofilms . In contrast, haney et al . Showed an increase in p. aeruginosa biofilm biomass in the presence of 0.2 mg / ml of superparamagnetic iron - oxide nanoparticles . In this study, influence of iron - oxide nanoparticles on biofilms formed on polymer brush coated biomaterial surface was evaluated . The study of combined effects of polymer brush coating and iron - oxide nanoparticles on biofilms is novel . A significant reduction (p <0.05) in biofilm growth on all the three bacteria was observed in the presence of iron - oxide nanoparticles compared to control (absence of iron - oxide nanoparticles). The highest reduction (p <0.05) was observed in the presence of iron - oxide nanoparticles at 0.15 mg / ml compared to other concentrations (0.01 mg / ml, 0.05 mg / ml, and 0.1 mg / ml) and control . At 0.15 mg / ml of iron - oxide nanoparticles, the highest inhibition (29 mm) was observed in s. aureus compared to e. coli and p. aeruginosa . Ros includes superoxide radicals, hydroxyl radicals, hydrogen peroxide, and singlet oxygen, which may cause chemical damage to proteins and dna in bacteria . Secondly, electrostatic interactions between nanoparticles and bacterial cell membranes or cell membrane proteins can result in physical damage, which ultimately leads to bacterial cell death . Other studies demonstrated that the small size of nanoparticles could contribute to their antibacterial effects [28, 29]. This study demonstrates that wettability of a biomaterial surface influences bacterial adhesion and biofilm growth . Polymer brush coated surfaces showed reduced bacterial adhesion compared to bare surfaces . A significant reduction in biofilm growth was observed due to the influence of iron - oxide nanoparticles on biofilms formed on polymer brush coated biomaterial surfaces . Thus combinational strategies such as polymer brush coating to biomaterial surface and influence of iron - oxide nanoparticles could significantly reduce biomaterial - associated infections.
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The mucosal surfaces lining the respiratory tract are continuously exposed to potentially infectious agents, as well as environmental toxics and antigens . The bronchial epithelium acts as a physicochemical barrier of the airway mucosa and is able to initiate inflammatory responses by releasing mediators upon exposure to stimuli such as cigarette smoke- or pathogen - derived molecules . One first - line mechanism of the airway immune defense against inhaled antigens and microorganisms consists of secretory immunoglobulin a (siga), the predominant immunoglobulin isotype in mucosal secretions [1, 2]. In the lamina propria, mucosal plasma cells produce iga molecules mainly as polymers, linked through a joining chain . Polymeric iga (piga) then binds to the polymeric ig receptor (pigr), a transmembrane glycoprotein selectively expressed on the basolateral surface of epithelial cells . The piga - pigr complexes are transported across epithelial cells up to the apical membrane where a proteolytic cleavage releases the main part of the extracellular domain of pigr, known as secretory component (sc), remaining bound to piga to form siga which assumes protective, antiinfectious functions within the mucosal lumen . Chronic obstructive pulmonary disease (copd) represents the fourth leading cause of death worldwide and results in large consumption of health care resources . This disorder characterized by progressive and mostly irreversible limitation of expiratory airflow is mainly caused by cigarette smoking, whereas only some (1520%) smokers will develop the disease . Among features of copd are epithelial changes (mucous hyperplasia) and chronic inflammation with recruitment / activation of leukocytes including neutrophils, macrophages, and cd8 t cells . However, the links between airway neutrophilic inflammation and impairment of epithelial functions remain poorly understood in copd . We previously showed that expression of pigr / sc was strongly decreased in the bronchial epithelium from patients with severe copd, and that this epithelial defect correlated with airflow limitation and with infiltration of submucosal glands by neutrophils . Neutrophils can be involved in various lung disorders notably through the release of oxidants, proteinases (including serine proteinases) and defensins, which have important effects on several epithelial functions such as mucus secretion, cytokine release, or receptor expression . More specifically, we next showed that activated neutrophils can induce in vitro a cleavage of epithelial pigr / sc through the release of serine proteinases (particularly neutrophil elastase), whereas a stimulatory effect on pigr / sc production by cultured bronchial epithelial cells was also observed . However, the mechanisms regulating this opposite effect of neutrophils on pigr / sc expression in airway epithelial cells remain unclear, in particular factors responsible for increased pigr / sc synthesis upon stimulation by neutrophils are not identified . The aim of the present work was therefore to evaluate the molecular determinants of the dual effect of neutrophils on pigr / sc expression in human bronchial epithelial cells . More particularly, the involvement of soluble mediators in the epithelial pigr / sc upregulation by neutrophils was investigated, as well as the role of proteinase / antiproteinase and redox balances and of intracellular mapkinase signaling pathways . Calu-3, a human (adenocarcinoma) bronchial epithelial cell line, was obtained from the atcc (rockville, md), and cultured in rpmi-1640 supplemented with 10% decomplemented fetal bovine serum, 100 iu / ml penicillin, 100 g / ml streptomycin and 2 mm l - glutamine (cambrex, rockland, me) and referred to as complete medium . 96-well plates were precoated for 2 hrs at 37c, with 3.1 g / ml vitrogen (cohesion, palo alto, ca), 10 g / ml fibronectin, and 50 g / ml bovine serum albumin in rpmi and followed by two washes with rpmi . To obtain a confluent bronchial epithelial layer, 50,000 calu-3 cells were seeded in this 96-well plate in 200 l of complete medium and cultured for 57 days until confluence . Polymorphonuclear neutrophils were obtained from whole heparinized blood from healthy donors by a one - step density gradient method (40 min centrifugation, 800 g, room temperature) using polymorphprep (axis shield, oslo, norway) following manufacturer's protocol . After washing, neutrophils were resuspended in rpmi-1640 and activated by 20 ng / ml il-8 (r&d system, minneapolis, mn) for 5 min followed by 1 nm formyl - methionyl - leucyl - phenylalanine (fmlp) (sigma aldrich, saint louis, mo) for 30 min at 37c . In all experiments, il-8/fmlp activated neutrophils were represented by activated neutrophils . Activated neutrophils alone were incubated (from 1.25 to 5 10 cells) in complete medium for 48 hrs to determine the production of cytokines including tgf-1 in supernatants . Confluent calu-3 bronchial epithelial cells were cultured for 48 hrs, with or without tgf-1 (20 ng / ml, r&d system). To evaluate the dose response to tgf-1, cells were incubated with increasing concentration from 0.2 to 80 ng / ml . After culture, pigr / sc production was measured in supernatants and cell lysates . Response effect of neutrophils on sc production by bronchial epithelial cells, confluent monolayers of calu-3 cells were incubated for 48 hrs with increasing numbers of activated neutrophils (from 0.3 to 15 10 cells) in the presence or not of 2.5 m human secretory leukocyte protease inhibitor (slpi, r&d system). Kinetics (from 2 to 72 hrs) of tgf-1 production by calu-3 epithelial monolayers alone, activated neutrophils (5 10 cells) alone or bronchial epithelial cells cocultured with neutrophils was also analyzed . Similarly, kinetics (from 1 to 48 hrs) of sc secretion by calu-3 epithelial layers was measured after incubation with tgf--1 (20 ng / ml) or activated neutrophils (5 10 cells). At the end of each culture, the required volume of supernatant was taken to measure elastase enzymatic activity, and 2 mm phenylmethylsulfonyl fluoride (pmsf, sigma aldrich) was then added to inhibit further serine proteinase activity . Epithelial cells were washed twice in pbs and lysed with 100 l triton (sigma aldrich) 0.1% w / v in pbs for 15 min on ice . Cell viability and activity of mitochondrial electron transport chain, as indicator for cytotoxicity, was determined by the capacity of cells to reduce mtt (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) to formazan . Confluent calu-3 epithelial cells were incubated with increasing number of neutrophils (from 0.3 to 15 10), with increasing concentrations of tgf- (from 0.2 to 80 ng / ml), with cse (from 0.1% to 10%) and with mapkinases inhibitors (pd, sp, and sb) for 48 hrs . 100 l of pbs containing 1 mg / ml mtt was added to each well and the plate was returned to 37c . To solubilize intracellular formazan produced by active mitochondria in living cells, 100 l of dimethylsulfoxide (dmso) was added per well . The absorbance at 550 nm was measured on a spectrophotometric plate reader and used for the calculation of relative cell viability where untreated cells represented 100% viability . Sc concentration was determined in culture supernatants and cell lysates from epithelial cells by sandwich elisa, as previously reported, by using affinity purified goat antihuman sc polyclonal antibody (developed in our laboratory, recognizing both soluble sc and membrane pigr / sc) as capture and detection (biotinylated) antibody . The reaction was revealed by streptavidine - hrp followed by tetramethylbenzidine (tmb) in the presence of hydrogen peroxide . Sensitivity of the immunoassay was ~0.2 ng / ml . Tgf- production was measured in supernatants from calu-3 cells, neutrophils or calu-3/neutrophils cocultures by elisa . A step of sample extraction (acidification) was carried out to allow for the release of tgf-1 from latent complexes, making it accessible for measurement in the immunoassay . A monoclonal antibody specific for tgf-1 had been coated onto the wells and extracted samples and standards were put into these wells followed by addition of biotinylated secondary antibody (tgf- immunoassay kit, biosource, nivelles, belgium). N - methoxysuccinyl - ala - ala - pro - val - p - nitroanilide (sigma chemical) was used as specific substrate, diluted at 1 mm in 0.1 m hepes buffer, ph 7.5, with 0.5 m nacl and 10% v / v dimethylsulfoxide . Supernatants (50 l) were incubated with 100 l of substrate solution at 37c in 96-well plates, and od increase was monitored at 415 nm using a microplate reader (biorad). Results were compared with standard curves obtained with serial dilutions of purified neutrophil elastase (elastin products co., owensville, ma), and expressed in nmole / l . Confluent bronchial epithelial layers were incubated for 48 hrs with tgf-1 (20 ng / ml) or with pmn (5 10) in the presence of the antioxidants n - acetylcystein or glutathione (sigma) at final concentration of 10 mm . The production of sc was measured in supernatants . After 30 min of stimulation by tgf- of a confluent monolayer in a 12-well plate, calu-3 cells were lyzed in 150 l of ripa buffer [16 mm tris base, 150 mm nacl, 6 mm edta, 10 g nonidet (np-40), 12 mm sodium deoxycholate, 3 mm sds, and ph 7.4] with 10 g / ml of protease cocktail inhibitor (roche), 1 mm pmsf, 1 mm naf and 1 mm na3vo4 . 40 l of protein lysate are diluted in 40 l of laemmli's sample buffer, heated at 100c for 5 min, loaded in a 12% sds - page gel and run at 180 v for 1 h. cell proteins were transferred onto a nitrocellulose membrane (hybond - c, amersham biosciences, uk) at 70 ma for 1.5 h at room temperature . The membrane was blocked with 5% bsa in tris - buffered saline with 0.1% tween 20 (tbst) for 1 h at room temperature, washed, and incubated with the primary ab diluted 1/1000 in tbst - bsa 1% (anti - phospho erk, anti - phospho p38 or anti - phospho jnk; cell signalling technology, beverly, ma) overnight at 4c . Membranes were then incubated for 1 h at room temperature with a secondary ab diluted 1/2000 in tbst - bsa 1% (anti - rabbit igg / hrp, cell signalling technology). Immunoreactive bands were developed using chemiluminescence (ecl, amersham biosciences, uk) and detected with the chemidoc xrs apparatus (bio - rad laboratories, hercules, ca). Calu-3 epithelial cells were pretreated for 1 hr with 50 m pd98059, 50 m sb203580 or 50 m sp600125 as selective inhibitors of extracellular signal - regulated kinase-1/2 (erk) mitogen - activated protein kinase (mapk), p38 mapk, and c - jun - n - terminal kinase (jnk) mapk, respectively (new england biolabs, beverly, ma). Calu-3 cells were then incubated for 48 hrs with tgf-1 (20 ng / ml) or neutrophils (5 10 cells), and production of sc in supernatants was determined by elisa . The differences between the groups were analyzed as appropriate by means of the mann whitney test, using graphpad prism 4.0 (graphpad prism, san diego, ca). To set up a model to examine effects of neutrophils on sc production by bronchial epithelial cells, confluent epithelial layers of calu-3 cells were incubated for 48 hrs with increasing numbers of activated neutrophils (figure 1(a)), with evaluation of cytotoxicity . 10 did not significantly affect epithelial cell viability according to mtt assay (viability 80% compared to control). However cytotoxicity was observed for 10 10 neutrophils (cell viability decreased by 20%). Sc production increased when epithelial cells were incubated with neutrophils, starting at 1.25 10 and with a maximal increase at 2.5 10 neutrophils (p <.01, figure 1(a)). At further increased neutrophil numbers (> 5 10), sc production was downregulated and tended to decrease at 15 10 neutrophils as compared to control (p = .08, figure 1(a)), consistent with cytotoxicity (mtt assay) and/or proteolytic cleavage . Elastase activity evaluated in supernatants from the same calu-3/neutrophils cocultures increased as expected with the number of neutrophils (figure 1(b)). These results indicated that this system allowed to address the two opposite effects of neutrophils on epithelial sc, and experiments were carried out to explore the mechanisms of sc upregulation by nontoxic numbers of neutrophils releasing elastase in a concentration range that could be observed in chronic airway neutrophilic diseases, for example, during exacerbations . To evaluate the role of serine proteinases on the regulation of sc production by neutrophils, calu-3 cells were cultured with increasing neutrophil numbers in the presence or not of secretory leukocyte protease inhibitor (slpi), a natural inhibitor of neutrophil elastase, at a concentration (2.5 m) almost completely suppressing elastase activity (> 90% inhibition). Sc production was measured in both epithelial supernatants and cell lysates (figure 2). In the presence of exogenous slpi, sc upregulation observed in epithelial supernatants at intermediate neutrophil numbers (p <.05 at 2.5 10 neutrophils) was further increased . In addition, the presence of slpi promoted an increase in pigr / sc in epithelial lysates (p <.05 at 0.6 10 neutrophils), while no effect was observed in this cell compartment in the absence of slpi (figure 2) or with slpi alone, without neutrophils (data not shown). Besides the effect of proteinases / antiproteinases on sc upregulation, involvement of other neutrophil (and/or epithelial)-derived soluble factors was assessed . Cytokines known to induce pigr transcription such as tnf- or il-1 were not detected in coculture supernatants (data not shown). In contrast, tgf-, which was detected in resting calu-3 cell supernatants at a similar level as in complete (serum - supplemented) medium, was increased upon neutrophil coculture (3-fold increase at 5 10 neutrophils, p <.05; figure 3(a)). In order to determine the respective role of epithelial cells and neutrophils in tgf- production, a time course of tgf- production by epithelial cells alone, activated neutrophils alone or epithelial cell / neutrophil cocultures was carried out (figure 3(b)). A trend for tgf- increase was observed in neutrophil and coculture supernatants as early as after 2 hrs, and further increased at later time points (p <.05 at 48 and 72 hrs, figure 3(b)) in cocultures . In contrast, epithelial cells began to secrete tgf- between 4872 hrs, with a 3-fold increase as compared to 2 hrs (p <.05). At each time point, tgf- in neutrophil supernatants was higher than the epithelial production, except at 72 hrs . These results indicated that neutrophils represented the main source of tgf- up to 48 hrs of coculture, with a modest (and late) contribution from epithelial cells . Tgf- had no significant cytotoxic effect on epithelial cell viability up to 80 ng / ml, with even increased mtt reduction at 80 ng / ml (not shown). Addition of recombinant human tgf-1 significantly increased pigr / sc production, in cell lysates (p <.05; figure 4(a)), and a trend was also observed for sc secretion in supernatants . In dose - response experiments, sc production upon tgf- stimulation increased from 1 ng / ml to reach a plateau ~20 ng / ml (figure 4(b)). The kinetics of sc production by bronchial epithelial cells incubated with tgf- or neutrophils, the main source of tgf-, was assessed (figure 4(c)). From 1 hr to 14 hrs of incubation, no difference was observed between the three conditions of stimulation, constitutive sc production starting to significantly increase from 24 hrs . Effects of tgf- and neutrophils had similar kinetics on sc production, with upregulation observed at 48 hrs (figure 4(c)). We examined whether the redox balance could affect tgf-- and neutrophil - induced sc upregulation . Calu-3 cells were cultured with tgf- or neutrophils in the presence or not of two antioxidants, n - acetylcysteine and glutathione . In the presence of these antioxidants, effects of neutrophils n - acetylcysteine and glutathione had no effect on tgf- production, whereas they did slightly reduce elastase activity presumably by reducing the ph (data not shown). The link between p38 mapk activation and redox balance in epithelial cells cocultured with activated neutrophils was assessed . Activation of p38 and erk was assayed in calu-3 cells cocultured with neutrophils in the presence or not of n - acetylcysteine (figure 5(b)). Upon neutrophil coculture erk was activated at 60 min, while p38 was activated as soon as after 5 min and also peaked at 60 min . In the presence of the antioxidant, p38 mapk activation was inhibited, whereas that of erk was not affected or even promoted . To analyse the intracellular mechanisms involved in sc upregulation and engaged in bronchial epithelial cells activated by tgf- or neutrophils, activation of erk, p38, and jnk mapkinase was assessed, as well as effects of their selective inhibition . For each inhibitor, specificity and absence of significant cytotoxicity was confirmed by western blot and mtt assay, respectively (data not shown). Activation of erk and p38 mapk was confirmed in calu-3 stimulated by tgf-, with a maximal effect at 5 min both for erk and p38 (figure 6(a)). The pharmacologic inhibitors did not significantly affect baseline sc secretion (figure 6(b)). While no significant effects were observed with erk or jnk pathway inhibitors (pd98059 and sp600125, resp . ), the p38 mapk inhibitor (sb203580) suppressed neutrophil- and tgf--driven sc upregulation (p <.05, figure 6(b)). Epithelial pigr / sc is the critical receptor for the transcellular routing of iga involved in frontline defense mechanisms at mucosal surfaces, including the airways . We previously observed that the bronchial epithelium of severe copd displays a strongly reduced pigr / sc expression, which correlated with airway obstruction and neutrophil infiltration . Our previous in vitro study showed that neutrophils could induce on one hand pigr / sc cleavage and on the other hand upregulation of sc production in epithelial cells, suggesting a dual effect of neutrophils on pigr / sc . This study shows that, in an in vitro coculture system set up to address the mechanisms of pigr / sc dual regulation by neutrophils, increased epithelial sc production was observed at intermediate numbers of neutrophils (corresponding to 10 nm elastase released), while further increasing neutrophils downregulated sc, presumably due to proteolytic cleavage (> 20 nm elastase;) and/or cytotoxic effects . In addition, sc upregulation was favoured in the presence of slpi, a natural elastase inhibitor, and associated with tgf- production by neutrophils . Moreover, we show for the first time that tgf- is able to stimulate epithelial pigr / sc production, in a dose - dependent manner . We also demonstrate that tgf-- and neutrophil - driven sc upregulation depends on the redox balance (cigarette smoke extracts and antioxidants both suppressing the effect) and that p38 map kinase represents a downstream, intracellular signalling pathway targeted by tgf- to induce pigr / sc production upon stimulation by neutrophils . Neutrophils are able to secrete a large range of soluble factors including proteinases and oxidants, as well as cytokines / chemokines and growth factors, involved in inflammatory responses and tissue remodelling . Among these factors produced by neutrophils, our data indicate that elastase and tgf- represent the major candidates to underlie, respectively, the down- and upregulation of pigr / sc in epithelial cells exposed to activated neutrophils . A robust production of tgf- was detected in our coculture supernatants, mainly arising from neutrophils . In addition, a dose - dependent increase in sc expression was observed upon exogenous tgf- stimulation . Tgf- produced in cocultures ranged from 0.5 to 1 ng / ml (figure 3), a concentration close to levels effectively upregulating sc production (figure 4(b)). In addition, maximal sc production was reached at 48 hrs of stimulation, both for tgf- and neutrophils . Moreover, functional experiments pointed to common molecular pathways (redox system, p38 mapk), further supporting that neutrophils could induce pigr / sc expression mainly by producing tgf-. While elastase is the major serine proteinase released by neutrophils involved in lung tissue breakdown due to cigarette smoking (know as emphysema), tgf- represents a multifunctional growth factor . It modulates cell proliferation and differentiation and induces synthesis of extracellular matrix proteins [10, 11] and is thereby involved in the development of tissue fibrosis . Tgf- has also been shown as chemotactic for neutrophils, while exerting prominent immunosuppressive functions . In addition, it has a unique role in mucosal tissues in promoting class switch recombination of mucosal plasma cells to iga, a critical ig for prevention of microbial infection and for control of the commensal flora [13, 14]. Here, we show that tgf- not only triggers iga synthesis but also promotes its transport across the epithelium, further supporting the view of this cytokine as a critical factor in mucosal homeostasis . Bronchial epithelial cells can also produce tgf- either constitutively or upon stimulation such as by cigarette smoke [15, 16], whereas ifn- inhibits epithelial tgf- release whilst promoting pigr / sc expression . Intriguingly, it was shown that tgf- expression is increased in small airway epithelium from smokers and patients with copd [17, 18], and was suggested as a factor downregulating slpi expression in these patients . A reduction in slpi secretion can also be induced by elastase, as recently reported . Of note, no production of slpi (or elafin) thus, it remains to investigate whether relationships between tgf- signalling and epithelial functions including production of pigr / sc and slpi are affected in copd . In healthy subjects, a balance between oxidants and antioxidants keeps the extracellular environment in a reduced state . Cigarette smoke, which contains massive amounts of oxidants, is the most important risk factor for the development of copd . Our results showed that glutathione or n - acetylcysteine, a glutathione precursor, inhibited the epithelial sc production induced by tgf- or by neutrophils . Tgf- is produced in an inactive form, complexed to the latency - associated peptide (lap), and this complex needs to be dissociated to release active tgf-. Reactive oxygen species (ros) are able to oxidize lap and to release active tgf-, this effect being prevented by oxidant scavenging . The main cellular sources of ros in the lung not only include activated phagocytes such as neutrophils but also structural cells such as endothelial cells, alveolar and bronchial epithelial cells [23, 24]. Accordingly, it is plausible that in our study gsh and nac prevented tgf- activation by neutrophil (or epithelial cell)derived oxidants, thereby inhibiting tgf-induced sc production . In addition, antioxidants could also affect epithelial intracellular signalling, including p38 mapk activation [25, 26]. Similarly, the natural antiproteinase slpi might modulate tgf-- and neutrophil - driven epithelial cell activation, such as nfb pathway . However, our data support that slpi further promotes sc upregulation upon neutrophil coculture not by affecting epithelial cell signalling but rather by inhibiting elastase activity . Intracellular kinases play a crucial role in the control of activation of (anti)inflammatory genes in the airways, in t - cell function and tissue remodelling . Map kinases control in airway epithelial cells the transcription of several genes involved in copd pathogenesis such as mucins and inflammatory mediators (e.g., tnf-, il-8, and matrix metalloproteinases). Neutrophil- and tgf--driven sc upregulation in epithelial cells was almost completely abolished by the inhibitor sb203580, indicating the requirement for p38 mapk activation, in contrast to erk and jnk pathways . In human bronchial epithelial cells, exposure to oxidants (peroxide) activates mapk cascades, particularly stress responsive p38 mapk [25, our data]. Thus, oxidative stress, caused by inflammatory cells and/or by cigarette smoke derivatives may upregulate epithelial pigr / sc production by activating p38 mapk . Increased numbers of p38 positive cells were previously observed in alveolar walls of patients with copd, as compared to smokers and nonsmoking controls . Moreover, p38 inhibitors are effective in several animal models of inflammatory disorders, including copd . For instance, the inhibitor sb2439063 reduced neutrophilia and expression of inflammatory mediators in a rat model of copd . However, it has been observed that cytokine - induced synthesis of elafin, another natural epithelial - derived neutrophil elastase inhibitor, is also dependent on p38 mapk [32, 33]. Thus, activation of p38 mapk pathway might be critical in the airways to both inflammatory responses and transactivation of protective epithelial genes, including local anti - proteinases and pigr / sc . In conclusion, this paper demonstrates that activated neutrophils trigger increased epithelial pigr / sc expression through redox- and p38 mapk - dependent mechanisms . It also highlights a novel mucosal function for tgf-, upregulating not only iga synthesis but also its receptor - mediated transepithelial transport, and further suggests that the redox balance plays an important role in modulating epithelial responses to neutrophils . Thus, this paper provides a molecular basis for the dual effect of neutrophils on mucosal / secretory immunity which consists of on the one hand on proteolytic cleavage of pigr / sc (and iga) by elastase and proteinase-3, and on the other hand on tgf--mediated activation of pigr / sc synthesis via oxidant- and p38-dependent pathways . These findings are relevant to airway cell biology and neutrophilic airway diseases, suggesting that imbalance between the two identified opposite regulatory mechanisms of pigr / sc may lead to downregulation of secretory iga immunity, as observed in smokers who developed severe copd . The cellular and molecular determinants of this (im)balance and fine tuning of pigr expression will be further investigated in primary cultures of airway epithelial cells from copd patients.
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Evidence is growing in support of a role for infections in the aetiology of childhood leukaemia, particularly for the most common subtype, acute lymphoblastic leukaemia (all). Two infection - related hypotheses have gained popularity and are currently supported by substantial, yet inconsistent, epidemiologic findings . Kinlen first proposed the population mixing hypothesis in response to the observed childhood leukaemia clusters occurring in the early 1980s in seascale and thurso, two remote and isolated communities in the uk that experienced a rapid influx of professional workers . He proposed that childhood leukaemia may result from an abnormal immune response to specific, although unidentified, infections commonly seen with the influx of infected persons into an area previously populated with non - immune and susceptible individuals . This hypothesis suggests a mechanism that involves a direct pathological role of specific infectious agents, presumably viruses, in the development of childhood leukaemia and that an immunizing effect may be acquired through previous exposure . Supportive data include several subsequent studies conducted by kinlen and others examining similar examples of population mixing including rural new towns, situations of wartime population change and other circumstances contributing to unusual patterns of personal contact . Currently, there is no molecular evidence implicating cell transformation by a specific virus . The delayed infection hypothesis proposed by greaves emphasizes the critical nature of the timing of exposure and is intended to apply mostly to common b - cell precursor all (c - all), which largely accounts for the observed peak incidence of all between 2 and 5 years of age in developed countries . He described a role for infections in the context of a two - hit model of the natural history of c - all, where the first hit or initiating genetic event occurs in utero during fetal haematopoiesis producing a clinically covert pre - leukemic clone . The transition to overt disease occurs, in a small fraction (1%) of pre - leukaemia carriers, after a sufficient postnatal secondary genetic event, which may be caused by a proliferative stress - induced effect of common infections on the developing immune system of the child . This adverse immune response to infections is thought to be the result of insufficient priming of the immune system usually influenced by a delay in exposure to common infectious agents during early childhood . With the assumption that improved socio - economic conditions may lead to delay in exposure to infections, the greaves hypothesis provides one plausible explanation for the notably higher incidence rates of all with its characteristic peak age between 2 and 5 years observed only in more socio - economically developed countries . Although different in hypothesized mechanism, both the population mixing and delayed infection hypotheses propose childhood leukaemia to be caused by an abnormal immune response to infection(s) acquired by personal contacts, and are compatible with available evidence . In some populations, it is possible that both mechanisms may be operating . Several previous epidemiological studies have used day - care attendance as an indicator of the increased likelihood of early exposure to infections, since it is well documented that in developed countries exposures to common infections, particularly those affecting the respiratory and gastrointestinal tracts, occur more frequently in this type of setting . The immaturity of children s immune systems in combination with the lack of appropriate hygienic behaviour is believed to promote the transmission of infectious agents in this social setting . In the current analysis, we took a meta - analytic approach to summarize the findings to date on the relationship between day - care attendance and risk of childhood all . Literature searches were conducted in pubmed to identify original research and review articles related to childhood leukaemia and day - care attendance and/or social contacts published between january 1966 and october 2008 . Child care, day care and social contact. In addition, the bibliographies of epidemiology publications on childhood leukaemia and infections were searched to identify studies that may not have been captured through the initial database search . This included the review published in 2004 by mcnally and eden on the infectious aetiology of childhood acute leukaemia (al). Among the studies identified, inclusion in the meta - analysis was limited to observational studies of case control or cohort design of any size, geographic location and race / ethnicity of study participants . When more than one publication from an individual study was available, either the most recent publication or the publication that performed the analysis most applicable to evaluating the delayed infection hypothesis was selected . Studies needed to have reported a relative risk (rr) or odds ratio (or) and confidence intervals (cis), or original data by disease status from which a measure of effect could be calculated . The outcome of interest was defined as clinically diagnosed leukaemia in children between the ages of 0 and 19 years . In the very few studies that did not distinguish between specific leukaemia subtypes, it was assumed that all was the primary subtype since it accounts for the majority (80%) of leukaemia diagnoses in children . The exposure of interest generally referred to as day - care attendance, which, in addition to formal day care, may have included preschool, nursery school, play groups, mother toddler groups and other early social contacts . A strict criterion for the meaning of regular attendance was not defined a priori since it was assumed that this would vary between studies . Of the primary studies identified, four were excluded for various reasons, including study emphasis on evaluating leukaemia prognosis and outcome, an earlier analysis of data from a study for which a more complete and recent publication is available, and not reporting a risk estimate for day - care attendance . After the exclusions, a total of 14 studies, all case control in design, were retained for the meta - analysis . For most studies, the ors and 95% cis for leukaemia, al, all or c - all among those who attended day care compared with those who did not attend day care were extracted . Among the few studies that did not provide this estimate, the or for a similar measure was extracted, including those for no deficit in social contacts, regular contact outside the home,> 36 months duration of day - care attendance, increasing index and family day - care measure, and social activity . In two instances, the reported or was recalculated to reflect the risk associated with the highest level of day - care attendance and/or social activity measure compared with the lowest . Furthermore, several studies reported risk estimates for stratified analyses by specific subtype of leukaemia, age at diagnosis, specific age of day - care attendance, or race / ethnicity; multiple estimates were extracted from these studies for the purposes of subgroup and sensitivity evaluations in the meta - analysis, including specific leukaemia subtypes, particularly all and c - all and timing of day - care attendance . In general, studies referred to the common precursor b - cell all subtype (cd10 and cd19 positive all) as c - all . Four studies defined c - all with an added criterion that specified an age range between 2 and 5 years . Risk estimates by specific diagnosis age groups were not extracted since there were only a few studies that provided this information and the age cut - points varied . For the one study that stratified by race / ethnicity, two separate risk estimates were included in the meta - analysis since the reported estimates were based on independent populations . The between - study heterogeneity was assessed using the q statistic, which tests the null hypothesis that the estimated effect is homogenous across all studies . Acknowledging that the eligible studies have been conducted independently and may represent only a random sample of the distribution of all possible effect sizes for this association, the random effects model was utilized, which incorporates an estimate of both between - study and within - study variation into the calculation of the summary effect measure . Compared with the fixed effects model, this method is more conservative and generally results in a wider ci . Finally, publication bias was evaluated visually using the funnel graph method that displays the distribution of all included studies by their point estimates and standard errors . In addition, the begg and mazumdar adjusted rank correlation test was used to test for correlation between the effect estimates and their variances which, if present, provides an indication of publication bias . The association with c - all was evaluated with a meta - analysis of 7 of the 14 studies . If a study reported multiple ors and 95% cis by timing of day - care attendance, the risk estimate associated with the earliest timing (e.g. Age 2 years) was used to be consistent with the delayed infection hypothesis . The effect of the timing of exposure was evaluated in subgroup meta - analyses of studies reporting risk estimates for early day - care attendance (age 2 years) and studies reporting risk estimates for day - care attendance anytime before diagnosis . Finally, a series of sensitivity analyses were conducted to evaluate the sources of study heterogeneity, namely, the influences of potential selection bias, and heterogeneity in disease classification and exposure definition . The analyses were conducted using the statistical software, stata version 9 . Literature searches were conducted in pubmed to identify original research and review articles related to childhood leukaemia and day - care attendance and/or social contacts published between january 1966 and october 2008 . Child care, day care and social contact. In addition, the bibliographies of epidemiology publications on childhood leukaemia and infections were searched to identify studies that may not have been captured through the initial database search . This included the review published in 2004 by mcnally and eden on the infectious aetiology of childhood acute leukaemia (al). Among the studies identified, inclusion in the meta - analysis was limited to observational studies of case control or cohort design of any size, geographic location and race / ethnicity of study participants . When more than one publication from an individual study was available, either the most recent publication or the publication that performed the analysis most applicable to evaluating the delayed infection hypothesis was selected . Studies needed to have reported a relative risk (rr) or odds ratio (or) and confidence intervals (cis), or original data by disease status from which a measure of effect could be calculated . The outcome of interest was defined as clinically diagnosed leukaemia in children between the ages of 0 and 19 years . In the very few studies that did not distinguish between specific leukaemia subtypes, it was assumed that all was the primary subtype since it accounts for the majority (80%) of leukaemia diagnoses in children . The exposure of interest generally referred to as day - care attendance, which, in addition to formal day care, may have included preschool, nursery school, play groups, mother toddler groups and other early social contacts . A strict criterion for the meaning of regular attendance was not defined a priori since it was assumed that this would vary between studies . Of the primary studies identified, four were excluded for various reasons, including study emphasis on evaluating leukaemia prognosis and outcome, an earlier analysis of data from a study for which a more complete and recent publication is available, and not reporting a risk estimate for day - care attendance . After the exclusions, a total of 14 studies, all case control in design, were retained for the meta - analysis . For most studies, the ors and 95% cis for leukaemia, al, all or c - all among those who attended day care compared with those who did not attend day care were extracted . Among the few studies that did not provide this estimate, the or for a similar measure was extracted, including those for no deficit in social contacts, regular contact outside the home,> 36 months duration of day - care attendance, increasing index and family day - care measure, and social activity . In two instances, the reported or was recalculated to reflect the risk associated with the highest level of day - care attendance and/or social activity measure compared with the lowest . Furthermore, several studies reported risk estimates for stratified analyses by specific subtype of leukaemia, age at diagnosis, specific age of day - care attendance, or race / ethnicity; multiple estimates were extracted from these studies for the purposes of subgroup and sensitivity evaluations in the meta - analysis, including specific leukaemia subtypes, particularly all and c - all and timing of day - care attendance . In general, studies referred to the common precursor b - cell all subtype (cd10 and cd19 positive all) as c - all . Four studies defined c - all with an added criterion that specified an age range between 2 and 5 years . Risk estimates by specific diagnosis age groups were not extracted since there were only a few studies that provided this information and the age cut - points varied . For the one study that stratified by race / ethnicity, two separate risk estimates were included in the meta - analysis since the reported estimates were based on independent populations . The between - study heterogeneity was assessed using the q statistic, which tests the null hypothesis that the estimated effect is homogenous across all studies . Acknowledging that the eligible studies have been conducted independently and may represent only a random sample of the distribution of all possible effect sizes for this association, the random effects model was utilized, which incorporates an estimate of both between - study and within - study variation into the calculation of the summary effect measure . Compared with the fixed effects model, finally, publication bias was evaluated visually using the funnel graph method that displays the distribution of all included studies by their point estimates and standard errors . In addition, the begg and mazumdar adjusted rank correlation test was used to test for correlation between the effect estimates and their variances which, if present, provides an indication of publication bias . The association with c - all was evaluated with a meta - analysis of 7 of the 14 studies . If a study reported multiple ors and 95% cis by timing of day - care attendance, the risk estimate associated with the earliest timing (e.g. Age 2 years) was used to be consistent with the delayed infection hypothesis . The effect of the timing of exposure was evaluated in subgroup meta - analyses of studies reporting risk estimates for early day - care attendance (age 2 years) and studies reporting risk estimates for day - care attendance anytime before diagnosis . Finally, a series of sensitivity analyses were conducted to evaluate the sources of study heterogeneity, namely, the influences of potential selection bias, and heterogeneity in disease classification and exposure definition . Table 1 presents selected characteristics of the 14 studies included in this meta - analysis . The studies, all case control in design, were published between 1993 and 2008 and were conducted in many different geographic areas . Most studies achieved a population - based ascertainment of cases utilizing a national registry or a regional network of all major paediatric oncology centres . A population - based control selection strategy was most common with the exception of three studies that selected hospital - based controls . Only 1 of the 14 studies utilized a records - based day - care assessment protocol, whereas the remaining studies relied on standardized questionnaires administered either in person, by telephone or by mail . All studies have accounted for major confounding factors such as age, sex, race and socio - economic status through a matched study design and/or statistical adjustment in the analysis . Of the 14 studies identified, 11 studies have reported either a statistically significant reduced risk associated with day - care attendance and/or social contact measures or provide some evidence of a reduced risk . Table 1select characteristics of studies included in the meta - analyses of day - care attendance and childhood leukemiaauthor, year (location)case ascertainmentcontrol selectiondata collectionselect resultsconfounding addressedpetridou et al ., 1993 (greece, attica and crete)125 leukaemia (attica) 11 leukaemia (crete)age 014 yearschildren s hospitals of the university of athens (198791) and the university of crete (199092)187 frequency matched children who attended the outpatient clinic of the hospitals where the children with leukaemia were treatedtelephone interviews with parents of childrenattendance at crche (yes / no) at any time leukaemia: 0.67 (0.41, 1.11)in infancy (for 3 months in the first 2 years of life) leukaemia: 0.28 (0.09, 0.88)frequency match: sex, age, hospitaladjustment: place of residence, social classroman et al ., 1994 (uk)38 allage 04 yearsdiagnosed 197289born in west berkshire or north hampshire, and were residents there when diagnosed112 individually matched children selected from hospital delivery registersparents were interviewedpreschool playgroup (yes / no) in the year before diagnosis (for 3 months) all: 0.6 (0.2, 1.8)individual match: sex, date of birth, mother s age, area of residence at birth, time of diagnosisadjustment: not specifiedpetridou et al ., 1997 (greece)153 leukaemiaage 014 yearsdiagnosed 199394nation wide network of paediatric haematologists / oncologists300 individually matched children hospitalized at the same time as the corresponding case for acute conditionsguardians of all subjects completed an interviewer - administered questionnaireday - care attendance (ever / never) leukaemia: 0.83 (0.51, 1.37)individual match: sex, age, geographic regionadjustment: maternal age at birth, maternal education, sibship size, birth order, persons per roomschuz et al ., 1999 (germany)1010 al (686 c - all)diagnosed 198094age 014 yearsnation wide german children s cancer registry at the university of mainz2588 matched children randomly selected from complete files of local offices of registration of residentsstructured questionnaire based on the us children s cancer groupday - care attendance not directly assesseddeficit in social contacts (yes / no, age 18 months excluded) al: 1.1 (0.9, 1.3) c - all: 1.0 (0.8, 1.2)analysis of alindividual match: date of birth, sex, districtadjustment: sesanalysis of c - alladjustment: sex, age, year of birth, study settingadjustment: ses, urbanizationdockerty et al ., 1999 (new zealand)97 alldiagnosed 199093age 014 yearsnew zealand cancer registry, public hospital admission / discharge computer system, and the children s cancer registry . Nationwide97 individually matched children randomly selected from the new zealand born and resident childhood population using national birth records209 solid cancer casesmothers interviewed in their homes using a questionnaire adapted from patricia mckinney and eve roman in the ukregular contact with other children from outside home at <12 months (yes / no, age <15 months excluded) all: 0.65 (0.36, 1.17)individual match: age and sexadjustment: sex, age, several others including social classinfante - rivard et al ., 2000 (canada)491 alldiagnosed 198093age 09 yearstertiary care centre similar to population - based ascertainment491 individually matched children chosen from the most complete census of children for the study yearsstructured questionnaire administered to mothers by phoneday - care attendance by age at entry entry 2 years old vs no all: 0.49 (0.31, 0.77)entry at> 2 years old vs no all: 0.67 (0.45, 1.01)individual match: age, sex, region of residence at diagnosisadjustment: maternal age, maternal educationneglia et al ., 2000 (usa)1744 all (633 c - all; excludes cases <1 year)diagnosed 198993age 014 yearschildren s cancer group member institutions throughout the usa1879 individually matched children randomly selected using the random digit dialing (rdd) methodologystructured interviewday - care attendance (age <1 year excluded) yes vs no all: 0.96 (0.82, 1.12) c - all: 0.96 (0.75, 1.24)day care before age 2 vs no all: 0.99 (0.84, 1.17) c - all: 1.05 (0.80, 1.37)individual match: age, race, telephone area code, exchange, sex (t - cell leukaemia only)adjustment: maternal race, education, family incomerosenbaum et al ., 2000 (usa)255 alldiagnosed 198091age 014 yearsfour clinical centers in a 31-county study region . Institutional tumour registries and department of paediatric haematology oncology records760 frequency matched children randomly selected through the live birth certificate registry maintained by the new york state department of healthself - administered questionnaire mailed to the parents of subjectstotal duration of out - of - home care (duration vs> 36 months) all: stay home: 1.32 (0.70, 2.52) 118 months: 1.74 (0.89, 3.42) 1936 months: 1.32 (0.70, 2.52)frequency match: sex, age, race, birth yearadjustment: maternal age, maternal education, birth year, maternal employment, breastfeeding, birth orderchan et al ., 2002 (hong kong)98 al (66 c - all)diagnosed 199497age 214 yearshong kong pediatric hematology and oncology study group228 children selected using rdd methodologyin - person interview using a structured questionnaire adapted from ukccs and translated into chineseindex and family day - care attendance (3-category variable) first year of life al: 0.96 (0.70, 1.32) child peak: 0.63 (0.38, 1.07) c - all: 0.93 (0.63, 1.36)matching: noneadjustment: age, number of children in household at reference dateperrillat et al ., 2002 (france)280 al (240 all)diagnosed 199599age 015 yearshospitals of lille, lyon, nancy and paris . Cases need to have resided in the hospital catchment area288 frequency matched children hospitalized in the same hospital as the cases, and residing in the catchment area of the hospitalin - person standardized questionnaireday - care attendance (age <2 years excluded) ever vs never al: 0.6 (0.4, 1.0)age started vs no day care> 12 months: 0.5 (0.3, 1.0) 712 months: 0.6 (0.2, 1.7) 6 months: 0.5 (0.3, 1.0)frequency match: age, sex, hospital, hospital catchment area, ethnic originadjustment: age, sex, hospital, ethnic origin, maternal education, parental professional categoryjourdan - da silva et al ., 2004 (france)473 al (408 all, 304 c - all)diagnosed 199598age 014 yearsnational registry of childhood leukaemia and lymphoma (nrcl)567 frequency matched children randomly selected using age, sex, and region quotas from a sample of 30 000 phone numbers representative of the french population on area of residence and municipality sizestandardized self - administered questionnaire on mothersday - care attendance (age <1 year excluded) ever vs never all: 0.7 (0.6, 1.0) c - all: 0.8 (0.6, 1.0)started at age <3 months vs never all: 0.6 (0.4, 0.8) c - all: 0.6 (0.4, 0.9)frequency match: age, sex, regionadjustment: age, sex, regiongilham et al ., <2 years)diagnosed 199196age 014 yearsnation - wide ascertainment through pediatric oncology units6238 individually matched children randomly selected from primary care population registersin - person interview with parents using a structured questionnairesocial activity in the first year of life (age <2 years excluded) any vs no social activity all: 0.66 (0.56, 0.77) c - all: 0.67 (0.55, 0.82)age started vs no day care all- <3 months: 0.71 (0.60, 0.85) 35 months: 0.71 (0.56, 0.90) 611 months: 0.76 (0.63, 0.92)individual match: sex, month and year of birth, region of residence at diagnosisadjustment: age at diagnosis, sex, region, maternal age, mother working at time of birth, deprivationma et al ., 2005 (usa)294 all (145 c - all; excludes case <1 year)diagnosed 19952002age 014 yearspopulation - based ascertainment from major paediatric clinical centres in northern and central california376 individually matched children randomly selected from statewide birth certificate files maintained by the california department of health servicespersonal interview with primary caretakerchild - hours of exposure at day care (age <1 year excluded) 5000 child - hours (1st year) vs 0 all- hispanic: 2.10 (0.70, 6.34) white: 0.42 (0.18, 0.99) c - all- hispanic: 2.53 (0.60, 10.7) white: 0.33 (0.11, 1.01)individual match: date of birth, sex, mother s race, hispanic statusadjustment: annual household income, maternal educationkamper - jorgensen et al ., 2008 (denmark)559 all (199 c - all)diagnosed 19892004age 015 yearsall cases of childhood leukaemia identified in a cohort of all children in denmark5590 individually matched children selected from population registersrecords - based data from three population - based registries: the nordic society of paediatric haematology and oncology, the danish civil registration system, and the childcare databasechild - care attendance in children during first 2 years of life (yes / no) all: 0.68 (0.48, 0.95) c - all: 0.58 (0.36, 0.93)individual match: date of birth, sex, birth cohortadjustment: several demographic characteristics was considered but none were major confoundersses, socioeconomic status; rdd, random digit dialing; ukccs, united kingdom childhood cancer study . Select characteristics of studies included in the meta - analyses of day - care attendance and childhood leukemia 125 leukaemia (attica) 11 leukaemia (crete) children s hospitals of the university of athens (198791) and the university of crete (199092) at any time leukaemia: 0.67 (0.41, 1.11) in infancy (for 3 months in the first 2 years of life) leukaemia: 0.28 (0.09, 0.88) frequency match: sex, age, hospital adjustment: place of residence, social class born in west berkshire or north hampshire, and were residents there when diagnosed in the year before diagnosis (for 3 months) all: 0.6 (0.2, 1.8) individual match: sex, date of birth, mother s age, area of residence at birth, time of diagnosis adjustment: not specified nation wide network of paediatric haematologists / oncologists individual match: sex, age, geographic region adjustment: maternal age at birth, maternal education, sibship size, birth order, persons per room nation wide german children s cancer registry at the university of mainz structured questionnaire based on the us children s cancer group day - care attendance not directly assessed individual match: date of birth, sex, district adjustment: sex, age, year of birth, study setting adjustment: ses, urbanization new zealand cancer registry, public hospital admission / discharge computer system, and the children s cancer registry . Nationwide 97 individually matched children randomly selected from the new zealand born and resident childhood population using national birth records 209 solid cancer cases individual match: age and sex adjustment: sex, age, several others including social class tertiary care centre similar to population - based ascertainment entry 2 years old vs no all: 0.49 (0.31, 0.77) entry at> 2 years old vs no all: 0.67 (0.45, 1.01) individual match: age, sex, region of residence at diagnosis adjustment: maternal age, maternal education 1744 all (633 c - all; excludes cases <1 year) children s cancer group member institutions throughout the usa yes vs no all: 0.96 (0.82, 1.12) c - all: 0.96 (0.75, 1.24) day care before age 2 vs no all: 0.99 (0.84, 1.17) c - all: 1.05 (0.80, 1.37) individual match: age, race, telephone area code, exchange, sex (t - cell leukaemia only) adjustment: maternal race, education, family income four clinical centers in a 31-county study region . Institutional tumour registries and department of paediatric haematology oncology records frequency match: sex, age, race, birth year adjustment: maternal age, maternal education, birth year, maternal employment, breastfeeding, birth order hong kong pediatric hematology and oncology study group first year of life al: 0.96 (0.70, 1.32) child peak: 0.63 (0.38, 1.07) c - all: 0.93 (0.63, 1.36) adjustment: age, number of children in household at reference date hospitals of lille, lyon, nancy and paris . Cases need to have resided in the hospital catchment area ever vs never al: 0.6 (0.4, 1.0) age started vs no day care> 12 months: 0.5 (0.3, 1.0) 712 months: 0.6 (0.2, 1.7) 6 months: 0.5 (0.3, 1.0) frequency match: age, sex, hospital, hospital catchment area, ethnic origin adjustment: age, sex, hospital, ethnic origin, maternal education, parental professional category 473 al (408 all, 304 c - all) national registry of childhood leukaemia and lymphoma (nrcl) ever vs never all: 0.7 (0.6, 1.0) c - all: 0.8 (0.6, 1.0) started at age <3 months vs never all: 0.6 (0.4, 0.8) c - all: 0.6 (0.4, 0.9) frequency match: age, sex, region adjustment: age, sex, region 1286 all (791 c - all; excludes cases <2 years) nation - wide ascertainment through pediatric oncology units any vs no social activity all: 0.66 (0.56, 0.77) c - all: 0.67 (0.55, 0.82) age started vs no day care all- <3 months: 0.71 (0.60, 0.85) 35 months: 0.71 (0.56, 0.90) 611 months: 0.76 (0.63, 0.92) individual match: sex, month and year of birth, region of residence at diagnosis adjustment: age at diagnosis, sex, region, maternal age, mother working at time of birth, deprivation 294 all (145 c - all; excludes case <1 year) population - based ascertainment from major paediatric clinical centres in northern and central california 5000 child - hours (1st year) vs 0 all- hispanic: 2.10 (0.70, 6.34) white: 0.42 (0.18, 0.99) c - all- hispanic: 2.53 (0.60, 10.7) white: 0.33 (0.11, 1.01) individual match: date of birth, sex, mother s race, hispanic status adjustment: annual household income, maternal education all cases of childhood leukaemia identified in a cohort of all children in denmark individual match: date of birth, sex, birth cohort adjustment: several demographic characteristics was considered but none were major confounders ses, socioeconomic status; rdd, random digit dialing; ukccs, united kingdom childhood cancer study . As shown in table 2, the 14 studies included a total of 6108 cases and generated a combined or estimate indicating that day - care attendance is associated with a reduced risk of childhood all (or = 0.76, 95% ci: 0.67, 0.87). Figure 1 provides a visual portrayal of the relationship between day - care attendance and the risk of childhood all . Three large studies conducted in germany, the usa and the uk appeared to carry a large proportion of the weight in the meta - analysis at 13% each . The combined risk estimates excluding each of these studies individually remained similarly reduced indicating that no one large study was able to completely explain the protective effect observed (data not shown). No remarkable evidence of publication bias was apparent from the funnel plot since the data points for these 14 studies were, in general, randomly distributed around the combined or estimate (plot not shown). This visual interpretation of the results was confirmed by the large p - value using the rank correlation method (p = 0.553). Table 2meta - analysis of studies examining the association between day - care attendance and risk of childhood allstudy, yearoutcome, age in yearsday - care definitiontimingcasesor95% ciwi (%) petridou et al ., 1993leukaemia, 014attendance at crche: yes / nobefore age 2 years1360.280.09, 0.881.2roman et al ., 1994all, 04pre school playgroup: yes / noyear before dx380.600.20, 1.801.3petridou et al ., 1997leukaemia, 014day care: ever / neverbirth to dx1530.830.51, 1.375.0schuz et al ., 1999 al, 1.514deficit in social contacts: yes / nobefore age 2 years9210.910.90, 1.3012.7dockerty et al ., contact outside home: yes / nofirst year of life900.650.36, 1.173.8infante - rivard et al ., 2000all, 09day care: entry at 2 years / neverat or before age 2 years4330.490.31, 0.775.6neglia et al ., 2000all, 114day care before age 2 years: yes / nobefore age 2 years17440.990.84, 1.1713.3rosenbaum et al ., 2000all, 014out - of - home care:> 36 months / nonebirth to dx1580.760.70, 2.523.3chan et al ., 2002al, 214index and family day care: 3-levelfirst year of life980.960.70, 1.328.5perrillat et al ., 2002al, 215day - care attendance: yes / nobirth to dx2460.600.40, 1.005.5jourdan - da silva et al ., 2004all, 114day - care attendance: yes / nobirth to dx3870.700.60, 1.0010.3gilham et al ., 2005all, 214social activity: any / nonefirst year of life12720.660.56, 0.7713.6ma et al ., 2005whiteall, 114day care first year of life: yes / nofirst year of life1360.770.43, 1.403.8ma et al ., 2005hispanicall, 114day - care attendance: yes / nobirth to dx1201.090.62, 1.904.1kamper - jorgensen et al ., 2008all, 015attendance to child care: yes / nobefore age 2 years1760.680.48, 0.957.9p - value (heterogeneity) = 0.014combined:61080.760.67, 0.87100.0percent weight assigned to each or in the random effects model.schuz et al . : changed reference to yes - deficit in social contacts by calculating the inverse of the or provided for no - deficit in social contacts; rosenbaum et al . : estimated the or for> 36 months by calculating the inverse of the originally provided or for stayed home.day-care attendance censored on reference date was used due to the low number of hispanic subjects attending day care during the first year of life.dx, diagnosis; wi, weight . Figure 1 forest plot displaying ors and 95% cis of studies examining the association between day - care attendance and risk of childhood all . The risk estimates are plotted with boxes and the area of each box is inversely proportional to the variance of the estimated effect . The dashed vertical line represents the combined risk estimate (or = 0.76), and the width of the diamond is the 95% ci for this risk estimate (0.670.87). Meta - analysis of studies examining the association between day - care attendance and risk of childhood all percent weight assigned to each or in the random effects model . : changed reference to yes - deficit in social contacts by calculating the inverse of the or provided for no - deficit in social contacts; rosenbaum et al . : estimated the or for> 36 months by calculating the inverse of the originally provided or for stayed home. Day - care attendance censored on reference date was used due to the low number of hispanic subjects attending day care during the first year of life . Forest plot displaying ors and 95% cis of studies examining the association between day - care attendance and risk of childhood all . The risk estimates are plotted with boxes and the area of each box is inversely proportional to the variance of the estimated effect . The dashed vertical line represents the combined risk estimate (or = 0.76), and the width of the diamond is the 95% ci for this risk estimate (0.670.87). We attempted to maintain a reasonable balance between maximizing the inclusion of studies and minimizing sources of heterogeneity, by relaxing the eligibility criteria to include estimates for broader leukaemia subtypes, other social contact measures and unspecified timing of exposure . The contribution of the influence of possible sources of heterogeneity on the combined risk estimate was evaluated . In subgroup meta - analyses presented in table 3 examining the influence of the timing of exposure, the combined or for seven studies reporting estimates for day - care attendance or social contacts before diagnosis showed a reduced risk of childhood all (or = 0.81, 95% ci: 0.70, 0.94). When the meta - analysis was limited to the nine studies that specifically evaluated day - care attendance at or before age 1 or 2 years, a similarly reduced risk of all (or = 0.79, 95% ci: 0.65, 0.95) was observed . Table 3subgroup meta - analyses of day - care attendance and risk of childhood all evaluating the influence of timing of day - care attendancestudy, yearday care any timeday care at age 2casesor95% ciwi (%) casesor95% ciwi (%) petridou et al ., 1994petridou et al ., 19971530.830.51, 1.377.8schuz et al ., 19999210.910.90, 1.3015.7dockerty et al ., 1999900.650.36, 1.176.5infante - rivard et al ., 20004330.490.31, 0.778.8neglia et al ., 200017440.960.82, 1.1238.017440.990.84, 1.1716.1rosenbaum et al . 20022460.600.40, 1.008.9jourdan - da silva et al ., 20043870.700.60, 1.0022.1gilham et al ., 20081760.680.48, 0.956.3combined:30800.810.70, 0.94100.051260.790.65, 0.95100.0p - value (heterogeneity):0.2770.001percent weight assigned to each or in the random effects model . Wi, weight.schuz et al . : changed reference to yes - deficit in social contacts by calculating the inverse of the or provided for no - deficit in social contacts; rosenbaum et al . : estimated the or for> 36 months by calculating the inverse of the originally provided or for stayed home. Subgroup meta - analyses of day - care attendance and risk of childhood all evaluating the influence of timing of day - care attendance percent weight assigned to each or in the random effects model . Schuz et al . : changed reference to yes - deficit in social contacts by calculating the inverse of the or provided for no - deficit in social contacts; rosenbaum et al . : estimated the or for> 36 months by calculating the inverse of the originally provided or for stayed home. A series of sensitivity analyses were conducted on the meta - analysis of the 14 studies to examine the influence of individual study characteristics on the combined or, namely, potential biases in the selection of controls, the categorization of leukaemia and the assessment of day - care attendance . Figure 2 presents a summary of these analyses showing that none of these factors was able to completely account for the reduced risk of all observed in the main analysis of the 14 studies . For example, in the evaluation of potential control selection bias, reduced risks were observed for the analyses excluding three studies that used hospital - based controls (or = 0.78, 95% ci: 0.68, 0.90) and excluding two studies that used random digit dialing (rdd) to select controls (or = 0.72, 95% ci: 0.63, 0.81). Similarly reduced combined ors were observed when excluding studies that included infants (<1 year of age) in the study population (or = 0.81, 95% ci: 0.70, 0.94), studies not specifically examining all (or = 0.74, 95% ci: 0.63, 0.87), and studies that did not define the exposure strictly as attendance at a day care or a similar type of setting (or = 0.74, 95% ci: 0.61, 0.88). Figure 2 plot showing results of sensitivity meta - analyses evaluating the influence of potential biases within individual studies on combined risk estimates . Plot showing results of sensitivity meta - analyses evaluating the influence of potential biases within individual studies on combined risk estimates . Table 4 presents the results of the meta - analyses evaluating the association between childhood c - all and day - care attendance . The analysis of c - all contained fewer numbers of studies compared with the analysis of all . Similar to the result from the meta - analysis of all, the combined or for the seven studies of c - all was also <1, although the ci was slightly wider (or = 0.83, 95% ci: 0.70, 0.98). The subgroup analyses among studies of day - care attendance before age 1 or 2 years and c - all generated results similar to those for all (data not shown). Table 4meta - analysis of studies examining the association between day - care attendance and risk of c - allstudy, yearageday - care definitiontimingcasesor95% ciwi (%) schuz et al ., 19991.514deficit in social contacts: yes / nobefore age 2 years6581.000.80, 1.2019.9neglia et al ., 200025day care before age 2 years: yes / nobefore age 2 years6331.050.80, 1.3716.3chan et al ., 2002214index and family day care: 3-levelfirst year of life660.930.63, 1.3611.4jourdan - da silva, 2004114day - care attendance: yes / nobirth to dx3040.800.60, 1.0017.0gilham et al ., 2005white25day care first year of life: yes / nofirst year of life740.490.19, 1.262.8-hispanic25day - care attendance: yes / nobirth to dx710.910.41, 2.053.8kamper - jorgensen et al ., 2008015attendance to child care: yes / nobefore age 2 years1010.580.36, 0.938.7p - value (heterogeneity) = 0.044combined:26980.830.70, 0.98100.0percent weight assigned to each or in the random effects model.day-care attendance censored on reference date was used due to the low number of hispanic subjects attending day - care during the first year of life.dx, diagnosis; wi, weight . Meta - analysis of studies examining the association between day - care attendance and risk of c - all percent weight assigned to each or in the random effects model . Day - care attendance censored on reference date was used due to the low number of hispanic subjects attending day - care during the first year of life . The evidence from a large and growing body of literature related to the exposure to infectious agents, as measured by day - care attendance, and the risk of childhood leukaemia was systematically evaluated using a meta - analytic approach . Heterogeneity between epidemiologic studies and their results is common and constitutes one of the major challenges in such a synthesis . Although the random effects model was used in this analysis to account for some of the between - study variation, we acknowledge the importance of interpreting results together with a thorough consideration of the potential sources of heterogeneity . All the studies included in this analysis were conducted with the a priori objective of testing the biologically plausible, delayed infection hypothesis, which specifies a predicted direction of risk, timing of the exposure and the most applicable subtype of leukaemia . Overall, the studies show consistency in support of a reduced risk associated with day - care attendance or social contacts during early childhood, with the vast majority of studies either reporting an effect in the hypothesized direction or no association . A quantitative assessment using meta - analysis indicates that day - care attendance is associated with a reduced risk of childhood all, as well as c - all . The reduction in risk persisted despite a thorough consideration of potential sources of study heterogeneity . We did not conduct a meta - analysis specifically in non - c - all or acute myeloid leukaemia due to the limited number of studies reporting results for these associations . Of the four studies that present data for non - c - all, three studies showed reduced ors, but lacked precision . Based on currently available data, it is difficult to determine whether the association applies to a specific subtype of all only or all in general . The subgroup meta - analysis by timing of day - care attendance did not suggest a stronger reduction in risk for day care specifically at or before age 1 or 2 years as might have been expected based on the hypothesis . However, a few individual studies have shown that the strongest reduction in risk occurs when day - care attendance is started <6 months of age . Although not formally evaluated in this meta - analysis, several individual studies that used detailed exposure assessment protocols demonstrated evidence of dose response effects . Strong trends were observed for increasing levels of child - hours of day - care attendance, levels of social activity and age at start of day care . We were not able to conduct a comparable meta - analysis of studies pertaining to the related mechanism of rural although it was not possible to analyse the role of population mixing in the same manner as was done for the delayed infection hypothesis, it is recognized that these two processes may be interrelated or occur simultaneously and that both mechanisms may be operating in a given population . Thus, the results observed for the analyses of studies providing data relevant to the timing of infection in early life cannot be interpreted as ruling out the possible role of population mixing, but rather lend further support to the role of immune related processes in the aetiology of all . One major consideration in the evaluation of study validity is the possibility of selection bias, a type of systematic error that occurs when there is differential selection of either the cases or controls on the basis of characteristics which may affect exposure status . One way this may arise is if cases and controls do not originate from the same source population . A population - based ascertainment of cases is considered favourable since a defined source population, from which controls may be selected, is easily identifiable . Other strategies of case ascertainment may be appropriate as well, as long as the source population can be clearly defined . As implemented in three of the included studies, selection of controls among the inpatient cohort of the same hospital as the case diagnosis can fulfill this requirement, but can introduce bias if the illnesses / conditions of the control group are related to the exposure under study . Also, it has been suggested that the use of rdd, a population - based method of control recruitment, may result in a control group biased with respect to certain population characteristics that may be associated with exposures of interest . Analysis excluding the three studies that selected hospital - based controls or the two studies that used rdd to recruit population - based controls produced similar results to those for the full set of studies . Similar types of systematic biases resulting in socio - economic differences between cases and controls have been implicated in other studies as well, including the large united kingdom children childhood cancer study (ukccs) and the northern california childhood leukemia study (nccls). Adjustments for these differences have been implemented in the analyses; however, the possibility of residual effects cannot be ruled out . To alleviate some of this concern, results of a subgroup analysis conducted in the nccls among matched cases and controls who had the same annual household income showed that the pattern of association with day - care attendance persisted . The potential for information bias in case control studies is of particular importance due to the retrospective nature of data collection, and the recall of past exposures may be influenced by disease status . Most studies collected exposure data based on respondent recall using a standardized questionnaire administered either in person, by telephone or by mail . Recall bias in the evaluation of c - all is expected to be less likely, since diagnoses of c - all are usually made between ages 2 and 5 years, and recall of early exposure histories may be easier for the primary caregiver . Although the influence of recall bias could not be formally evaluated in these meta - analyses, one records - based day - care study conducted by kamper - jorgensen et al . In denmark reported a reduced risk of childhood all associated with childcare attendance during the first 2 years of life . Several subtype specific analyses performed in this study showed the strongest association in b - cell precursor all and c - all . In addition to potential biases associated with the ability of respondents to accurately recall past events, there was variation between studies in the extent of exposure assessment and categorization of individual exposures to infectious agents . Control study conducted in germany that used a deficit in social contacts variable based on the assumption that children were likely to have attended day care if during the first 2 years of their life both parents were in full - time work . The assumption made in the formulation of this social contact variable most likely contributed some non - differential misclassification, which tends to bias findings towards one of no effect . Their analysis did not indicate an association between deficit in social contact and al or c - all . In contrast, in the ukccs, gilham et al . Created a hierarchical variable that reflected a child s overall social activity based on interview data incorporating information on frequency of regular activity with children outside the home, frequency of attendance at a day nursery or nursery school, and number of other children in attendance . These analyses indicated that social activity / day - care attendance is associated with a reduced risk of childhood all ., in the first publication on day - care attendance from the nccls, constructed a child - hours of exposure variable incorporating information on the number of months attending a day care, mean hours per week at this day care and the number of children exposed to at this day care . They reported that children who had more total child - hours of exposure had a reduced risk of all . These results were later confirmed in a follow - up analysis using a larger study population . In non - hispanic white children, children in the highest category of child - hours during infancy had a reduced risk of all and c - all compared with children who did not attend day care with strong evidence of a dose response effect . This association was not observed in hispanic children, which, as noted by the authors, had different socio - economic and demographic characteristics, including larger family size and different day - care utilization patterns . Although these types of refined exposure assessment strategies that account for duration, frequency and size of the day - care facility serve as examples for future studies, results from these analyses may have contributed to study heterogeneity . In a meta - analysis of 10 studies that strictly defined the exposure as attendance at a day care or other similar types of settings, a reduced risk estimate was observed . Current evidence suggests that different subtypes of leukaemia, defined by both immunophenotypic and molecular characteristics, may be associated with distinct aetiological mechanisms . To minimize the bias associated with misclassification of the phenotype, most studies specifically evaluating the infectious hypothesis have reported results by subtype - specific leukaemia such as c - all, and have excluded infants since there is evidence suggesting these leukaemias may be associated with a causal mechanism involving transplacental chemical carcinogenesis . This is not expected to be a major source of error, as observed in the sensitivity analysis, since infant leukaemias comprise only a very small proportion of all leukaemia diagnoses (<5%). It is believed that the hypothesis on infections, particularly the delayed infection hypothesis, is most relevant to all and its most common subtype, c - all . Limiting the meta - analysis to only those studies providing risk estimates for specific subtypes resulted in a reduced risk associated with both all and c - all . The ukccs recently published results from the first records - based study examining the relationship between clinically diagnosed infections in the first year of life and childhood all . Contrary to what is expected based on the delayed infection hypothesis and what was observed in this meta - analysis of day - care attendance, the results of this well - designed records - based study showed evidence of an increased risk of childhood all and c - all associated with clinically diagnosed infections in the first year of life . It is possible that these contrasting results reflect one of many mechanisms involved in the aetiology of childhood all . The authors explain that their findings may indicate that a dysregulated immune response to infections during the first few months of life leads to an increased risk of all . Alternatively, from a methodological perspective, it has been suggested that these contrasting results may be an indication that previous studies using self - reported data on infections and social contacts, many of which have found a reduced risk of all, may be biased due to differential recall / reporting between cases and controls . Although more studies are needed to evaluate this apparent discrepancy, it is important to note at this juncture that infection based on clinical diagnosis may reflect a different infectious disease experience of the child compared with a self - reported infectious disease history, as mothers may not seek medical attention for all of the common infections experienced by the child . Although still susceptible to recall bias, surrogate measures of exposure to infections such as day - care attendance and birth order, are recognized as strong alternative measures to testing the delayed infection hypothesis, since they are highly associated with common childhood infectious diseases and have the added advantage of capturing a child s asymptomatic infections . It is not known to what extent recall bias may have affected results of previous day - care studies, but there is evidence from a recent denmark study also showing strong evidence of a reduced risk associated with a records - based assessment of day - care attendance . Overall, this meta - analysis of existing epidemiological data provides strong support for an association between exposure to common infections in early childhood and subsequent risk of all . As an indirect measure of exposure to infections, the ability of day - care attendance to serve as a surrogate measure may vary depending on characteristics of the facility attended and the child s pattern of attendance . Epidemiologic studies have shown that the transmission and development of infectious diseases are highly influenced by the age of the child, frequency and duration of attendance, structure and size of the facility . Future epidemiologic studies of childhood leukaemia should attempt to obtain this type of detailed information on the facilities attended to refine the exposure classification . Although inconsistent, there is evidence from studies of other surrogate measures of exposure to infections including birth order, parental social contacts in the workplace, and other immune - related factors (e.g. Vaccination and breastfeeding history), that support a role for infections and immune response in the aetiology of childhood leukaemia . The causal significance of the role of infections in childhood all would be strengthened by identification of a plausible biological mechanism for the conversion of pre - leukemic cells following infection and by incorporation of genetic biomarkers of susceptibility and immune response into further epidemiological studies . The protective effect of early infection on risk of subsequent childhood all parallels the similarly protective impact of parasitic infections on type i diabetes in both animal models and children . An important implication of these hygiene-related hypotheses and supportive data is that some form of prophylactic intervention in infancy may ultimately be possible . Grants from the us national institute of environmental health sciences [grant numbers ps42 es04705, r01 es09137] and the children with leukaemia foundation, uk . Funding to pay the open access publication charges for this article was provided by the us national institute of environmental health sciences [grant numbers ps42 es04705, r01 es09137].
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The sulfur chemistry of transition metals represents a challenging and exciting research topic in the field of inorganic and structural chemistry, which has actually been thoroughly explored by several authors. (1) because of the peculiar features of sulfur species, such as high polarizability, large negative charge, coordination versatility, and manifold apticity, sulfur - based ligands often present a chameleonic behavior with respect to their chemical, redox, and electronic properties(2) as well as to their coordination behavior . Metalsulfur - based compounds find application in different technological fields such as catalysis,(3) photovoltaic materials,(4) magnetic resonance imaging and contrast agents,(5) semiconductor technology,(6) energy storage technology,(7) corrosion prevention, and tribology. (8) most of these applications have been reviewed by stiefel in a dedicated text,(9) while the relevance of sulfur in the field of life science, where many important electron - transfer proteins and enzymes involve the coordination of sulfur - containing ligands to different metals, has been extensively reported. (10) a further intriguing aspect of metalsulfur chemistry is the peculiar electronic structure characterizing ms bonds, which has been the topic of a few reviews. (11) particularly interesting is the formation of polynuclear complexes with metalsulfur bonds because the electron polarizability, which give rises to ms bridges and to the extended ms - based networks, allows the formation of a delocalized electronic structure, which is, in turn, responsible for the outstanding properties of these polynuclear compounds . A wide variety of sulfur ligands have been used to prepare polynuclear sulfur clusters with most of the metals . In this wide and manifold field, thiocarboxylic acids represent a versatile class of sulfur - based ligands and monothio- and dithiocarboxylates have been often reported as ligands in metal mono- and polynuclear complexes . In the past, we explored the synthesis of different early - transition - metal oxo clusters (with omo moieties)(25) by the reaction of metal alkoxides with carboxylic acids, normally obtaining polynuclear structures . As demonstrated by these works, this early - transition - metal oxo cluster chemistry is mainly based on hydrolysis and condensation reactions involving the starting metal alkoxide precursor, which undergoes substitution reaction by the carboxylates . The cleaved alcohol further reacts with an excess of carboxylic acid in an esterification reaction. (26) the in situ formed water accounts for the hydrolysis / condensation reactions, leading to the formation of the omo - based metaloxo inorganic core, in a sui generis solgel reaction . These polynuclear structures are typically obtained by reaction of the corresponding metal alkoxide with a carboxylic acid . They are characterized not only by different metals but also by manifold nuclearity, structures and connectivity modes, and different functional groups, which can enable, in a further synthetic step, their embedding into a matrix through reaction with suitable precursors . Some review articles(25) have extensively described the chemistry of these polynuclear oxo clusters, whereas the mechanism leading to their formation and their ligand exchange dynamics has also been the topic of thorough studies . Cluster chemistry of zirconium is an established and mature field of research,(27) especially as far as zirconium halide clusters(28) are concerned . More recently, several authors have focused on oxo and carboxylato clusters of zirconium also as precursors for the corresponding nanostructured oxide. (29b) in the present work, we used monothioacetic acid to coordinate zirconium (added as butoxide), generating polynuclear clusters, with a behavior in some way related to what was observed with the homologue carboxylic acids that we employed in the past . All reactions and manipulations were carried out under an argon atmosphere using standard schlenk or septum / cannula techniques . Zr(obu)4 (80%) in n - butanol (purchased by abcr gmbh, karlsruhe, germany) and anhydrous n - butanol (99+%; purchased by aldrich, milan, italy) were stored under an argon atmosphere and not further purified . Monothioacetic acid (taa; 96%; purchased by aldrich, milan, italy) was used as received . Two samples were characterized through raman spectroscopy: crystalline compound zr4(3-o)2(-socch3)2(socch3)8(obu)2 (zr4), synthesized with a molar ratio zr / taa of 1:8, and monothioacetic acid as the reference . They were put in capillary tubes under an argon atmosphere in order to avoid air contact . Raman spectrum of liquid monothioacetic acid: 2919 (s, sh, ch), 2574 (s, sh, sh), 1706 (m, c = o in c(o)sh), 1427 (w), 1134 (w), 999 (w), 839 (w), 629 (s, sh), 450 (s, sh), 329 (w), 145 (w). The h and c nmr spectra were obtained at 298 k as c6d6 solutions with a bruker dmx-400 avance spectrometer operating at 0 400.13 and 100.61 mhz, respectively . Details on the experiments (cosy, tocsy, noesy, hmqc, and bird) performed and chemical shift assignments are reported in the supporting information . Crystals of zr3, zr4, and zr6 suitable for single - crystal x - ray diffraction were taken directly from reaction mixtures, selected in perfluoropolyether oil, mounted on a bruker axs kappa diffractometer with an apex ii ccd area detector, and measured in a nitrogen stream at 100 k. graphite - monochromated mo k radiation (= 71.073 pm) was used for all measurements . The structures were solved with direct methods and then refined by the full - matrix least - squares method based on f using the program package shelxtl (bruker axs). Selected bond lengths are summarized in table 2 for zr3 and in table 3 for zr6 and zr4 . Zr3 [zr3(3-s3cch3)2(s2cch3)6]2buoh: disordered buoh was found and refined in channels between the cluster molecules; a distinction between the c and oh end was not possible . Therefore, all atoms were refined as c atoms . Zr6 [zr6(3-o)5(-socch3)2(-o2cch3)(socch3)11(buoh)]: two of the unbridging socch3 groups were refined disordered over two positions with occupancies 0.89:0.11 and 0.74:0.26 . This could be improved when additionally an s atom with occupancy 0.06 (and o131 with occupancy 0.94) was refined on the position of the residual electron density maximum, which was situated 69 pm from o131 and 257 pm from zr5 . However, the anisotropic parameters of this s atom were nonpositive definite . Therefore, -o2cch3 was substituted by -monothioacetate by around 6%, but it was not implemented in the structure refinement . The buoh group was refined disordered over two positions (0.57:0.43), although there are probably at least three different positions . The highest residual electron density peak (2.45) was found at 216 pm from c074 of the buoh . This could be explained by partial occupation by a small molecule, e.g., monothioacetic acid . Zr4 [zr4(3-o)2(-socch3)2(socch3)8(obu)2]: the buo ligand was refined with very small anisotropic parameters, even for the terminal c atoms . The zro distance was 187.2(2) pm, which is in good agreement with values reported in the literature for zrobu coordination (see the discussion for zr6). Ccdc 779090 (for zr3), 779091 (for zr4), and 779092 (for zr6) contain the supplementary crystallographic data for this paper . These data can be obtained free of charge from the cambridge crystallographic data centre via www.ccdc.cam.ac.uk/data_request/cif . The solution for exafs analysis was prepared with the same procedure as that employed for the synthesis of the clusters, using an alkoxide / taa molar ratio of 1:6 . The exafs measurements were performed at beamline c1 of the hamburger synchrotronstrahlungslabor (hasylab) at desy (hamburg, germany). They were performed at the zr k - edge (17998.0 ev) using a si(311) double crystal monochromator . A detailed description of the experiments and the procedure adopted for data evaluation(31) and processing are reported in the supporting information . The solution for exafs analysis was prepared with the same procedure as that employed for the synthesis of the clusters, using an alkoxide / taa molar ratio of 1:6 . The exafs measurements were performed at beamline c1 of the hamburger synchrotronstrahlungslabor (hasylab) at desy (hamburg, germany). They were performed at the zr k - edge (17998.0 ev) using a si(311) double crystal monochromator . A detailed description of the experiments and the procedure adopted for data evaluation(31) and processing are reported in the supporting information . To an 80% solution in buoh of zr(obu)4 (2.67 g, 5.6 mmol) monothioacetic acid (3.2 ml, 44.6 mmol) was added at 298 k (molar ratio zr / taa = 1/8). After the addition of taa, evolution of h2s was observed, which was removed from the schlenk - tube through repeated venting . The reaction mixture was then allowed to stand at room temperature for 7 days, resulting in the separation of yellow, rectangular crystals . The nmr spectra of the oxothio cluster zr4 were recorded as c6d6 solutions because of the instability of these species in deuterated acetone and dimethyl sulfoxide . Nevertheless, besides core - bonded n - butoxy and monothioacetate peaks, the spectra showed as most relevant signals those pertaining to n - butyl acetate, likely due to the presence of adventitious water . The spectra showed as the most relevant signals those due to n - butyl acetate, reported below . Nmr: 0.854 (m, 3h), 1.285 (m, 2h), 1.494 (m, 2h), 1.842 (s, 3h), 4.045 (m, 2h). C nmr: 14.36, 20.01, 21.12 (acetyl ch3), 31.66, 64.62, and 170.61 (co). H nmr: 0.97, ca . 1.2, ca . 1.6, ca . 4.2 . H nmr: 2.41, 2.43, 2.51, 2.55, 2.62, 2.67, 2.69 . C nmr: 36.6 and 45.9 (ch3), 220.0, 222.2, 226.4, 230.2, 231.4, 232.8, 233.0, 234.0, 261.3, 265.2, 267.0, and 268.7 (thiocarbonyl). The raman spectrum of the oxothio cluster zr4 (figure 4) was recorded on the crystals, whereas that of the monothioacetic acid was recorded directly on the liquid specimen . The obtained spectra were analyzed by using literature data. (33) raman data of zr4: 2914 (s, sh, ch), 1464 (w), 1352 (w), 1178 (m), 707 (s, sh), 574 (m, br), 531 (w), 380 (w), 246 (m, sh), 206 (m). To an 80% solution in buoh of zr(obu)4 (1.60 g, 3.3 mmol) monothioacetic acid (1.9 ml, 26.7 mmol) was added at 288 k (molar ratio zr / taa = 1/8). After the addition of taa, evolution of h2s was observed, which was removed from the schlenk - tube through mild venting . The reaction mixture was then allowed to stand 30 days at 278 k, resulting in the separation of yellow, rectangular - shaped crystals . From the same reaction batch of zr6, maintained at 278 k, after 60 days red crystals separated from the reaction mixture . The same crystal formed exclusively when h2s was added (see below) to the reaction system of the synthesis of zr6 . It is worth noting that, in all of the reactions of zr(obu)4 with taa, the development of h2s was observed, which was removed from the reaction batches by venting . To an 80% solution in buoh of zr(obu)4 (2.07 g, 4.3 mmol), prepared at 263 k was added monothioacetic acid (1.8 ml, 26.0 mmol) at 263 k (molar ratio zr / taa = 1/6) after the addition of taa and h2s, further evolution of h2s was not observed as long as the temperature system was maintained at lower than 268 k. at a temperature of 268 k, considerable evolution of gas (h2s) was observed, which was removed from the schlenk - tube through venting . After 30 min, the reaction mixture was allowed to stand at room temperature for 3 h and then at 278 k for 30 days, resulting in the separation of dark - red crystals, identical with those of zr3 . As demonstrated by previous works, this early - transition - metal oxo cluster chemistry is mainly based on hydrolysis and condensation reactions involving the starting metal alkoxides precursor, which undergoes substitution reaction by the carboxylates . The alcohol further reacts with an excess of carboxylic acid in an esterification reaction,(28) and the in situ formed water accounts for the hydrolysis / condensation reactions, leading to the formation of the omo - based metaloxo inorganic core, in a sui generis solgel reaction . In this study, we aimed to explore the possibility of extending the well - known oxo cluster chemistry to the formation of analogous thiooxo and dithio cluster structures . The research in this framework is driven by the impact on the basic structural and inorganic chemistry of metalsulfur compounds as well as, as was already outlined, by possible applications in a number of fields based on compounds characterized by different electronic structures, with respect to their oxygen - based homologues . In this framework, the formation of heteroleptic complexes, with s, o or s, s ligands chelating the same metal ion, turns out to be particularly interesting because the smo and sms fragments are expected to be characterized by electronic properties significantly different from those of the omo one . Concerning zirconium, coucouvanis et al . Explored the synthesis and structural characterization of different zrs polynuclear clusters, both trinuclear and hexanuclear, whereas other chalcogenido complexes of zirconium have been reported by howard et al. (38) and kekia and rheingold. (39) however, to the best of our knowledge, both monothioacetic and dithioacetic acid have not yet been employed for the synthesis of thiozirconium polynuclear clusters . Therefore, we started our studies by reacting monothioacetic acid with zirconium n - butoxide at room temperature and, in the absence of a solvent, by slightly changing the reaction conditions (molar ratios, reaction temperature, etc .) In different experiments . In table 4, the experimental conditions for the obtainment of the different clusters are summarized, together with the other reaction conditions, leading to the formation of crystals that were, however, not structurally resolved . As described above, the reactions were performed by varying the zr / taa molar ratio and by adding ex situ h2s to the reaction batch (see table 4). Moreover, further experiments were carried out to investigate how modification of the other experimental conditions (temperature, solvent, solvent removal, etc .) In particular, we first changed the temperature at which the reaction occurred in the range from 15 to + 27 c . The effect of this variation was, upon a temperature decrease, mainly to slow down the formation of the solid precipitate . At low temperature (15 c), the observed growth of the crystals was extremely slow, whereas at room temperature (27 c), sudden precipitation of a microcrystalline solid was instead observed . Concerning the addition of a solvent, one attempt was carried out by using anhydrous thf [molar ratios: zr(obu)4/taa / thf = 1/4/1 at 20 c]. In this case the reactions were accordingly carried out without the addition of solvent(s) because the presence of a liquid phase was already provided by the nature of the precursors, both liquid [taa and 80% zr(obu)4 in butanol]. As far as the removal of excess butanol and the liquid phase is concerned, this led invariably to degradation of the formed crystals . About the kinetics of crystal formation, some general considerations can be made . (i) the formation of different crystals occurs always in the same sequence: first, the yellow rectangular - shaped crystals (zr6) and, after a time span of about 60 days, red crystals (zr3). (ii) the formation of zr4 seems to be favored by lower temperatures; the formation of the zr3 crystals containing only sms bonds does require longer time . (iii) the ex situ addition of h2s promotes the selective formation of red crystals of zr3 . From the latter two points, it can be concluded that the selective formation of zr3 is triggered by the presence of s either added ex situ as h2s or formed in situ upon hydrolysis of monothioacetic acid (see scheme 2sm in the supporting information). This latter reaction requires longer times, thus explaining the slower formation of the zr3 crystals . It should be pointed out that all of the obtained crystals are highly sensitive to air and humidity and rapidly hydrolyze upon exposure to air . For this reason, we restricted our analyses to only the samples reported in table 4 . In all of the syntheses, the fast development of h2s, which had to be removed from the schlenk - tube by outgassing, was observed upon the addition of monothioacetic acid to zirconium butoxide (see section 4.5). As discussed in section 4.5, three new polynuclear complexes were characterized by single - crystal x - ray diffraction . In the following, the crystallographic symmetry of the cluster is c2, with the 2-fold axis passing through zr2 (figure 1). However, the molecular symmetry of the cluster is d3, with a 3-fold axis through the cme groups of the s3cch3 ligands . Three zr atoms form a nearly equilateral triangle and are capped by two 3-s3cch3 ligands . The zrzr, zrs, and sc distances are in the range 385.97(3)387.04(3), 264.36(3)266.24(4), and 182.96(14)183.44(14), respectively . Each zr atom is coordinated by four s atoms from the two capping 3-s3cch3 ligands and four s atoms from the two chelating s2cch3 ligands, which leads to the coordination number 8 for all zr atoms . The presence of a 3-s3cch3 ligand with similar coordination behavior was previously found in a nickel complex [ni3(3-s3cch3)(s2cch3)3],(40) although the sc distances for the 3-s3cch3 ligand are in a broader range there (176199 pm). The origin of 3-s3cch3 and s2cch3 ligands in zr3 will be discussed in section 4.5 . It has the same space group but different cell parameters [a = 2007.6(2) pm, b = 1675.62(19) pm, c = 1552.0(3) pm, = 125.247(1), v = 4263.9(11) pm]. The core of this oxo cluster (figure 2) is formed by a planar star - shaped pentagon with one zr atom in the center, five zr atoms in the tips, and five o atoms, each bridging three zr atoms, with one of them being the central zr atom . The maximum deviations of zr and o atoms in the star from the plane calculated through all zr atoms are only 7.24(4) and 6.5(4) pm, respectively, and the sum of the bond angles around the 3-o atoms is close to 360. the zrcenterzrtip, zrcenter3-o, and zrtip3-o distances are in the ranges 330.13(8)341.15(9), 206.9(4)222.5(4), and 197.7(4)209.8(4) pm, respectively . In this cluster, the variable coordination behavior of the zr atoms is noteworthy; each zr atom has a different coordination environment (table 5). The connection between the individual zr atoms is mediated mainly through oxo bridges; only zr1 and zr2 are connected also by two 2-socch3 ligands on both sides of the star plane [distance zr1o = 208.0(4) and 209.2(4) pm; zr2s = 274.66(17) and 281.63(16) pm], and zr5 and zr6 are connected by a -o2cch3 (to 5% -socch3; for details, see the experimental part) ligand in the star plane [distance zro = 217.4(4) and 218.9(5) pm]. There is also a weak interaction between the o atom of a socch3 chelating ligand on zr4 and the adjacent zr5 [distance zr5o10 = 271.5(4) pm]. The distance is much longer than the similar one in zr4, maybe also because of the higher coordination number of zr5 (zr6) than that of zr1 (zr4). This ligand lies also in the zr plane [deviations are 26.1(5) pm for o10 and 9.65(18) pm for s10]. The zrtipo and zrtips distances are in ranges of 221.7(5)230.7(8) and 265.87(16)276.03(17) pm, respectively . M(thiocarboxylate)x (x = 2, 3) coordination has not often been reported for transition - metal complexes . The presence of a buoh ligand (rather than buo) was confirmed by the rather long distance zr3o71/o75 = 222.0(8)/217.9(10) pm, although the h atom was not found in the residual electron density (for details, see the experimental part). For comparison, typical zro distances of the zror groups are in the range 195200 pm, while a zro distance of 228.6(5) pm was found for the coordinated n - butanol molecule in zr6o4(oh)4(methacrylate)8(isobutyrate)4(buoh). (32) this cluster is built from two zr2(socch3)5(obu) units connected by two oxo bridges and related by an inversion center (figure 3). Its crystallographic symmetry is ci, but the molecular symmetry is almost c2h if we do not take into account the buo ligands . Its core is formed by four zr atoms, which lie exactly in a plane, and two o atoms, which connect three zr atoms each and are located 0.23(19) pm off the zr plane . Furthermore, zr1 and zr2 are connected by the o atom of a bridgingchelating -socch3 ligand, which is also approximately located in the zr4 plane [4.4(2) pm off the zr plane for o4 and 1.19(10) pm for s4]. The zro distances in the core lie in the range 203.5213.1 pm . In the -socch3 ligand, the zro distance to the adjacent zr1 is even shorter than that to zr2 [distance zr2o4 = 234.13(19) pm, zr1o4 = 228.6(2) pm, and zr2s4 = 276.40(8) pm]. Besides this, all zr atoms are coordinated by two chelating socch3 ligands, zr1 additionally by a terminal obu ligand . This results in coordination numbers of 7 and 8 for zr1 and zr2, respectively . The zro and zrs distances for unbridging socch3 ligands are in the ranges 224.3(2)224.7(2) and 266.76(8)270.11(9) pm, respectively . The distance zr1o11 = 187.2(2) pm shows that the buo ligand is deprotonated because the distance zr1o11 is much shorter than the equivalent bond for the buoh ligand in zr6 . To the best of our knowledge, such a coordination of thiocarboxylate ligands, where o is bridging and s coordinates to the parent metal, has, to our knowledge, not yet been reported for transition - metal complexes . Monothioacetic acid and the crystalline zr4 cluster were additionally analyzed by raman spectroscopy performed under an argon atmosphere . The assignment of the peaks was carried out on the basis of (i) the very few references present in the literature on raman spectra of compounds containing zrs bonds,(31) (ii) a comparison with monothioacetic acid, and (iii) consideration of the cluster structure . Both the spectra of zr4 and taa present a sharp and intense band at about 29142919 cm, ascribed to ch stretching . On the contrary, the sharp peak corresponding to the sh stretching at 2574 cm in the spectrum of taa is not present in the spectrum of the cluster zr4, in agreement with the crystal structure, which evidences deprotonation of monothioacetic acid upon coordination to the zr atoms . Accordingly, in the zr4 spectrum also c = o stretching of the undissociated acid at 1706 cm disappears upon coordination . Instead, a sharp band at 1179 cm is present, which can be ascribed to cs stretching in the monothioacetate species. (31) the band at 706 cm would correspond, accordingly, to cs deformation . The most interesting zone is that in the range 380200 cm, where the metaloxygen and metalsulfur stretchings are expected . By comparison with the spectrum of the reference acid and with the literature data,(31) the band at 246 cm in the cluster spectrum is ascribed to the zrs eg phonon frequency, whereas that at 291 cm could be assigned to zro . The assignment of this latter band is, however, challenging because at 300 cm also the symmetric zrs stretching would be expected;(31) in fact, both bonds (zrs and zro) are present in the tetranuclear cluster structure (see figure 3). The solution behavior of the cluster zr4 was investigated by h and c nmr mesurements . Most of the resonances are attributed to n - butyl acetate (see the experimental section). An additional set of four, less intense, broad resonances (see the experimental section) is attributed to n - butoxy units reasonably bonded to the metal core . Moreover, another set of less intense and broader h nmr resonances in the ranges 2.342.44 and 2.632.67 ppm correlate in the hc hmqc heteronuclear one - bond correlation map with broad c nmr resonances at 36.6 and 45.9 ppm, respectively, and in the hc hmbc multiple - bond heteronuclear correlation spectrum with two sets of c nmr resonances detected in the ranges of 220235 and 260270 ppm as well, as shown in figure 5 . We suggest that these very low field signal sets are due to thioacetate moieties engaged in different coordination situations with the metal core of the cluster . To the best of our knowledge, there are no reports on the c chemical shift values of the carbonyl moiety in thioacetate salts or thioacetate coordination compounds, likely because of the instability of this kind of subunit . Application of the principal component analysis,(41) as developed by tasic and rittner,(42) allowed us to calculate for the (thio)carbonyl c nucleus a downfield shift of ca . 30 ppm on going from methyl acetate to methyl thioacetate . By applying this procedure to the zirconium oxo clusters recently published by some of us,(27a) where it is reported that the carbonyl c nuclei of chelating acetates resonate at 184.7 ppm, we estimated for a chelating thioacetate a rough value of ca . 215 ppm, which is in acceptable agreement with the 220235 ppm values found for the cluster . Moreover, in agreement with the literature,(43) we attribute the lowest - field hc (260270 ppm) to the chelating thioacetates bearing a bridging carbonyl o atom observed in the crystal structure . This additional coordination should further deshield the c nuclei and make the coordinative bond stronger . This is confirmed by the absence of exchange peaks in the phase - sensitive noesy measurements (see figure 1sm in the supporting information) between the h nuclei of the corresponding methyl groups and those of the other methyl groups belonging to the chelating thioacetates, which, in contrast, exhibit intense exchange correlations, as was already found and discussed . In order to achieve a deeper insight into the mechanism of the reaction of zirconium butoxide with monothioacetic acid in a 1:6 molar ratio, time - dependent exafs measurements were carried out during the first 20 h after the mixing of the two reactants . In figure 6, the consecutive x - ray absorption near - edge structure (xanes) spectra are shown, together with zr(obu)4 and zr4 as references . Zirconium xanes spectra offer the possibility of gaining insight into the oxidation state by inspection of the edge position and the near coordination number around the x - ray absorber by inspection of the white line (first resonance after the edge step). As outlined in a previous work, zr k - edge xanes spectra show a split white line for coordination numbers of 6 (octahedral) in the nearest - neighbor shells, while only a single resonance can be found for higher coordination numbers. (46) this is also evident in figure 6 . The reference spectrum of zr(obu)4 shows the expected splitting because this compound is present as octahedrally coordinated dimeric species. (47) in contrast, the spectrum of zr4, whose nearest - neighbor coordination is composed of five o and two s atoms, exhibits only a single white line . Although the white - line shape in the course of the reaction of zirconium butoxide with monothioacetic acid differs from the final product, only a single white line is still present over the whole period of 20 h; i.e., also these samples show a higher nearest - neighbor coordination number than 6 . However, because of the rather broad white line, a mixture of six and higher coordinated zirconium centers is likely . While the xanes analysis is restricted mostly to the nearest - neighbor coordination, evaluation of the exafs spectra, which are shown in figure 7, allows more detailed insights into the reaction mechanism . The results of fitting of the experimental spectra with theoretical models are summarized in table 6 . The changes observed within the individual shells over the course of the reaction are summarized in figure 8 . All spectra of the reaction were fitted with models consisting of zro, zrs, and zrzr contributions . Zrc shells were not included in the first because of the problematic backscattering properties of carbon. (48) the first measurement was started approximately 10 min after mixing of the reactants; thus, the initial transformation of the starting compound zr(obu)4 into an sulfur - containing species can be considered as rather fast . From figure 7, it is evident that in the following time period the most significant changes in the k(k) spectra occur in the ranges 56, 78, and 1011, while this is the case in the fourier - transformed function between 2.5 and 3 and around 3.5 . Fitting the spectra at the beginning required a short oxygen shell at around 1.9, which is in very good agreement with the zro1 shell found in zr4 . It has to be admitted that from the exafs data it is not clear whether a mixture of sulfur - containing species and zr(obu)4 or only one complex with a short zro1 contribution is present . However, because zrs and zrzr contributions according to the final structure of the zr4 cluster are already detected, the second case is more likely . However, over the course of the reaction, the short zro1 contribution vanishes and only one zro shell remains . Although the coordination number of the second oxygen shell (zro2) is higher than that in zr4, its distance agrees quite well with the final cluster . The overall oxygen coordination is slightly reduced from 6 to 5 when only one zro contribution can be found after 726 min but increases again to 6.7 after an additional 380 min, which can be considered as the first indication of another rearrangement . Surprisingly, also a reduction of the zrs coordination number is observed in the first 20 h of the reaction from 0.7 to 0.1 and only a minor contribution remains at the end of the measurements . The sulfur shell at the beginning of the reaction is therefore more related to the final zr4 cluster than that after 20 h. the zrzr contributions show the opposite behavior because after 10 min the zrzr1 coordination number is higher than that in the final zr4 cluster and diminishes over the course of the reaction . The coordination number in the zrzr2 shell is constantly slightly higher than that in zr4, from which a more condensed cluster structure, likely a square pyramid or a tetrahedral cluster, can be deduced. (49) from the exafs measurements, it is therefore clear that in the first 20 h of the reaction the final cluster is not yet formed (and actually its crystallization requires a longer time). Moreover, at the beginning, the shell structure of the final cluster is already present with shorter and longer zro contributions, a zrs and two zrzr shells, but this precluster structure of zr4 is then subject to strong changes in the first 20 h of the reaction . At the end of the measurements, only the zirconium core exhibits a close relationship to zr4, while the oxygen and sulfur shells indicate completely different structures in this time interval, which likely undergo further rearrangements and coordination / hydrolysis reactions to give the observed final structure . Xanes spectra recorded for the references zr(obu)4 and zr4 and over the course of the reaction of zr(obu)4 with 6 equiv of taa at 278 k recorded at the times indicated in the graph . Experimental k(k) spectra (left) and their corresponding fourier - transformed functions (left) of the references zr(obu)4 and zr4 also recorded over the course of the reaction of 1 mol of zr(obu)4 with 6 mol of taa at 278 k. the spectra were shifted for clarity . Summary of the kinetic changes in the individual neighbor shells over the course of the reaction according to table 6 . Fermi energy, i.e., shift to account for discrepancies between the experimental and theoretical functions . A particularly intriguing task in the investigation of these thio and oxothio clusters was to unravel the reaction steps leading to the formation of these polynuclear complexes starting from zr(obu)4, which, according to recent exafs studies, evidenced in solution a dinuclear structure, with the zr atoms in an octahedral coordination . The chemistry of monothioacetic acid, which is much more acidic (pka = 3.3) than the oxygen homologue acetic acid (pka = 4.76), is extensively reviewed in the literature . Thiocarboxylic acid undergoes tautomerism, with the thiol form (i) being the predominant one and, at lower temperature and in polar solvents, the thion form (ii) prevailing (see scheme 1sm in the supporting information). The hydrolysis of monothioacetic acid to give acetic acid and hydrogen sulfide, according to the reactions shown in scheme 2sm in the supporting information, has been thoroughly investigated by different authors(52) and the heat of hydrolysis has been determined, pointing out that the hydrolysis rate and the extent of conversion to acetic acid are both enhanced by acidic media and low ph. (52b) in the reactions described in this paper, monothioacetic acid can interact both with the coordinated buo moiety of zr(obu)4 as well as with buoh coordinated to the alkoxide in the commercial formulation . It also needs to be taken into account that in most of our reactions monothioacetic acid is employed in a large excess . The esterification reaction between the alcohol and monothioacetic acid generates h2o, which can be considered the starting reagent for a large series of reactions . In fact, on the basis of the experimental evidence (development of h2s) and literature data,(53) it is reasonable to consider the following side reactions, which account for the formation of both acetic and dithioacetic acids whose anions are present in two of our clusters . The plethora of described reactions and equilibria (tautomerism and hydrolysis of monothioacetic acid, hydrolysis and condensation, coordination, rearrangements), often simultaneously occurring in the reaction batch, and the lack of analytical tools to reliably follow these processes dramatically complicates the picture . As described at the beginning of the results and discussion section, different experiments were carried out to try to elucidate the reaction sequence, and on the basis of the obtained results and of previous knowledge, some mechanistic hypothesis for the formation of the different species can be proposed . Concerning the zr4 cluster, characterized by the s, o coordination of thiocarboxylate ligands, where o is bridging and s coordinates to the metal, it is reasonable to assume that its formation proceeds along the reaction pattern similar to that already proposed for the homologous oxo clusters (see scheme 1). The above scheme accounts also for the presence of 3-o, found in both zr4 and zr6, and of the acetate anion, found in zr6 . As far as this last point is concerned, even though we have no experimental evidence, it has to be highlighted that the acetate ion is present only in zr6 . This finding is in agreement with the fact that the formation of zr6 seems to occur in a second step, after the formation of zr4 . Actually, time - resolved exafs data suggest that, after a first fast coordination of monothioacetate, a complex sequence of events, likely involving also one or more rearrangements of the first formed structure, occur . These rearrangements would account for the formation of a more complex (and quite unsual) structure than that observed in zr6, in which the structural motifs are similar to those observed in zr4, as explained below . Moreover, scheme 1 and equilibria shown in schemes 1sm and 2sm in the supporting information account also for the evolution of h2s, which develops in the first stages of the reaction, and it is assumed to be a key step for the formation of the other structure observed, zr3 . In fact, in the thiocluster zr3, it is possible to observe the presence only of sulfur - based ligands, actually dithioacetates and the peculiar ethane-1,1,1-trithiolate moiety, [3-s3cch3]. While the formation of dithioacetate ions is related to the equilibrium (1b) of scheme 2sm in the supporting information, the presence of the [3-s3cch3] trianion is more complicated to explain, even though the participation of h2s or s in its formation seems evident . It was worth noting that the ethane-1,1,1-trithiolate moiety has been previously found as a ligand in a nickel(ii) cluster obtained by bonamico et al. (40) by the reaction of nickel bis(dithioacetate) with cs2, whereas kniep and reski(54) reported the formation of a 1:1 adduct of asi3 with hexathiaadamantane (shown in figure 9) by the reaction of asi3 with monothioacetic acid . This hexathiaadamantane group was also obtained by the reaction of monothioacetic acid with zncl2, as reported by stetter. (55) hexathiaadamantane obtained by the reaction of taa with asi3, by kniep and reski. (54) although it was not possible, on the basis of the available experimental evidences, to propose a complete mechanism for the formation of this unusual 3-s3cch3 ligand, it is reasonable to assume that the species ch3cs2 and s are involved . It can be argued that, in the presence of these species, a first cluster structure with sulfide ligands forms to which, in a second step, the dithioacetate ligands attach . This hypothesis seems to be strengthened by the experimental evidence that the formation of zr3 requires longer time, after the formation of zr4 (first obtained product) and zr6 (second product), i.e., after that extended hydrolysis reaction occurred, with the formation of h2s . Moreover, it has also to be highlighted that whenever h2s was added as a coreactant to the reaction mixture the selective formation of zr3 was observed, thus confirming that s is actually involved in the formation of this thiocluster . The relevance of hydrolytic processes in the formation of the reported zirconium clusters is strengthened by the experimental evidence that the crystals of zr4 are formed before those of zr6, whose formation requires the occurrence of complete hydrolysis reaction . Its formation can be explained by assuming the aggregation of dimeric and trimeric units (analogues to those leading to the formation of zr4), in the presence of acetic acid in the reaction mixture . By considering the structure of zr6, it is possible to evidence the presence of a repeating structural unit, observed also in the zr4 structure, thus strengthening the assumption of the preliminary formation of the structural units leading to zr4, which presents exclusively monothioacetate ligands and which, under suitable conditions, can rearrange to give zr6 . The reaction of zirconium butoxide with monothioacetic acid resulted in three crystalline polynuclear complexes, which are characterized by different cluster cores and different sulfur - containing and sulfur - free ligands . The clusters evidence that the reactions in this system are by far more complex than the corresponding reactions with carboxylic acids because of the simultaneous equilibria of the precursor acid . In particular, the presence of coexisting equilibria of monothioacetic acid, leading to the formation of h2s and of the dithioacetate species strongly affects the occurrence of the reactions and the nature of the final product . In fact, two of the formed clusters (zr4 and zr6) are characterized by the s, o -coordination of thiocarboxylate ligands, and it is reasonable to assume that their formation occurs following a reaction pattern similar to that already observed in the case of oxo clusters . On the contrary, as far as the thiocluster zr3 is concerned, which is characterized by the presence of only sulfur - based ligands (dithioacetates and the peculiar ethane-1,1,1-trithiolate moiety, [3-s3cch3]), a completely different reaction pattern is expected to take place . In this case, the involvement of h2s or s [either free or coordinated to (and activated by) the metal atom] to its formation could be argued . These species would lead to the formation of a first cluster structure based only on sulfide ligands to which the dithioacetate ligands formed by hydrolysis of monothioacetic acid coordinate in a second step . Accordingly, the formation of this thiocluster requires more time (necessary for the extended hydrolysis reaction, leading to the release of h2s), and it is favored in the presence of an excess of h2s in the reaction batch . It might be interesting, in this regard, to compare the relatively well - known and established oxo cluster chemistry with these new thioxo and dithio clusters . Apart from the already highlighted analogies in coordination modes, nuclearities, and polyhedra arrangements and connectivities, there are also remarkable differences . First of all, the most striking difference, which can be traced back by the hybrid nature of the chosen ligand, bearing both sulfido and oxo teeth, is the presence of concurring equilibria, involving monothioacetic acid and accounting for the presence in solution of monothio- and dithioacetate species which could all be detected in the three different structures, as well as the presence of both sulfido and oxo bridges . In the homologue oxo clusters the ligands, both chelating and bridging, are in all cases carboxylates, and the bridges the manifold nature of the species present in solution remarkably complicates the overall picture, thus making particularly difficult to propose a reaction mechanism . The only conclusions we can draw in that regard are summarized in schemes 1 and 2 . In conclusion, the described thio and oxothio clusters evidence as the well - established chemistry of the homologous oxo clusters (based on omo bonds) can actually be extended to the sulfur - based species, and polynuclear clusters in which both smo and sms bridges are present can be obtained . However, it should be pointed out that, in this case, the existence of different equilibria involving the bidentate ligand remarkably affects the fate of the reaction, leading to the formation of species completely different from the structural and coordination points of view with respect to those observed in the case of the oxo clusters . Indeed, although some recurring structural motifs are similar to those observed in the case of oxo clusters, such as the presence of chelating and bridging modes for the bidentate (thio- and dithioacetate) ligands, completely new coordination fashions and geometries are observed . On the basis of these considerations, it can be highlighted that the presented results, although several questions and chemical issues still remain open, pave the way for the investigation of a still relatively unexplored field of research, i.e., the polynuclear chemistry of early transition metals with sulfido and oxo / sulfido ligands . The obtained results and structures contribute to shedding light on the already outlined complex chemistry of these systems and also to triggering new research on further metals such as hafnium, titanium, and vanadium as well as on further ligands (e.g., dithioacetic acid, thiomethacrylic acid, etc . ),
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Ulcerative colitis (uc) is an idiopathic disorder that involves a proximal extension of chronic inflammation in the colonic mucosa . Major manifestations of uc include bloody diarrhea, rectal bleeding, frequent stools, tenesmus, and pain in the lower abdomen, all of which undergo repeated relapses following exacerbation and remission of the disorder [1, 2]. Although the exact etiology of uc is yet to be fully understood, the general consensus is that a combination of the following factors leads to its development: genetic risks of maternal inheritance, environmental variability, and dysbiosis [3, 4]. Treatment of the disease mainly consists of conventional pharmaceutical approaches, as well as surgical interventions for those who do not respond to the initial therapeutics . For uc patients exhibit an impaired quality of life, treatment is mostly aimed at restoring it, through induction and maintenance of the remission phase . Drugs currently in use for patients with uc can be categorized as anti - inflammatory drugs, immunosuppressants, pro / antibiotics, and biologic agents . However, in most cases, the underlying mechanisms are still not clear, and long - term efficacy is achieved in only approximately one - third of the patients . Current options are also hindered by undesired adverse effects such as metabolic changes, increased risk of infections or lymphomas, or even higher mortality . Therefore, it is imperative that effective complementary and alternative measures be developed to remedy such defects . Animal models of uc have proven to be useful in terms of understanding the pathophysiology of uc, not to mention testing the in vivo potency of possible therapeutic agents . While murine models are most frequently used, a uc - like phenotype is induced via chemical administration or bacterial infection [4, 7]. Dextran sulfate sodium (dss), trinitrobenzene sulfonic acid (tnbs), oxazolone, and acetic acid are major colitogenic substances that cause injury to the intestinal epithelium . The most widely used is the dss, since it is relatively simple in regard to administration (usually through drinking water), besides control of dosage and duration . Rather than directly causing luminal inflammation per se, dss exposes the lamina propria and submucosa to enteric antigens and bacteria by breaking down the epithelial barrier . Many of the consequent inflammatory responses resemble those of the human uc, as evidenced by exhibition of characteristic features such as weight loss, diarrhea, fecal occult blood, and intestinal shortening [9, 10]. Histopathologic features of dss - induced colitis include mucin depletion, epithelial degeneration, and neutrophil infiltration . The pathogenesis of dss - induced colitis involves defective alterations in the tight junction complex and marked increases in the expression of inflammatory cytokines [10, 11]. With such keen resemblance to the human - derived uc, the preclinical model of dss - induced colitis has been validated as relevant for the translation of murine data into human disease [1, 7] and thus has been adopted in this study . The use of complementary and alternative medicine (cam) in patients with inflammatory bowel disease (ibd) is quite common, with a rate over 40% in north america . Herbal therapy is the single most used modality of cam, followed by homeopathy, naturopathy, and dietary supplements [13, 14]. The primary reason behind such unconventional medicine use has been reported to be direct disease - related benefits, in addition to psychological factors such as the need for greater autonomy or emphasis on holistic approaches [14, 15]. Unpurified herbal decoctions contain biologically active compounds that may display antioxidative, anti - inflammatory, and prebiotic properties [13, 16]. In this study, we have tested the effects of oral angelica acutiloba kitagawa (aak) extract on a dss - induced colitis model . The japanese aak belongs to the same genus as the korean a. gigas and the chinese a. sinensis, all of which have been substituted by one another for angelicae radix (angelica root). While a. gigas is well - known for its active ingredient decursin and decursinol angelate, aak is differentiated by a relatively low content of both, and a relatively high content of essential oils, organic acids, and polyphenols like z - ligustilide, n - butylidenephthalide, ferulic acid, and chlorogenic acid . Previously reported bioactivities of the aak extract include antitumor, anticomplementary, immunostimulatory, antidiabetic, antiobesity, and, most importantly, a considerable anti - inflammatory activity [17, 2426]. Here we demonstrate for the first time that orally administered aak extract significantly suppresses intestinal inflammation in the dss - induced acute colitis model . Our findings may represent a novel efficacious and reasonable confrontation for the standard treatment of uc, as its cost of nonadherence and opportunity loss is substantial, and still on the rise . Male, 6-week - old c57bl/6 mice weighing at an average of 23 g (1 g) were obtained from central lab animal inc . Mice were housed in a pathogen - free facility with a light / dark cycle of 12 h, under ambient temperature (2123c) and humidity (5560%). All animals had access to food and water ad libitum and were acclimated for 2 weeks before the experiment . Dried root of aak (500 g) was obtained from kyung hee herb pharm (seoul, korea). Briefly, the sections were immersed in 30% etoh and extracted 2 times for 2 h each, using a reflux extractor (glhmp - f1000, global lab, siheung, korea). The extract was further concentrated with an evaporator (rotavapor r-220, bchi labortechnik ag, flawil, switzerland), followed by a filtration step and subsequent freeze - drying (lp30, ilshin biobase co., yangju, korea) at 5 mm torr (yield = 21.3%). For chromatographic separation, 100 mg of freeze - dried sample powder was dissolved in 1 ml meoh, sonicated for 30 min, and filtered through a pvdf membrane . Chromatographic separation of the extract was performed using a separations module (2690, waters, ma, usa) and 5 m column (nucleosil 100 - 5 c18, macherey - nagel, dren, germany). Using solvents a (water) and b (acetonitrile), the gradient program was run as follows: 03 min (20% b), 38 min (30% b), 818 min (30% b), 18 - 19 min (50% b), 1940 min (50% b), and 40 - 41 min (90% b), at 1 ml / min flow velocity and 330 nm wavelength . Quantitative content of decursin (#kp013, npc biotech, sejong, korea) was analyzed via a photodiode array detector (996, waters, ma, usa) interfaced to the separations module (figure 1). After adaptation, mice were randomly divided into the following 4 groups (46 mice / group): normal control, colitic control, aak 100, and aak 500 . Colitis was induced with dss as previously described . Briefly, 3650 kda dss (#0216011080, mp biomedicals, ca, usa) was dissolved in sterile tap water at a final concentration of 4% (wt / vol) and presented to mice as drinking water for 7 consecutive days . Normal controls were administered sterile tap water by oral gavage once per day, with free access to drinking water; colitic controls were also administered sterile tap water by oral gavage once per day, but with free access to dss - treated drinking water; aak 100 and aak 500 mice were, respectively, administered 100 and 500 mg / kg of body wt aak by oral gavage once per day, with free access to dss - treated drinking water . The doses of aak were chosen on the evidence of previously - demonstrated protective effects of angelica root in multiple inflammatory disease models [22, 2931]. Mice were monitored daily for changes in body weight, stool consistency, and stool blood . Body weight on any particular day was calculated as the relative percentage weight to that of day 1 . Stool consistency was evaluated macroscopically using the following 4-point scale: 0, formed and hard; 1, formed but soft; 2, loose stool; 3, watery . Traces of occult blood in feces were assessed using hemoccult sensa single slides (#38078, beckman coulter, ca, usa) in the following 4-point scale: 0, hemoccult negative (); 1, hemoccult positive (+); 2, hemoccult positive (+ +); 3, gross bleeding on the anus site . After sacrifice, the colon was removed and measured in length for evaluation of shortening . After 6 h receiving the last gavage, mice were anaesthetized with avertin . Upon dissection, the full colon was also removed and measured in length, from the cecocolic junction to the proximal rectum . Following extraction of the cecum, the colon was then washed in phosphate buffered saline (pbs), and the inflamed distal region was cut and fixed in 4% formaldehyde for histological examination . The remaining mid - proximal portion was dissected into fragments and stored at 80c for further analysis . Fixed distal colon tissue samples were embedded in paraffin using a tissue processor (tp1020, leica biosystems, nussloch, germany). Paraffin blocks and 4 m section slides were made via conventional methods . Upon hematoxylin and eosin (h&e) staining, representative regions of the longitudinal and transverse sections were viewed using light microscopy at 100x/400x magnification, respectively . Briefly, a total score ranging from 0 (healthy) to 12 (extensive injury) was determined by the sum of respective scores for the following parameters: (1) epithelial erosion, (2) goblet cell depletion, (3) inflammatory cell infiltration, and (4) crypt deformation . Scoring of each parameter was defined as follows: 0, none; 1, mild; 2, moderate; 3, severe . Colon tissue samples were thawed and total rna was extracted using the r&a - blue kit (#17501, intron biotech ., eluted rna samples were quantified with the nanodrop spectrophotometer (thermo scientific, de, usa) at 260 nm absorbance, and 2 g of rna was subsequently reverse - transcribed using the primescript cdna synthesis kit (#6110a, takara, shiga, japan) according to the manufacturer's protocol . Real - time quantitative pcr was carried out on reaction tubes (#4358293, applied biosystems, ca, usa) and caps (#4323032, applied biosystems, ca, usa) in a stepone real - time pcr system (applied biosystems, ca, usa) with 100 ng of cdna, the sensifast sybr hi - rox kit (#bio-92005, bioline, london, uk), and previously described primers for the targeted genes [33, 34]. Relative gene expression was calculated via the comparative ct (2) method, and mouse glyceraldehyde 3-phosphate - dehydrogenase (gapdh) was used as an endogenous control for normalization . Upon whole blood collection via cardiac puncture, the serum was isolated by centrifugation at 1,500 rpm for 20 min at 4c . Determination of serum ige levels was performed using the bd opteia mouse ige elisa set (#555248, bd biosciences, ca, usa), according to the manufacturer's protocol . After the final step, plate was read with a microplate reader (versamax, molecular devices, ca, usa) at 450 nm absorbance . All statistical analyses were performed using the graphpad prism 5 (graphpad software inc ., ca, usa) software . Statistical significance for differences between curves was tested using two - way analysis of variance (anova), followed by bonferroni correction . Statistical significance for differences between means was tested using one - way anova, followed by newman - keuls multiple comparison test . Data are presented as the mean sd, and p values of <0.05, <0.01, and <0.001 were considered statistically significant . Upon receiving dss, mice developed acute colitis, which was evidenced by a reduction in average food intake and body weight . Oral administration of aak (500 mg / kg / day) resulted in a higher daily average food intake compared to colitic controls with vehicle treatment (figure 2(a)). Accordingly, significant reduction in body weight loss was observed in mice with aak (500 mg / kg / day) administration, on days 5, 6, and 7 (figure 2(b)). No significant differences between low - dose (100 mg / kg / day) aak - treated mice and colitic controls were observed, in both average food intake and body weight . Changes in stool consistency and occult blood were observed in mice upon exposure to dss . As early as day 2, mice receiving dss developed soft stools with mild traces of diarrhea and tested positive in the guaiac test . The severity of manifestations progressively intensified towards termination of experiment, where mice exhibited watery feces and gross bleeding on the anus site . In line with the effects on food intake and body weight loss, oral administration of aak (500 mg / kg / day) resulted in a significant suppression of diarrhea events on days 3, 6, and 7 (figure 2(c)). Oral aak (500 mg / kg / day) also significantly protected against presence of occult blood on days 6 and 7 (figure 2(d)). Again, no significant differences between low - dose (100 mg / kg / day) aak - treated mice and colitic controls were observed, in the scores of both stool consistency and occult blood . Immediately after the termination of experiment, colitic controls with dss had a significantly shortened colon length (5.1 0.4 cm), compared to normal controls with vehicle treatment (7.3 0.5 cm). Mice with oral aak (500 mg / kg / day) administration had a significantly well - retained colon length (6.6 0.3 cm) compared to colitic controls . As for the low - dose (100 mg / kg / day) aak group, average colon length (5.1 0.6 cm) was analogous to that of colitic controls (figures 3(a) and 3(b)). In the case of spleen weight, oral aak (500 mg / kg / day) administration showed a trend of reduction (68.9 3.8 mg), although it did not reach statistical significance compared to colitic controls (84.1 3.3 mg) (figure 3(c)). Histological assessment of distal colon samples revealed that dss - treated mice exhibited loss of surface epithelium, villous blunting, crypt distortion or loss, goblet cell loss, and inflammatory cell infiltration . While normal controls showed mild or no signs of histological damage (score 0.5 0.6), colitic controls had marked severe morphological changes throughout the epithelium and mucosa, with a dense transmural inflammatory cell infiltration (score 10.5 2.4). The colons of oral aak - treated mice showed substantial protection against such damage . At high - dose (500 mg / kg / day) aak administration, mucosal architectures and goblet cells were relatively well - preserved, with a sparse distribution of inflammatory cells in the lamina propria (score 4.7 1.9). Low - dose (100 mg / kg / day) administration of aak did exhibit signs of amelioration, but not to the same extent (score 8 0.8) (figure 4). To evaluate the mitigative effects of aak on dss - mediated immune response, expression of inflammatory cytokines, inducible enzymes, matrix metalloproteinases (mmps), and tight junction - related proteins ifn-, il-1, il-6, il-12, and il-10, compared to normal controls . Administration of aak (500 mg / kg / day) was able to significantly suppress the elevation of all these cytokines (figure 5). Regarding the expression of inducible enzymes inos and cox-2, oral aak (500 mg / kg / day) significantly suppressed both, which were elevated upon dss administration . Similar patterns of amelioration were observed in the expression of mmp-9 and mmp-14 (figure 6). While degraded epithelial integrity was evident in the marked decrease of occludin and zo-1 expression upon dss administration, oral aak (500 mg / kg / day) was capable of restoring it . In the case of icam-1, an increase was present upon dss administration, which was successfully reduced to the basal level in oral aak (500 mg / kg / day)-treated mice (figure 7). In dss - treated colitic controls, serum total ige levels were upregulated by approximately twofold (484.4 44.4 ng / ml), compared to that of normal controls (168.6 60.2 ng / ml). In mice with oral aak (500 mg / kg / day) administration, serum total ige levels were significantly downregulated (224 172.3 ng / ml) (figure 8). Clinical response to standard therapy among uc patients is discrete, depending on the combination of factors that eventually led to the disease . Despite recent advances in therapeutic regimen, a considerable portion of patients with uc still fail or lose response to conventional nonbiologic agents, which results in varied complications such as fibrostenosis that may require surgical intervention . Approximately 11% of patients who use immunomodulators such as azathioprine or methotrexate discontinue administration due to allergic reactions, nausea, or pancreatitis . In the case of corticosteroids, 55% of patients end up in cessation of therapy as a result of recurring acne, facial swelling, and osteoporosis . While biological agents (e.g., anti - tnf monoclonal antibodies) emerge as an alternative option, they have also been held by secondary responses that include the risk of hypersensitivity, immunogenicity, infection, and congestive heart failure . Blockade of a single cytokine as such may give rise to an alternative compensatory pathway and thus is ineffective in many cases . Herbal decoctions comprise a number of components that modulate the expression of multiple cytokines simultaneously and therefore could prove as an effective complementary or alternative modality for uc patients . Nonadherence to treatment is yet another problem in the standard management of uc, which is caused by inconveniences following administration of large number of tablets, fear of side effects, or doubts in the need for medication during disease quiescence . As phytotherapy is highly accepted by uc patients for its efficacy, low cost, and relative safety, it may also prove as a promising confrontation for such patient - derived nonadherence issues . In this study, we demonstrated the effects of aak extract in a dss - induced acute colitis model . Roots of the angelica plant are known to be rich in polysaccharides that exert immunomodulatory and anti - inflammatory [24, 25] effects . The polysaccharides from a. sinensis, which is largely similar in composition to that of aak, showed protective effects in tnbs and dnbs - induced rat models of uc [29, 40, 41]. While the root extracts from aak have been reported to suppress inflammatory mediators in mouse macrophages, analogous effects on a murine uc model are not yet confirmed . Hence this study was aimed at investigating the effects of aak extract on a dss - induced model of uc . As previously reported, intestinal inflammation in the dss - induced model of uc is primarily manifested as symptoms comparable to those of human - derived uc, such as anorexia, body weight loss, diarrhea, fecal occult blood, and colon shortening [9, 10, 42]. In patients with ibd, decreased appetite in uc patients has been reported to be the result of increased serum leptin levels, which in turn sends a negative feedback signal to the hypothalamus [44, 45]. As for animal models, a comparable elevation in serum leptin, as well as the consequent loss of body weight, has been observed in the early stages of tnbs - induced intestinal inflammation in rats . In line with these reports, the colitic group in our study exhibited a decrease in average food intake and body weight over the days of dss administration . The ethanol extract of aak was able to attenuate anorexia and subsequent weight loss, as evidenced by a relatively well - maintained food consumption and body weight at the terminal stage of experiment . Events of diarrhea and fecal occult blood were also significantly suppressed during the same period, both of which represent impaired epithelial barrier function and mucosal damage [47, 48]. For unknown reasons, the colonic longitudinal muscle undergoes hypertrophy and subsequent contraction during chronic uc . This contraction produces a diffuse or segmental narrowing of the lumen, which in turn causes shortening of the colon, namely, the lead pipe sign . In this study, colonic length was significantly retained upon oral administration of aak, compared to colitic controls . In animal models of uc, splenomegaly is validly indicative of intestinal inflammation, and mitigative effects of therapeutic substances have often been confirmed by the suppression of spleen enlargement [50, 51]. The imbalance between pro- and anti - inflammatory cytokines disrupts ease of inflammation and further leads to an elongation of the disease . In line with such reports, cytokine - based therapies such as anti - tnf- factors have recently been adopted in the uc - treatment algorithm . The murine dss - induced colitis model exhibits a distinct cytokine profile with elevated levels of tnf-, ifn-, il-1, il-6, and il-12 . While the models used in this study displayed similar patterns, our data suggest that oral administration of aak significantly suppresses mrna expression of such inflammatory cytokines in the inflamed colonic tissue . The proinflammatory cytokines tnf- and ifn- play a pivotal role in the pathogenesis of uc, by altering the tight junction and inducing apoptosis of intestinal epithelial cells [5355]. In caco-2 cell monolayers, tnf--induced epithelial permeability was mediated by the expression of myosin - light chain kinase (mlck). In the apical membrane of t84 cells, ifn- induced redistribution of occludin, claudin-1 and claudin-4, away from the tight junction domain . The therapeutic potency of targeting these cytokines have been demonstrated in actual clinical trials with anti - tnf- antibodies, as well as in in vivo studies where colitis showed attenuation upon dss stimulation in ifn- mice . In the inflamed mucosa of ibd patients, key antigen - presenting cells (apcs) produce considerable amounts of proinflammatory cytokines such as il-1, il-6, and il-12 . When compared to healthy subjects, patients with uc manifested a significant reduction in the ratio of il-1 receptor antagonist to il-1, which implies the augmented activation of il-1 system in uc . In a previous study using dss - induced model of colitis, mice deficient in il-1-converting enzyme (caspase-1) exhibited attenuated signs of intestinal inflammation, suggesting the therapeutic relevance of il-1 family blockade . In experimental colitis, macrophages and cd4 t cells of the lamina propria produce an increased amount of il-6 [62, 63]. Upon binding with soluble il-6r (sil-6r), the il-6-sil-6r complex induces proinflammatory cytokine production in intestinal apcs and t cells, by binding to the gp130 surface molecule [36, 62]. The therapeutic potentials of il-6 blockade have been confirmed in a previous study by yamamoto et al ., where administration of anti - il-6r monoclonal antibody to a murine model of colitis resulted in reduced disease activity and suppression of proinflammatory cytokines . Il-12 is a heterodimeric cytokine produced by monocytes and macrophages in response to bacteria or their products [65, 66]. By inducing ifn- synthesis and th1 cell differentiation, il-12 contributes to the breakdown of tolerance against luminal antigens in human and murine colitis . Since ifn- upregulates il-12 production in macrophages, the established loop between il-12 and ifn- further contributes to the perpetuation of intestinal inflammation . As neutralizing antibodies to il-12 have shown suppressive effects on chronic intestinal inflammation, targeting il-12 in uc may also prove as an effective means of treatment [69, 70]. The anti - inflammatory cytokine il-10 is known to exhibit immunoregulatory properties by blocking the production of proinflammatory cytokines in monocytes and macrophages . Restoration of endogenous il-10, as well as administration of its exogenous components, has been proposed as a possible treatment for ibd . However, such is not the case in this study, as the level of il-10 expression was upregulated upon dss administration and downregulated by aak treatment . Similar patterns have been reported in previous studies with murine [34, 73] and porcine models of intestinal inflammation . One possible explanation may be the rapid return to a homeostatic cytokine milieu upon treatment, as the endogenous production of il-10 may have been insufficient to downregulate proinflammatory cytokines, and thus local inflammation [71, 74]. In further studies, the expression of anti - inflammatory cytokines besides il-10, such as il-4, should be examined to confirm the contribution of other endogenous factors . Taken together, the suppressive effects of aak on inflammatory mediators of the above imply its possibility as a potent complementary and alternative therapy against uc . Oral administration of aak also significantly suppressed the mrna expression of inducible enzymes and mmps in the inflamed tissues of dss - induced colitic mice . Elevated expressions of inos and cox-2 in the morbid colonic mucosa indicate production of nitric oxide (no) and prostaglandin e2 (pge2), two of the bioactive agents involved in colonic injury . Recent studies have suggested a synergetic action of both enzymes, as inhibition of no synthesis resulted in a reduced secretion of pge2 . Combined blockage of inos and cox-2 reportedly had a cytoprotective effect under uc conditions, and analogous data were drawn in the present study using aak . Mmps are endopeptidases that degrade the extracellular matrix (ecm) components during active flares of uc . Mmps are capable of shedding molecules such as tnf-, il-1, and il-6 from macrophage surfaces . In this study, mucosal recovery of aak - treated mice was evident in our histological data, which was also confirmed by the restored mrna expression of tight junction proteins zo-1 and occludin . Lastly, our study demonstrated the suppressive effects of aak on total serum ige concentrations . Patients with uc have reported suffering from allergic diseases such as asthma, allergic rhinitis, or eczema, twice as much as matched healthy controls . In another study by d'arienzo et al ., total serum ige levels were accordingly higher in uc patients, compared to healthy controls . Our results indicate that oral aak extract may also have beneficial effects in treating the secondary atopic manifestations following uc . Taken together, this study suggests that oral administration of aak exerts a protective effect against the pathogenesis of acute colitis, by improving major symptoms such as anorexia, weight loss, diarrhea, occult blood, and colon shortening . Furthermore, we demonstrated that such attenuative effects are partially mediated by the suppression of multiple inflammatory markers, including cytokines, inducible enzymes, mmps, and tight junction - related proteins . These findings provide the possibilities for a more sustainable approach towards uc, as conventional therapeutics with a single - cytokine target are likely to have limitations due to development of compensatory pathological pathways . In the present study, aak mostly regulated apc - derived cytokines . However, recent studies have suggested that nonimmune cells, such as epithelial cells or stromal fibroblasts, may also produce inflammatory cytokines in ibd . In view of such findings, we may hypothesize that aak elicits an innate immune cell - mediated response, while targeting the epithelium itself at the same time . However, as our data do not confirm a predominant activation of either mechanism, further in vivo and in vitro experiments are required to define the exact mode - of - action responsible for the protective activities of aak.
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Breast cancer is the second most common cancer among women in the united states, with approximately 54,010 new cases of carcinoma in situ (noninvasive cancer) and 207,090 cases of invasive cancer diagnosed per year . In addition, breast cancer is the second leading cause of cancer death in women, with about 39,840 deaths occurring per year.1 five - year survival rates vary by stage: the 5-year survival rate for patients with stage 0 through stage iia breast cancer ranges from 92 to 100%, while the 5-year survival rate for patients with stage iv metastatic breast cancer is approximately 20%.1 thus, the goal of therapy for invasive breast cancer is primarily palliative that is, aimed at alleviating and controlling symptoms as well as improving quality of life.2 treatments in common use for invasive breast cancer include cytotoxic chemotherapy, hormonal treatments, and targeted therapies / biologics, which can be administered as single agents or in combination with each other.3 given that current chemotherapies have advantages or disadvantages relative to each other, it would be useful to understand how these influence patient preferences for treatment . For example, one recent study evaluated preferences for health states associated with chronic lymphocytic leukemia treatments using the standard - gamble method.4 other studies have also examined patient preferences for select groups of chemotherapy toxicities.5,6 however, to date, no study has evaluated the importance of toxicities relative to other characteristics such as efficacy and administration . Conjoint analysis, which is increasingly being used in evaluating medical interventions, involves respondents making trade - offs among product features (attributes) such as mode of administration and risk of adverse events.7,8 the resulting data, or utilities, enable the assessment of the relative importance of each treatment attribute; specifically, they show the influence that each attribute has on overall treatment preferences . The goal of the present study was to use conjoint analysis to capture patient preferences for attributes associated with chemotherapies for invasive breast cancer . Specifically, the survey aimed to assess the trade - offs that breast cancer patients are willing to make among chemotherapies according to different toxicity, regimen, and efficacy profiles . This was a cross - sectional, internet - based survey assessing the chemotherapy treatment preferences of women with breast cancer . All participants were women with united states residency; at least 18 years of age; had a diagnosis of stage i through iv breast cancer; had received chemotherapy treatment within the past 5 years; and provided informed consent and health insurance portability and accountability act- compliant authorization.9 participants completed the survey at home and worked at their own pace . However, research team members were available via telephone or email to answer questions or provide technical help if necessary . The study followed the tenets of the declaration of helsinki, and the protocol was approved by a commercial institutional review board (independent irb; plantation, fl). This research was implemented following published methodological guidelines for conjoint studies.10 the survey design and analysis are detailed below . Twelve attributes of eight invasive breast cancer chemotherapy treatments were identified from a comprehensive literature review, the common toxicity criteria grading system, a detailed assessment of breast cancer forum discussions, and consultation with clinical experts . These attributes included selected grade iii / iv toxicities, efficacy (survival advantage), and regimen . Attributes were described in lay terminology so that they could be easily understood by patients . Each attribute was presented in different levels that represented the full range of possibilities across available breast cancer treatments . Based on the literature and clinician input, potential ranges for the incidence of each of the toxicities were identified, and these were used as the most favorable and least favorable levels, respectively . This involves a hybrid approach, in which the data are collected in phases.11,12 the respondent completes the aca computer - assisted questionnaire, and the questionnaire is modified during the responding process on the basis of the respondent s previous choices . Thus the interview can focus on just those attributes that the respondent considers most important and those attribute levels regarded as most relevant . In the first phase, respondents were asked to rate the levels of each attribute in terms of acceptability on a 7-point scale, from 1, not at all acceptable, to 7, the second phase (paired comparison questions) elicited treatment preferences by asking respondents to make trade - offs among attributes and choose from a pair of hypothetical treatments . In each of these questions, the profiles of two hypothetical treatments labeled simply chemotherapy a and chemotherapy b were presented with different levels of the same three attributes (no chemotherapies were named in the survey). Respondents used a 7-point scale to indicate not only their preference, but the strength of their preference; response options ranged from strongly prefer a to strongly prefer b . The profiles presented to respondents in this second phase were customized based on responses to previous questions . The survey was pilot - tested with respect to wording and comprehension in a sample of four breast cancer patients; no major changes were necessary . The conjoint data were analyzed using sawtooth software ssi web (v 6.4; sawtooth software, sequim, wa). Analysis of aca data involved the combination of the initial rating (the acceptability questions in the first phase) and the paired comparison questions . Specifically, using the information from the initial rating questions, prior utilities were calculated . For example, desirability values 3, 2, and 1 would be converted to 1, 0, and 1, respectively . These initial estimates are part - worths, where within each attribute the values have a mean of zero, and differences between values are proportional to differences in desirability ratings or rank orders of preference . With respect to the paired comparison questions, a column vector was created for the dependent variable as follows: the respondents answers were zero - centered, where the most extreme lowest value was given 4, and the most extreme highest value was + 4 . Each pair s question contributed a row to both the independent variable matrix and dependent variable column vector . Ordinary least - square estimates of the n attribute levels were computed by regression of the dependent variable column vector on the matrix of independent variables . The part - worth estimates based on the prior and paired comparison phases were normalized to have equal sums of differences between the best and worst levels of each attribute across all attributes . The two vectors of part - worths were added together, yielding final utilities for each attribute level for each respondent . The absolute values of the final utilities were arbitrary; what was important was the magnitude of difference between them . Whereby the utility weight of the attribute level falling in the middle had a value that approximated zero and the more favorable level was higher, or positive, and the less favorable level was lower, or negative . Finally, the bayes approach was applied to the data to further refine the precision of the utility estimates . The utilities enable the calculation of the relative importance of each attribute for each respondent in influencing treatment decisions . Specifically, the relative importance was calculated for each respondent by dividing the range for each attribute (utility of highest level utility of lowest level) by the sum of ranges of all attributes for the respective individual and multiplying it by 100 . These estimates indicate how much the difference in importance between the best and worst levels of each attribute affects the decision to choose a treatment . These are ratio data, meaning for example that an attribute with an importance of 10% is twice as important as an attribute with an importance of 5% . Using the mean utility weights for the attribute levels, we compared the percentages of patients who would prefer the following scenarios differing with respect to selected attributes, while holding all other attributes constant: (1) three levels of efficacy (no additional survival, 1 month additional survival, and 3 months additional survival); (2) three regimens (most convenient [oral], least convenient [21-day cycle; 3-hour infusion on days 1, 8, and 15], and one in between [28-day cycle; 610 minute infusion on days 1, 8, and 15]); and (3) a 0% versus a 10% chance of each toxicity . Twelve attributes of eight invasive breast cancer chemotherapy treatments were identified from a comprehensive literature review, the common toxicity criteria grading system, a detailed assessment of breast cancer forum discussions, and consultation with clinical experts . These attributes included selected grade iii / iv toxicities, efficacy (survival advantage), and regimen . Attributes were described in lay terminology so that they could be easily understood by patients . Each attribute was presented in different levels that represented the full range of possibilities across available breast cancer treatments . Based on the literature and clinician input, potential ranges for the incidence of each of the toxicities were identified, and these were used as the most favorable and least favorable levels, respectively . This involves a hybrid approach, in which the data are collected in phases.11,12 the respondent completes the aca computer - assisted questionnaire, and the questionnaire is modified during the responding process on the basis of the respondent s previous choices . Thus the interview can focus on just those attributes that the respondent considers most important and those attribute levels regarded as most relevant . In the first phase, respondents were asked to rate the levels of each attribute in terms of acceptability on a 7-point scale, from 1, not at all acceptable, to 7, the second phase (paired comparison questions) elicited treatment preferences by asking respondents to make trade - offs among attributes and choose from a pair of hypothetical treatments . In each of these questions, the profiles of two hypothetical treatments labeled simply chemotherapy a and chemotherapy b were presented with different levels of the same three attributes (no chemotherapies were named in the survey). Respondents used a 7-point scale to indicate not only their preference, but the strength of their preference; response options ranged from strongly prefer a to strongly prefer b . The profiles presented to respondents in this second phase were customized based on responses to previous questions . The survey was pilot - tested with respect to wording and comprehension in a sample of four breast cancer patients; no major changes were necessary . The conjoint data were analyzed using sawtooth software ssi web (v 6.4; sawtooth software, sequim, wa). Analysis of aca data involved the combination of the initial rating (the acceptability questions in the first phase) and the paired comparison questions . Specifically, using the information from the initial rating questions, prior utilities were calculated . For example, desirability values 3, 2, and 1 would be converted to 1, 0, and 1, respectively . These initial estimates are part - worths, where within each attribute the values have a mean of zero, and differences between values are proportional to differences in desirability ratings or rank orders of preference . With respect to the paired comparison questions, a column vector was created for the dependent variable as follows: the respondents answers were zero - centered, where the most extreme lowest value was given 4, and the most extreme highest value was + 4 . Each pair s question contributed a row to both the independent variable matrix and dependent variable column vector . Ordinary least - square estimates of the n attribute levels were computed by regression of the dependent variable column vector on the matrix of independent variables . The part - worth estimates based on the prior and paired comparison phases were normalized to have equal sums of differences between the best and worst levels of each attribute across all attributes . The two vectors of part - worths were added together, yielding final utilities for each attribute level for each respondent . The absolute values of the final utilities were arbitrary; what was important was the magnitude of difference between them . Whereby the utility weight of the attribute level falling in the middle had a value that approximated zero and the more favorable level was higher, or positive, and the less favorable level was lower, or negative . Finally, the bayes approach was applied to the data to further refine the precision of the utility estimates . The utilities enable the calculation of the relative importance of each attribute for each respondent in influencing treatment decisions . Specifically, the relative importance was calculated for each respondent by dividing the range for each attribute (utility of highest level utility of lowest level) by the sum of ranges of all attributes for the respective individual and multiplying it by 100 . These estimates indicate how much the difference in importance between the best and worst levels of each attribute affects the decision to choose a treatment . These are ratio data, meaning for example that an attribute with an importance of 10% is twice as important as an attribute with an importance of 5% . Using the mean utility weights for the attribute levels, we compared the percentages of patients who would prefer the following scenarios differing with respect to selected attributes, while holding all other attributes constant: (1) three levels of efficacy (no additional survival, 1 month additional survival, and 3 months additional survival); (2) three regimens (most convenient [oral], least convenient [21-day cycle; 3-hour infusion on days 1, 8, and 15], and one in between [28-day cycle; 610 minute infusion on days 1, 8, and 15]); and (3) a 0% versus a 10% chance of each toxicity . Of 121 participants who completed the conjoint survey, 108 (89%) were included in the final data analysis . Thirteen participants were excluded because their responses in the first section of the survey were illogical (eg, a participant rating a 24% chance of fatigue as more acceptable than a 0% chance) in at least two instances . Table 2 reports the demographic and clinical characteristics of the study population who were included in the analysis . The mean age was 50 years, with a mean time of 80 months since cancer diagnosis; 60% had attained at least a college - level education . With respect to treatment experience, about 35% of patients were on chemotherapy at the time of their participation in the study, and the mean time since the sample s last chemotherapy administration was approximately 19 months . The mean utilities for each attribute level are presented in table 3 (the actual values are arbitrary; the magnitude of the differences among them are what should be evaluated). In general, the utilities for each attribute level were ordered in the direction that was expected, where the most favorable attribute level had the highest utility and the least favorable level had the lowest . The efficacy attribute had the highest utility value for its most favorable level (additional survival benefit of 3 months) and lowest utility value for its least favorable level (no additional survival benefit), with means of 97.05 and 83.77, respectively . This finding indicates that patients view a change in survival advantage from 0% to 3 months as more important than a change from the least favorable to the most favorable level for all other attributes . Overall, the most important attributes in driving treatment preferences were survival advantage, neutropenia / hospitalization, and toxicities such as nausea / vomiting, fatigue, and diarrhea . Least important attributes were the toxicities of mucositis / stomatitis, myalgia, and hand - foot syndrome . In general, the difference between the most inconvenient to the most convenient administration regimen was less important than a 13% chance or more of severe toxicities, but more important than a 10%12% chance of severe toxicities . The exception to this finding was myalgia, which had a maximum level of 15% chance of occurring; the difference between 0% versus 15% chance of myalgia was still less important than the difference between the most inconvenient and most convenient regimen (percentage importances = 8.8% versus 3.2%). In sensitivity analyses, we created scenarios for each toxicity (ie, each attribute that was a side effect) in which we compared preferences for a 0% risk of that toxicity versus a 10% risk of that toxicity, holding the risk of all other toxicity attributes constant . Specifically, we obtained preference estimates for two product profiles in which one toxicity differed by 10% and the remaining attributes were held constant; we repeated this for each side effect . Figure 2 shows the percentages of patients who would prefer a treatment given a 0% chance versus a 10% chance of each toxicity occurring . The findings show that patients most preferred to avoid neutropenia / hospitalization, diarrhea, nausea / vomiting, and sensory neuropathy (ie, they most preferred no risk of these toxicities relative to no risk of the other toxicities). An additional analysis found no substantial differences in relative importance estimates of toxicities between those who had experienced the side effect versus those who had not (data not shown). Figure 3 presents the percentages of patients preferring each of three regimens that differ in efficacy (survival advantage) and the percentages of patients preferring three regimens, ie, an oral regimen, a regimen somewhat in the middle with respect to convenience (28-day cycle; 610 minute infusion on days 1, 8, and 15), and the most inconvenient (21-day cycle; 3-hour infusion on days 1, 8, and 15). The findings show that the difference between a 1-month and 3-month survival advantage substantially influenced patient preferences; the percentages of patients preferring each of these regimens increased from 5.7% to 93.7%, respectively . Also, an oral regimen was highly preferred; 76.6% of patients would choose an oral regimen versus 18.9% who would choose the middle regimen . To the best of our knowledge, this study is the first to assess patient preferences for invasive breast cancer treatments considering a variety of treatment features . Specifically, this study assessed the trade - offs that breast cancer patients are willing to make among the risks of severe toxicities, administration regimen, and efficacy when choosing a chemotherapy . Among the chemotherapy attributes evaluated in this study, those that were most influential in driving patient preferences for treatment were improved survival, the risk of neutropenia leading to hospitalization, and the risks of severe fatigue, nausea and vomiting, and diarrhea . Least important were the risks of severe myalgia, mucositis, and hand - foot syndrome . The study findings are useful in better understanding patient preferences in oncology and may enhance the medical decision - making process . For most patients, the difference between having no additional survival advantage and having a survival advantage of 3 months was about three times more important than an approximate 10% versus 0% risk of experiencing several severe toxicities, including severe myalgia, mucositis, hand - foot syndrome, and motor neuropathy . In addition, the incremental improvement in survival was one - and - a - half times as important as an approximate 15%20% versus 0% risk of having severe nausea and vomiting, fatigue, diarrhea, or sensory neuropathy . The finding of the high relative importance of improved efficacy is consistent with previous conjoint analyses of medical therapies13,14 and a review of previous literature,15 which found that patients are willing to accept the risk of serious adverse events in exchange for improved efficacy . For example, johnson et al12 evaluated the treatment perceptions among patients with multiple sclerosis, and they found that most patients indicated they are willing to accept risks of life - threatening adverse events in exchange for improvements in their health outcomes . This study builds upon previous work that has examined rankings among chemotherapy side effects.16 specifically, sun et al16 evaluated preferences for 27 different side effects among patients with ovarian cancer . They found that severe nausea and vomiting was the least preferred toxicity and that numbness in hands / feet similarly, the current study showed that patients would most want to avoid the chance of severe nausea and vomiting and the chance of severe numbness in the arms and legs (sensory neuropathy) among the various potential toxicities evaluated, assuming that they each had an equal chance of occurring . Our study found no substantial differences in the relative importance of toxicities between those who had experienced the toxicity versus those who had not . This finding differed from a previous study that examined utility (time trade - off) estimates for chemotherapy - related ototoxicity, nephrotoxicity, and neurotoxicity among ovarian cancer patients.17 specifically, the study found that the most favorable assessment of a particular toxicity was reported from individuals who experienced the selected toxicity . This may be attributable to the fact that patients were asked to assume that the toxicity was permanent; as such, patients with experience may have felt they could better tolerate the effect than nave patients . In contrast, in the current study, each of the side effects had a specified duration, and thus this may not have been significant enough to translate into differences between those experienced versus not experienced with each toxicity . Moreover, our findings were consistent with the finding of no substantial differences observed in relative importance of eleven of 12 side effects between hiv treatment - experienced, who may have experienced treatment side effects, versus treatment - nave patients.18 the current study also found that a more convenient administration regimen was more important to patients than severe side effects when the respective risk was less than 13% . Similarly, improved convenience has been found to be influential in treatment choices in other studies.13,14 for example, in a study of patients with idiopathic thrombocytopenic purpura, patients were willing to accept significant treatment - related risks in exchange for improvements in treatment efficacy and convenient administration.13 our study was not without limitations . First, each of our toxicity attributes was described using durations from a few days to 6 months after treatment end, and these durations represented clinical averages . These average durations may not be reflective of individual experiences, however, as our discussions with clinicians revealed that side effect experience is highly variable between patients . In addition, the participants in our sample did not represent the full range of breast cancer patients; the majority were well - educated, most were caucasian, and they were all healthy enough to complete a computer - based survey at home . It is unclear whether the preferences of severely ill cancer patients (eg, those who are bedridden) or other ethnic groups would be different from those expressed by patients in our sample . Because of these limitations, while our study has yielded important findings with implications for the medical community, future research is necessary to evaluate the generalizability of our conclusions . In summary, this survey evaluated a comprehensive set of severe toxicities and other features that are observed with chemotherapies for invasive breast cancer . Our study showed that, despite the risk of serious toxicities, a small incremental survival advantage is highly influential in patient preferences for chemotherapy . The findings from this research may be useful in incorporating patients views into medical decision making processes, patient education, cost resource allocations, and drug development . A better understanding of patients preferences may help to improve patient satisfaction and compliance with treatment regimens.
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All available dna sequences for coi (108 head and 58 clothing lice), 18s rrna (10 head and 12 clothing lice), ef-1 (25 head and 9 clothing lice), and rpii (25 head and 10 clothing lice) for p. humanus and the outgroup p. schaeffi (chimpanzee louse) were downloaded from genbank (available as supplementary table s1, supplementary material online). All sequences were aligned by hand using se - al v2.01a11 (http://tree.bio.ed.ac.uk/software/seal/), with the 18s rdna aligned to secondary structure (gillespie 2004; gillespie et al . Substitution rates (table 2) for the four genes were estimated in beast v.1.5.3 (drummond and rambaut 2007). Rates were calibrated by placing an exponential prior distribution (lower bound = 5 ma, mean = 5.5 ma) on the divergence of p. humanus (human) and p. schaeffi (chimpanzee) lice that reflects conservatively recent estimates for the divergence of their hosts (kumar and hedges 1998). Each gene was analyzed using a range of substitution and clock models, as well as tree priors, with posterior estimates made from the model that best fit the data as determined by marginal likelihoods estimated in the program tracer v1.5 (http://tree.bio.ed.ac.uk/software/tracer). Markov chains were run for at least 100 million generations, sampled every 10,000 generations, and the first 10% of samples were discarded as burn - in . Models of substitution, likelihood scores, and mean substitution rates per gene calculated in beast . Between site rate variation partitioned by codon position (cp). Multilocus bayesian i m coalescent analysis was performed on the p. humanus sequences using the program i m (hey 2005). All analyses used the hky substitution model, whereas priors on model parameters were broad uniform distributions conservatively estimated from preliminary runs . Markov chains were run for> 200 million generations and replicated 8 times to ensure convergence . A louse generation time of 21 days (18 generations per year) and the substitution rates in table 2 were used to convert parameter estimates from mutational to demographic units.
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In the aging population the number of alzheimer disease (ad) patients is expected to increase . However, the diagnostic accuracy of the clinical criteria is relatively low (sensitivity 80% and specificity of 70%)). With this in mind, biological markers in body fluids are urgently needed to sustain diagnosis, as they are an objective tool and reflect ongoing processes . Biomarkers can aid not only in early diagnosis or in differential diagnosis but also in estimation of prognosis and, ideally, monitoring progression of this disease . The concentrations of amyloid - beta(1 - 42) (a42), total tau (tau), and tau phosphorylated at position 181 (ptau) in cerebrospinal fluid (csf) of ad patients can be used as biomarkers . Several laboratories measure these three biomarkers in csf, and a major challenge is to translate the technology from the lab to clinical practice . To reach this goal, the technique should be robust and laboratories should be adequately experienced . In addition, results obtained in different centres should be comparable to the highest possible degree . The comparability of results between different centres is crucially dependent on the performance of the biomarker tests in the various institutions, and this can be assessed with an external quality assessment scheme . No such scheme was available and that is why we took the initiative in 2004 to send samples to a number of laboratories with previous experience in performing these csf biomarker assays, with their own elisa assays . The results revealed large variation in the concentrations of the three biomarkers between the different laboratories and a difference in variation at each evaluated time point . Overall variation for tau was slightly better in 2008 than in 2004, since the mean interlaboratory cv was 21% in 2004 and 16% in 2008 . For ptau the mean between - laboratory cv increased slightly, that is, from 13% in 2004 to 15% in 2008 . The largest overall change was seen for a42, where the between - laboratory variation increased from 31% to 37% . The introduction of other elisa methods appeared to be responsible for this overall increase in variation . Laboratories that used the innotest assays improved the between - laboratory variation of a42 from 30% in 2004 to 22% in 2008, suggesting that experience and standardization of assay procedure may contribute significantly to reduce between laboratory variation . With the aim to improve between - laboratory performance we set out to identify the specific differences in procedures between laboratories . For this, we organised a hands - on workshop at the end of 2009 . 26 participants from 17 different international centres with previous experience in performing the assays were divided in 14 groups, and every group performed the a42, tau, and ptau assays . Identical samples containing pooled anonymised csf samples, with concentrations of these biomarkers covering concentrations observed in controls, in ad patients and an intermediate value, were provided to the groups . Standard curves were diluted by each group according to the protocol, starting with dissolving the solid powder . All groups analysed the same samples, and the assays were performed simultaneously in the same laboratory and used the protocol (incubation procedures) as provided by the manufacturer . One exception was the incubation temperature of the tau assay, which should be in an incubator at 25 2c, and was at room temperature due to practical reasons . During the performance of the assays, the two persons in every group discussed their usual laboratory practice in performing the assays and differences were recorded . The standard curves were calculated using a 5-parameter logistic (5pl) curve fitting on the biorad microplate manager software . The mean and standard deviations of the concentrations in the pools were calculated per group . Table 1 lists the items that were noticed to vary among the laboratories participating in the workshop . The items involved procedures for pipetting, incubation, washing, finishing, and sample handling . Several items listed in table 1 were standardised during the workshop due to its setup . Therefore, we were able to evaluate the inter - assay variation under circumstances that excluded several items that varied between the laboratories (table 2), providing an indication of the relative contribution of these sources of error . The cause was identified as lack of experience (group 3) and an error in the calculation of the dilution of the standard to make the standard curve (group 11). For tau, no outliers were identified while, for ptau, the outcomes of the concentration of the highest pool were deviating in group 14 . The intra - assay variation based on the concentrations of the unknowns was high for pool 1 and 2 of abeta but on average was below 10% for tau and ptau . The principle aim of this study was to address the issue of procedural differences as a source of variation between outcomes of csf biomarker analysis . One of the results of workshop was a list with differences in procedures among the labs, containing 23 items . Examples of these items are the use of polypropylene plate for sample predilution, the inclusion of 1500 pg / ml standard in the curve, the use of 50 l h2so4 in the a42 assay, and the use of 5pl curve fitting and incubation temperature of 25 2c for total tau . The type of pipette is not prescribed by the manufacturer, neither was the mode of pipetting . We do not expect wiping off the tips, which was historically done with specific types of tissues; inverse pipetting and the pipette brand are factors inducing much variation, as long as one mode of pipetting of the samples is consistently used during the assays . The use of a single pipette versus using a multichannel pipette may influence the time needed to fill an entire plate and can be important when incubation time is short, such as for a42 . For preparation of standards a new tip for each concentration is required . During pipetting in the plate, however, the magnitude of this effect, if any, should be tested, to provide a better basis for recommendation . Incubation temperatures, incubation in the dark, and shaking were other items that varied between the laboratories . Whether this variation in incubation conditions is relevant for the current tests is not known but likely . This should be tested, and explicit information regarding influence of shaking and temperature requirements should be provided in assay protocols . Regarding exclusion of the 1500 pg / ml standard in the curve, we did a recalculation of the pools for one of the groups on a curve fitting without 1500 pg / ml standard and the outcomes differed by 7.6%, 9.4%, and 7.2% for pool 1, 2 and 3 from the original data . Washing procedures (number of washing steps, volume, purity of washing solutions) are important issues influencing variation and background . This can lead to removing antibody - antigen complexes from the plate when washing too much, as well as insufficient removal of unbound complexes, thereby causing high background . Furthermore, incomplete dissolution of crystals from the washing buffer may lead to aberrant buffer concentration when only part of the buffer is used . The difference in volume of the stop - solution h2so4 probably does not influence the outcomes, as long as it is performed for all samples in one test similarly, as the ph of the reaction mixture is only marginally influenced by variation in the volume of the stop solution . Sample handling (storage at 4c or repeated freezing) can be very important as well, specifically for instable proteins . For the current proteins, a42, tau, and ptau, a previous study has shown that repeated freezing and storage at 4c do not have a significant effect on tau concentrations while a42 concentrations may be reduced due to storage for a few days at 4c . Results from another study, however, reported no effect of storage temperatures on a42 concentrations (blennow, personal communication). Closing the vial is important to avoid losing the contents upon accidentally falling, and to avoid contamination . For scientific purposes, ideally, the influence of each of the procedures listed in table 1 on the intra- and interassay variation is systematically tested . The current study excluded several potential sources of error, indicated in the last column of table 1, such as differences in incubation temperatures, washing, variation in standardised curve fitting, and lot - to - lot variation . The inter - assay variation in results obtained during the workshop was below the limits for intra - assay variation for all assays, with a few exceptions (table 2), suggesting that standardisation indeed leads to reduced inter - assay variation . This may be caused by increased experience as a42 assay was started with during the workshop and the participants may have needed some time to get used to the laboratory . Alternatively, the observed variation may be the normal variation for these assays, as these differences were similar to what has been reported before in and what we observe in our own laboratory . The importance of lot - to - lot variation is stressed by the results in table 3, showing the reduction of inter - assay variation in our laboratory when we started using multiple assays from the same batch, purchased at once . The variation could be reduced if for instance clear quality control criteria and who - approved standards would be available . The issue of quality control is currently addressed in a large multicenter study, and our initiative has been adopted by the alzheimers association . Standardisation of csf collection and biobanking procedures would be another strategy to tackle pre - analytical variation . We recently published such guidelines for standardised csf collection and biobanking protocols, that was based on a broad consensus between multiple centers . Adherence to these guidelines will reduce variation induced by pre - analytical factors as well and increase the quality of studies aimed at discovery and validation of novel biomarkers . In conclusion, the evaluation of the workshop showed that even under standardised conditions as in this workshop, with the same protocol and laboratory circumstances, inter - assay variation is comparable to intra - assay variation and is acceptable for the large majority of groups . The influence of several items that varied between the labs should be studied and protocols should be adapted accordingly . Provision of information on the influence of specific items in the data sheets might be needed to convince the users of this requirement . Reduction in variation is most critical for csf concentrations around the cutoff points especially if these guide decision making in individual patients . Efforts for standardisation and the establishment of international reference laboratories and reference values will ultimately increase the reliability of the assays . This will provide a basis to include these biomarkers assays more prominently in the diagnostic workup of alzheimer's disease.
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Rheumatologic diseases manifest themselves in varying combinations of symptoms and signs, particularly at early stages, and therefore make differential diagnosis a challenge, especially for nonrheumatologists including general practitioners . Since diagnosis at an early stage and adequate treatment improve prognosis, assistance in establishing diagnosis is desirable . Given the substantial progress in computer science in the last years, the idea of computers taking the role of diagnostic support is not far - fetched . Software applications have affected decision processes in clinical routine, for example, in controlling depth of anesthesia or in detecting drug interactions . Software tools to support physicians in the diagnostic process have been developed in almost every field of medicine . A widely utilized type is the so - called expert system, defined as artificial intelligence program designed to provide expert - level solutions to complex problems . There are three different approaches to knowledge - based systems depending on the form of knowledge representation: rule based, case based, and model based . In rule based reasoning, the knowledge is expressed by rules, often if then rules . The rules can be newly developed or can be extracted from decision tables or decision trees . In case of based reasoning, the inference engine searches the knowledge base for similar cases . Finally, models, that is, biochemical or biophysical, can also form the knowledge . The approaches to intelligent computing systems are artificial neuron nets, genetic algorithm, and fuzzy systems . Artificial neuron nets are built like biologic intelligent nervous systems and are regarded as learn - like . Genetic algorithm mimics the process of natural evolution and is mainly used in search processes . Fuzzy systems are usually based on rules, but the reasoning is approximate to cope with uncertainty and imprecision because the rules are given varying truth - value using fuzzy sets . Thus, linguistic certainty or frequency levels, such as probable or seldom, derived from medical texts or experts can be incorporated into the knowledge base . The probability of a diagnosis is calculated with the accuracy of a test or a clinical finding and the prevalence of the disease . Different methodologies are often combined, which are then called hybrid expert systems . Already in 1959, ledley and lusted anticipated the use of computers in supporting decisions and proposed different mathematical models to emulate the reasoning in medical diagnosis . They were archetypes for following expert systems, but they also demonstrated the challenges in the development of such tools . It was used for diagnosis and therapy of bacterial infection and has become the probably best - known expert system in medicine . It was designed to assist physicians in the diagnosis of complex and multiple diseases in internal medicine covering more than five hundred diseases . The problems encountered in developing internist-1 and its successors showed that a comprehensive knowledge base is needed for a correct diagnosis of complex diseases in internal medicine . Several somewhat outdated review articles explored the development and application of expert systems in medicine in general [4, 10, 14]. In 1991, bernelot moens and van der korst reviewed the literature assessing computer - assisted diagnosis of rheumatic diseases . Meanwhile, also in rheumatology new expert systems have emerged and earlier expert systems have been improved to meet the many demands of modern rheumatology: establishing an early diagnosis with the highest probability to allow for a better outcome with the help of a prompt treatment . Besides an overview of characteristics, comprehensiveness, and validation of existing diagnostic expert systems in rheumatology, this systematic review seeks to point out whether the current expert systems fulfill the expectations of clinicians in daily practice and finally what the characteristics of an optimal system would be . The research questions were as follows: what information is currently available on diagnostic expert systems in rheumatology, how do these systems work, what is their validity and their applicability in daily practice, and finally what is an optimal diagnostic expert system expected to be . In the optimal scenario we anticipated to find comprehensive reports on each individual diagnostic expert system including information on the precise diagnostic algorithm, the targeted diseases, a well - described validation cohort, and predictive values for diagnostic performance . The data would allow for a statistical comparison of the expert systems . In a suboptimal scenario, only descriptive reports of expert systems will be found allowing for a comprehensive overview of the past developments without statistical comparability . Medline, embase, and cochrane library were searched using the following medical subject heading (mesh) terms: rheumatic diseases, rheumatology, arthritis, computer assisted diagnosis, and expert systems . All publications referring to diagnostic expert systems in rheumatology or in a rheumatic subfield were included . Reviews, editorials, and literature which described an expert system only used for education of healthcare providers and therefore not used in diagnostics were excluded . Also, literature that referred to an expert system used for identifying solely the stage of a disease and hence not used for diagnosing a disease itself was excluded . Records which described an expert system applied only to image analysis literature referring to data mining strategies using index diagnoses or solely epidemiological variables was excluded as well . Figure 3 shows a flow diagram of the selection of studies . In case of uncertainties, inclusion or exclusion year of the last update of the system, number of considered rheumatic diseases, targeted diseases, information to feed the expert systems (history, clinical exam, laboratory analyses, and imaging studies), methodology of the inference mechanism, and embedding of accepted disease criteria sets such as the american college of rheumatology (acr) or the european league against rheumatism (eular) criteria were extracted using standard forms . For the description of the validation method and the performance, the following information was extracted from the articles: number of cases used for the validation, determination of the resulting diagnosis, identification of the correct diagnosis, the reference diagnosis, percentage of correctly identified cases, sensitivity and specificity, positive predictive values, negative predictive values, positive likelihood ratio, and negative likelihood ratio . One record describing an expert system developed solely for education and one record referring to an expert system that was not designed for clinical use were excluded . Six reviews or editorials were excluded [6, 15, 1922]. In the case of repeated reports, either the original or the more comprehensive article was included in the evaluation leading to a final number of 38 original articles (figure 3). In these 38 articles, 25 different expert systems and their successors or further developments are presented . Most of the articles shown in this review presented the development and the methodology of expert systems . The number of considered diseases varies from one to 170 . Both the amount and the nature of information to feed the expert systems vary according to the targeted disease group and inference mechanisms . The following methodologies of expert systems were observed: rule based, case based, model based, artificial neuron nets, fuzzy systems, bayes' theorem, and other not further described algorithms or calculation tools (figure 1). 19 of the 25 expert systems (76%) were validated . The number of cases used for the validation varied widely between 32 real cases and 12 000 simulated patients . Different units of measurement were selected to report the performance of the expert systems, mostly the percentage of correctly diagnosed cases, sensitivity and specificity . The proportion of correctly diagnosed cases the diagnostic accuracy was between 43.1 and 99.9% . Values for sensitivity and specificity ranged from 62 to 100, and 88 to 98%, respectively . Positive or negative predictive values and likelihood ratios were only surveyed for two expert systems [26, 27]. Binder et al . Showed a positive likelihood ratio of 12.1 (95% ci 7.7019.1) and a negative likelihood ratio of 0.187 (95% ci 0.0990.351). Excluding this last report the reference standards were chosen differently: diagnoses according to established criteria, consensus diagnoses, discharge diagnoses, and diagnoses provided by a rheumatologist were used most often as reference . Three expert systems presented certain criteria for the determination of the resulting diagnosis when several diagnoses were presented as a result or when a probability value was added to the diagnosis . An article that reports on the applicability of a rheumatological expert system in clinical routine could not be identified in the published literature . First, an overview over 25 different diagnostic expert systems designed for rheumatology is given . Second, it is shown that the different designs and validation methods of the expert systems hinder the comparison of their performances . Third, we found no publications reporting on the routine application of an expert system in rheumatology . Artificial intelligence has achieved enormous progress in its development and computers have outclassed human beings in various fields, such as computer chess or ibm's watson winning on the quiz show jeopardy! Given this progress in technology and the time period covered by this systematic review of over forty years, the low number of identified expert system is surprising . The reasons would be either low interest in supportive software or, more likely, the difficulties encountered in simulating the complex human diagnostic process . Reported that developers of knowledge - based systems regarded pharmacovigilance, intensive care monitoring, and support for guidelines and clinical pathways as the most promising fields of knowledge - based systems . In other words, systems covering clearly defined decision rules or comparing databases . First of all, nonspecific findings occurring in multiple rheumatic diseases are common and consequently complicate the knowledge representation in expert systems . Second, there is a lack of epidemiological data concerning the prevalence and incidence of rheumatic diseases as well as sensitivity and the specificity of single findings in diseases . Third, even if available for a large population, such data vary greatly amongst ethnic groups and regions becoming an increasing problem in times of global migration . Fourth, for many of the disease - specific findings, there are no internationally established standardized cut - off values . And finally, many rheumatic diseases can coexist with each other in overlap syndromes . Nevertheless, the growing understanding of diseases and the corresponding findings or symptoms will facilitate the representation of medical knowledge and decision processes in the future . In consequence of the variation in the method of validation, first, the result of the expert systems to be compared with the reference diagnosis was presented in different ways . Some expert systems indicated a probability value of the calculated resulting diagnosis, and others present a hypotheses list . Final diagnoses in rheumatology often remain descriptive or incomplete and evolve over time as many of the rheumatic disorders present atypically and do not completely fulfill a diagnostic criteria set at the beginning . This issue is met by the presentation of the results as a hypotheses list or probability values, which can, as an important advantage, multiply the user's own differential diagnosis and lead to more focused testing . Yet for example, the diagnostic accuracy is erroneously high if a diagnosis at a low position in the hypotheses list or a diagnosis with a low probability value is accepted as a correct resulting diagnosis during the validation process . Second, there is a lack of widely accepted reference standards for the correct diagnosis to compare the resulting diagnosis with . Some authors used diagnoses in medical records or discharge diagnoses as a comparator assuming the correctness of their peers, some chose the consensus of rheumatologists, and others used diagnoses according to official diagnostic criteria sets . The latter is probably the most reliable way; however, even if international consensus criteria exist, there are still many different criteria sets especially for rare diseases where the superiority of one set over the other and in particular the threshold for a diagnosis remains a matter of debate . In addition, many of these criteria sets were established to obtain homogenous cohorts in clinical trials leading to a low sensitivity in early or mild disease . Another approach was the assessment of the interobserver variability by hernandez et al . And martn - baranera et al . . Here, the distance between the resulting diagnoses of clinicians and renoir was calculated without setting a reference diagnosis . By this means the uncertainty of the final diagnosis and the error proneness of clinicians were taken into account . The transferability of expert systems to the general population (the external validity) can be tested with a validation in a developer - independent clinical setting . Only ai / rheum, cadiag, and rheuma [29, 39, 40, 49] were validated this way, resulting in a lack of data on the transferability to daily practice of most of the presently available expert systems . Besides the internal and external validity, the following features are, according to kawamoto et al ., highly associated with an expert system's ability to improve clinical practice: the availability at the time and location of decision making, the integration into clinical workflow, and the provision of recommendations rather than a pure assessment . The wider use of computers in clinical routine, such as the possible use of tablet computers on ward rounds, will facilitate the integration into clinical workflow and enhance the availability at the time and location of decision making . The need of more detailed documentation for quality assurance may have a positive influence as well . Are the only authors who reported the clinical use of their diagnostic expert system cadiag-4/rheuma - radio . The expert system was incorporated in the medical information system of the respective clinic . For the lack of accessibility of diagnostic support, the universally present search engines for the world wide web have become a popular alternative with an astonishing accuracy as shown by tang and ng and lombardi et al . . And schewe and schreiber regarded the time required for data input as the most limiting factor . Considering the smaller amount of input data and consequently the shorter input time, specialized and restricted expert systems like the laboratory results analyzing system presented by binder et al . Kaplan presented a system with a provisional hypothesis list, which updates after every further input . Here, the data input is limited; hence, there is a risk of missed diagnoses due to the less thorough questioning . The required time for data input would decrease if the expert system was compatible with the institutional medical information system and consequently could allow direct access to all electronically stored patient data comprising patient history, physical exam, imaging studies, and laboratory analyses . . Then again the data input and the required time depend on an intuitive user interface, which boegl et al . Believed to have the biggest influence on the clinical success . The reason for the absence of expert systems in clinical use hitherto has been discussed in detail in the literature . Mandl and kohane claimed that health information technology in general was in arrears compared to other industries . Also they took the health information technology products as too specific and incompatible with each other . They stated that the lack of acceptance by the medical staff is the main problem in the application of knowledge - based systems in medicine . The different points of view of developers and clinicians show that a better cooperation is necessary . Expert systems have to be adapted to clinical problems and to clinical workflow . On the other hand, clinicians should become more aware of the supportive possibilities of expert systems . In spite of computerized assistance, the user of the expert system needs rheumatologic fundamentals for the detection and the correct description of rheumatologic findings . Cadiag, ai / rheum, renoir, rheumexpert, and mesicar were specifically developed for the assistance of nonrheumatologists [31, 37, 39, 48, 50]. These systems were designed to remind the nonspecialist of rare diseases or to indicate the cases which needed immediate treatment . Yet, an expert system's outcome highly depends on the entry of correct parameters . Therefore, educational parts were added to some of the expert systems to increase the user's diagnostic skills . These educational parts explain certain symptoms or show photographs of findings [30, 42, 51]. Also, some systems provided a link to literature, such as medline, for further information [30, 42]. A widely accepted system ideally covers the demands of generalists and specialists offering an easy understandable handling and not being too basic at the same time . The integration of widely accepted diagnostic criteria sets such as the acr or eular criteria into the diagnostic process would increase the acceptance and credibility of an expert system . It also reduces the influence of individual diagnostic strategies of the developers . Nevertheless, only six of the identified expert systems reported the integration of such criteria sets into their expert database . The downside of diagnostic criteria originating primarily from classification criteria for the inclusion into clinical trials, however, is the generally low sensitivity in early disease . This insensitivity of some criteria, such as the 1987 ara criteria for rheumatoid arthritis, forced leitich et al . To modify the criteria using fuzzy sets to gain different levels of sensitivity . The recent development of official diagnostic criteria, which are more dedicated to the diagnosis in an early stage of the disease, will make their use in the design of expert systems more attractive . Furthermore, some methodologies are ill suited to the use of diagnostic criteria, such as a mere probabilistic approach like bayes' theorem or artificial neural network . These systems extract their knowledge base from patient data, such as symptoms and clinical findings, and the corresponding diagnoses assuming a correctness of the chosen diagnosis . Diagnostic criteria cannot be included in these systems without the combination with another methodology or an adaption of the reasoning process like the review of symptom weighing . Other ways of knowledge representation facilitate the usage of official diagnostic criteria, like rule - based reasoning though the minority of the articles presenting a rule - based expert system reported an integration of official diagnostic criteria . Although a thorough systematic search has been performed in the most relevant databases, some reports could have been missed if written in other languages than english or german . As most of the current literature is published in english at least as an abstract, we are confident that we did not miss relevant articles on diagnostic expert systems in rheumatology . The number of expert systems which have remained unpublished because of their expected commercial use or the abortion of the system at an early stage is hard to estimate . The reported expert systems showed a great variety in diseases spectrum, methodology, and validation status . This made a statistical comparison of the systems impossible . And finally, the important topic of patient reported outcomes which are of increasing importance not only in clinical trials and patient's follow - up but also in the diagnostic process was beyond the scope of this review . In conclusion, this systematic review shows that the many attempts made for an ideal expert system in rheumatology in the past decades have not yet resulted in convincing validated tools allowing for reliable application in daily practice . Nevertheless, the demand in support by expert systems is pressing as the knowledge about the rheumatic diseases increases and the therapeutic options especially in early disease stages are growing constantly . An ideal diagnostic expert system in rheumatology the expert system would allow for universal integration into the clinical workflow as well as rapid and intuitive data input . Since rheumatologic diagnoses cannot always be definite, the resulting diagnosis would have a probabilistic grade to indicate uncertainty . The system would also have an educational component to improve the nonexpert's ability to recognize pathological findings . Finally, accepted diagnostic criteria sets would be applied to increase the general validity of the system's diagnostic process . Based on the demand of such a tool and the progress made hitherto it seems to be a matter of time until new and promising expert systems enter clinical practice.
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Ocular inflammation is common after ophthalmic surgery, particularly after surgical removal of cataracts combined with intraocular lens (iol) implantation . This inflammatory response includes the metabolism of arachidonic acid to prostaglandins and leukotrienes and the recruitment of neutrophils and macrophages to the site of surgical trauma . This process eventually manifests as a mild iritis, corneal edema, and increased cells and proteins (flare) in the anterior chamber of the eye, accompanied by hyperalgesia . While recent advances in surgical techniques (smaller incisions), more efficient phacoemulsifiers, and improved viscoelastics have improved cataract surgery outcomes, postoperative inflammation and pain remain a major source of discomfort for patients . If left untreated, postoperative inflammation can lead to suboptimal vision results or complications such as cystoid macular edema (cme) [1, 36]. As surgical techniques have improved, ocular inflammation following cataract surgery is managed by topical anti - inflammatory drugs such as non - steroidal anti - inflammatory drugs (nsaids) and/or corticosteroids . Both are effective in resolving postoperative inflammation and pain, increasing patient comfort, and decreasing the risk of complications [1, 711]. They inhibit phospholipase a2 in the inflammatory cascade, which converts membrane phospholipids to arachidonic acid, thereby inhibiting the cyclooxygenase and lipoxygenase pathways and the formation of all eicosanoids . Corticosteroids suppress both the early (capillary dilation, increased vascular permeability, recruitment of leukocytes) and late (deposition of fibrin, proliferation of inflammatory cells and chemokines) phases of inflammation [1316]. However, they are also associated with side - effects, including steroid - induced intraocular pressure (iop) elevation, lowered resistance to infection, risk of cataract formation, and decreased wound healing [1620]. Of these, increased iop is the most significant side - effect for the post - cataract patient, and is thought to be due to structural and biochemical changes in the trabecular meshwork causing increased resistance to aqueous humor outflow . Steroid - induced iop elevation has been reported to occur in 1836% of patients, termed steroid responders, [15, 22] with risk factors including a history of glaucoma, a familial predisposition toward glaucoma, or high myopia [22, 23]. Older corticosteroids, such as prednisolone and dexamethasone, are associated with a greater impact on iop compared to newer corticosteroids . Recent research indicates that nsaids may have a synergistic effect with corticosteroids, particularly for the prevention of cme . In most instances, it is therefore vital for ophthalmic surgeons to be able to provide patients with a corticosteroid option that offers high efficacy yet does not result in an increase of iop to clinically significant levels . Loteprednol etabonate is a novel corticosteroid produced by retrometabolic design . In retrometabolic drug design, an inactive and nontoxic metabolite of a reference compound is utilized as a starting point for conversion to a therapeutically active, metabolically labile compound . Loteprednol etabonate was designed starting with cortienic acid, an inactive metabolite of prednisolone . Structurally, loteprednol etabonate differs from prednisolone in that the ketone at the carbon-20 (c-20) position is replaced with a chloromethyl ester and the 17-hydroxyl group is replaced with a carbonate moiety (fig . 1). After exerting its effects, loteprednol etabonate is rapidly metabolized by tissue esterases to cortienic acid etabonate and then to cortienic acid, thereby limiting any potential adverse effects associated with its use . Preclinical studies demonstrated that loteprednol etabonate is highly lipophilic and has strong binding affinity to glucocorticoid receptors . Indeed, its lipophilicity was found to be 10 times greater while its binding affinity to the glucocorticoid receptor was found to be 4.3 times greater than that of dexamethasone [27, 28]. Approved in the united states in 1998 for the treatment and prevention of various steroid - responsive ocular inflammatory conditions as well as for the treatment of postoperative ocular inflammation, loteprednol etabonate has since received marketing approval in various countries across europe, latin america, the middle east, north africa, and asia.fig . 1loteprednol etabonate (i) and its inactive metabolites, cortienic acid etabonate (ii) and cortienic acid (iii) loteprednol etabonate (i) and its inactive metabolites, cortienic acid etabonate (ii) and cortienic acid (iii) the objective of this paper was to review the available published clinical data on loteprednol etabonate suspension 0.5% in the treatment of postoperative inflammation and pain, and to assess its efficacy and safety along with that of other corticosteroids formally studied and approved for the treatment of postoperative inflammation, namely, difluprednate and rimexolone . Publications on loteprednol etabonate were identified through medline searches (1950 onwards) using the terms loteprednol, postoperative pain, inflammation, cataract, and cataract surgery . In order to identify publications about other corticosteroids currently used in postoperative pain and inflammation, the terms rimexolone, difluprednate, prednisolone, dexamethasone, fluorometholone, cataract, cataract surgery, postoperative, and postsurgical inflammation were also searched . Only ophthalmic studies were included, and validity was assessed based on dosage form (topical only), indications for use, study endpoints, and year of publishing . The search was limited to english language, peer - reviewed primary studies and any reviews published in the last 5 years . Additional references were obtained by searching reference lists of identified articles . As no direct head - to - head studies comparing loteprednol etabonate to rimexolone or to difluprednate were found, insights on comparative safety and efficacy of loteprednol etabonate, rimexolone, and difluprednate were drawn from vehicle - controlled studies or from studies in which these newer corticosteroids were compared to older corticosteroids such as dexamethasone and/or prednisolone acetate . The efficacy and safety of loteprednol etabonate ophthalmic suspension 0.5% for the treatment of postoperative inflammation has been demonstrated by several studies over the last decade (table 1). A double - masked, vehicle - controlled evaluation of the efficacy and safety of loteprednol etabonate for postoperative inflammation after cataract removal with iop implantation was conducted in 1998 by the loteprednol etabonate postoperative study group . In this study, 203 patients with an anterior chamber inflammation (aci) severity 3 (09 scale) on the day following cataract surgery were randomized to either loteprednol etabonate or vehicle administered four times daily in the operated eye for 14 days . Resolution of aci, defined as 5 cells and none - to - trace flare, was observed in 55% of patients in the loteprednol etabonate group and 28% of patients in the vehicle group (27% difference, p <the rate for individual signs of cell and flare as well as supportive signs and symptoms of chemosis, erythema, bulbar injection, ciliary flush, pain, photophobia, tearing and discomfort, all favored the loteprednol etabonate group (p <0.05). From a safety perspective, both treatment groups exhibited a mean decrease in iop (12 mmhg) when compared with baseline . No patients in the loteprednol etabonate group versus a single patient in the vehicle group exhibited a clinically significant increase in iop (10 mmhg). Stewart et al . Conducted an identical study to evaluate the efficacy and safety of loteprednol etabonate in controlling aci . In this study, 227 post - cataract patients with aci severity 3 (09 scale) on the day following surgery were randomized to loteprednol etabonate or vehicle . Resolution of aci at the final visit was observed in 64% of patients in the loteprednol etabonate and 29% of patients in the vehicle groups (35% difference, p <0.001). Again, the resolution rate for cells and flare individually as well as supportive signs and symptoms of chemosis, erythema, bulbar injection, ciliary flush, pain, photophobia, tearing, and discomfort all favored the loteprednol etabonate group (p <0.05). As in the first study, mean iop decreased in both treatment groups relative to baseline by 12 mmhg . A clinically significant increase in iop (10 mmhg) was observed in three patients in the loteprednol etabonate group . Comstock and usner further explored the efficacy of loteprednol etabonate in resolving pain and discomfort in these studies . The treatment effect for pain and discomfort was 31 and 43%, respectively, for the first study and 24 and 30%, respectively, for the second study . Analysis of pooled data indicated that the proportion of at - risk patients with resolution of pain at the final visit was 84% for the loteprednol etabonate group and 56% for the placebo group (p <0.05). Similarly, resolution of discomfort at the final visit was 79% for the loteprednol etabonate group and 42% for the placebo group (p <0.05).table 1studies demonstrating the efficacy and safety of loteprednol etabonate (le) 0.5% for postoperative inflammationstudy parametersstewart et al . Stewart comparatorvehiclevehicle1% (pa)fluorometholone acetate 0.1% (fa)no . Of patients2032272030treatment duration (weeks)2242patients with resolution of aci at final visit (%) le group64le group55le group60le group60vehicle29vehicle28pa group50fa group100mean iop at final visitmean decrease in iop of 12 mmhg for both treatment groupsmean decrease in iop of 12 mmhg for both treatment groupsle group12 3 mmhgnot reportedpa group16 1 mmhgclinically significant increases in iop (10 mmhg)n = 3 for loteprednol etabonate; n = 0 for vehiclen = 0 for loteprednol etabonate; n = 1 for vehiclenot reportednot reportedaci anterior chamber inflammation, iop intraocular pressure, le loteprednol etabonate, pa prednisolone acetate, fa fluorometholone acetateresolution of aci defined as anterior chamber cell count <5 and flare grade 0 (none) studies demonstrating the efficacy and safety of loteprednol etabonate (le) 0.5% for postoperative inflammation aci anterior chamber inflammation, iop intraocular pressure, le loteprednol etabonate, pa prednisolone acetate, fa fluorometholone acetate resolution of aci defined as anterior chamber cell count <5 and flare grade 0 (none) three small prospective studies further compared the efficacy and/or safety of loteprednol etabonate with that of other corticosteroids . Stewart compared the efficacy and safety of loteprednol etabonate 0.5% and fluorometholone acetate 0.1% in the treatment of postoperative inflammation . A total of 30 post - cataract patients were enrolled in this randomized, double - masked, parallel - group study . All patients instilled a single drop of the assigned study medication four times daily for 14 days . At the final visit, no statistically significant differences in flare, anterior segment cell, or conjunctival hyperemia were observed between the two treatment groups . Compared the efficacy and safety of loteprednol etabonate and prednisolone acetate 1%, in the treatment of postoperative inflammation following cataract surgery . Twenty patients were randomly assigned to loteprednol etabonate or prednisolone, instilled four times daily for the first week, tapering to once daily by week 4, in this randomized double - masked study . Patients from both groups achieved a similar resolution of postoperative inflammation (conjunctival hyperemia, corneal edema, aqueous cells, flare), with 60% of patients in the loteprednol etabonate group and 50% of patients in the prednisolone group achieving significant resolution of inflammation by the final visit . Despite the study s small sample size, treatment with loteprednol etabonate had less effect on iop elevation than prednisolone . The mean (sd) iop on the final visit was 12 (3) mmhg in the loteprednol etabonate group compared with 16 (1) mmhg in the prednisolone group . Oban and kocak also compared the safety of loteprednol etabonate 0.5% and prednisolone acetate 1% in 40 patients after uncomplicated phacoemulsification surgery . Treatments were administered five times daily from the first day postoperatively, and patients were evaluated at 1 day, 1 week, and 1 month thereafter . At all postoperative visits, the mean iop was lower in the loteprednol etabonate group than in the prednisolone group . The authors concluded that loteprednol etabonate 0.5% use after cataract surgery is associated with a smaller increase in iop than prednisolone use . As indicated above, in most instances, topical corticosteroids are used in combination with nsaids for the treatment of pain and postoperative inflammation . Compared the use of loteprednol etabonate 0.5% alone, loteprednol etabonate 0.5% in combination with indomethacin 0.1%, and dexamethasone disodium phosphate 0.15% in the treatment of postoperative inflammation following uncomplicated cataract surgery . Patients were divided into three groups, the first of which included 79 patients administered indomethacin 0.1% from 3 days before to 2 weeks after surgery and loteprednol etabonate for 4 weeks after surgery . The second and third groups comprised 81 patients treated with loteprednol etabonate 0.5% for 4 weeks and 78 patients treated with dexamethasone for 4 weeks, respectively . All three therapeutic regimens were effective in preventing postoperative ocular inflammation at postoperative weeks 1 and 4 with very mild aci and very low corneal fluorescein staining in all 3 groups . Two cases of iop elevation (both in the dexamethasone group) and three cases of cme (loteprednol etabonate group = 1, and dexamethasone group = 2) were observed . The authors concluded that there was better control of iop and prevention of cme in the combination group but that larger confirmatory studies were needed . The above studies establish the efficacy and/or safety of loteprednol etabonate 0.5% for the treatment of postoperative inflammation in prospective, randomized, double - masked studies . Additional published studies provide insight into the safety of long - term treatment with loteprednol etabonate and safety of loteprednol etabonate in steroid responders . Howes et al . Studied the systemic pharmacokinetics, systemic effects, and iop effects of loteprednol etabonate 0.5% after chronic ocular administration in a double - masked study . Healthy individuals aged 1944 years were randomized to receive either loteprednol etabonate (n = 10) or vehicle (n = 4) instilled in each eye every 2 h while awake (8 times a day) on days 0 and 1 and four times daily on days 2 through to 42 . Blood samples were collected at 0, 15, 30, 60, and 120 min after the first and eighth doses on day 0 and after the fourth dose on day 42, and once on days 7, 14, and 28 . Plasma levels of loteprednol etabonate and cortienic acid etabonate were below the level of quantitation (1 ng / ml) in all samples collected, while cortisol levels were all within normal range indicating a lack of hypothalamic pituitary adrenal axis suppression . None of the patients exhibited a clinically significant increase in iop (10 mmhg) over the 6 week study period . Compared the effects of a combination of loteprednol etabonate and tobramycin (le / t) with that of dexamethasone and tobramycin (dm / t) on iop in healthy volunteers over a 4 week treatment period . A total of 306 volunteers were randomized to receive either le / t or dm / t administered every 4 h for 28 days . Clinically significant increases in iop (10 mmhg) were observed in three (1.95%) subjects treated with le / t compared with 11 (7.48%) subjects treated with dm / t (p = 0.028). Assessed the long - term safety of loteprednol etabonate across all development studies of loteprednol etabonate (0.2 and 0.5% suspension) in a large retrospective analysis . These development studies included the use of loteprednol etabonate for postoperative inflammation as well as other ocular inflammatory conditions (seasonal allergic conjunctivitis, giant papillary conjunctivitis, and uveitis). The proportion of patients exhibiting clinically significant increases in iop (10 mmhg) was 0.5, 1.7, and 6.7% with vehicle, loteprednol etabonate, and prednisolone acetate 1% (used as a comparator in some studies), respectively . Excluding patients that continued to wear contact lenses (allowed in giant papillary conjunctivitis studies), the proportions of patients showing clinically significant increases in iop were 1.0, 0.6, and 6.7% for vehicle, loteprednol etabonate, and prednisolone acetate 1%, respectively . In studies with loteprednol etabonate 0.5%, the proportion of patients with a clinically significant increase in iop was 2.1% if patients wearing contact lenses were included and 0.8% if these patients were excluded . In addition to the above studies on the safety of loteprednol etabonate with long - term use, two studies in steroid responders support the relative lack of impact of loteprednol etabonate on iop . In a retrospective review, holland et al . Evaluated data from 30 post - penetrating keratoplasty and post - keratolimbal allograft patients who, after experiencing increased iop to 21 mmhg, were switched from prednisolone acetate 1% to loteprednol etabonate 0.5% . Results showed a mean (se) reduction of iop from 31.1 (1.13) mmhg on prednisolone acetate to 18.2 (1.37) mmhg on loteprednol etabonate (p <0.001) and no allograft rejection . Bartlett and colleagues examined the safety of loteprednol etabonate in a crossover study in 19 known steroid responders . Subjects received either loteprednol etabonate or prednisolone acetate 1.0% for 42 days followed by a washout period of 14 days prior to being crossed over to the other treatment . During treatment with loteprednol etabonate, iops were within the normal range, with a mean iop elevation of 4.1 mmhg over the 42 day period (ns vs baseline). In contrast, the subjects iops were significantly greater compared to baseline during treatment with prednisolone acetate; mean iop elevations of 5.9, 7.7, and 9.0 mmhg were observed by days 14, 28, and 42, respectively (p <0.05 for all). Taken together, the available clinical efficacy and safety studies of loteprednol etabonate 0.5% for the treatment of postoperative inflammation, combined with safety studies on the long - term use of loteprednol etabonate or use of loteprednol etabonate in steroid responders suggest that loteprednol etabonate is a potent and safe topical corticosteroid with a low propensity to increase iop relative to older corticosteroids such as prednisolone acetate or dexamethasone . Rimexolone is a c-20 ketone steroid similar to prednisolone with the 17-hydroxyl group replaced with a methyl group and an additional methyl group at the c16 position . While it has been granted regulatory approval in the united states, rimexolone has not received approval in all countries in the european union . Bron et al . Assessed the safety and efficacy of rimexolone 1% ophthalmic suspension compared with placebo for reducing postoperative inflammation after cataract surgery and iol implantation . In this study, 182 post - cataract patients were randomized to rimexolone 1% or placebo instilled four times daily for 14 days postoperatively . As was the case in studies with loteprednol etabonate, inclusion criteria included a grade 3 (09 scale) for the sum of cells and flare combined on the day following cataract surgery . By the final visit, the proportion of patients with resolution of inflammation (5 cells and none - to - trace flare) was 59.7 and 27.6% for the rimexolone and placebo group, respectively (32% difference; p <0.0001). Supportive measures of ocular discomfort, corneal edema, bulbar conjunctival erythema, anterior vitreous reaction and the physician s impression all favored treatment with rimexolone (p <0.05). The authors reported no perceptible change in iop in either group at any visit but indicated that the study was not designed to show differences in iop . In this study, 197 patients with combined cell and flare severity 3 for cells and flare combined (09 scale) 24 h after cataract surgery were randomized to rimexolone 1% or placebo four times daily for 14 days ., 59.7% of patients in the rimexolone group compared to 32.1% of patients in the placebo group had their inflammation resolved by the final visit (27.6% difference, p <0.001). Secondary measures of bulbar conjunctival erythema, and the physician s follow - up impression also favored rimexolone treatment compared to placebo treatment (p <0.05) although there was no between - group difference in the presence of ciliary flush or anterior vitreous reaction . Mean iop decreased relative to baseline (postoperative day 1) in both treatment groups, although two patients in each treatment group experienced a clinically significant increase in iop (10 mmhg). Based on the treatment effects noted in these studies, the clinical efficacy observed with rimexolone appears similar to that observed in vehicle - controlled studies with loteprednol etabonate . Several published studies compared the clinical efficacy and safety of rimexolone 1% to prednisolone acetate 1% . Compared the efficacy of rimexolone to prednisolone, administered four times daily for 15 days postoperatively for the treatment of postoperative ocular inflammation in 80 post - cataract patients (baseline inflammation severity not specified). Both treatments were effective in reducing postoperative inflammation, with no between - group differences in anterior chamber cell count or flare severity at any postoperative visit (days 1, 3, 8, 15, 18). However, conjunctival hyperemia was worse in the rimexolone group on days 1 and 3 (p <0.05), while corneal edema was worse in the prednisolone group on day 8 (p <0.05). There were also no between - group differences in iop, with mean (sd) final visit iops of 12.96 (3.2) mmhg and 11.65 (2.86) mmhg, respectively . Also evaluated the anti - inflammatory efficacy of rimexolone with that of prednisolone acetate 1%, both administered four times daily for 15 days, in patients (n = 48) undergoing cataract extraction by phacoemulsification . There was no difference between treatment groups in mean inflammation scores (aqueous cell, flare, and conjunctival hyperemia individually) at postoperative follow - up visits except for cells at postoperative day 3 which were lower in the prednisolone group . Although mean iop decreased in both groups relative to postoperative day 1, a significant difference between treatments was found at postoperative day 3, with mean (sd) iops of 10.9 (1.3) mmhg and 11.9 (1.9) mmhg in the rimexolone and prednisolone groups, respectively . There were no differences between treatments in mean iop at days 7 or 15 . Furthermore, hirneiss et al . Compared the clinical efficacy and safety of rimexolone with that of prednisolone and ketorolac tromethamine in 45 patients after cataract extraction . There was no difference between treatments in control of aqueous cells, but prednisolone was more effective than rimexolone in controlling flare and conjunctival hyperemia . Notably, one patient from the prednisolone group was discontinued for a marked early increase in iop . Finally, leibowitz et al . Studied the iop - increasing potential of rimexolone 1.0% with that of fluorometholone alcohol 0.1% in a double - masked, two - way crossover study of 45 otherwise healthy steroid responders . Following verification of steroid responder status with either dexamethasone or prednisolone acetate, subjects were randomized to either rimexolone or fluorometholone administered every 2 h for 2 days followed by four times daily for 40 days . On completion of the 6 week study duration or on exhibiting an increase in iop of 10 mmhg, the study treatment was stopped; subjects completed a 1 month washout and then received the alternate treatment for another 6 weeks . There was no significant difference between rimexolone and fluorometholone in the number of subjects demonstrating an increase in iop of 10 mmhg or in the number of weeks required to achieve a 10 mmhg increase . Rimexolone treatment resulted in a mean increase in iop of 7.5 mmhg in patients previously observed to have a mean increase of 11.8 mmhg with dexamethasone (p = 0.001), and a mean increase of 6.2 mmhg in patients previously observed to have a mean increase of 12.1 mmhg with prednisolone acetate (p <0.001). As noted above, a similar cross - over study with loteprednol etabonate resulted in a mean iop elevation of 4.1 mmhg over a six - week period (ns vs baseline) compared to 9.0 mmhg for prednisolone acetate (p <0.05 vs baseline). Table 2 compares the efficacy and safety parameters evaluated in the above - mentioned studies on rimexolone.table 2studies demonstrating the efficacy and safety of rimexolone 1% for postoperative inflammationstudy parametersbron et al . Comparatorplaceboplacebopapapa and ketorolacno . Of patients182197804845treatment duration (weeks)2up to 2224patients with resolution of aci at final visit (%) rimexolone60% rimexolone60% not reportednot reportednot reportedvehicle28% placebo32% mean iop at final visitno perceptible changes in iopmean decrease in iop in both treatment groups compared to baselinerimexolone13.0 3.2 mmhgrimexolone11.6 1.4 mmhgrimexolone13.25 mmhgpa group14.60 mmhgpa group11.7 2.9 mmhgpa group10.8 1.3 mmhgketorolac group13.73 mmhgclinically significant increases in iop (10 mmhg)not reportedn = 2 for rimexolone;not reportednot reportednot reportedn = 2 for placeboaci anterior chamber inflammation, iop intraocular pressure, pa prednisolone acetateresolution of aci defined as anterior chamber cell count <5 and flare grade 0 (none)the study did not report the percentages of patients with resolution of aci; instead, mean cell and flare at study visits were reported studies demonstrating the efficacy and safety of rimexolone 1% for postoperative inflammation aci anterior chamber inflammation, iop intraocular pressure, pa prednisolone acetate resolution of aci defined as anterior chamber cell count <5 and flare grade 0 (none) the study did not report the percentages of patients with resolution of aci; instead, mean cell and flare at study visits were reported in summary, vehicle - controlled studies with rimexolone suggested similar treatment effects compared to loteprednol etabonate for the control of postoperative inflammation with minimal iop impact . However, contrary to comparative studies with loteprednol etabonate, studies comparing rimexolone with prednisolone acetate suggest rimexolone s clinical efficacy may not be as robust as that of prednisolone . Difluprednate ophthalmic emulsion, 0.05%, has also been approved for postoperative anti - inflammatory use in the united states . Like loteprednol etabonate, difluprednate is a derivative of prednisolone . Structural modifications include the addition of fluorine atoms at both the c-6 and c-9 positions, a butyrate ester at the c-17 position and acetate ester at the c-21 position (fig . 2). However, difluprednate retains the c-20 ketone moiety of prednisolone . Evaluated the safety and efficacy of difluprednate 0.05% versus placebo for postoperative inflammation in two identical double - masked, placebo - controlled studies . Patients (n = 438) with anterior chamber cell grade 2 one day after ocular surgery were randomized to difluprednate twice daily, difluprednate four times daily or placebo (n = 110 in each study) for 14 days followed by a 14 day tapering period . Primary assessment included the resolution of anterior chamber cells, proportion of patients with clinical response, and absence of pain / discomfort . For comparative purposes, only the proportion of patients with clinical response (defined as 5 cells and no flare) is summarized here . By day 15, clinical response was observed in 72.7% of patients receiving difluprednate twice daily and 71.0% of patients receiving difluprednate four times daily compared to approximately 27% of patients receiving placebo (difference ~45%, p <0.0001 vs placebo). Proportions for pain - free patients and secondary measures of photophobia, chemosis, corneal edema, and conjunctival injection were all significantly better in the difluprednate groups compared to placebo groups . In terms of safety, mean iop remained within normal range in all treatment groups; however, three patients (3%) in each of the difluprednate treatment groups experienced clinically significant increases in iop compared with two patients (1%) in the placebo group . Studied the clinical efficacy of difluprednate administered twice daily for managing ocular inflammation and pain following cataract surgery . Patients (n = 121) were randomized to receive either difluprednate or placebo twice daily for 16 days; this was followed by a 14 day tapering period . This study differed from previous studies in that dosing was initiated 24 h before ocular surgery . Resolution of cells (5 cells) and grade 0 flare was observed in 74.7% of difluprednate patients compared to 42.5% of placebo patients (difference = 32%; p = 0.0006). Significant differences were also observed between the two groups in the proportions of patients that were free of ocular pain and discomfort (difference = 34.6%; p = 0.0004). As in the study by korenfeld three subjects (3.7%) in the difluprednate group had a clinically significant increase in iop (10 mmhg). Further details of the above - mentioned trials are provided in table 3.fig . 2structures of loteprednol etabonate (a), rimexolone (b) and difluprednate (c)table 3studies demonstrating the efficacy and safety of difluprednate for postoperative inflammationstudy parameterskorenfeld et al . Duration15 days16 day treatment periodpatients with a clinical response (%) difluprednate bid72.7difluprednate74.7% difluprednate qid71placebo42.5mean iop at final visitno significant changes from baseline for the difluprednate group reportednot reportedclinically significant increases in iop (10 mmhg)n = 3 for difluprednate bid;n = 3 for difluprednaten = 3 for difluprednate qid;n = 2 for placeboaci anterior chamber inflammation, bid twice daily, qid 4 times daily, iop intraocular pressurethe treatment period was followed by a 2 week tapering period before treatment was stoppedclinical response defined as anterior chamber cell count <5 and flare grade 0prior to commencement of dose - tapering structures of loteprednol etabonate (a), rimexolone (b) and difluprednate (c) studies demonstrating the efficacy and safety of difluprednate for postoperative inflammation aci anterior chamber inflammation, bid twice daily, qid 4 times daily, iop intraocular pressure the treatment period was followed by a 2 week tapering period before treatment was stopped clinical response defined as anterior chamber cell count <5 and flare grade 0 prior to commencement of dose - tapering in summary, while the above placebo - controlled studies suggest difluprednate has a similar treatment effect to loteprednol etabonate, the impact of difluprednate on iop may be greater . Indeed, cable reported significant elevations of iop in patients undergoing uncomplicated cataract surgery administered difluprednate twice daily following surgery . In this retrospective chart review of 100 consecutive patients, all patients had a history of open - angle glaucoma, but were not known steroids responders, and the average increase in iop was 17.8 mmhg . The iop increases were managed by the discontinuation of difluprednate, and the administration of topical glaucoma medication if required . In contrast, iop of 10 mmhg was seen on average in 1.4% of loteprednol etabonate treated patients in vehicle - controlled clinical studies and as few as 1.7% of long - term users of loteprednol etabonate [35, 46]. Figure 3 compares the resolution of cells and flare in clinical studies of loteprednol etabonate 0.5%, rimexolone 1%, and difluprednate 0.5% for postoperative inflammation following uncomplicated cataract surgery, based on vehicle - controlled studies.fig . 3resolution of cells and flare in clinical studies of loteprednol etabonate 0.5%, rimexolone 1%, and difluprednate 0.5% for postoperative inflammation following uncomplicated cataract surgery . Resolution of cells and flare was defined as 5 cells and none - to - trace flare in loteprednol etabonate and rimexolone studies and <5 cells and no flare in difluprednate studies resolution of cells and flare in clinical studies of loteprednol etabonate 0.5%, rimexolone 1%, and difluprednate 0.5% for postoperative inflammation following uncomplicated cataract surgery . Resolution of cells and flare was defined as 5 cells and none - to - trace flare in loteprednol etabonate and rimexolone studies and <5 cells and no flare in difluprednate studies as evidenced by a comprehensive review of the data from published studies, loteprednol etabonate is effective in resolving anterior chamber cells and flare as well as in reducing postoperative pain and discomfort . Based on the available data, loteprednol etabonate offers efficacy similar to older corticosteroids such as prednisolone acetate, with a much - reduced effect on iop, thereby presenting an improved safety profile as compared to these older compounds . Extensive searches through the available literature have demonstrated a lack of direct head - to - head studies comparing loteprednol etabonate to other newer corticosteroids (such as rimexolone and difluprednate) formally approved for this indication . In order to address this gap, we compared the relative safety and efficacy of loteprednol etabonate with these newer corticosteroids across vehicle - controlled studies and by examining data from studies in which these compounds were compared to older corticosteroids . Based on our results, loteprednol etabonate provides similar efficacy to rimexolone and difluprednate by offering similar rates of resolution of ocular inflammation . The use of loteprednol etabonate, however, seems to be associated with fewer clinically significant increases in iop (10 mmhg), thereby reducing the risk of corticosteroid - induced ocular hypertension and eventual corticosteroid - induced glaucoma . While these results provide significant insights into the effects of these newer corticosteroids, high quality, active - controlled, randomized clinical trials between these compounds are needed to assess their comparative safety and efficacy.
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Rathke's cleft cysts (rccs) are non - neoplastic congenital lesions that develop from an epithelial remnant of rathke's pouch, a normal component of pituitary development that gives rise to the anterior lobe, pars intermedia, and pars tuberalis of the gland . Topographically, rccs usually occur in the sellar fossa with or without suprasellar involvement, but they can arise from every part of the craniopharyngeal duct, which is a part of rathke's pouch11,14). Intracranial ectopic rccs are exclusively found in the suprasellar region, likely a result of pouch remnants within the pars tuberalis above the diaphragma sellae3). In rare instances, rccs may reside in the sphenoid sinus or clivus regions8). In this report, the author presents a case of a rcc located in the retrosellar and prepontine area . A 41-year - old woman presented with an acute - onset headache, which progressively worsened over the following 3 days . This patient had signs of meningeal irritation with increased cerebrospinal fluid (csf) cell number and protein levels . The basal values of pituitary hormone including the gonad, thyroid, and adrenal axes were normal on laboratory evaluation . Computed tomography (ct) scan revealed a nonenhancing homogeneous hyperdense mass confined to the retrosellar region . Mean hounsfield unit (hu) for the central lesion was 105.1 hu . There was no bony abnormality in the sella turcica and the clivus . Magnetic resonance (mr) imaging showed an ovoid and well - demarcated mass without contrast enhancement displaying isointensity on t1-weighted images and hypointensity on t2-weighted (fig . This 25 mm - sized lesion was located just dorsal to the pituitary gland and occupied the preopontine cistern . Differential diagnosis included; craniopharyngioma, pituitary adenoma, neurenteric cyst, dermoid, and rcc . A pterional craniotomy disclosed a gray - whitish tumor, which was not attached to the infundibulum, and extended up the third ventricle . On approaching the pituitary region, the cyst was observed between the optic nerve and carotid artery . The thin capsule was opened, yellow - whitish mucinous contents evacuated, and the cyst elevated with further dissection . There was no evidence of previous hemorrhage at surgery (fig the tumor was gradually decompressed and totally excised through the space lateral to the carotid artery . The cyst was entirely located behind the dorsum sellae, there was no invasion into the diaphragm . Pathological examination revealed the typical features of a rcc with columnar epitheliums and cystic contents (fig . Postoperative ct scans showed no evidence of residual cyst in the prepontine cistern (fig . With the increased use and evolution of brain mr imaging technology more rccs are being discovered; however, symptomatic cases are rare and accurate preoperative diagnoses can be difficult . Excluding pituitary adenomas, rccs are the most common lesion in the sellar and suprasellar region . Microscopically, they are typically composed of simple or pseudostratified columnar or cuboidal epithelium, often with ciliated or mucin - secreting goblet cells . Neuroembryologically, the origin and pathogenensis of ectopic rcc is a subject of controversy, however, it is not surprising to assume that it is similar to the mechanism for ectopic occurrence of the pituitary adenoma or craniopharyngioma2,5). Ectopic locations include the nasopharynx, the sphenoid sinus, the clivus, and the posterior fossa7,11,15). It has been proposed that rccs originate from epithelial cell remnants of the obliterated craniopharyngeal duct along the path traversed by rathke's pouch and that location is determined by the embryological events of the sellar and/or suprasellar region . The pars tuberalis, while developing from the ventral portion of rathke's pouch, rotates to come in contact with the neuroectodermal layer of the developing ventral cerebral vesicle in the early embryonic period . As a result of different rates of rotation of the pars tuberalis, rathke's pouch cells predominantly spread through sellar and suprasellar region, but few cells can be located outside the sella turcica3,8). Failure of ectopic pouch remnants beyond the pituitary fossa to regress further during development can lead to cystic dilation and formation of a symptomatic rcc . On ct scans, the majority of rccs are homogenous, nonenhancing, and of low - density, as are many other well - defined sellar and suprasellar lesions, including craniopharyngiomas, arachnoid cysts, and pituitary adenomas13). However, cases of mixed density rccs with iso-, hypo-, and hyperdense foci have been reported in the literature15). The cyst in this report differed from ordinary rccs because of its high density on ct, which was ascribed to higher protein content14). In this case, based on the ct hu density values, relatively acute hemorrhage (60 - 70 hu) or calcification (1000 hu) was thought unlikely and ruled out . The major differential consideration for a hyperdense rcc includes the classic colloid cyst and the rare " white " epidermoid cyst; because they may appear hyperattenuated on nonenhanced ct scans6,16). However, colloid cysts should not be mistaken for rccs since they typically occur only at the foramen of monro . Unlike rccs, epidermoid cysts usually have an irregular configuration of the cyst wall and may calcify, and are usually located off midline . Variable mr imaging characteristics of rccs were presumed in part to be due to cholesterol contents, cell debris, mucopolysaccharides, and rarely, hemorrhage within the cysts10,16). Uncommon rccs like the this case that have high- and isointensity on t1- and hypointensity on t2-weighted images are more likely to induce earlier symptoms, when the cysts are smaller than their csf - like intensity counterparts12,17). A rcc with low viscosity fluid may be confused with suprasellar arachnoid cysts, since they typically have the same signal intensity as csf . It has been suggested that the presence of waxy nodules is the most reliable diagnostic indicator of rccs4). In the present case, the intracystic nodule was easily observed on t2-weighted images, but detection of the nodule on t1-weighted images was difficult because of similarities in the cyst fluid and nodule intensities . While t2 hypointensity is not observed in every rcc case, this is in contrast to pituitary adenomas where hypointensity on t2 would not be expected unless there were hemorrhages . Rcc shows no enhancement or is limited to the cyst wall; conversely, craniopharyngiomas typically have nodular, globular, or rim enhancement on mr scanning6). In the case of ectopic rccs in the prepontine region, the major differential diagnosis is neurenteric cyst or dermoid cyst because commonly they have a similar location15). However, dermoid cysts have the same imaging characteristics as fat because they contain liquid cholesterol . The best diagnostic clue for a neurenteric cyst is a lobulated, nonenhancing, slightly hyperintense mass in front of the medulla on both t1-weighted and t2-weighted imagings13). Rccs may compress structures within and/or adjacent to the sella, which can elicit symptoms including visual impairment, pituitary endocrine dysfunction, and headache . Rarely, they also cause abscess formation, chronic inflammation, hyponatremia, cavernous sinus syndrome, diabetes insipidus, or meningitis1). This patient presented with severe headache of acute - onset suggestive of leakage of the cystic contents . Acute symptoms that mimicked pituitary apoplexy were noted in up to 11.0% of reported cases9). For treating symptomatic sellar and suprasellar rccs, although they are rare, the transcranial approach may be required for complex cysts or entirely supra- and retrosellar lesions, which was evident in the present report . Regardless of the surgical approach chosen, treatment of retro- and suprasellar rccs is more difficult owing to their intimate proximity to the optic chiasm and pituitary stalk3). The author reports a rare case of a completely retrosellar type of rcc that was not attached to the pituitary fossa . The unique mr findings of an intracystic nodule with low signal intensity on t2-weighted image may be an indicator in differentiating rccs from other parasellar cystic lesions.
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According to a who survey conducted in 2005/2006, finnish schoolchildren's tooth brushing frequency was one of the lowest in europe . Thirty - seven percent of finnish 11-year - old boys and 55% of girls reported brushing their teeth more frequently than once a day . The figures were similar among 13- and 15-year - olds (boys 35% and 39% and girls 55% and 61%, resp . ). All values were clearly below the european mean values: 11-year - olds 56% and 67%, 13-year - olds 55% and 69%, and 15-year - olds 54% and 74% for boys and girls, respectively . Only children in lithuania, greece, turkey, and malta brushed their teeth more seldom than the finns . Children in switzerland and in finland's neighboring country sweden brushed their teeth most often . The time of eruption of molars, particularly the first permanent molars, is considered as a time of high caries risk for decaying [24]. It has been reported that intensive dental care at eruption time has resulted in reduction in the amount of visible plaque, need for sealants and fillings, and consequently, amount of frequent recalls . An example of an excellent outcome of oral health promotion comes from nex, denmark . In nex, a community - based program was designed and launched during period 1987 - 1988 to improve oral health by focusing on nonoperative caries treatment of children and adolescents aged 018 years and improving their self - care, that is, tooth brushing, especially at times of tooth eruption . Dmft values in the nex community have sunk well below the national average during the past decades, that is, after the intervention started [5, 6]. The total costs of the dental services decreased approximately by 15% during the period from 1987 to 1999 . In finland, after a community - based oral health intervention on schoolchildren, a survey revealed that tooth brushing frequency, knowledge, and attitudes of schoolchildren in terms of oral health improved . However, it was concluded that to have an optimum outcome, oral health promotion should be a continuous process rather than a short - term intervention . In finland, dental care is free up to the age of 18 and children are invited to regular examinations by a dentist or a dental assistant or an oral hygienist at individual recall intervals . After the recession in the 1990s, resources for oral health promotion in the municipalities were limited . However, in a mid - sized finnish city laukaa, authorities wanted to still keep on prioritizing oral health promotion, and in 2008, the city launched a still on - going health education project the tooth brushing school . The tooth brushing school was based on ideology and methods used in nex . The aim of the project was to have all children less than 12 years of age and their parents / care givers living in laukaa attend the tooth brushing school . In addition, in all laukaa schools, oral health lessons were, and still are, organized every year with a specific theme . Examples of earlier themes are snacking; the little ones follow the big ones' example; and good for mouth, good for you . The oral health section of the municipal health services of laukaa has also introduced its own hamster mascot, the hamsu hamster (hampaat ja suu, meaning teeth and mouth). There is also a website (http://www.hamsu.net/) where children can get more information about dental care together with their parents . The aim of this study was to evaluate the outcome of a community - based oral health promotion project based on an individual as well as a public approach on schoolchildren's tooth brushing and other oral health behaviors . We hypothesized that schoolchildren's tooth brushing habits can be influenced by lessons at school and simple individual instructions, especially if the parents become involved . During the school year 2009 - 2010, an intense oral health promotion was carried out among all schoolchildren in laukaa, finland . Some children in laukaa, finland had been invited to tooth brushing school in the summer of 2008 and even more of them during the school year 2009 - 2010 . In finland, all children under 18 years of age are entitled to dental care without charges by the municipality of their residence . All expenses of this project were covered by the municipality of laukaa . In the tooth brushing school, children were first asked about their oral habits and then given a chance to inspect their own teeth using a hand mirror assisted by a dental assistant (pn). Children were demonstrated what a clean tooth looks and feels like and taught how to clean surfaces covered with plaque . In other cases, the child came to the tooth brushing school as often and as many times as needed, sometimes even every other week . Children were accompanied in the brushing school by their parents, who heard and saw what their children were taught . In the autumn of 2009, all children who were not absent from school answered a questionnaire on their oral health behavior . Children in grades 1 and 2 assisted by their parents answered the questionnaire at home and children in grades 36 at school . A total of 1,185 out of 1,554 children (76%) answered the questionnaire (table 1). In the analyses, the children were divided into two groups according to their grades: children in grades 14 and children in grades 5 - 6 . In the autumn of 2010, again all children who were not absent from school were asked to answer a questionnaire on their oral health behavior again . Out of 1,567 children, 1,293 (84%) answered this questionnaire . To compare the answers of the same groups of children in 2009 and 2010, for the analyses, the children were divided into two groups according to their grades: children in grades 25 and children in grade 6 . The questionnaires were similar to children in all grades . In 2009 and 2010, there were eight variables in the questionnaire with seven response alternatives describing the frequency of the behavior . Three to four times a day or more frequently to less than twice a month or never . Questions in 2009 were how often do you brush your teeth; how often do you use dental floss; how often do you use tooth pick; how often do you use fluoride toothpaste; how often do you use nonfluoride toothpaste; how often do you use fluoride tablets; how often do you use xylitol products; and finally, how often do your parents help you with tooth brushing? In 2010, gender and school grade were also included in the questionnaire . A new question was do your parents remind you of brushing your teeth? The alternatives given were yes, no, and i do not know . The following questions of 2009 were not included in 2010: how often do you use non - fluoride toothpaste and how often do your parents help you with tooth brushing? The question how often do you use xylitol products? Was divided into two different questions as follows: how often do you use xylitol lozenges and how often do you use xylitol chewing gum? The answers were recorded into two categories as follows . Brushing frequency was recoded into those brushing at least twice a day and the rest . Use of dental floss was recoded into those flossing at least 2 - 3 times a week and the rest . Use of fluoride toothpaste was recoded into those using it at least twice a day and the rest . Fluoride tablet use was recoded into those using them once a day and the rest . Use of any xylitol products, gum, or lozenge was recoded into those using them at least three to four times a day and the rest . The answers to the questionnaire in 2009, and 2010 were compared using cross - tabulation . In 2009, the data had been collected combing the children in classes 14 and 5 - 6 . In the year 2010, the class of each respondent was registered . To compare results in 2009 and 2010, those in class 7 attend a secondary school and did not participate in this study in 2010 . The main results on oral health behaviors in 2009 and 2010 as well as in different grades in 2010 were presented graphically . Also, oral health behaviors of different groups were analyzed by cross - tabulation . Statistical significance of the differences between the groups was evaluated using chi - squared test . Effect of different background factors was evaluated using binary logistic regression analysis, 95% confidence intervals . Goodness of fit of the model in these data was tested using hosmer and lemeshow test ., chicago, il, united states) was used for the statistical analysis and for producing graphics . The data did not contain any personal identification of the patients; therefore, neither consent from the patients nor children, nor approval of an ethical committee was needed (practices for research permits, oulu university hospital, finland, 2009). Tooth brushing and flossing frequency as well as use of fluoride tablets increased significantly among the schoolchildren during the study period 2009 and 2010 (p <0.05). Frequency of using fluoride tooth paste practically remained the same (60.5% in 2009 versus 60.9% in 2010) (figure 1). Both in 2009 and in 2010, younger children brushed their teeth and used fluoride toothpaste more frequently than the older ones (figure 2). However, the older children (grades 5 - 6) used significantly more frequently dental floss and had fluoride tablets than the younger ones (grades 14) (p <0.05) (figure 2). The older children also used significantly more often xylitol products than the younger children did (p = 0.001). Children in lower grades got significantly more often daily parental help in tooth brushing in 2009 (21.4%), and parents reminded them of tooth brushing in 2010 (76.4%) significantly more often than older children (2.6% and 57.2%, (p <0.001)). The results in 2010 showed a slight trend of decreasing tooth brushing frequency by age (table 2) both among girls and boys when investigated grade by grade . Nevertheless, use of dental floss, fluoride tablets, and xylitol lozenges increased until decreasing again between the grades 5 and 6 . Significantly bigger proportion of children in the upper grades (55.3%) reported using xylitol chewing gum two times a day or more often compared to children in the lower grades (43.0%) (p <0.001). Overall, girls brushed their teeth twice a day (72.0%) significantly more often than boys (57.3%). Girls also used toothpaste, dental floss, and xylitol gum significantly more often than boys (p <0.05). Boys had significantly more often parental guiding than girls in every age group (p <0.05). According to the results in 2010, low tooth brushing frequency (daily or less frequently) was significantly affected by male gender and age (poorer towards grades 5 - 6). Tooth brushing education had a small impact on tooth brushing frequency, whereas parental reminding can be considered protective for children in lower grades (table 3). Tooth brushing frequency increased significantly among the schoolchildren between the years 2009 and 2010, which shows that even a simple intervention targeted to groups and individuals of schoolchildren can be beneficial on their health behavior . We can also see a slight trend that tooth brushing frequency declines towards grades 5 and 6 among both girls and boys compared to younger children . These findings support the idea that oral health intervention should be given to all children but have focus on older children, especially boys . School health surveys are carried out in finland every other year . In the 2008/2009 survey, 43% of 1416-year - olds reported brushing their teeth twice a day or more often; the results show a slight improvement compared with the 2004/2005 survey (40%). Both in 2009 and in 2010, tooth brushing frequencies in laukaa are well above those national averages, but again school - health surveys present mainly habits of older school - children . And it may well be that also in laukaa the figures of this study group could be worse when the children grow older, if their oral health habits keep their worsening course even after grade 6 . Anyhow, reason for delightful situation among the grades 26 in 2010 may be the beneficial effect of the years of oral health education organized in the laukaa community on children's oral health . It has also been shown that municipalities which have stated goals and have a strong focus on caries prevention have improvement in caries experience data compared to communities without such strategies . The numbers are big and the response rates exceed 70% of the total age group in both years . It is likely that some children were off school on the day the questionnaire was answered or sent home . Information about the number of children unable or refusing to answer the questionnaire was not available nor were the possible causes for refusals . This study is practice - based and was originally not designed to be a research . Therefore, the questionnaire used had not been validated and similar forms were used for children of different ages . The children in grades 1 and 2 filled the forms at home with the help of their parents which makes the results more reliable for them . Even if parents may not be aware of their children's fear, they can be expected to know about 1st and 2nd graders tooth brushing habits . A deficiency of this study is also the lack of ids of the respondents which hinders comparing results on the survey in 2009 and 2010 at individual basis, as well as having information on attendance in oral health promotion program at individual basis . Furthermore, it would have been valuable to compare dmfs or cpi values from patient records because the questionnaire does not tell anything about the quality of brushing, which is another main point besides frequency that tooth brushing school seeks to improve . Unfortunately, patient records contained no information of participation in tooth brushing school and again no ids were collected, and thus oral health data could not be collected . Therefore, the effect on oral health could not be analyzed like it has been done in nex . In our study, boys were lazier brushers than girls (57.2% and 72%). This supports the findings of the finnish school health survey and kajaani, another city with a long history of continuous oral health promotion . In kajaani, 74% of 12 - 13-year - old girls and 66% of 15 - 16-year old girls reported brushing their teeth at least twice a day . For boys in those age groups, proportions of those brushing their teeth twice a day were 58% and 51%, respectively . This again speaks for oral health promotion; low profile projects can be effective, when resources are limited for big ones . It is interesting that the proportion of those using toothpaste twice a day is lower than the proportion of those brushing their teeth twice a day . It can be speculated why children do not always use toothpaste when brushing their teeth . 1994, it is beneficial for schoolchildren to brush their teeth twice a day with fluoride toothpaste . Here, it may be possible that not all children understood the question in the questionnaire about using fluoride toothpaste correctly . In the 2009 questionnaire, there were separate questions on the use of fluoride toothpaste and non - fluoride toothpaste, which could have confused children . In the 2010 questionnaire, the non - fluoride toothpaste alternative, however, was erased because most of the toothpastes in finland contain fluoride . 2004 showed in their study that children whose parents had favorable attitudes towards controlling their children's tooth brushing had favorable oral health habits . In the present study, in 2009, a year after the tooth brushing school intervention started, children in grades 14 got significantly more often parental help than older children . Parental help may improve the level of oral hygiene and thus provide long - lasting benefits for the child . Unfortunately, the same question was not used in the 2010 questionnaire, and thus we cannot compare the responses in the two years . In 2010, 66.8% of the girls and 73.2% of the boys were reminded of brushing their teeth daily by their parents . In every age group, boys were more often reminded about tooth brushing by their parents than girls . According to poutanen et al . 2007, the parents' role model is extremely important to children and its effect is slightly stronger on boys than on girls . Children in grades 14 were significantly more often reminded about tooth brushing than children in grades 5 - 6 . One explanation for this difference may be that children in grades 1 - 2 answered the questionnaire at home with their parents and children in grades 36 at school . The effect of parental reminding can be considered most effective for children in the low grades . Xylitol products are commonly used in finland . Over 30% of the children in laukaa used xylitol products at least 3 to 4 times a day . Use of xylitol products was more common among older children and girls than younger children and boys . Regular use of both xylitol gum and candies is reported to reduce caries occurrence by about 50% compared with the control group . Xylitol chewing gum has been shown to be equally effective with sealants in caries prevention . Advantages of xylitol products are that they are freely available and can be bought without prescription . Use of xylitol is also cost - effective because it does not require oral health resources . Analyzing an outcome by any oral health promotion project targeted to children is challenging; was the improvement or deterioration in oral health habits only coincidence, or caused by something else going on in the society, or simply by the growing age of a child? However, this should not hinder the health professionals from promoting oral health . To be effective, the promotion should not be only one project, but rather an on - going process like the one reported in nex . In conclusion, this study gives interesting information about oral health habits of 713-year - olds, when most of the previous studies in finland and elsewhere have been conducted among older children . It can also be concluded that oral health promotion to schoolchildren is beneficial for their health behavior . Our findings emphasize a need of booster - programs targeted to 11 - 12-year - olds and especially boys . It is also noteworthy that intervention may be more effective on younger children (710-year - olds) compared to older children; maybe because of the activity of the parents, which should not be neglected as a resource in oral health promotion.
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Prostate cancer is a neoplasm with a variable natural history that ranges from indolent to aggressive (1). Several clinical features of prostate cancer including tumor stage, gleason score, serum psa, and biopsy criteria are used in routine clinical practice to categorize the patients into groups of low, intermediate, high, or very high risk for tumor recurrence following local therapy . Patients with more risk criteria usually suffer substantial disease related mortality (1, 2). However, up to 30% of men undergoing radical prostatectomy will relapse often as a result of micrometastatic disease present at the time of surgery (2). Currently, there is much debate on the use of inherited genetic information in clinical applications including risk assessment . Therefore, a critical research question is if genotype adds information to risk prediction beyond that of traditional risk factor (3). The challenge is to identify those patients at risk for relapse and to better understand the molecular abnormalities that define tumors at risk of relapse (2). Attempts to explore genetic correlation with tumor behavior have found alterations in a number of candidate genes associated with prostate cancer . However, no single gene has been shown to have sufficient prognostic utility to warrant clinical implementation . This study attempts to evaluate relationship between clinical parameters of prostate cancer (psa, gleason sore, and metastase) and expression of nkx3.1, amacr, tmprss2- erg, erg, and spink1 genes . Patients with prostate cancer were selected from individuals who referred to the urology department of uro - oncology research center (uorc) from june 2011 to march 2013 . All the patients were new cases of the disease with no medical history of surgery or therapy regarding prostatic problem . They were first visited by an urologist and underwent imaging and laboratory tests according to the standard diagnostic approaches . Thirty four tissue samples were obtained via open radical prostatectomy, and in 9 patients with metastatic disease, tissue sample was obtained via needle biopsy . One replicate was stained by hematoxylin and eosin, and examined by a pathologist for detection of malignant changes and evaluation of tumor grade expressed as the gleason score . The tissue samples were instantly immersed in rnalater solution (qiagen) and kept at room temperature for 24 hours . Then, these tissue samples were transferred into liquid nitrogen container for long - term storage . Total rna containing small rnas (e.g.mirnas) was extracted and purified from tissue sample (50 gr) using mirneasy mini kit (qiagen, cat . Cdna synthesis was performed using oligo dt or random hexamers (protoscipt kit, new england biolab, ned #e 6300s) to convert mrna or non - coding rnas, respectively . Exon - exon junction spanning primers and taqman probe were designed using primer express v.3 software (applied biosystems) and verified to be specific for their targets by blast analysis on ncbi website . Nkx3.1 amacr, spink1, erg, and tmprss2-erg gene fusion were assigned as target genes . Serially ten - fold diluted plasmids were used as template in triplicate real - time pcr experiments . Relative mrna expression was normalized to the geometrical mean of the ct values determined for gapdh gene using comparative ct method . Correlation between the expression of each gene and psa level, gleason score and presence of metastase was evaluated and analyzed using spss software . Patients with prostate cancer were selected from individuals who referred to the urology department of uro - oncology research center (uorc) from june 2011 to march 2013 . All the patients were new cases of the disease with no medical history of surgery or therapy regarding prostatic problem . They were first visited by an urologist and underwent imaging and laboratory tests according to the standard diagnostic approaches . Thirty four tissue samples were obtained via open radical prostatectomy, and in 9 patients with metastatic disease, tissue sample was obtained via needle biopsy . One replicate was stained by hematoxylin and eosin, and examined by a pathologist for detection of malignant changes and evaluation of tumor grade expressed as the gleason score . The tissue samples were instantly immersed in rnalater solution (qiagen) and kept at room temperature for 24 hours . Then, these tissue samples were transferred into liquid nitrogen container for long - term storage . Total rna containing small rnas (e.g.mirnas) was extracted and purified from tissue sample (50 gr) using mirneasy mini kit (qiagen, cat . No . Cdna synthesis was performed using oligo dt or random hexamers (protoscipt kit, new england biolab, ned #e 6300s) to convert mrna or non - coding rnas, respectively . Exon - exon junction spanning primers and taqman probe were designed using primer express v.3 software (applied biosystems) and verified to be specific for their targets by blast analysis on ncbi website . Nkx3.1 amacr, spink1, erg, and tmprss2-erg gene fusion were assigned as target genes . Serially ten - fold diluted plasmids were used as template in triplicate real - time pcr experiments . Relative mrna expression was normalized to the geometrical mean of the ct values determined for gapdh gene using comparative ct method . Correlation between the expression of each gene and psa level, gleason score and presence of metastase was evaluated and analyzed using spss software . A total number of 43 specimens were studied, from which 9 were obtained from patients with metastatic cancer . The psa levels were less than 10 ng / ml, 10 - 20 ng / ml and more than 20 ng / ml in 12, 17, 14 patients, respectively . Gleason score was lower than 7, equal to 7, or above 7 in 10, 20, and 4 patients, respectively . As can be seen, the expression of amacr decreased in metastatic prostate cancer (p = 0.02). The expression of other genes showed no difference between metastatic and non - metastatic tumors (p> 0.1). No relationship was detected between the expression of the five genes and the gleason score (p> 0.1). Up to now, genetic criteria have no role in risk classification, prognosis, and treatment planning of prostate cancer . Establishing a relationship between gene expression and clinical parameters (psa, gleason score and clinical stage) can help us in treatment planning . It may be possible to predict clinical behavior of prostate cancer based on gene expression analysis of primary tumor . Such predictive strategies would allow for the rational treatment and application of postsurgical therapeutics to high risk individuals . Here, we appraise five important genes in prostate cancer: nkx3.1 is an androgen - regulated homeodomain gene whose expression is predominantly localized to prostate epithelium and regulates prostate epithelial proliferation . Nkx3.1 is located on chromosome 8p 21.2, a region that shows loss of heterozygosity (loh) in 12 - 89% of high grade prostatic intraepithelial neoplasia (pin) and 35% to 89% of prostatic adenocarcinomas (4, 5). Bethel cr et al . Reported that the frequency of loh on chromosome 8p increases with advanced prostate cancer grade and stage (6) although we detected no relationship between the expression of nkx3.1 and psa level, gleason score and metastatic disease . . Showed that the expression of nkx3.1 was highly specific of prostate cancer and breast cancer, with little or no staining in a large number of other tumor type (7). Amacr is a key enzyme in the beta - oxidation catabolic pathway of fatty acids and is known to be upregulated in several cancers including prostate cancer (8, 9). Amacr is highly expressed in the majority of prostate cancer samples, about 6 times higher than the level expressed in the bph samples (9). Amacr is abundantly expressed and recognized as a standard tissue biomarker capable of highly sensitive and specific diagnosis of prostate cancer . The value of amacr and its variants is to develop diagnostic biomarkers that will complement the diagnostic capability of psa, while addressing the limitation of psa, specifically its low specificity (8). Sequence variants of amacr have been previously investigated to find their association with the risk of prostate cancer (10). Although jun luo et al . Reported that both untreated metastases and hormone refractory prostate cancer were generally strongly positive for amacr (9), amacr expression only decreased in metastatic patients in our study . Also reported that the amacr expression shows a decrease in metastatic prostate cancer when compared with localized disease that is associated with cancer specific survival (11). Luo et al . Found no relationship between amacr ihc score and gleason grade, pathological stage, patient age, or preoperative serum psa (9). The difference may be due to several factors including study design, tumor localization, ethnic background, and detection methods . Several studies have investigated the prevalence and clinical value of tmprss2-erg in prostate cancer . The median prevalence in both clinically localized and castration resistant prostate cancer is around 40 - 50%, with a lower frequency reported in high grade prostatic intraepithelial neoplasia (12). Studies analyzing the association between tmprss2-erg states and clinicopathological parameters such as gleason score and prognostic value have indicated conflicting results . Moreover, the clinical impact is still unclear, since while some authors have suggested a worse prognosis for fusion versus non - fusion cancers (13), other either found a favorable prognostic association (14, 15) or did not find any association with clinical outcome (16, 17). In fact, erg protein expression is the active form of the gene product and hence could be a better method in documenting any prognostic significance (13). Observed that erg prevalence significantly increased from high grade pin (17.5%) to pt2 tumors (27.5%) then to pt3 (43%) and metastases (53%) (13). They showed that the association of erg fusion with both advanced stage and better outcome in their study could be explained by the fact that fusion positive and fusion negative prostate cancer are likely to progress via different molecular pathways (13). . Showed that the androgen driven events causing tmprss2-erg fusion and other rearrangements of androgen- dependent genes in prostate epithelial cells of young patients preferentially lead to low grade (and not high grade) prostate cancer . This finding may help explain the slight but significant predominance of low grade cancer in young patients (18). A study by fleischmann et al . Showed that tmprss2-erg fusion was not overrepresented in high risk population of metastasizing prostate cancer . Investigation of tmprss2-erg fusion prevalence in primary tumor and corresponding metastases might help further elucidate the biological role of this mutation (19). Studies on the prognostic relevance of tmprss2-erg fusion in cancers treated by radical prostatectomy have shown consistent results . Based on survival curves, some studies have suggested that tmprss2-erg fusion is a poor (20) and conversely a good (14) prognostic factor or some other studies have reported that it is of no prognostic value (21). Moreover, in a large cohort study with more than 2800 prostate cancer patients, the surrogate marker for tmprss2-erg fusion, erg protein expression, did not predict survival (22). These conflicting results may be due to differences in cohort size and composition, therapy, clinical endpoints, mechanisms of fusion (deletion vs. translocation), and detection methods . The serin peptidase inhibitor kazal type1 (spink1), also known as pancreatic secretory trypsin inhibitor (psti) or tumor associated trypsin inhibitor (tati), is an extracellular secreted protein, protecting pancreas from auto - digestion by preventing premature activation of pancreatic protease (23). Additionally, spink1 is described to play an important role in the prevention of apoptosis in benign tissue (23). Several studies suggested spink1 expression in malignancies including breast cancer (24), colorectal cancer (25), hepatocellular carcinoma (26), and prostate cancer (27, 28). The expression of spink1 has been described to define a subset of aggressive erg - fusion negative prostate cancer (29). Moreover, spink1 was detectable non - invasively in the serum (30) and urine (29) in patients with prostate cancer and pre - clinical models utilize spink1 as therapeutic target in spink1 positive erg fusion negative prostate cancer (27). The analysis of a large and well - defined prostate cancer collection excluded the expression of spink1 as a prognostic biomarker in prostate cancer (23). Found no associations between this gene and psa recurrence, neither in all cancerous cases nor in the relevant subgroup of fusion negative prostate cancers . Genetic criteria can include new information to clinical data for better risk assessment and treatment planning, and may identify patients at risk of relapse and metastasis . A decrease in the expression of amacr may be a sign of poor prognosis and an increase in the risk of metastasis . We found no relationship between nkx3.1, tmprss2-erg, erg, and spink1 expression and clinical findings . Due to different genetic findings in various studies, it seems necessary to design more studies with matched control expect for genetic information in order to define the exact role of genetic factors . Nkx3.1 is an androgen - regulated homeodomain gene whose expression is predominantly localized to prostate epithelium and regulates prostate epithelial proliferation . Nkx3.1 is located on chromosome 8p 21.2, a region that shows loss of heterozygosity (loh) in 12 - 89% of high grade prostatic intraepithelial neoplasia (pin) and 35% to 89% of prostatic adenocarcinomas (4, 5). Bethel cr et al . Reported that the frequency of loh on chromosome 8p increases with advanced prostate cancer grade and stage (6) although we detected no relationship between the expression of nkx3.1 and psa level, gleason score and metastatic disease . Showed that the expression of nkx3.1 was highly specific of prostate cancer and breast cancer, with little or no staining in a large number of other tumor type (7). Amacr is a key enzyme in the beta - oxidation catabolic pathway of fatty acids and is known to be upregulated in several cancers including prostate cancer (8, 9). Amacr is highly expressed in the majority of prostate cancer samples, about 6 times higher than the level expressed in the bph samples (9). Amacr is abundantly expressed and recognized as a standard tissue biomarker capable of highly sensitive and specific diagnosis of prostate cancer . The value of amacr and its variants is to develop diagnostic biomarkers that will complement the diagnostic capability of psa, while addressing the limitation of psa, specifically its low specificity (8). Sequence variants of amacr have been previously investigated to find their association with the risk of prostate cancer (10). Although jun luo et al . Reported that both untreated metastases and hormone refractory prostate cancer were generally strongly positive for amacr (9), amacr expression only decreased in metastatic patients in our study . Also reported that the amacr expression shows a decrease in metastatic prostate cancer when compared with localized disease that is associated with cancer specific survival (11). Luo et al . Found no relationship between amacr ihc score and gleason grade, pathological stage, patient age, or preoperative serum psa (9). The difference may be due to several factors including study design, tumor localization, ethnic background, and detection methods . Several studies have investigated the prevalence and clinical value of tmprss2-erg in prostate cancer . The median prevalence in both clinically localized and castration resistant prostate cancer is around 40 - 50%, with a lower frequency reported in high grade prostatic intraepithelial neoplasia (12). Studies analyzing the association between tmprss2-erg states and clinicopathological parameters such as gleason score and prognostic value have indicated conflicting results . Moreover, the clinical impact is still unclear, since while some authors have suggested a worse prognosis for fusion versus non - fusion cancers (13), other either found a favorable prognostic association (14, 15) or did not find any association with clinical outcome (16, 17). In fact, erg protein expression is the active form of the gene product and hence could be a better method in documenting any prognostic significance (13). Observed that erg prevalence significantly increased from high grade pin (17.5%) to pt2 tumors (27.5%) then to pt3 (43%) and metastases (53%) (13). They showed that the association of erg fusion with both advanced stage and better outcome in their study could be explained by the fact that fusion positive and fusion negative prostate cancer are likely to progress via different molecular pathways (13). Showed that the androgen driven events causing tmprss2-erg fusion and other rearrangements of androgen- dependent genes in prostate epithelial cells of young patients preferentially lead to low grade (and not high grade) prostate cancer . This finding may help explain the slight but significant predominance of low grade cancer in young patients (18). A study by fleischmann et al . Showed that tmprss2-erg fusion was not overrepresented in high risk population of metastasizing prostate cancer . Investigation of tmprss2-erg fusion prevalence in primary tumor and corresponding metastases might help further elucidate the biological role of this mutation (19). Studies on the prognostic relevance of tmprss2-erg fusion in cancers treated by radical prostatectomy have shown consistent results . Based on survival curves, some studies have suggested that tmprss2-erg fusion is a poor (20) and conversely a good (14) prognostic factor or some other studies have reported that it is of no prognostic value (21). Moreover, in a large cohort study with more than 2800 prostate cancer patients, the surrogate marker for tmprss2-erg fusion, erg protein expression, did not predict survival (22). These conflicting results may be due to differences in cohort size and composition, therapy, clinical endpoints, mechanisms of fusion (deletion vs. translocation), and detection methods . The serin peptidase inhibitor kazal type1 (spink1), also known as pancreatic secretory trypsin inhibitor (psti) or tumor associated trypsin inhibitor (tati), is an extracellular secreted protein, protecting pancreas from auto - digestion by preventing premature activation of pancreatic protease (23). Additionally, spink1 is described to play an important role in the prevention of apoptosis in benign tissue (23). Several studies suggested spink1 expression in malignancies including breast cancer (24), colorectal cancer (25), hepatocellular carcinoma (26), and prostate cancer (27, 28). The expression of spink1 has been described to define a subset of aggressive erg - fusion negative prostate cancer (29). Moreover, spink1 was detectable non - invasively in the serum (30) and urine (29) in patients with prostate cancer and pre - clinical models utilize spink1 as therapeutic target in spink1 positive erg fusion negative prostate cancer (27). The analysis of a large and well - defined prostate cancer collection excluded the expression of spink1 as a prognostic biomarker in prostate cancer (23). Found no associations between this gene and psa recurrence, neither in all cancerous cases nor in the relevant subgroup of fusion negative prostate cancers . Genetic criteria can include new information to clinical data for better risk assessment and treatment planning, and may identify patients at risk of relapse and metastasis . A decrease in the expression of amacr may be a sign of poor prognosis and an increase in the risk of metastasis . We found no relationship between nkx3.1, tmprss2-erg, erg, and spink1 expression and clinical findings . Due to different genetic findings in various studies, it seems necessary to design more studies with matched control expect for genetic information in order to define the exact role of genetic factors.
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The arithmetic, geometric and harmonic means, as well as the standard deviation (sd), as the measures of variability, are the ones most frequently used descriptive statistics in the calculation of pharmacokinetic (pk) (cocchetto et al . 1980; lam et al . 1985; roe and karol 1997; griffin et al . 1999 a common problem in the analysis and interpretation of comparative pharmacokinetic parameters is the high value of sd (davit et al . 2008; haidar et al . 2008; van peer 2010). In extreme cases, the high value of sd makes it impossible to make the right decision as to the fate of the study . This problem concerns many types of studies, from preclinical studies, pilot pharmacokinetic studies to bioequivalence (be) (riley 2001; chien et al . One way to solve the problem of comparative analysis of data burdened with high values of sd is to optimize the sample size of the group in the study, which consists of a precise determination of the number of subjects or animals on the basis of intrasubject variability (fda 2006; ramirez et al . However, even in be studies, in some cases, to determine the correct number of subjects, a pilot study is needed or even a two - stage study model (ema 2010). A high value of variability (cv%) of observed concentrations or pk parameters, especially often makes it impossible to properly interpret the outcome of the study in case of research concerning new drugs, for which their value of variability of key pk parameters is unknown . This situation is further complicated by the fact that research in the early stages of drug discovery and pilot studies is usually conducted on a small number of subjects (for example first in man). The scatter of results described by mean values and sd is the result of many factors . To a large extent it depends on the fate of the drug in the organism, such as absorption, distribution, re - distribution, metabolism and elimination . One of the factors standing outside of a living organism is the scatter of results, which comes from the precision of the used analytical method . Currently, the limit value for the cv% of precision for the calibration curve excluding the lower limit of quantitation point (lloq) is 15%, while in the point equal to lloq, this value can be 20% (fda 2001). In relation to incurred sample analysis for classic drugs, as the acceptable range of differences for repeated analysis, the range 20% is proposed (ema 2009; rozet et al . This means that every bioanalytical result introduces an error to the pharmacokinetic calculations as well as the chosen research model human, laboratory animal or cells in the in vitro studies (jansen et al . The problem of analysis of pk after per os administration of the drug is high variability in the c t values from first sampling to the phase of distribution . The common factor influencing maximum concentration (cmax) and last concentration (clast) values is the spread of analysis result, which determines the precision of the analytical method . All three, absorption, distribution and elimination, processes which in point of time corresponding to cmax occur simultaneously . In case of a single administration of the drug in the elimination phase, the values of the concentration can be observed, which illustrate almost exclusively elimination processes (excluding the redistribution phenomenon), until the interval between the end of the distribution phase and the value equal to \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$t_{\hbox{max}} + t_{1/2kel} \times 3 $$\end{document} (emea 2001; veng - pedersen 2001; fda 2006; hc 2010). It can be assumed that the factors that affect the cmax relative standard deviation (cmax, cv%), resulting from simultaneously occurring elimination, are to some extend dependent on the value of last concentration relative standard deviation (clast, cv%) minus the error resulting from the precision of the analytical method (cv%, an). Pk studies are usually conducted in the conditions of good laboratory practice and good clinical practice, or in accordance with the principles of these quality systems . It can be therefore assumed that the sum of errors connected to the subject, experimental animal, used formulation or bioanalytical method is constant, while keeping the experiment conditions, controlled by the quality system . It can also be assumed that in each pk study, a minimum range of sd for c t is possible to achieve . In relation to a single administration, the closest value in many cases could be the lowest value of cv% for the last points of sampling in the elimination phase . In this phase of the study, the deviations from the mean are usually the lowest in the whole series, as the elimination phase is the dominant one and no other process, which is characterized by high variability (for example absorption) influences the sd of the analyzed concentrations . Taking the above into account, the aim of this study was to propose a method of transformation of c t, which would allow significantly reducing the sd value of observed concentrations, without the statistically significant influence on the value of the mean and median for each sampling point . In the presented study, the lowest value of relative standard deviation (rsd%) of concentrations observed in the elimination phase and the value of precision of the used analytical method were used to optimize the arithmetic and geometric mean and the value of sd obtained after single oral administration of itraconazole, which is characterized by high variability of pharmacokinetic parameters . A single dose of 100 mg of itraconazole was administered orally (sporanox 100 mg tab ., janssen pharmaceuticals) for male subjects 20 to 40 years old, with a body mass index 20 to 25 kg / m . Blood samples were collected just prior to administration and at 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 6.0, 8.0, 12.0, 24.0, 36.0, 48.0, and 72.0 h after the administration . The concentration analyses were performed using tandem mass spectrometry, using the method described previously (grabowski et al . The study was approved by independent ethics committee of district council of physicians, baniowa 3, warsaw (resolution no . Itraconazole is a drug with high intrasubject variability, and the formulation belongs to the group of high variability drug product (hvdp). Therefore, the majority of pharmacokinetic profiles began and ended at different time points (different time of absorption delay and concentration with values <lloq in last sampling points). T profiles that were chosen were those which originated from different subjects and those having identical number of indicated concentrations in the same interval . T profiles were obtained this way between 1.5 and 48 h after the drug administration (table 1).table 1concentrations of itraconazole administered orally at a single dose of 100 mg (sporanox 100 mg tab ., janssen pharmaceuticals) for 10 male subjects . Data before (148 h sampling points) and after transformation (136 h sampling points)time (h)12345678910 drug concentration before transformation (ng ml) 1.58.778.7611.843.446.544.443.764.703.0217.752.036.0315.4217.898.3324.536.2111.0148.305.5031.862.570.6775.3631.7613.6729.508.4726.3585.5810.9968.103.064.6380.3822.5820.9647.8115.3730.0176.8913.1266.203.555.6069.6822.6736.4838.2520.9429.8060.4118.6557.944.057.9861.1931.0040.2955.2833.0032.1777.4724.2653.524.557.0662.7742.7415.3661.9733.9143.9477.4724.1348.265.047.8256.8635.5045.2547.7437.7064.7280.0732.2144.126.038.9639.4928.5029.7932.5437.1254.6871.1333.4924.798.031.7125.1416.3122.5530.3127.7532.5442.8626.5618.4212.024.0419.7811.5412.6316.1815.3421.7231.6918.8313.0224.012.2611.925.115.867.177.4010.3611.184.626.1536.06.234.545.053.413.974.025.627.415.184.1048.03.135.263.393.143.173.643.224.593.182.92 drug concentration after transformation (ng ml) 1.56.756.759.124.238.045.464.625.783.7113.672.027.7518.9622.0010.2418.897.6413.5437.206.7624.542.554.4358.0439.0616.8136.2810.4232.4165.9113.5252.453.049.7861.9027.7725.7836.8218.9036.9159.2216.1450.983.542.8253.6627.8844.8647.0425.7536.6546.5222.9444.624.044.6547.1338.1349.5542.5740.5839.5659.6629.8441.224.543.9448.3452.5618.8947.7341.7054.0459.6629.6837.175.058.8143.7943.6655.6558.7146.3749.8461.6739.6154.266.047.9130.4135.0536.6440.0245.6542.1154.7841.1930.498.024.4230.9220.0627.7323.3421.3725.0633.0132.6622.6512.018.5115.2314.1915.5319.9018.8716.7324.4114.5016.0124.09.449.186.287.218.829.107.988.615.687.5636.04.805.583.894.194.884.944.335.713.995.0448.0 3.135.263.393.143.173.643.224.593.182.92 data are not subject to transformation concentrations of itraconazole administered orally at a single dose of 100 mg (sporanox 100 mg tab ., janssen pharmaceuticals) for 10 male subjects . Data before (148 h sampling points) and after transformation (136 h sampling points) data are not subject to transformation the source of variability in cmax point and for concentrations illustrating clast are different and are the result of different processes, which are subject to the drug molecule in the two time points . Components that generate the cmax, cv% value are inter alia: variability resulting from the absorption process (cv%, abs), variability resulting from the distribution process (cv%, dist), variability resulting from the elimination process (cv% el) and cv% an . Cv% an which in this case is expressed by the precision of the method designated for the value equal to lloq . The main components that generate the clast, cv% value are cv% el and cv% an . Both cv% el and cv% an to a large extent influence the value of cmax, cv%, as the drug elimination process is simultaneous to the processes of distribution and absorption . Therefore, it was assumed that1\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$c _ {{{\hbox{max}}, {\text{cv \%}}}} \approx {\text{cv \%}} _ {\text{abs}} {\text {+ cv \%}} _ {\text{dist}} {\text {+ cv \%}} _ {\text{el}} {\text {+ cv \%}} _ {\text{an}} $$\end{document}while in the case of a point in the elimination phase:2\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$c _ {{{\hbox{max}}, {\text{cv}}\,\%}} \approx {\text{cv}}\,\%_{\text{el}} + {\text{cv}}\,\%_{\text{an}} $$\end{document}subtracting eq . 2 from eq . 1 the value 3\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$c_{{{\text{max, cv\,\%}}}} = {\text{cv\,\%}} _ {\text{abs}} {\text {+ cv\,\%}} _ {\text{dist}} $$\end{document}is obtained, which allows to observe the cmax value, without the factors responsible for the variability of the qualifying process and the analytical method . Adopting the above assumptions, a scheme of data transformation was proposed, which is illustrated by the following example: the arithmetic mean (ma) for the sampling point equal to 1.5 h is 7.30 ng ml, concentration value (cn) for one of the subjects in the analyzed series is before the transformation 8.77 ng ml (\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$c_{n}> m_{a} $$\end{document}); the lowest value of variability in the elimination phase is the value obtained for the sampling point in the 48th hour and equals to 21.26% (clast, cv%); cv% an for the lloq value is 7.06%; cmax, cv% in the analyzed group is 30.82% therefore4\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$x = c_{\text{last, cv\,\%}}-(c_{\text{last, cv\,\%}})\times {\text{cv\,\%}}_{\rm an}/100$$\end{document}which in the case of the analyzed point is 19.76%, 5\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$y = c_{\text{max, cv\,\%}}-(c_{\text{max, cv\,\%}})\times {\text{cv\,\%}}_{\rm an}/100$$\end{document}which in the case of the analyzed point is 28.64%, 6\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$y_{1} = (y \times c_{n}) /100 $$\end{document}represents the percentage of concentration value before the transformation (cn) calculated with the value of the variability cmax, cv% reduced with cv% an, which in the case of the analyzed point gives the value of 2.51 ng ml.7\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$x_{1} = (y_{1} \times x)/100, $$\end{document}represents the percentage of value y1 calculated with the value clast, cv% reduced with cv%, an, which in the case of the analyzed point gives the value of 0.496 ng ml . The value of cn after the transformation of (cnt) is8\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$c_{{n{\text{t}}}} = c_{n} + (y_{1} - x_{1}), $$\end{document}if9\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$c_{n} <m_{a}\; {\text{and}}\;c_{{n{\text{t}}}} = c_{n} - (y_{1} - x_{1}) $$\end{document}if \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$c_{n}> m_{a} $$\end{document}. In the case of the analyzed concentration point \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$c_{n}> m_{a} $$\end{document} therefore\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$c_{{n{\text{t}}}} = 8.77 - (2.51 - 0.496) \, $$\end{document}, which after transformation gives the concentration equal to \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$c_{{n{\text{t}}}} = 6.75 $$\end{document} ng ml . The transformation of all of the concentration points was made in an analogous way, excluding the series of concentration, which were the source for clast, cv% . In developed form, used formulas take the form: if \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$c_{n} <m_{a} $$\end{document}, then cnt takes the value:10\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\begin{gathered} c_{{n{\text{t}}}} = c_{\text{n}} + [(((c_{{{\text{max, cv\,\%}}}} - (c_{{{\text{max, cv\,\%}}}} \times {\text{cv\,\%}} _ {\text{an}}) /100) \times c_{n}) /100) - ((((c_{{{\text{max, cv\,\%}}}} - (c _ {{{\hbox{max}}, {\text{cv\,\%}}}} \times \\ {\text{cv\,\%}} _ {\text{an}}) /100 \times c_{n}) /100) \times (c_{{{\text{last, cv\,\%}}}} - (c_{{{\text{last, cv\,\%}}}} \times {\text{cv}}\,\%_{\text{an}}) /100))/100)] \\ \end{gathered} $$\end{document}if \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$c_{n}> m_{a} $$\end{document}, then cnt takes the value:11\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\begin{gathered} c_{{n{\text{t}}}} = c_{n} - [(((c_{{{\text{max, cv \%}}}} - (c _ {{{\hbox{max}}, {\text{cv \%}}}} \times {\text{cv \%}} _ {\text{an}}) /100) \times c_{n}) /100) - ((((c_{{{\text{max, cv \%}}}} - (c_{{{\text{max, cv \%}}}} \times \\ {\text{cv \%}} _ {\text{an}}) /100 \times c_{n}) /100) \times (c_{{{\text{last, cv \%}}}} - (c_{{{\text{last, cv \%}}}} \times {\text{cv}} \%_{\text{an}}) /100))/100)] \\ \end{gathered} $$\end{document} non - compartmental modeling was used to estimate pharmacokinetic parameters of itraconazole . Pharmacokinetic calculations were performed with the use of phoenix winnonlin 6.3 (certara l.p . ). The area under the c t curve (auc) from time 0 to the last concentration time point and for infinity (auc0-tlast; auc0-inf) as well as area under first moment of concentration time curve (aumc) from time 0 to the last concentration time point (aumc0-tlast), were determined by the trapezoidal method . Mean residence time (mrt0-tlast) from time 0 to the last concentration time point was calculated using the standard formula \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${\text{mrt}} _ {{0 {\text {- tlast}}}} {\text {= aumc}} _ {{0 {\text {- tlast}}}} / {\text{auc}} _ {{0 {\text {- tlast}}}}. $$\end{document} the elimination rate constant (kel) was determined by linear regression of the last three points on the c t curve . In relation to calculations of t1/2kel in the specified population it is recommended to conduct the analysis with the harmonic mean and the proper value of pseudo sd (lam et al . T data, the data distribution is inclined according to the log - normal model . Thus, the geometric mean (mg) and the corresponding coefficient of variation are the factors of descriptive statistics for t1/2kel, which are considered to be more appropriate than the arithmetic mean (ma) (keene 1995; senn 2002; gad 2009). In relation to t1/2kel, the harmonic mean and the value of pseudo sd were calculated . In relation to the other parameters as tool for measurement of central tendency, median (m) and his standard deviation (sdm) were used . A statistical analysis of ma, mg, m and their sd (sda; sdg; sdm) was performed using microsoft office excel software . The percent of relative standard deviation (cv%) was calculated using formula \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${\text {cv\,\%}}\;=\;{\text{sd}}/{\text{m}}\times{100} $$\end{document} raw and transformed data correlations were confirmed by sign test and all pharmacokinetics correlations were confirmed by student - t test . Differences with p <0.05 were regarded as statistically significant . In the presented study, the lowest value of relative standard deviation (rsd%) of concentrations observed in the elimination phase and the value of precision of the used analytical method were used to optimize the arithmetic and geometric mean and the value of sd obtained after single oral administration of itraconazole, which is characterized by high variability of pharmacokinetic parameters . A single dose of 100 mg of itraconazole was administered orally (sporanox 100 mg tab ., janssen pharmaceuticals) for male subjects 20 to 40 years old, with a body mass index 20 to 25 kg / m . Blood samples were collected just prior to administration and at 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 6.0, 8.0, 12.0, 24.0, 36.0, 48.0, and 72.0 h after the administration . The concentration analyses were performed using tandem mass spectrometry, using the method described previously (grabowski et al . The study was approved by independent ethics committee of district council of physicians, baniowa 3, warsaw (resolution no . Itraconazole is a drug with high intrasubject variability, and the formulation belongs to the group of high variability drug product (hvdp). Therefore, the majority of pharmacokinetic profiles began and ended at different time points (different time of absorption delay and concentration with values <lloq in last sampling points). T profiles that were chosen were those which originated from different subjects and those having identical number of indicated concentrations in the same interval . T profiles were obtained this way between 1.5 and 48 h after the drug administration (table 1).table 1concentrations of itraconazole administered orally at a single dose of 100 mg (sporanox 100 mg tab ., janssen pharmaceuticals) for 10 male subjects . Data before (148 h sampling points) and after transformation (136 h sampling points)time (h)12345678910 drug concentration before transformation (ng ml) 1.58.778.7611.843.446.544.443.764.703.0217.752.036.0315.4217.898.3324.536.2111.0148.305.5031.862.570.6775.3631.7613.6729.508.4726.3585.5810.9968.103.064.6380.3822.5820.9647.8115.3730.0176.8913.1266.203.555.6069.6822.6736.4838.2520.9429.8060.4118.6557.944.057.9861.1931.0040.2955.2833.0032.1777.4724.2653.524.557.0662.7742.7415.3661.9733.9143.9477.4724.1348.265.047.8256.8635.5045.2547.7437.7064.7280.0732.2144.126.038.9639.4928.5029.7932.5437.1254.6871.1333.4924.798.031.7125.1416.3122.5530.3127.7532.5442.8626.5618.4212.024.0419.7811.5412.6316.1815.3421.7231.6918.8313.0224.012.2611.925.115.867.177.4010.3611.184.626.1536.06.234.545.053.413.974.025.627.415.184.1048.03.135.263.393.143.173.643.224.593.182.92 drug concentration after transformation (ng ml) 1.56.756.759.124.238.045.464.625.783.7113.672.027.7518.9622.0010.2418.897.6413.5437.206.7624.542.554.4358.0439.0616.8136.2810.4232.4165.9113.5252.453.049.7861.9027.7725.7836.8218.9036.9159.2216.1450.983.542.8253.6627.8844.8647.0425.7536.6546.5222.9444.624.044.6547.1338.1349.5542.5740.5839.5659.6629.8441.224.543.9448.3452.5618.8947.7341.7054.0459.6629.6837.175.058.8143.7943.6655.6558.7146.3749.8461.6739.6154.266.047.9130.4135.0536.6440.0245.6542.1154.7841.1930.498.024.4230.9220.0627.7323.3421.3725.0633.0132.6622.6512.018.5115.2314.1915.5319.9018.8716.7324.4114.5016.0124.09.449.186.287.218.829.107.988.615.687.5636.04.805.583.894.194.884.944.335.713.995.0448.0 3.135.263.393.143.173.643.224.593.182.92 data are not subject to transformation concentrations of itraconazole administered orally at a single dose of 100 mg (sporanox 100 mg tab ., janssen pharmaceuticals) for 10 male subjects . Data before (148 h sampling points) and after transformation (136 h sampling points) data are not subject to transformation the source of variability in cmax point and for concentrations illustrating clast are different and are the result of different processes, which are subject to the drug molecule in the two time points . Components that generate the cmax, cv% value are inter alia: variability resulting from the absorption process (cv%, abs), variability resulting from the distribution process (cv%, dist), variability resulting from the elimination process (cv% el) and cv% an . Cv% an which in this case is expressed by the precision of the method designated for the value equal to lloq . The main components that generate the clast, cv% value are cv% el and cv% an . Both cv% el and cv% an to a large extent influence the value of cmax, cv%, as the drug elimination process is simultaneous to the processes of distribution and absorption . Therefore, it was assumed that1\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$c _ {{{\hbox{max}}, {\text{cv \%}}}} \approx {\text{cv \%}} _ {\text{abs}} {\text {+ cv \%}} _ {\text{dist}} {\text {+ cv \%}} _ {\text{el}} {\text {+ cv \%}} _ {\text{an}} $$\end{document}while in the case of a point in the elimination phase:2\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$c _ {{{\hbox{max}}, {\text{cv}}\,\%}} \approx {\text{cv}}\,\%_{\text{el}} + {\text{cv}}\,\%_{\text{an}} $$\end{document}subtracting eq . 2 from eq . 1 the value 3\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$c_{{{\text{max, cv\,\%}}}} = {\text{cv\,\%}} _ {\text{abs}} {\text {+ cv\,\%}} _ {\text{dist}} $$\end{document}is obtained, which allows to observe the cmax value, without the factors responsible for the variability of the qualifying process and the analytical method . Adopting the above assumptions, a scheme of data transformation was proposed, which is illustrated by the following example: the arithmetic mean (ma) for the sampling point equal to 1.5 h is 7.30 ng ml, concentration value (cn) for one of the subjects in the analyzed series is before the transformation 8.77 ng ml (\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$c_{n}> m_{a} $$\end{document}); the lowest value of variability in the elimination phase is the value obtained for the sampling point in the 48th hour and equals to 21.26% (clast, cv%); cv% an for the lloq value is 7.06%; cmax, cv% in the analyzed group is 30.82% therefore4\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$x = c_{\text{last, cv\,\%}}-(c_{\text{last, cv\,\%}})\times {\text{cv\,\%}}_{\rm an}/100$$\end{document}which in the case of the analyzed point is 19.76%, 5\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$y = c_{\text{max, cv\,\%}}-(c_{\text{max, cv\,\%}})\times {\text{cv\,\%}}_{\rm an}/100$$\end{document}which in the case of the analyzed point is 28.64%, 6\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$y_{1} = (y \times c_{n}) /100 $$\end{document}represents the percentage of concentration value before the transformation (cn) calculated with the value of the variability cmax, cv% reduced with cv% an, which in the case of the analyzed point gives the value of 2.51 ng ml.7\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$x_{1} = (y_{1} \times x)/100, $$\end{document}represents the percentage of value y1 calculated with the value clast, cv% reduced with cv%, an, which in the case of the analyzed point gives the value of 0.496 ng ml . The value of cn after the transformation of (cnt) is8\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$c_{{n{\text{t}}}} = c_{n} + (y_{1} - x_{1}), $$\end{document}if9\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$c_{n} <m_{a}\; {\text{and}}\;c_{{n{\text{t}}}} = c_{n} - (y_{1} - x_{1}) $$\end{document}if \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$c_{n}> m_{a} $$\end{document}. In the case of the analyzed concentration point \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$c_{n}> m_{a} $$\end{document} therefore\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$c_{{n{\text{t}}}} = 8.77 - (2.51 - 0.496) \, $$\end{document}, which after transformation gives the concentration equal to \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$c_{{n{\text{t}}}} = 6.75 $$\end{document} ng ml . The transformation of all of the concentration points was made in an analogous way, excluding the series of concentration, which were the source for clast, cv% . In developed form, used formulas take the form: if \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$c_{n} <m_{a} $$\end{document}, then cnt takes the value:10\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\begin{gathered} c_{{n{\text{t}}}} = c_{\text{n}} + [(((c_{{{\text{max, cv\,\%}}}} - (c_{{{\text{max, cv\,\%}}}} \times {\text{cv\,\%}} _ {\text{an}}) /100) \times c_{n}) /100) - ((((c_{{{\text{max, cv\,\%}}}} - (c _ {{{\hbox{max}}, {\text{cv\,\%}}}} \times \\ {\text{cv\,\%}} _ {\text{an}}) /100 \times c_{n}) /100) \times (c_{{{\text{last, cv\,\%}}}} - (c_{{{\text{last, cv\,\%}}}} \times {\text{cv}}\,\%_{\text{an}}) /100))/100)] \\ \end{gathered} $$\end{document}if \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$c_{n}> m_{a} $$\end{document}, then cnt takes the value:11\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\begin{gathered} c_{{n{\text{t}}}} = c_{n} - [(((c_{{{\text{max, cv \%}}}} - (c _ {{{\hbox{max}}, {\text{cv \%}}}} \times {\text{cv \%}} _ {\text{an}}) /100) \times c_{n}) /100) - ((((c_{{{\text{max, cv \%}}}} - (c_{{{\text{max, cv \%}}}} \times \\ {\text{cv \%}} _ {\text{an}}) /100 \times c_{n}) /100) \times (c_{{{\text{last, cv \%}}}} - (c_{{{\text{last, cv \%}}}} \times {\text{cv}} \%_{\text{an}}) /100))/100)] \\ \end{gathered} $$\end{document} pharmacokinetic calculations were performed with the use of phoenix winnonlin 6.3 (certara l.p . ). The area under the c t curve (auc) from time 0 to the last concentration time point and for infinity (auc0-tlast; auc0-inf) as well as area under first moment of concentration time curve (aumc) from time 0 to the last concentration time point (aumc0-tlast), were determined by the trapezoidal method . Mean residence time (mrt0-tlast) from time 0 to the last concentration time point was calculated using the standard formula \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${\text{mrt}} _ {{0 {\text {- tlast}}}} {\text {= aumc}} _ {{0 {\text {- tlast}}}} / {\text{auc}} _ {{0 {\text {- tlast}}}}. $$\end{document} the elimination rate constant (kel) was determined by linear regression of the last three points on the c t curve . In relation to calculations of t1/2kel in the specified population it is recommended to conduct the analysis with the harmonic mean and the proper value of pseudo sd (lam et al . T data, the data distribution is inclined according to the log - normal model . Thus, the geometric mean (mg) and the corresponding coefficient of variation are the factors of descriptive statistics for t1/2kel, which are considered to be more appropriate than the arithmetic mean (ma) (keene 1995; senn 2002; gad 2009). In relation to t1/2kel, the harmonic mean and the value of pseudo sd were calculated . In relation to the other parameters as tool for measurement of central tendency, median (m) and his standard deviation (sdm) were used . A statistical analysis of ma, mg, m and their sd (sda; sdg; sdm) was performed using microsoft office excel software . The percent of relative standard deviation (cv%) was calculated using formula \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${\text {cv\,\%}}\;=\;{\text{sd}}/{\text{m}}\times{100} $$\end{document} raw and transformed data correlations were confirmed by sign test and all pharmacokinetics correlations were confirmed by student - t test . Differences with p <0.05 were regarded as statistically significant . The lowest values of cv% for raw data (rd) were noted for the sampling point 48 h after the administration of the drug, and the value was 20.61% (table 1). It was used to transform data in the rest of the time points (1.535 h). The concentration values after the transformation (td) are presented in the table 1 . Image of differences between rd and td for the largest fluctuation of c t curve is presented in fig . 1 . On the basis of rd and td, cv% was calculated for ma, sda, mg, sdg, m and sdm, which is presented in the table 2 . In relation to the value of ma, mg and m between the rd and td data no statistically significant differences (p> 0.05) were found between the mean values of individual points of concentration and the rd and td data . The sda value and sdg were significantly lower (p <0.05) in relation to the rd group.fig . 1concentration time data of itraconazole administered orally at a single dose of 100 mg for 10 male subjects before (solid line) and after transformation (dashed line). A represents arithmetic mean and standard deviation, b shows geometric mean and standard deviation and c shows median and standard deviationtable 2mean (n = 10), standard deviation, and relative standard deviation of raw data before and after transformationtime (h)raw data (rd)transformed data (td) m a sda rsd m g sdg rsd m sdm rsd m a sda rsd m g sdg rsd m sdm rsd1.57.304.6663.816.224.5573.235.624.6682.906.812.9443.156.342.8344.645.622.9552.572.020.5114.3770.0916.1114.3388.9616.6614.3786.3018.759.6251.2716.439.4157.3116.339.6659.132.542.0529.6570.5131.5430.0295.1830.6329.6596.7937.9319.7352.0232.1719.5860.8834.3420.1758.743.043.8026.4560.4035.9726.2973.0938.9126.4567.9838.4216.4242.7435.0015.9545.5636.8616.4244.543.541.0418.5345.1537.1418.0148.4937.3718.5349.6039.2810.4326.5537.889.9926.3839.7310.4626.324.046.6216.9836.4243.8316.3537.2946.9116.9836.1943.297.8718.1842.667.4917.5741.907.8718.794.546.7618.9340.4942.5818.4443.3146.1018.9341.0743.3712.1828.0941.4411.7228.2845.8412.1826.585.049.2014.5529.5847.4513.9229.3346.5014.5531.3051.247.6314.9050.717.2614.3145.088.4018.646.039.0513.9535.7237.2113.3635.9035.3113.9539.5140.437.7119.0839.777.3518.4735.848.0222.368.027.427.6427.8726.467.3127.6327.167.6428.1426.124.7018.0025.754.4817.3824.744.7019.0012.018.486.1933.5117.635.9333.6617.516.1935.3717.393.1317.9817.162.9717.3416.373.1319.1024.08.202.9435.807.732.8336.547.292.9440.317.991.2816.097.891.2215.517.771.4018.0436.04.951.2224.534.831.1624.024.801.2225.344.740.6313.284.700.6012.724.840.6312.9948.0 3.560.7621.263.500.7220.613.200.7623.68 p value>0.050.0350.006>0.050.0350.009>0.050.0390.010 p - value between raw (1.536 h) and transformed data (1.536 h) m a arithmetic mean (ng ml); sd a standard deviation of ma, m g geometric mean (ng ml), sd g standard deviation of mg, m median (ng ml), sd m standard deviation of m, rsd relative standard deviation (%) value of c last, cv% data which are not transformed concentration time data of itraconazole administered orally at a single dose of 100 mg for 10 male subjects before (solid line) and after transformation (dashed line). A represents arithmetic mean and standard deviation, b shows geometric mean and standard deviation and c shows median and standard deviation mean (n = 10), standard deviation, and relative standard deviation of raw data before and after transformation p - value between raw (1.536 h) and transformed data (1.536 h) m a arithmetic mean (ng ml); sd a standard deviation of ma, m g geometric mean (ng ml), sd g standard deviation of mg, m median (ng ml), sd m standard deviation of m, rsd relative standard deviation (%) value of c last, cv% data which are not transformed the results of the key calculations of pharmacokinetic parameters are shown in table 3 . The analysis of sdg pharmacokinetic parameters in rd and td groups indicated more than twice lower value of sdg in td group . The cv% (\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${\text{sd}}_{a} /m_{a} \times 100 $$\end{document}) of pharmacokinetic parameters, such as kel, t1/2kel, tmax, cmax, auc0-tlast, auc0-inf, aumc0-last and mrtinf calculated on the basis of td was lower by 21.25, 15.44, 17.43, 9.47, 9.85, 1.59, 10.39 and 2.69%, respectively, than cv% obtained for the pk parameters in rd group but not statistically significant (p> 0.05). The cv% (\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${\text{sd}}_{g} /m_{g} \times 100 $$\end{document}) of pharmacokinetic parameters kel, t1/2kel, tmax, cmax, auc0-tlast, auc0-inf, aumc0-last, and mrtinf calculated on the basis of td was significantly lower (p <0.05) by 39.40, 30.75, 44.13, 59.42, 53.77, 51.82 and 38.83%, respectively, from cv% obtained for the pk parameters in rd group . The ratio of mg in the rd group to mg in the td group ranged between 0.935 and 1.041 and was not statistically significant (p> 0.05).table 3pharmacokinetics parameters of itraconazole (administered orally at a single dose of 100 mg for ten male subjects) calculated using data before and after transformationstat . K el (h) t 1/2kel (h) t max (h) c max (ng ml)auc0-tlast (ng h ml)auc0-inf (ng h ml)aumc0-tlast (ng h ml)mrt0-tlast (h)pharmacokinetics analysis using raw concentrations (rd)ma 0.0324.33 4.0559.06632.48755.788,916.2614.21sda 0.0110.511.3018.20184.64171.382,336.140.89rsd38.5643.1932.1130.8229.1922.6826.206.25 mg 0.0322.543.8556.37610.57740.338,662.6414.19sdg 0.0110.131.2517.48176.53163.322,230.720.84rsd39.9444.9432.4931.0028.9122.0625.755.94m0.0324.334.0559.06632.48755.788,916.2614.21sdm 0.019.971.2317.27175.16162.582,216.260.84rsd36.5840.9730.4729.2427.6921.5124.865.93pharmacokinetics analysis using transformed concentrations (td)ma 0.0320.484.5554.94607.90705.078,610.9714.19sda 0.018.671.2115.33159.98157.342,021.770.86rsd29.6242.3226.5227.9026.3222.3223.486.08 mg 0.0321.074.4254.49602.87697.748,546.8014.18sdg 0.015.290.996.8680.57107.761,060.430.52rsd24.2025.1122.5012.5813.3715.4412.413.63m0.0321.694.5554.94607.90705.078,610.9714.19sdm 0.015.250.996.8480.42107.511,058.490.52rsd23.3624.2221.6712.4513.2315.2512.293.63 h harmonic mean, m a arithmetic mean, sd a standard deviation of ma, m g geometric mean, sd g standard deviation of mg, m median, sd m standard deviation of m; rsd relative standard deviation (%), k el elimination rate constant; t 1/2kel half life in elimination phase, t max time to reach maximum concentration, c max maximum concentration, auc 0-tlast area under the curve between zero and last concentration, auc 0-inf area under the curve from zero to infinity, aumc 0-last area under the first moment curve between zero and last concentration, mrt inf mean residence time (aumc0-tlast / auc0-tlast) pharmacokinetics parameters of itraconazole (administered orally at a single dose of 100 mg for ten male subjects) calculated using data before and after transformation h harmonic mean, m a arithmetic mean, sd a standard deviation of ma, m g geometric mean, sd g standard deviation of mg, m median, sd m standard deviation of m; rsd relative standard deviation (%), k el elimination rate constant; t 1/2kel half life in elimination phase, t max time to reach maximum concentration, c max maximum concentration, auc 0-tlast area under the curve between zero and last concentration, auc 0-inf area under the curve from zero to infinity, aumc 0-last area under the first moment curve between zero and last concentration, mrt inf mean residence time (aumc0-tlast / auc0-tlast) this manuscript presents the method of data transformation calculated on the basis of pk parameters expressed as ma, mg and m. until now, only a few methods of transforming the values of pk parameters were proposed (abdallah 1998; fujita et al . Frequently, these methods were based on data transformation through the normalization of pharmacokinetic parameters, value of the dose or physiological parameters (body weight, dose, body surface, normalization etc . ), (sathirakul et al . 2003; sathyan et al . Normalization and scaling of pharmacokinetic data are also used in the allometric analysis, in scaling either concentrations, time or pharmacokinetic parameters (mahmood 2005). These methods, however, do not use the variation values obtained in the study to transform data . In the c t data sequences standard deviation in individual time points within the population is different and ranges from low to high . This is true for both, the analysis of the variability within and between subjects . In the analyzed case, the phase of itraconazole absorption is a subject of large fluctuations . After a single administration of the drug, the low values of deviation for individual c usually equal to or a bit higher than the lloq value, the variability of the sd value often does not exceed 10% . This occurs because these are the concentrations which usually represent only the process of elimination that is not affected by the factors responsible for the variability of the kinetics of absorption and distribution of the drug . In relation to a single administration, the exceptions are the cases of redistribution of the drug in the late elimination phase (davis et al . 2010). Also, in the analyzed case the volatility calculated for each sampling point was the lowest at a point closest to the lloq value of the analytical method . It happens differently in the case of concentrations analyzed in relation to oral administration, while the drug is still present in the stomach or the process of absorption has begun in the intestines . Depending on many factors, the variability in this phase can be very high (duquesnoy et al . 1998; tubic et al . The proposed method allows to achieve a reliable picture of the pharmacokinetic profile, free of substantial interference in the average values of obtained concentrations and in the values of pharmacokinetic parameters with a simultaneous decrease in the value of sd . T data at points, in which the sd is particularly high . Reducing the value of sd for studies such as first in man, pilot or the rare ones, due to the difficulty of collecting sufficient number of subjects, can help to make a decision about the further direction of the research.
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Failure of a total knee arthroplasty (tka) is often associated with loss of bone from either the proximal aspect of tibia or the distal aspect of the femur . These defects are usually classified as contained or uncontained.1 the reconstruction of large uncontained defects represents a major challenge to the revision surgeon . Options for reconstruction2 of large defects include metal augments, custom prostheses, massive autogenous bone - grafts and massive allografts . Metal augments are recommended only for small to medium sized defects in elderly low demand patients . Custom prostheses are costly, time consuming to manufacture and may not fit as well as planned or at all at the time of surgery . Autogenous bone - grafting is not suitable for large defects or in situations where structural support is required . Allografts provide a biological solution, have the advantage of easy fashioning to fit irregular defects, restore bone stock and have relatively low cost . Use of femoral head allograft, complete distal femur or proximal femur allograft has been reported in some studies34; however, no study has reported use of both distal femur and proximal tibia allograft in a single revision total knee arthroplasty . We report the first successful use of both complete distal femoral and proximal tibia massive allografts in the reconstruction of large femoral and tibial uncontained defects during revision total knee arthroplasty . A 70-year - old male patient, farmer by occupation, presented with a swollen, painful and unstable right knee with primary complaint of inability to bear weight . Passive range of motion was painful and limited to 10 degrees of hyperextension to 50 degrees of flexion in april 2005 . The patient also had a periprosthetic supracondylar fracture of femur following a trivial fall, for which open reduction and internal fixation was done elsewhere eight years back with condylar blade plate . The skin revealed thick scar tissue on both anterior and lateral aspects of the knee due to secondary wound healing after previous surgeries . Anteroposterior and lateral radiographs [figure 1] revealed severe osteolytic bone defects on femoral and tibial aspects along with primary total knee prosthesis and condylar blade plate in situ . Infection was ruled out preoperatively with bone scan and laboratory markers (erythrocyte sedimentation rate, c- reactive protein). We decided to reconstruct the knee with structural distal femur and proximal tibia allografts and constrained fixed - bearing revision knee prosthesis (nexgen, lcck, zimmer, warsaw). Anteroposterior and lateral x - rays of right knee showing extensive osteolysis on both femoral and tibial aspects we used the previous midline incision and a medial parapatellar approach . We removed much necrotic, granulomatous tissue (biopsy proven) which usually occurs due to extensive polyethylene wear . After debridement, the whole proximal tibia [figure 2] presented a massive uncontained defect, classified as type iii according to anderson orthopaedic research institute classification.5 the whole distal femur also presented a large uncontained type iii bone defect involving both medial and lateral femoral condyles [figure 2]. However, femoral attachment of lateral collateral ligament was deficient, and ligament was identified with a piece of bone . The intraoperative photograph shows the defect after thorough debridement both proximal tibial and distal femoral cadaveric allografts [figure 3] with a diameter smaller than that of the host bone were selected so that they could be placed within the host cortical shell . The allografts were taken from our own cadaveric bone bank, where they underwent radiation with 2.5 megarads and were stored at 70c . Two surgical teams were used during the management of the case, for optimal efficiency . One team prepared the allograft, making appropriate femoral cuts and cementing in a long - stemmed revision femoral component . Cultures were taken from the knee as well as the allograft to rule out any existing infection . Step - cut prepared distal femoral and proximal tibial allografts both the recipient distal femur and proximal tibia were step - cut with minimal bone resection . On the side table, step - cut was made in both allografts to match that of the host bone and instrumented with trial implants . The implant allograft composite was put for trial in situ, and the cuts were fine tuned as required . The revision knee components were cemented [figure 4] on the back - table into the distal femoral and proximal tibial allografts with a long stem . Care was taken during cementing to avoid getting cement on the interface between the graft and the host . Once the cement had set, the construct was implanted with full assembly, matching the two step - cuts . The residual host femur, with its ligaments and other soft tissues attached, was wrapped around the allograft host junction to serve as a living bone graft [figure 5]. Extensor mechanism alignment and tracking was checked, and the wound was closed in layers . Fully prepared composite allograft with lcck implant intraoperative photograph showing invagination of composite allografts into host bone prophylactic antibiotics consisted of an intravenous first - generation cephalosporin for five days, followed by oral antibiotics for seven days . Partial weight bearing was allowed at four weeks, and full weight bearing was started after eight months when there was radiographic evidence of union on both sides . No wound complication was noted during the entire follow up . At the end of five years of follow up (i.e., in april 2010), the patient was walking full weight bearing and had complete incorporation of allograft and host bone with no signs of osteolysis [figure 6]. His active range of motion range of motion (rom) was 0 - 110 degrees . Anteroposterior and lateral x - rays of the same patient at five - year follow - up showing good allograft incorporation we removed much necrotic, granulomatous tissue (biopsy proven) which usually occurs due to extensive polyethylene wear . After debridement, the whole proximal tibia [figure 2] presented a massive uncontained defect, classified as type iii according to anderson orthopaedic research institute classification.5 the whole distal femur also presented a large uncontained type iii bone defect involving both medial and lateral femoral condyles [figure 2]. However, femoral attachment of lateral collateral ligament was deficient, and ligament was identified with a piece of bone . The intraoperative photograph shows the defect after thorough debridement both proximal tibial and distal femoral cadaveric allografts [figure 3] with a diameter smaller than that of the host bone were selected so that they could be placed within the host cortical shell . The allografts were taken from our own cadaveric bone bank, where they underwent radiation with 2.5 megarads and were stored at 70c . Two surgical teams were used during the management of the case, for optimal efficiency . One team prepared the allograft, making appropriate femoral cuts and cementing in a long - stemmed revision femoral component . Cultures were taken from the knee as well as the allograft to rule out any existing infection . Step - cut prepared distal femoral and proximal tibial allografts both the recipient distal femur and proximal tibia were step - cut with minimal bone resection . On the side table, step - cut was made in both allografts to match that of the host bone and instrumented with trial implants . The implant allograft composite was put for trial in situ, and the cuts were fine tuned as required . The revision knee components were cemented [figure 4] on the back - table into the distal femoral and proximal tibial allografts with a long stem . Care was taken during cementing to avoid getting cement on the interface between the graft and the host . Once the cement had set, the construct was implanted with full assembly, matching the two step - cuts . The residual host femur, with its ligaments and other soft tissues attached, was wrapped around the allograft host junction to serve as a living bone graft [figure 5]. Extensor mechanism alignment and tracking was checked, and the wound was closed in layers . Fully prepared composite allograft with lcck implant intraoperative photograph showing invagination of composite allografts into host bone prophylactic antibiotics consisted of an intravenous first - generation cephalosporin for five days, followed by oral antibiotics for seven days partial weight bearing was allowed at four weeks, and full weight bearing was started after eight months when there was radiographic evidence of union on both sides . No wound complication was noted during the entire follow up . At the end of five years of follow up (i.e., in april 2010), the patient was walking full weight bearing and had complete incorporation of allograft and host bone with no signs of osteolysis [figure 6]. His active range of motion range of motion (rom) was 0 - 110 degrees . Anteroposterior and lateral x - rays of the same patient at five - year follow - up showing good allograft incorporation few options are available to the surgeon for reconstruction of massive bone defects surrounding a failed total knee arthroplasty (tka). The application of allografts in revision tka is an attractive option . The use of femoral head allografts for the management of large bone defects in revision tka has been reported . Very few studies have also reported using either massive distal femoral or proximal tibial allograft for large defect reconstruction in revision tka.34 our patient presented a unique problem, having severe bone loss on both femoral and tibial aspects . Using both massive distal femoral and proximal tibial allografts proved to be a successful mode of treatment with distinct advantages . Placing the allograft within the vascularized host bone will add some structural support to the allograft and provide an environment by which the allograft may eventually incorporate . One major concern with the use of allografts is the risk of infection . In a large series by lord et al.,6 the risk of infection with the use of small allografts (femoral heads) was negligible; and with the use of larger allografts, acceptable (4%-5%). Some authors have recommended plate and screw fixation of the allograft in addition to invagination of the graft, to ensure rotational stability . Using a stemmed component and avoiding the use of plate fixation decreases the need for extensive soft tissue dissection, avoids weakening of the graft and may decrease the risk of infection by preserving the soft tissue envelope.7 another concern about the use of massive allografts is their performance in the long term, especially in view of reports showing resorption and failures of allografts used in revision total hip arthroplasty at longer follow ups . However, use of allografts in anatomically matching sites (proximal tibia for proximal tibia, distal femur for distal femur) allows for accurate orientation of trabeculae along the lines of stress . This technique requires that the allograft is appropriately shaped for intussusception into the host bone with stable struts of host bone maximally contacting step - cuts in the graft.8 the limitation in terms of applicability of this study in practice could be the non - availability of such allografts at many centers . Newer trabecular metal wedges and sleeves may be an option; however, they are very costly . These allografts are free for patients at our institute and provide a biological and effective alternative . There should be a stimulus to establish more and more cadaveric allograft retrieval centers to deal with the complex and often neglected cases of bone loss, which is particularly common in the indian scenario . The use of dual structural massive allografts provides a stable and durable reconstruction of this uncommon presentation with both femoral and tibial large bone deficiencies encountered during a revision tka . At a five - year follow - up, we did not find any infection, graft failure or loosening of implant, in spite of using two massive structural allografts in a single revision tka . Further follow up with large number of patients is necessary to determine the long term fate of these allografts.
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Wound healing defects and associated soft tissue damages are becoming more important in the orthopedic practice . This is due to the significant increase of prosthesis implantations, including knee prosthesis implantations and the demographic development of society . The demands on a long lifetime of the prostheses are consistently increasing due to the increasing life expectancy of the patients receiving prostheses . Standardizations in endoprosthetics, increased experience of surgeons and the further development of prostheses has led to the consistent improvement of long - term results and lifetime of artificial joints, so that prostheses are implanted and replaced in younger patients as well . A secure and stable coverage with soft tissue is a basic requirement for good short - term and long - term results regarding the prosthesis . Without a sufficient coverage with vital soft tissue with a good blood supply, the risks of infection of the prosthesis and reduced capacity of the new joint exist . This is especially important for the knee because there is little soft tissue coverage in general which contributes to the increased risk for wound healing defects due to the surgical wound leading to a reduced blood supply . Transcutaneous measurements of oxygenation have shown that the level of oxygenation near the surgical scar necessary for wound healing is only reached after two to three days [. Besides the negative impact of the surgery itself on the primary wound healing, various other exogenous factors play a role in wound healing . The most important factors are rheumatoid arthritis, diabetes, long - term cortisone intake, malnutrition, nicotine use, hypothyroidism and existing multiple scars in the area of the surgery,, . Laing et al . Have described five stages of soft tissue defects after knee endoprosthetics 1992: stage 0: redness near the wound without wound dehiscence or development of necrosis stage 1: only superficial skin necrosis or tension blisters, deeper layers not affected; no fistula stage 2: extensive skin necrosis with fistula towards the knee, deeper layers of tissue not affected stage 3: joint fistula with dehiscence of deeper layers; a small part of the joint prosthesis is visible stage 4: widespread tissue necrosis with wound dehiscence and visible prosthesis stage 0: redness near the wound without wound dehiscence or development of necrosis stage 1: only superficial skin necrosis or tension blisters, deeper layers not affected; no fistula stage 2: extensive skin necrosis with fistula towards the knee, deeper layers of tissue not affected stage 3: joint fistula with dehiscence of deeper layers; a small part of the joint prosthesis is visible stage 4: widespread tissue necrosis with wound dehiscence and visible prosthesis for stages 0 and 1, a conservative treatment with fixation of the knee and bed rest for the patient is favoured and the soft tissue damage usually heals well without surgical treatment . For stage 2 and above, surgical measures are preferred . In stage 2, the superficial skin necrosis should be removed and temporary soft tissue coverage and vac - therapy should occur . Permanent soft tissue coverage can occur in the case of asepsis . Depending on size, a split - skin (mesh - graft) or full - skin graft can be chosen . 1)), a muscle flap is necessary, because the deeper damages cannot be treated with mere skin transplants . The benefits of muscle flaps, e.g. A gastrocnemius rotation flap, are evident: because the muscle flap is supplied with blood, it can be placed on infected wounds; this leads to a significant improvement of wound healing . This is due to secure soft tissue coverage of possibly exposed bone and prosthetic material . It is also due to the transportation of immunocompetent cells to the site of infection with the normal blood supply which leads to an improvement of wound healing and defence against infection . For this stage, the elevation and use of one gastrocnemius muscle belly, medial or lateral, is sufficient (figure 2 (fig . The lateral gastrocnemius muscle flap plasty was first described by barford and pers and the medial gastrocnemius muscle flap plasty was first described by ger . The medial head is often longer and wider than the lateral head which is why it is usually used . Surales medialis and laterialis, the elevation of an isolated flap and the coverage of almost the entire knee (figure 3 (fig . The muscle belly should be lifted without a skin graft because this could cause problems with the closure of the skin near the location of extraction . For the dermal closure of the implanted muscle flap, split - skin (mesh - graft) with a thickness of 0.3 to 0.5 mm the muscular aponeurosis and parts of the fascia of the muscle should be removed, so that it can heal well (figure 4 (fig . An area of up to 60 square centimetres can be covered this way . The muscle coverage with split - skin (mesh - graft) is also indicated (figure 5 (fig . If there is an intact suralis muscle, the expected muscular deficit regarding plantar flexion is remarkably small, and there is little aesthetic defect involved with muscle flap plasty . 16 patients were treated for stage 3 soft tissue damage and seven patients were treated for stage 4 soft tissue damage . Accordingly, 16 patients received one - headed muscle flaps and seven patients received two - headed muscle - flaps with split - skin grafts from the ipsilateral thigh . Out of 23 patients, dehiscence or necrosis at the split - skin graft occurred in three patients, and this required repeated split - skin grafts . One patient experienced dehiscence near the extraction point of the muscle which was successfully treated with split - skin graft after vac - therapy . Four patients experienced recurring infection of the prosthesis with development of a fistula and large soft tissue destruction which was successfully treated with elaborate surgical measures in one case . The extremity could not be saved in three cases, and this resulted in above - knee amputation . Due to the increasing number of implantation of knee endoprostheses and the associated number of soft tissue damages, a standardized procedure for the treatment of these complications is necessary . In the case of deep soft tissue damages with fistulas reaching the prosthesis or exposing the prosthesis, the gastrocnemius muscle flap is a good method for secure coverage of the prosthesis with well - perfused tissue . Despite the high rate of complications of almost 35% which required at least another surgery, this method of soft tissue reconstruction is a necessary skill for surgeons involved with the implantation of knee endoprostheses.
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The study protocol was approved by the tianjin hospital ethics committee, and informed consent of all patients was obtained . The inclusion criteria were 1) the injury time of hip fractures was <24 h before admission and fracture diagnosis by x - ray examinations in our emergency department, 2) patient participated in physical examinations each year and blood glucose was normal (<6.1 mmol / l) with ordinary values of glycosylated hemoglobin (hba1c), 3) a pair of lower limb venous vascular ultrasound examinations confirmed no deep venous thromboembolism (dvt), 4) osteoporosis was confirmed by bone density examination, and 5) orthopedic surgery was not completed within 1 week after admission to the hospital or no operation was performed (conservative treatment). The exclusion criteria were 1) the time of hip fracture injury was> 24 h before admission; 2) diagnosed type 1 or type 2 diabetes; 3) need of emergency operation within 1 week after hip fracture; 4) the occurrence of dvt, pulmonary embolism, or thyroid disease; and 5) renal / liver failure . Blood and urine routine, liver and kidney function, calcium, phosphorus, and serum cardiac enzyme concentrations, insulin level, and serum lipids were tested after admission . Electrocardiography (ecg), cardiorespiratory function test, ultrasonic cardiography, chest x - ray, lower limb venous vascular ultrasound, and bone mineral density measurements of the bilateral hip were conducted . Closed reduction, bone traction treatments, and prevention of dvt were administered to all patients with hip fractures on the day of admission (10) and they continued to take their cardiac medications . All patients who met the inclusion criteria were recorded with a standardized form throughout the study course . The fasting blood glucose (fbg) was examined at 6:00 a.m. every morning from the 1st to the 7th day after admission, and ecgs were measured at admission and from the 1st to the 7th day after admission at 10:00 a.m. every morning . If clinical symptoms indicated and/or electrocardiographic changes suggested a myocardial infarction, additional blood samples were drawn from the antecubital vein in order to examine creatine kinase (ck), ck - mb isoenzyme, and troponin - i values . Ami diagnosis criteria met at least two of following: 1) the ck - mb isoenzyme and/or troponin - i concentration elevated above the hospital laboratory's myocardial infarction threshold; 2) persistent st - t segment changes, new q waves, or left bundle branch block occurred; 3) serious precordial chest pain lasting for 30 min or more . Stress hyperglycemia diagnosis criteria were as follows: 1) patients did not have a previous diabetes history, 2) admission or in - hospital fasting plasma glucose levels of 7 mmol / l or more in two or more tests (13), and 3) normal hba1c values ., chicago, il) software was applied by a professional statistician to complete the analyses . The period of follow - up interviews was 3 months after discharge, and mortality was recorded by the investigators via telephone calls and/or outpatient clinic visits . Consecutive hip fracture patients (n = 1,257) were selected for the analysis during a 5-year period from february 2007 to february 2012 . The study protocol was approved by the tianjin hospital ethics committee, and informed consent of all patients was obtained . The inclusion criteria were 1) the injury time of hip fractures was <24 h before admission and fracture diagnosis by x - ray examinations in our emergency department, 2) patient participated in physical examinations each year and blood glucose was normal (<6.1 mmol / l) with ordinary values of glycosylated hemoglobin (hba1c), 3) a pair of lower limb venous vascular ultrasound examinations confirmed no deep venous thromboembolism (dvt), 4) osteoporosis was confirmed by bone density examination, and 5) orthopedic surgery was not completed within 1 week after admission to the hospital or no operation was performed (conservative treatment). The exclusion criteria were 1) the time of hip fracture injury was> 24 h before admission; 2) diagnosed type 1 or type 2 diabetes; 3) need of emergency operation within 1 week after hip fracture; 4) the occurrence of dvt, pulmonary embolism, or thyroid disease; and 5) renal / liver failure . Blood and urine routine, liver and kidney function, calcium, phosphorus, and serum cardiac enzyme concentrations, insulin level, and serum lipids were tested after admission . Electrocardiography (ecg), cardiorespiratory function test, ultrasonic cardiography, chest x - ray, lower limb venous vascular ultrasound, and bone mineral density measurements of the bilateral hip were conducted . Closed reduction, bone traction treatments, and prevention of dvt were administered to all patients with hip fractures on the day of admission (10) and they continued to take their cardiac medications . All patients who met the inclusion criteria were recorded with a standardized form throughout the study course . The fasting blood glucose (fbg) was examined at 6:00 a.m. every morning from the 1st to the 7th day after admission, and ecgs were measured at admission and from the 1st to the 7th day after admission at 10:00 a.m. every morning . If clinical symptoms indicated and/or electrocardiographic changes suggested a myocardial infarction, additional blood samples were drawn from the antecubital vein in order to examine creatine kinase (ck), ck - mb isoenzyme, and troponin - i values . Ami diagnosis criteria met at least two of following: 1) the ck - mb isoenzyme and/or troponin - i concentration elevated above the hospital laboratory's myocardial infarction threshold; 2) persistent st - t segment changes, new q waves, or left bundle branch block occurred; 3) serious precordial chest pain lasting for 30 min or more . Stress hyperglycemia diagnosis criteria were as follows: 1) patients did not have a previous diabetes history, 2) admission or in - hospital fasting plasma glucose levels of 7 mmol / l or more in two or more tests (13), and 3) normal hba1c values . Chicago, il) software was applied by a professional statistician to complete the analyses . The period of follow - up interviews was 3 months after discharge, and mortality was recorded by the investigators via telephone calls and/or outpatient clinic visits . Patients (n = 1,257) who met the inclusion criteria were enrolled in this clinical observation . Among the patients, the average age was 75.20 7.71 years and 64.52% were women . The most prevalent comorbidity was hypertension (57.68%), followed by coronary artery disease (cad) or family history of cad (49.90%), dyslipidemia (elevation of ldl cholesterol [ldl - c], 49.27%; triglyceride, 39.14%; and low levels of hdl cholesterol [hdl - c], 21.40%), obesity (37.63%), and smoking (28.16%). The frequency of stress hyperglycemia was 47.89% (602/1,257) and that of ami was 9.31% (117/1,257). Figure 1 shows the correlation between time of fracture and fbg . In the stress hyperglycemia group, repeated anovas showed that at each time point in the stress hyperglycemia group, the blood glucose values were significantly different (p <0.05), whereas in the non - hyperglycemia patients, they did not change (p = 0.10) (fig . The incidence of ami in the stress hyperglycemia group was significantly higher (12.46% [75/602]) than in the non - hyperglycemia group (6.41% [42/655], p <0.05). The number of amis was correlated with the occurrence of hyperglycemia and was significantly higher 3 days after injury in the hyperglycemia group (p <0.05), whereas the temporal distribution of ami in the non - hyperglycemia group was not different between 3 and> 3 days after injury (table 2). In all patients with ami, nstemi events occurred more often than stemi (62.39% [73/117] vs. 37.61% [44/117]), but stemi significantly prevailed in the stress hyperglycemia group (p <0.05). In table 3, risk factors for ami and their significance are listed . A multiple logistic regression analysis using a forward stepwise (conditional) method revealed that stress hyperglycemia is an independent risk factor for the development of ami (relative risk [rr] 2.130 [95% ci 1.4313.172]). Patients with stress hyperglycemia after hip fracture were 2.130-fold more likely to develop an ami than the non - hyperglycemia individuals . Characteristics of myocardial infarction in the two groups at the end of 3 months follow - up, there were no differences in mortality in patients with versus without stress - induced hyperglycemia (6.98% [42/602] vs. 4.43% [29/655], p = 0.051). In our study, we found that the frequency of stress hyperglycemia was 47.89% and the incidence of ami was 9.31% in patients after hip fractures . The incidence of ami in the stress hyperglycemia group was 12.46%, whereas the overall ami incidence in patients without hip fractures in china is 2.6% (14), indicating that the incidence of ami in patients after hip fractures appears higher than overall ami incidence in china . Others have reported that the increased incidence of ami after hip fracture was related to osteoporosis (4,14). Osteoporosis and cardiovascular diseases share common risk factors like poor general health status, lifestyle, nutrition, hormone secretion, vitamin d deficiency, systemic inflammation - related (e.g., c - reactive protein, interleukin-6, or tumor necrosis factor) diseases, and medications through different mechanisms both in bone and arteries (1525). Patients with osteoporotic fractures are likely to have coexisting blood vessel diseases, but in our study, we found that particularly stress hyperglycemia plays a vital role, because the incidence of ami in the stress hyperglycemia group was 2.130 higher than in the non - hyperglycemia group . In patients with hip fractures, stress hyperglycemia was the sole significant independent risk factor for the development of ami (95% ci 1.4313.172). Acute fractures induce stress hormone secretion of glucocorticoid, glucagon, adrenaline, thyroxin, somatotropin, and others, which is called the stress response . During the stress response, activation of serial hormones induces insulin resistance, resulting in hyperglycemia and the associated risk factors present then in patients with acute traumata (26). It is well known that stress hyperglycemia is associated with oxidative stress, inflammatory responses, damaging of coronary microcirculation, and markedly worsened signal transduction pathways of endogenous cardioprotective responses . Hyperglycemia can induce adp - induced platelet aggregation and increase plasma catecholamine, which is associated with vulnerable plaque evolution, the promotion of microcircular dysfunctions, and thrombogenesis (2737). Our present work indicates that for patients without a history of diabetes, stress - induced hyperglycemia plays a key role in the risk of developing ami . Our present analysis revealed that most (62.67% [47/75]) amis occurred within the first 3 days after hip fractures, which is in accordance with the literature (2). In the stress hyperglycemia group, blood glucose peaked at 23 days after hip fracture and then declined gradually, indicating a coincidence of ami with the peak time of fbg . The cause of a more frequent stemi occurrence in stress - induced hyperglycemia might be that raised glucose levels contribute to platelet activation and thereby enhanced platelet - mediated thrombogenesis (38), which develops into completely occlusive thrombi . In addition, our study showed stress hyperglycemia ranges of 6.19.7 mmol / l after hip fractures for the first time, and we recommend that stress - induced hyperglycemias after hip fracture should be identified early . A limitation of our study was that we did not investigate an effect of inflammatory factors and/or stress hormones on fbg levels, which lead to increased ami risk . In addition, we did not compare the results of this study with patients suffering from diabetes, and we did not find differences between the stress hyperglycemia and no hyperglycemia groups regarding comorbid disorders and/or drug administrations, e.g., statin against hypertension . Therefore we cannot completely rule out that other factors also contributed to the incidence of myocardial infarctions . Moreover, patients were not accurately diagnosed for diabetes in the longer term, and because blood glucose levels largely fluctuate, we did not specifically treat the hyperglycemias . Up until now, there has been no clear agreement on whether it is necessary to control glucose levels in these patients . In addition, we only show the significance of hyperglycemia on the incidence of ami during admission; we did not show clinical outcome data at follow - up . However, this study underlines the importance of understanding the indication for adopting appropriate methods to identify stress - induced hyperglycemia in correlation with ami after hip fracture . We recommend that in patients, even without previous diabetes, fbgs and ecgs should be monitored for at least the first 7 days after hip fractures . This might be helpful for the endocrinologist, cardiologist, and orthopedic surgeon to timely detect amis . We conclude that stress - induced hyperglycemia increased the risk of ami in patients with hip fractures.
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Duchenne muscular dystrophy (dmd) is a progressive x - linked neuromuscular disease, affecting 1 in 3600 live male births . Classically untreated boys lose ambulation by 9.5 years (range 612), with respiratory, cardiac and orthopedic complications following in the second decade and premature death . Recent studies have, however, demonstrated that there is a new natural history of the disease [13], mainly related to improvements in standards of care, and glucocorticoid (gc) treatment . The effect of gc has also been confirmed by cochrane reviews concluding that gc treatment should be considered the gold standard as demonstrated by placebo controlled studies that are not available for any other treatment . Recent longitudinal studies have clearly shown a delay in loss of ambulation in boys treated with gc compared to untreated boys . The outcome appears to be also related to the regime of gc with a reported median age at loss of ambulation of 12 years for boys on intermittent regime and of 14.5 years for those on daily treatment . While in the past gc treatment was often only started at the time when dmd boys were showing more difficulties in getting up from the floor or climbing stairs, the recently published standards of care suggest that gc treatment should be started earlier, ideally between 4 and 6 years, with some studies suggesting that gc should be started even before the age of 4 years . There is even less agreement on the time when gc treatment should be discontinued . For many years in several centers the treatment was discontinued at the time boys lost ambulation as it was felt that the risk of gaining weight in patients who were less active was bigger than the possible beneficial effects . A few recent studies, however, have reported a possible beneficial effect of gc, but no systematic study has been performed using a scale assessing functional abilities . This is probably also related to the paucity of clinical tools assessing upper limb function in dmd . The performance of upper limb (pul) test, recently developed as part of an international effort to provide a disease specific assessment for upper limb function in dmd, has proved to be a reliable tool, also suitable in a multicentric setting, for both ambulant and non - ambulant dmd boys and young adults . The pul allows to follow the proximal to distal progression of involvement observed in dmd by assessing various functional abilities in three domains (shoulder, elbow, distal). The aim of this study was to establish the possible effect of gc treatment on upper limb function by using the pul in a cohort of non - ambulant dmd boys and adults . The patients included in this study are part of a larger prospective longitudinal study aimed at assessing upper limb function in a larger cohort of ambulant and non - ambulant dmd boys and adults involving 13 tertiary neuromuscular centers . All clinical evaluators were trained by the same lead physical therapist to ensure standardization of equipment, assessment procedures and scoring . Informed consent was obtained from each patient . As we aimed to establish the possible effect of gc in patients who maintained it after loss of ambulation compared to those who stopped gc at the time when they lost ambulation, in this study we only included non - ambulant patients who had lost ambulation for at least two years . Patients were retrospectively subdivided into three subgroups: a) those who were still on gc after loss of ambulation, b) those who stopped gc at the time they lost ambulation; c) those who were never on gc or who had them while ambulant for less than a year . In order to make the groups comparable we did not include 4 patients above the age of 26 years as they were all untreated . The pul includes 22 items with an entry item to define the starting functional level, and 21 items subdivided into shoulder level (4 items), middle level (9 items) and distal level (8 items) dimensions . For weaker patients a low score on the entry item means high - level items do not need to be performed . Scoring options vary across the scale between 01 and 06 according to performance . Each dimension can be scored separately with a maximum score of 16 for the shoulder level, 34 for the middle level, and 24 for the distal level . A total score can be achieved by adding the three level scores (max total score 74). Baseline pul was compared across the 3 gc groups adjusting for age using a global test based on a repeated measures anova, considering shoulder, middle and distal pul assessments as repeated measures on the same subject, with age (<18 and 18 years) and gc subgroups as factors . This global test approach gives a unique p value for a difference across gc subgroups of pul assessments . Post - hoc comparisons using an anova model were run separately for assessing the impact of age and gc subgroup on shoulder, middle and distal assessments . Twelve - month change was evaluated as a% decrease from baseline; patients with pul = 0 at baseline were excluded from the analysis of change; patients with a pul increase over 12 months were set as stable patients (decrease = 0). The patients included in this study are part of a larger prospective longitudinal study aimed at assessing upper limb function in a larger cohort of ambulant and non - ambulant dmd boys and adults involving 13 tertiary neuromuscular centers . All clinical evaluators were trained by the same lead physical therapist to ensure standardization of equipment, assessment procedures and scoring . Informed consent was obtained from each patient . As we aimed to establish the possible effect of gc in patients who maintained it after loss of ambulation compared to those who stopped gc at the time when they lost ambulation, in this study we only included non - ambulant patients who had lost ambulation for at least two years . Patients were retrospectively subdivided into three subgroups: a) those who were still on gc after loss of ambulation, b) those who stopped gc at the time they lost ambulation; c) those who were never on gc or who had them while ambulant for less than a year . In order to make the groups comparable we did not include 4 patients above the age of 26 years as they were all untreated . The pul includes 22 items with an entry item to define the starting functional level, and 21 items subdivided into shoulder level (4 items), middle level (9 items) and distal level (8 items) dimensions . For weaker patients a low score on the entry item means high - level items do not need to be performed . Scoring options vary across the scale between 01 and 06 according to performance . Each dimension can be scored separately with a maximum score of 16 for the shoulder level, 34 for the middle level, and 24 for the distal level . A total score can be achieved by adding the three level scores (max total score 74). Baseline pul was compared across the 3 gc groups adjusting for age using a global test based on a repeated measures anova, considering shoulder, middle and distal pul assessments as repeated measures on the same subject, with age (<18 and 18 years) and gc subgroups as factors . This global test approach gives a unique p value for a difference across gc subgroups of pul assessments . Post - hoc comparisons using an anova model were run separately for assessing the impact of age and gc subgroup on shoulder, middle and distal assessments . Twelve - month change was evaluated as a% decrease from baseline; patients with pul = 0 at baseline were excluded from the analysis of change; patients with a pul increase over 12 months were set as stable patients (decrease = 0). Their age ranged between 11.1 and 26.9 years (mean 16.95; sd 3.52). Forty - eight were still on gc: 7 of the 48 were on daily steroids (mean dose 0.45 mg / kg / day) and 41 on intermittent (mean dose 0.49 mg / kg / day). The mean age when they stopped ambulation between the two subgroups was similar (11.2 and 11.1 years respectively). The remaining 18 were gc nave or had gc while ambulant for less than a year . The mean total scores were 47.92 in the gc group, 36 in those who stopped gc at loss of ambulation and 30.5 in the gc nave group (table 1). Baseline pul significantly increased passing from shoulder (mean = 1.85) to middle (mean = 19.14) to distal (mean = 20.21, p <0.01) domains (fig . 1 and table 1). Baseline pul was significantly higher in those who never stopped gc than in those who stopped gc or never used gc (global test p <0.001) and was lower in patients with age higher than 18 years (global test, p <0.001). While in the shoulder pul was close to zero for all the patients (71% in those who never stopped, 80% in those who stopped and 100% in those who never used gc, p = 0.006), the difference was very evident in the middle region (mean in those who never stopped = 24.02, mean in those who stopped = 15.20 and mean in those who never used gc = 11.61, p <0.001). The trend is still present in the distal pul that is less affected (mean in those who never stopped = 21.17, mean in those who stopped = 19.32 and mean in those who never used gc = 18.89, p = 0.04). Overall, shoulder pul was = 0 in 79% of patients, while middle pul was = 0 in 4 (4%) patients and distal pul was higher than 0 in all the patients . The percentage decrease in the middle pul was 10% in patients still using gc as compared to 34% and 36% in those who stopped and never used gc respectively (p <0.001). The change was lower (mean = 4.5%) and not significantly different among the gc groups in the distal region (p = 0.77). The mean changes were 3.79 in the gc group, 5.52 in those who stopped gc at loss of ambulation and 4.44 in the gc nave group . 2 show details of the changes in the whole cohort and in the subgroups subdivided according to gc treatment . The mean total scores were 47.92 in the gc group, 36 in those who stopped gc at loss of ambulation and 30.5 in the gc nave group (table 1). Baseline pul significantly increased passing from shoulder (mean = 1.85) to middle (mean = 19.14) to distal (mean = 20.21, p <baseline pul was significantly higher in those who never stopped gc than in those who stopped gc or never used gc (global test p <0.001) and was lower in patients with age higher than 18 years (global test, p <0.001). While in the shoulder pul was close to zero for all the patients (71% in those who never stopped, 80% in those who stopped and 100% in those who never used gc, p = 0.006), the difference was very evident in the middle region (mean in those who never stopped = 24.02, mean in those who stopped = 15.20 and mean in those who never used gc = 11.61, p <0.001). The trend is still present in the distal pul that is less affected (mean in those who never stopped = 21.17, mean in those who stopped = 19.32 and mean in those who never used gc = 18.89, p = 0.04). Overall, shoulder pul was = 0 in 79% of patients, while middle pul was = 0 in 4 (4%) patients and distal pul was higher than 0 in all the patients the percentage decrease in the middle pul was 10% in patients still using gc as compared to 34% and 36% in those who stopped and never used gc respectively (p <0.001). The change was lower (mean = 4.5%) and not significantly different among the gc groups in the distal region (p = 0.77). The mean changes were 3.79 in the gc group, 5.52 in those who stopped gc at loss of ambulation and 4.44 in the gc nave group . 2 show details of the changes in the whole cohort and in the subgroups subdivided according to gc treatment . The issue of continuing gc treatment after loss of ambulation has not yet reached a full consensus with, until recently, relative lack of available information on efficacy and safety data from the literature . This is also acknowledged by the dmd care guidelines published in 2010 that identify the use of gc therapy in non - ambulatory individuals as an area in need of further research . In the last few years a few studies have provided more evidence of the efficacy of gc treatment on cardiac and respiratory functions and on strength and other functional measures, but many clinicians still feel that there is not enough evidence to justify the use of gc after loss of ambulation . This is mainly due to the concern that the side effect such as increased weight gain following a reduction of physical activity secondary to loss of ambulation may outweigh the possible beneficial effect . Our results, using the pul, a tool specifically developed to assess upper limb function in dmd, suggest that gc has a beneficial effect on upper limb function after loss of ambulation . The gc treated group had an overall better baseline function than the untreated ones . Not surprisingly those who had been on gc until loss of ambulation had better baseline scores than the gc nave group . The overall difference was more obvious on the middle domain with gc treated patients having significantly higher scores than in the other groups, especially between the age of 12 and 18 years . In this age range most gc treated patients were still able to perform most of the activities exploring functional aspects from the elbow and also had some functional activities at shoulder level that were less present in those who stopped gc steroids at loss of ambulation and even less in the gc nave group . This trend was confirmed, even if to a lesser extent, also in the patients older than 18 years . The difference between the gc treated and untreated groups was also obvious in the 12-month changes as the treated group had a slightly slower deterioration than the untreated ones . This again was most obvious in the middle domain and between 12 and 18 years . In contrast at shoulder level the treated group appeared to have more negative changes compared to the untreated ones . This is, however, only due to the fact that the treated group had better baseline shoulder subscores and was therefore more likely to lose points related to the residual shoulder activity while the untreated groups had much lower baseline shoulder subscores (0 in the nave group) and therefore were more unlikely to lose any further point . The pul did not appear to be equally sensitive to capture the differences in the distal domain as most of the items in the distal domain assessing very distal activities such as different pinches were still present even in the older untreated boys . One of the limitations of this study is that the results in the non - ambulant cohort were part of a larger study also including ambulant boys, and the study was not prospectively designed with a randomized approach to establish the value of different regimens of gc . Even if patients were not prospectively randomized, however, there were no obvious differences in age between the two main groups . The mean ages of the patients who were still on steroids and those who stopped at loss of ambulation were similar, or if anything the latter were even slightly older . Our data provide for the first time evidence that continuing gc after loss of ambulation had a beneficial effect on upper limb function even if most patients were on an intermittent regime and on a relatively low dose as after loss of ambulation the dose is often not adapted to the weight . The effect of gc was more obvious in the patients between 12 and 18 years and at shoulder and elbow levels . This issue is highly relevant if we consider that the activities assessed at middle domain include activities that are essential for functional activities such as self - feeding or ability to perform positional transfers . Maintaining these activities or slowing the progression leading to their loss is therefore likely to have a significant impact on their activities of daily living and on their overall quality of life . Further studies, using a more systematic prospective approach, will help to clarify the possible effects of different regime steroids or dosage or weight that were not systematically explored in our study.
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Accuracy of dose delivery in radiotherapy is stringent requirement for the expected outcome in patients . The dose delivery methods became very precise with digital linear accelerators, their monitoring electronics and automation in terms of use of record / verify systems . The measured dose in water at reference point in phantom is an absolute parameter for planning the treatment monitor units (mu), both in conventional and intensity modulated / image guided treatments . Therefore, traceability of dose accuracy depend mainly on the calibration factor of the ion chamber / dosimeter provided by the accredited secondary standard dosimetry laboratories (ssdls), coming under the network of the international atomic energy agency (iaea) calibration laboratories . With the experience gained in maintaining an accredited laboratory elsewhere in the past, an attempt has been made to scrutinize the database in terms of surveillance of dosimetry standards at our radiotherapy clinic in the present center . Objective of our present study, is referred to our earlier report on status of beam level dose delivery, and correlate to realization of true absorbed dose in field conditions, in the user institution . As there is no telecobalt machine available for surveillance of dosimetry methods, an attempt was earlier made to study the efficacy of an extrapolation chamber as a the iaea dosimetry protocol (trs 398) highlights the uncertainties involved in various steps of dissemination of measurement parameters . The present report evaluates our reference dosimeters in terms of reproducibility in absorbed dose standards . The department has three reference class dose-1 electrometers (iba, gmbh, scanditronix wellhofer, germany) used for absorbed dose calibration in linear accelerator beams and one unidos electrometer (ptw, germany) for high dose rate brachytherapy measurements with suitable chambers . All electrometers have calibration factor 1.00 as per certificates provided from accredited ssdls, (iba, scanditronix wellhofer, nurenberg, germany; ptw, freiburg, germany). Calibration factor for absorbed dose estimation in terms of gray / coulomb in water were obtained during 2003, 2009, and 2013 from the same ssdl (iba chambers from iba, gmbh calibration laboratory; ptw chamber calibrated by ptw, freiburg). A strontium-90 check source (qsa global gmbh, germany) of nominal activity 30 mbq (0.8 mci) is used to periodically check the sensitivity variations of the 0.6 cc farmer chambers (fc 65) routinely . This provides a dose rate of about 1.25 mgy / s at the position of the chamber; requiring about 400 s for an accumulated reading of 500 mgy . Postal dose tl intercomparison program of iaea, vienna, started in our hospital in 2008, after (sultanate of) oman became a member state . And started participating in postal dose tl intercomparison program since 2008 . So far we participated in three intercomparisons, for three high energy photon beams 6 mv (two), 15 mv (one). The procedure followed here was to measure the absorbed doses in water (10 cm depth) on the day of tl irradiations, and use the dose / mu for calculating the mu required for 2 gy dose . The stated dose by the user institution and iaea dose estimates in their dosimetry laboratory at seibersdorf, austria, validate existing dosimetry at the hospital . The method of maintaining a constant cgy / mu in our linac photon and electron beams was explained in a previous report . When the daily check of output / mu showed drift beyond 2% limits retuning is carried out by the local service personnel . The agreement of measured absorbed dose using three chambers / electrometer combinations was checked using a 5 cm depth perspex, isocenter test tool (sun nuclear, usa) in 6 mv x - ray beam from clinac 600 cd linear accelerator (varian, usa). 100 mu with 10 10 cm field size at 100 cm fsd were delivered . Absorbed dose / mu was calculated from the charge measured, multiplied by nd, w calibration factor, corrected for temperature pressure . The doses were not converted into dose to water, and compared as it is . Three fc 65 chambers are connected one by one to different electrometers, their response and measured values were compared . The extent of agreement between dose estimates at the same reference point under constant geometry by two different ion chambers calibrated at different laboratories (a and b) is also checked . Table 1 lists the nd, w values of four dosimeters during first and subsequent calibrations . Slight changes in the nd, w factors in gray / coulomb are observed, effective on different dates of calibration . Over a period of about 9 years, a mean variation of about 0.2% increase was observed for the iba chambers (one to three), but the ptw chamber showed a nearly consistent response . Table 2 highlights the results of chamber response to a strontium-90 beta check source, indicating the long - term stability of the ion chambers to the constant geometry beta source . The mean variation of the chamber response with corresponding electrometer, remained well within 1% for all three chambers used for beam level measurements . Calibration factors nd, w for thimble (fc 65) chambers from different laboratories strontium-90 check source, chamber response for reading 500 mgy it could be observed from table 3, that the results of iaea postal tld intercomparison for three photon beams agreed well with our reported doses, over the period 2008 - 2012 representing the status of beam level dosimetry in this institution . Tables 4 and 5 show the measured absorbed doses in phantom at constant geometry, using chambers and electrometers which obtained calibration factors from same accredited laboratory . It is observed clearly that all the chamber / electrometer calibrations had an agreement of 0.13% standard deviations in realized absorbed doses . From table 6 it could be appreciated that nd, w factors obtained from two different calibration laboratories (in contrast to table 5 inference) do not realize the absorbed dose exactly, but agree within 0.6% (in perspex cylindrical phantom) and with deviations as high as 1.5 - 2.0% in solid water, water equivalent phantom . Absorbed dose estimates by iaea postal tld intercomparison dose estimate in cylindrical perspex phantom with nd, w corrected dose value from electrometers on gy display for different ion chambers estimate of absorbed doses with dosimeters traceable to different laboratories the above report enumerated calibration factor, nd, w, variability over a 9 year period, as is typical at the user level, and the extent of agreement / disagreement in application of nd, w factor in realizing dose were described . Comparison of calibration factors for all dosimeters showed an upward trend in the nd, w factors [table 1] which indirectly imply that they show slight under - estimation of dose, as the time progresses . Though it is negligible percentage per year, the slight change in effective volume of ionization is not ruled out . The international dosimetry protocol(trs 398, iaea) indicates relative standard uncertainty in dose estimates using nd, w for cobalt reference, a value of 0.9% for telecobalt beams, and 1.5% for high energy linear accelerator photon beam, calibrated at an user institution . Another report indicate uncertainties 0.8% for standards laboratory, total user beam level 2% with an overall uncertainty of 2.2% for the application of nd, w calibration factor . The status of beam level dosimetry is highlighted in table 3 for period 2008 - 2012 indicate a mean of 0.03%, maximum of + 1.4%, minimum of 1.4% for three photon beams for nine irradiations . However, beam output and energy stability have been evaluated since patient treatment with linacs began in 2005, and have been documented in a previous report . Table 1 has significance in a user clinic of megavoltage beams for treatment, as the nd, w factors form the basis for status of calibration over a long period of time . The drift in the calibration factor with a long time (8 - 10 years) observed in table 1 is in agreement with the values indicated by tomas kron viz . An experience in a previous institution cited elsewhere showed that the departmental secondary standards showing about 1 - 1.5% variations in 10 years period . It was indicated by us earlier that a constant value of linear accelerator beam output cgy / mu is used in the department, and continuous monitoring of the beams are carried out to quantify routine variations . If changes beyond acceptable limits are observed, the original cgy / mu is restored such that dose delivered to patients are within 2% limits from the mu point of view . All the three dosimeters (iba, germany) in table 1 are reference class dosimeters, and therefore the changes in nd, w factor should be realized as genuine changes representing some response change in chambers, as electrometer calibration remained same . The realization of dose in terms of cgy / mu is within 0.13% standard deviation for different chamber / electrometer combinations by taking gray / coulomb calibration, and within same 0.13% standard deviation by taking gray / reading calibrations [tables 4 and 5]. A careful scrutiny of strontium-90 check source response for these three chambers could not exactly correlate to the actual drift observed in nd, w factors over many years . It was highlighted that nd, w factors obtained from different laboratories (three chambers calibrated by iba compared with ptw calibrated chamber) [table 6] did not estimate absorbed doses with good agreement, exactly in solid water phantoms (in contrast to table 5 inference) with deviations as high as 1.5 - 2.0% . But agreement was seen (within 0.6%) in perspex cylindrical phantom and in water phantom (last four entries) [table 6]. In oman, we do not have an ssdl at present, though there are efforts to establish one in the university hospital, with iaea assistance . Even then, our center will continue to be the only institution where high energy photons / electron sources are available . This overview has highlighted the point that about 2% uncertainty limit exist in maintaining patient dosimetry from medical physics point of view; which is the achievable limit as per international standards.
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Fifteen children and adolescents with asds (asperger and high functioning autism) aged eight to 17 years old were compared with 15 age matched normally developing individuals . Participants with asds were diagnosed by a child and adolescent psychiatrist based on dsm - iv criteria (17). Visual memory tasks were assessed in all participants using some tests from cambridge neuropsychological test automated battery (cantab):paired associates learning (pal): in this test, some boxes are displayed on the screen and opened in a randomized order . Error occurs when the participant selects a box not containing the target stimulus (18, 19).pattern recognition memory (prm): this test has two phases . In the first phase, the participant is presented with a series of 12 colored visual patterns for three seconds . 12 paired novel and old patterns are presented in which the participant is required to choose between a pattern they have already seen and a novel pattern (20).this test is scored using two indices: a) mean correct latency; b) number correct.spatial recognition memory (srm): this test has also two phases . In the first phase, the participant is presented with a white square which appears at five different locations on the screen . The participant is told to memorize the locations of the square . In the second phase, the participant sees a series of five pairs of squares, one of which is in a place previously seen in the presentation phase . This test has two indices for scoring: a) mean correct latency; b) number correct.delayed matching to sample (dms): in this test, one target (a complex visual pattern) and four different patterns are presented in four different time intervals . The four patterns are presented simultaneously or 0, 4, and 12 seconds after target presentation . The participant is instructed to touch the pattern which exactly matches the target (21). Paired associates learning (pal): in this test, some boxes are displayed on the screen and opened in a randomized order . Error occurs when the participant selects a box not containing the target stimulus (18, 19). Pattern recognition memory (prm): this test has two phases . In the first phase, the participant is presented with a series of 12 colored visual patterns for three seconds . 12 paired novel and old patterns are presented in which the participant is required to choose between a pattern they have already seen and a novel pattern (20). This test is scored using two indices: a) mean correct latency; b) number correct . Spatial recognition memory (srm), the participant is presented with a white square which appears at five different locations on the screen . In the second phase, the participant sees a series of five pairs of squares, one of which is in a place previously seen in the presentation phase . This test has two indices for scoring: a) mean correct latency; b) number correct . Delayed matching to sample (dms): in this test, one target (a complex visual pattern) and four different patterns are presented in four different time intervals . The four patterns are presented simultaneously or 0, 4, and 12 seconds after target presentation . The participant is instructed to touch the pattern which exactly matches the target (21). The test is scored using four indices: a) mean correct latency; b) total corrects; c) prob error given correct: this index indicates the probability of error following a correct; d) prob error given error: in this index, the probability of error is illustrated following a previous error . The raven's progressive matrices (rpm) test was administered to evaluate participants' intellectual abilities (22). Asperger syndrome diagnostic scale (asds) was used to confirm the asperger's diagnosis . This scale is a 50-item, " yes " or " no " questionnaire, designed to identify children and adolescents aged 5 to 18 with asperger syndrome . These questions cover five different aspects of mental states including cognition, language skills, social interactions, sensorimotor, and maladaptive behaviors (23). The participants were firstly interviewed by a child and adolescent psychiatrist and diagnosed as having autism spectrum disorders based on dsm - iv criteria . Next, they were evaluated using the asds to confirm the diagnosis and were then examined using neuropsychological tests . Independent samples t test was used to compare the two groups in terms of different variables . Iq was considered as covariate and then the two groups were compared again to eliminate the iq effect . In this research, all statistical analyses were performed using the statistical package for the social sciences (spss), version 14 . Fifteen children and adolescents with asds (asperger and high functioning autism) aged eight to 17 years old were compared with 15 age matched normally developing individuals . Participants with asds were diagnosed by a child and adolescent psychiatrist based on dsm - iv criteria (17). Visual memory tasks were assessed in all participants using some tests from cambridge neuropsychological test automated battery (cantab):paired associates learning (pal): in this test, some boxes are displayed on the screen and opened in a randomized order . Error occurs when the participant selects a box not containing the target stimulus (18, 19).pattern recognition memory (prm): this test has two phases . In the first phase, the participant is presented with a series of 12 colored visual patterns for three seconds . 12 paired novel and old patterns are presented in which the participant is required to choose between a pattern they have already seen and a novel pattern (20).this test is scored using two indices: a) mean correct latency; b) number correct.spatial recognition memory (srm): this test has also two phases . In the first phase, the participant is presented with a white square which appears at five different locations on the screen . The participant is told to memorize the locations of the square . In the second phase, the participant sees a series of five pairs of squares, one of which is in a place previously seen in the presentation phase . This test has two indices for scoring: a) mean correct latency; b) number correct.delayed matching to sample (dms): in this test, one target (a complex visual pattern) and four different patterns are presented in four different time intervals . The four patterns are presented simultaneously or 0, 4, and 12 seconds after target presentation . The participant is instructed to touch the pattern which exactly matches the target (21). Paired associates learning (pal): in this test, some boxes are displayed on the screen and opened in a randomized order . Error occurs when the participant selects a box not containing the target stimulus (18, 19). Pattern recognition memory (prm): this test has two phases . In the first phase, the participant is presented with a series of 12 colored visual patterns for three seconds ., 12 paired novel and old patterns are presented in which the participant is required to choose between a pattern they have already seen and a novel pattern (20). This test is scored using two indices: a) mean correct latency; b) number correct . Spatial recognition memory (srm): this test has also two phases . In the first phase, the participant is presented with a white square which appears at five different locations on the screen . The participant is told to memorize the locations of the square . In the second phase, the participant sees a series of five pairs of squares, one of which is in a place previously seen in the presentation phase . This test has two indices for scoring: a) mean correct latency; b) number correct . Delayed matching to sample (dms): in this test, one target (a complex visual pattern) and four different patterns are presented in four different time intervals . The four patterns are presented simultaneously or 0, 4, and 12 seconds after target presentation . The participant is instructed to touch the pattern which exactly matches the target (21). The test is scored using four indices: a) mean correct latency; b) total corrects; c) prob error given correct: this index indicates the probability of error following a correct; d) prob error given error: in this index, the probability of error is illustrated following a previous error . The raven's progressive matrices (rpm) test was administered to evaluate participants' intellectual abilities (22). Asperger syndrome diagnostic scale (asds) was used to confirm the asperger's diagnosis . This scale is a 50-item, " yes " or " no " questionnaire, designed to identify children and adolescents aged 5 to 18 with asperger syndrome . These questions cover five different aspects of mental states including cognition, language skills, social interactions, sensorimotor, and maladaptive behaviors (23). The participants were firstly interviewed by a child and adolescent psychiatrist and diagnosed as having autism spectrum disorders based on dsm - iv criteria . Next, they were evaluated using the asds to confirm the diagnosis and were then examined using neuropsychological tests . Independent samples t test was used to compare the two groups in terms of different variables . Iq was considered as covariate and then the two groups were compared again to eliminate the iq effect . In this research, all statistical analyses were performed using the statistical package for the social sciences (spss), version 14 . The mean age of children and adolescents with asds was 12.80 (sd=3.23) while the mean age of control group was 10.53 (sd= 3.04). With regards to iq, youths with asds had significantly lower iqs compared to normal children and adolescents (p<0.01) (table 1). Demographic variables in children with asds and normal group in regards to paired associates learning test (pal), individuals with asds had poorer performance on this task compared to normal group (table 2). There was a significant difference between the two groups on " first trial memory score " (t = 2.092, p = 0.046), total errors adjusted (t = 17.662, p = 0.015) and " total trials adjusted " (t = 15.794, p = 0.013). Results of visual memory tests in children with asds compared with normal group without considering the iq as the covariance while iq was considered as covariate, none of the pal variables showed any significant difference between the two groups . In terms of pattern recognition memory (prm) and spatial recognition memory (srm) tests, significant differences were found between the two groups on " number correct " variable in prm (t = 6.390, p = 0.045) and srm (t = 1.026, p = 0.005) tests . Considering iq as covariate the differences were eliminated . With regards to delayed matching to sample (dms) task, it was found that children with asds had worse performance compared to normal youths on dms task (table 2). Children and adolescents with asds had less " total corrects " (t = 0.786, p = 0.002) and more " prob error given correct " (t = 0.277, p = 0.012) and " prob error given error " (t = 13.834, p = 0.019) compared to normal group . By eliminating the iq effect, no significant difference was found between the two groups in terms of dms variables . Some studies investigated visual perception and memory in children with autism spectrum disorders (asds), but their findings are not consistent . Because these experiments were mostly based on investigating meaningful shapes, the current study was designed to evaluate visual memory using meaningless shapes in children and adolescents with asds . We found that youths with asds had poorer performance on paired associate learning test which is mediated by temporal lobe compared to normal youths . It was also found that asds group had poorer performance on recognizing meaningless patterns on prm task, which is sensitive to impairment of temporal lobe . Youths with asds were poorer on visuospatial memory task, which is mediated by frontal lobe (24). They were also poor in matching the patterns to the sample based on the findings of delayed matching to sample task . This task is sensitive to impairment of frontal lobes and is assumed to be mediated by temporal lobe (18). These findings are inconsistent with the results of some other studies which indicated that children and adolescents with asds have intact object recognition and visual memory (25, 26) or showed that youths with asds had a better ability to recognize objects (27). This inconsistency may be due to different tasks that were used in our study as compared to the others . The visual memory tasks used in previous studies were mostly based on recognizing objects which may have been particularly interesting to children and adolescents with asds, so they could have recognized them easier than otherwise normal developing youths . Therefore, it can be gathered that the findings of this study could show the visual memory ability of children and adolescents with asds with better precision . On the other hand, these findings are consistent with the results of ameli's study on autistic individuals comparing the meaningful and meaningless pictures (8). They found that individuals with autism have poorer performance on meaningless visual memory patterns whereas they are as good as normal people on meaningful pictures (8). Moreover, when we considered iq as a covariance, the significant difference disappeared and the visual memory ability in both groups proved to be the same . By eliminating the iq effect, our findings will be consistent with prior study's results which showed no significant difference between normal youths and asds group . This means that this ability may be related to global cognitive ability, which is totally lower in youths with asds compared with normal children in the same age group . In fact, children and adolescents with asds have deficits in different aspects of cognition including the intellectual ability, visual memory, visuospatial recognition, and executive functioning . Based on these findings, we can conclude that youths with asds have poorer performance on meaningless pattern recognition as well as the visuospatial memory compared to normal age matched children and adolescents . One limitation of this study was the difference between the two groups in terms of their iqs . Since the intellectual abilities of youths with asd are mostly lower than their age matched normal group, matching the iqs of the two groups was rather difficult . The findings of this study suggest that asds children and adolescents are not superior in visual memory ability compared to normal youths.
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Tuberculosis (tb) is a contagious, infectious disease, due to mycobacterium tuberculosis (mt), which usually lasts throughout the life course and determines the formation of tubercles in different parts of the body . Mt has very ancient origins: it has survived over 70,000 years and it currently infects nearly 2 billion people worldwide; with around 10.4 million new cases of tb each year, almost one third of the world's population are carriers of the tb bacillus and are at risk for developing active disease . Tb has always been associated with a high mortality rate over the centuries, and also nowadays, it is estimated to be responsible for 1.4 million tb deaths, among infectious diseases after human immunodeficiency virus (hiv). Due to its infectious nature, complex immunological response, chronic progression and the need for long - term treatment, tb has always been a major health burden; in more recent years, the appearance of multi - drug resistant forms and the current tb - hiv epidemic, associated with its severe social implications, treating and preventing tb have represented a permanent challenge over the course of human history [4, 5]. It has been hypothesized that the genus mycobacterium originated more than 150 million years ago . Mycobacterium ulcerans, causing infections since ancient times, requires specific environmental conditions as reflected nowadays in its distribution worldwide . Three million years ago, an early progenitor of mt might have infected early hominids in east africa and 20.000 - 15.000 years ago, for the first time, the common ancestor of modern strains of mt might have appeared [8, 9]. Egyptian mummies, dating back to 2400 bc, reveal skeletal deformities typical of tuberculosis; characteristic pott's lesions are reported and similar abnormalities are clearly illustrated in early egyptian art [10, 11]. The first written documents describing tb, dating back to 3300 and 2300 years ago, were found in india and in china respectively [12, 13]. The ancient hebrew word schachepheth is used in the biblical books of deuteronomy and leviticus in order to describe tb; in the same period, in the andean region, archeological evidence of early tb, including pott's deformities, was provided by peruvian mummies, suggesting that the disease was present even before the colonization of the first european pioneers in south america [15 - 18]. In the ancient greece tb hippocrates described phtisis as a fatal disease especially for young adults, accurately defining its symptoms and the characteristic tubercular lung lesions . Excellent discoveries of the early scientists who studied tb were made in the same period: in greece, isocrates was the first author supposing that tb was an infectious disease, while aristotle suggested the contagious nature of " king's evil " in pigs and oxes . In roman times, tb is mentioned by celso, aretaeus of cappadocia and caelius aurelianus, but it is not recognized as sharing the same etiology of extrapulmonary manifestations such as scrofula, pott's disease and tb lupus . According to the greek clarissimus galen, who became personal physician of the roman emperor marcus aurelius in 174 ad, the symptoms of tb include fever, sweating, coughing and blood stained sputum; he recommended fresh air, milk and sea voyages as successful treatments for the disease [20 - 22]. After the decline of the roman empire, tb was widespread in europe in the viii and xix centuries, as witnessed by several archaeological findings . The byzantine doctors aetius of amida, alexander of tralles and paul of aegina described the pulmonary and glandular forms of tb, while in the arabic empire, avicenna supposed the contagious nature of tb . In the middle ages, scrofula, a disease affecting cervical lymph nodes, was described as a new clinical form of tb . The illness was known in england and france as " king's evil ", and it was widely believed that persons affected could heal after a royal touch . In the 12 century, william of malmesbury reported complementary treatments including visits at royal tombs, the kings' touch or the use of a coin - talisman . The practice of the king's touch established by english and french kings continued for several years . Queen anne was the last english monarch to use this practice (1712), george i put an end to it in 1714, while in france it continued up to 1825 . In the middle ages, moreover, the french surgeon guy de chauliac (1363) for the first time proposed a healing intervention for the cure of the " king's evil " . Guy de chauliac was also strongly in favour of the removal of scrofulous gland with an engraving, as recommended by paul of aegina, who advised the surgical removal of the diseased gland, taking care not to harm vessels or nerves of which the neck region is rich . As far as concerns the contagious nature of tb, a clear definition was first given by girolamo fracastoro in the sixteenth century . The exact pathological and anatomical description of the disease was illustrated in 1679 by francis sylvius, in his work opera medica, in which he describes tubercles, their progression to abscesses, cavities and empyema in the lungs and in other sites of consumptive patients . Short afterwards, in italian health law, in particular in an edict issued by the republic of lucca in 1699, there is the first official reference to the infectious nature of the disease . In 1735 the health board of the republic ordered the compulsory notification and isolation of consumptives, forbidding their admission in public hospitals, and establishing specific places for their treatment . In 1720, for the first time, the infectious origin of tb was conjectured by the english physician benjamin marten, in his publication " a new theory of consumption " . For the early eighteenth century both terms consumption and phthisis were used in the 17 and 18 centuries, until in the mid-19 century johann lukas schnlein coined the term " tuberculosis " . In the 18 century in western europe, tb had become epidemic with a mortality rate as high as 900 deaths per 100,000 inhabitants per year, more elevated among young people . For this reason, tb was also called " the robber of youth " . During the industrial revolution, the diffusion of particularly problematic social conditions, such as extremely deprived work settings, poorly ventilated and overcrowded housing, primitive sanitation, malnutrition and other risk factors, up to a third of english tradesmen and employees died of tb, whereas the same proportion decreased to a sixth in the upper class . The extreme anemic pallor of people affected by tb was at the origin of the new term " white plague ", coined during the 18 century [20, 35]. One hundred years later, tb was defined as " captain of all these men of death " because of its epidemic proportions in europe and north america, determining one in four deaths . At the beginning of the 19 century, there was a large scientific debate about different theories concerning the etiopathological origin of phthisis, arguing whether it might be considered: an infectious disease as generally considered in southern europe an hereditary one as stated in northern europe or a form of cancer . On the other hand, the discussion was about scrofula, tubercles, and phthisis as separate disease entities or manifestations of the same illness . In 1793, the caseous necrosis, " cheese - like ", phthisic abscesses were named " tubercles " by the scottish pathologist matthew baille . In 1810, the french physician gaspard - laurent bayle of vernet described the disseminated " miliary " tb in his work recerches sur la phthisie pulmonaire, recognizing tb not only as a disease affecting the lung, but a generalized one, clinically defined by coughing, difficulty in breathing, fever and purulent expectoration [37, 38]. In 1819, the french theophile laennac identified the presence of consolidation, pleurisy and pulmonary cavitation as pathognomonic signs of pulmonary or extrapulmonary tb . Tuberculosis most commonly affects the respiratory tract, but it could also infect gastrointestinal, bones, joints, nervous systems, lymph nodes, genitourinary tract and skin with inflammatory infiltration, caseation, necrosis, abscesses, fibrosis, formation of tubercles and calcification [39, 40]. He described their first appearance in the lungs, in their " miliary " (" millet seed - like ") form, their progressing to larger tubercles containing " cheese - like " (" caseous ") material, their breakdown into pus, and eventually forming cavities and empyema . Extra - pulmonary phthisic tubercles were recognized in the intestines, liver, meninges and other organs, as also described by sir percivall pott, a british surgeon that in 1779 defined as " pott's disease " the vertebral collapse and spinal cord paralysis caused by tb infection [33, 36, 37, 41, 42]. In 1843, the german physician philipp friedrich hermann klencke succeeded in the experimental reproduction of human and bovine forms of tb, causing generalized tb in rabbits, through a successful inoculation of material from a miliary tubercle into their liver and lungs . In 1849 lebert, publishing his work traite pratique des maladies scrofuleuses et tuberculeuses, suggested that the " king's evil " was a childhood disease that might cause suppuration and ulceration of different body's sites such as skin, ears, eyes, joints, bones, with a different pathogenesis from tb . The first successful remedy against tb was the introduction of the sanatorium cure, described for the first time in 1854 in the doctoral dissertation " tuberculosis is a curable disease " by hermann brehmer, a botany student suffering himself from tb, who reported his healing after a travel to the himalayan mountains . Afterwards brehmer founded an institution in gorbersdorf, a mountain town situated in a fir forest, in order to cure patients with continuous fresh air and good nutrition . The subsequent sanatoria were built with the same setup and permitted to cure a lot of tb patients in the next decades . The infectious nature of tb was demonstrated in 1865 by jean - antoine villemin, a french military surgeon at the army medical school . He formulated his hypothesis observing that tb was more frequent among soldiers who stationed for long times in barracks than among those in the field . He also highlighted how healthy army recruits coming from the countryside often became consumptive some months after the beginning of their service . Villemin's experiments consisted in inoculating a rabbit with " a small amount of purulent liquid from a tuberculous cavity " removed at autopsy from an individual died of tb . As described in villemin's work cause et nature de la tuberculose: son inoculation de l'homme au lapin, the inoculated animal remained alive and no disease signs were discovered, but at autopsy, three months later, extensive tb was evident . Villemin suggested that phthisis could be similar to glanders, an infectious disease in horses [20, 34, 36, 37, 47]. Preuves rationelles et exprimentales de sa spcificit et de son inoculabilit, dated 1868, in which he stated the presence of tblike illnesses in different animals . The author also noticed that more crowded urban areas had a higher prevalence of tb and that some parts of the world, like new zealand and australia, seemed to have not known tb until the arrival of pioneers . Some years later, in 1867, theodor albrecht edwin klebs was one of the early scientists to try to isolate the tb bacillus, sowing tuberculous material on egg white, stored in sterile flasks . In his experiments, the culture was quickly muddy and it was possible to recognize mobile bacilli, which, after inoculation into the peritoneal cavity, caused the disease in guinea pigs . Using the methylene blue staining recommended by paul ehrlich, he identified, isolated and cultivated the bacillus in animal serum . Robert koch presented this extraordinary result to the society of physiology in berlin on 24 march 1882, determining a milestone in the fight against tb . In the decades following this discovery, the pirquet and mantoux tuberculin skin tests, albert calmette and camille gurin (bcg) vaccine, selman waksman streptomycin and other anti - tuberculous drugs were developed . Koch contributed also to the elucidation of the infectious etiology of tb and for his scientific results, he was awarded the nobel prize in medicine in 1905 [33, 51]. Nowadays tb is still a major public health problem, for this reason a combined strategy, based on improving drug treatment, diagnostic instruments, and prevention strategy, is necessary, in order to eradicate m. tuberculosis by the year 2050, as committed by the world health organization (who).
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The success of a root canal filled tooth depends mainly on the extent of re - cleaning and re - shaping followed by the complete filling of the root canal system . Gutta - percha is the most commonly used material for filling the root canals, and it should be removed when retreatment is indicated . There are various methods that are followed to remove gutta - percha from the canal system; these include hand files, rotary files, as well as ultrasonic instruments . Studies have shown that none of the re - treatment procedures are able to completely clean the root canal walls, particularly in the apical third, where microorganisms generally persist . It is considered that the combined use of different techniques is more effective in the complete removal of gutta - percha . It has been reported in various studies that the use of ni - ti instruments for the purpose of gutta - percha removal during re - treatment is safe, fast, and efficient; ni - ti also maintains the shape of the root canal and its use also avoids the apical extrusion of debris . Few similar previous studies have contradicted the abovementioned findings; these studies have reported that the manual use of hedstrom files is more effective in the removal of gutta - percha when compared to ni - ti rotary systems during retreatment procedures . In addition, it should be noted that, many studies have not concluded the efficacy of one single rotary ni - ti system in the removal of root canal filling material . All the ni - ti rotary systems that were studied showed no significant difference among them in removing gutta - percha . The purpose of the present study is to compare manual and automated instrumentation techniques for the removal of root canal filling material as well as to compare the efficiency among automated systems, of which ni - ti system is especially designed for endodontic re - treatment procedures . Thirty extracted single rooted teeth of similar length were selected . The sample size for the study soft tissue remnants were cleaned by immersing the tooth in 3% sodium hypochlorite for 24 h. the samples were decoronated at the cementoenamel junction with a diamond disc (d&z, berlin, germany), leaving the root length to be approximately 18 mm in length [figure 1]. Standardized decoronation of samples cleaning and shaping of the canal system was done using the protaper system (dentsply maillefer) according to the manufacturer's instructions using an x - mart (dentsply). The canals were filled in increments using obtura ii and hand plugger was used to condense and plug the thermoplastic gutta - percha . Postoperative radiograph were obtained to determine the quality of root fillings [figure 3]. The specimens were sealed with temporary filling material (cavit, 3 m espe dental). The samples were stored at 37c for 30 days, and then, they were divided into three groups of 10 samples each, and each group was treated using a different technique . I was removed using hedstrom files; file sizes from #45 to #30 were used . The filling material was removed in a crown - down technique by using the file sizes in a reverse sequence . Protaper retreatment files d1, d2, and d3 were used in a crown - down technique . D1 is used for cervical debridement, followed by d2 at the middle one thind and d3 is worked to working length of the canal . Root canal filling material in group iii was removed using race files, sequence were used as suggested by the manufacturer (9 instruments, tapers range from 2 to 10%). All the samples were digitally radiographed after the re - treatment procedure with their respective instruments . A standard exposure time of 0.08 seconds and a standard distance of 5 cm was maintained . The digitized images were analyzed by dividing the canal into coronal, middle, and apical areas [figure 3]. Using standardized scoring criteria, the radio - opacity of the canals in each of the three divided areas were scored and evaluated [figure 4]. Thirty extracted single rooted teeth of similar length were selected . The sample size for the study soft tissue remnants were cleaned by immersing the tooth in 3% sodium hypochlorite for 24 h. the samples were decoronated at the cementoenamel junction with a diamond disc (d&z, berlin, germany), leaving the root length to be approximately 18 mm in length [figure 1]. Standardized decoronation of samples cleaning and shaping of the canal system was done using the protaper system (dentsply maillefer) according to the manufacturer's instructions using an x - mart (dentsply). The canals were filled in increments using obtura ii and hand plugger was used to condense and plug the thermoplastic gutta - percha . Postoperative radiograph were obtained to determine the quality of root fillings [figure 3]. The specimens were sealed with temporary filling material (cavit, 3 m espe dental). The samples were stored at 37c for 30 days, and then, they were divided into three groups of 10 samples each, and each group was treated using a different technique . The canal filling material in group i was removed using hedstrom files; file sizes from #45 to #30 were used . The filling material was removed in a crown - down technique by using the file sizes in a reverse sequence . Protaper retreatment files d1, d2, and d3 were used in a crown - down technique . D1 is used for cervical debridement, followed by d2 at the middle one thind and d3 is worked to working length of the canal . Root canal filling material in group iii was removed using race files, sequence were used as suggested by the manufacturer (9 instruments, tapers range from 2 to 10%). All the samples were digitally radiographed after the re - treatment procedure with their respective instruments . A standard exposure time of 0.08 seconds and a standard distance of 5 cm was maintained . The digitized images were analyzed by dividing the canal into coronal, middle, and apical areas [figure 3]. Using standardized scoring criteria, the radio - opacity of the canals in each of the three divided areas were scored and evaluated [figure 4]. The mean and standard deviation [table 1] of the three groups were analyzed first followed by performing the t - test along with analysis of variance test (two - tailed p value) among the three groups to determine the significant difference [table 2]. Mean and standard deviation of the three study groups t - test and analysis of variance test with two - tailed p value among the three study groups when the results were analyzed it was noted that there was a significant difference (p <0.05) between the effectiveness of manual method using hedstrom files to that of using rotary files . Both the rotary systems were effective in removing the root canal filling material from the canal walls . No significant difference (p> 0.05) was observed between protaper retreatment to that of the race file system in removing gutta - percha from the canal walls . When the cervical, middle, and apical thirds were analyzed separately, it was observed that all the groups performed well in removing the filling material from the canal walls at the cervical region . However, the significance of difference (p <0.05) was recorded when instrumentation was performed at the middle and apical third regions . The overall time taken to perform the procedure is also comparatively less when using rotary system to that of the manual method . The use of the protaper retreatment files was faster than the race files because of the less number of files (3 files) that are used for the procedure compared to the 9 files for the race file system . The choice of treatment for a failing root canal treated tooth is either a surgical procedure (apical surgery or extraction) or nonsurgical retreatment, out of which the latter is the most preferred . Filling material left after retreatment procedure may harbor necrotic tissue and bacteria, which could lead to a persistent disease and reinfection of the root canal system . The present study was undertaken to determine the effectiveness of gutta - percha removal technique in a root canal retreatment procedure . Complete removal of the old root canal filling material along with good debridement is very important for a successful root canal retreatment procedure . Many methods are used during endodontic retreatment, which include endodontic hand files, endodontic rotary files, ultrasonic files, and chemicals such as chloroform, zylene, turpentine, and many others . In this scenario, there are a number of endodontic rotary file systems introduced by different manufactures promising an effective filling material removal from the root canals . Some manufactures have even introduced exclusive root canal filling material removal systems such as the protaper retreatment files . However, few studies point out that the effectiveness of these exclusive retreatment systems is same to that of rotary system . A study compared the effectiveness of protaper and mtwo to that of retreatment system from the same manufactures, the protaper retreatment and mtwo retreatment systems; the study concluded that there was no significant difference among the study groups, and protaper and mtwo were as effective as protaper retreatment and mtwo retreatment file systems . All these systems to some extent challenge the conventionally used hand hedstrom files, which is / was used by many clinicians for gutta - percha removal during retreatment procedures . Hence, this increases the necessity for a clinician to access and know the best technique he can employ in the removal of gutta - percha with a rotary or hand file and sometimes the use of both the rotary and hand might become necessary . This study helps in evaluating the efficiency of two such rotary systems, the protaper retreatment files and race rotary files, along with hedstrom files . In the present study, single rooted premolar teeth were selected to reduce the variation in the effectiveness of the technique among the different study groups . The samples were shaped with protaper rotary system and obturated with thermoplastic gutta - percha so that they receive a relatively uniform quantity of filling material in their canals . The samples of group i were re - treated with hedstrom files, group ii were re - treated with protaper retreatment files and group iii with race rotary system . The primary outcome of the study is that none of the systems or the technique used were effective in a total or 100% removal of the gutta - percha form the root canals of the samples . Hulsmeann et al ., who also studied the cleaning ability of rotary instruments in retreatment, concluded that there was no system that was 100% effective in gutta - percha removal . The present study used a unique scoring criterion to determine the effectiveness of the gutta - percha removal not just in the whole of the root canal but also helped in determining the exact effectiveness of the different techniques at different parts of the root canal system . The canals were divided into three parts as the apical, middle, and the cervical thirds, and depending on the presence of residual filling materials the scoring was done . When the groups were compared at these different levels, the effectiveness of the techniques varied between the rotary and the hand methods . Group ii and group iii which represented the rotary systems did not show much difference in their efficiency in removing filling material from the middle and cervical one third of the canals . Both the rotary ni - ti files were very effective at the middle and cervical one third regions . The effectiveness of the rotary at these areas can be attributed to the greater tapers of the files at these areas, like that of race files used were 10% and 8% at the cervical areas, thus engaging more of the filling materials during the cleaning process . Files such as the race sized 10.40 and 8.35 are made of stainless steel; it is also concluded in a few studies that stainless steel files have a higher cutting efficiency than ni - ti files . Few recent studies have established that effectiveness of protaper retreatment files in gp removal, a study that compared protaper retreatment files to that of race, k3 and hedstrom files showed its efficacy in retreatment procedures . It is also estimated that the protaper retreatment system worked faster than mtwo retreatment files in removing root canal obturation material, both retreatment systems were considered to be effective, reliable, and fast . There are a few studies like those by hulsmann et al . And betty et al . That advocate the use of endodontic hand files for the removal of endodontic filling material . Studies by rodig et al . Also support the effectiveness of hand files in the removal of gutta - percha . In the present study, samples of group i, where the method of filling material removal was hedstrom files, showed less or no residual gutta - percha at the apical one third region . This might be because of the availability of a greater number of files with large tip sizes in the hedstrom file system, thus providing the comfort to use one size larger than the original size used in the initial cleaning of the canal, thus, helping in engaging the whole of the filling material from the area . The hand files also provide an unparallel tactile feel to the operator thus helping in understanding a better engagement of the filling material at the apical region . Unfortunately, one limitation of this study is that, an in - vitro study does not provide the same conditions as that of an in - vivo study, even though all the steps were taken to reduce as much errors as possible . A standardized method of root canal preparation was employed to minimize the variation among the study groups in relation to the quantity of gutta - percha that will be removed in the study . The obturation of the samples was performed by thermoplastic obturation technique to attain a homogenous mass of gutta - percha, which eliminates pools of entrapped sealer in the filling and also eliminates any loose filling at the apical third . The limitation of the study points toward the need of further research on the subject and technique of gutta - percha removal . Further, research should be oriented in reproducing more in - vivo conditions for more accurate results and to help the clinician in implementing the techniques for a more effective retreatment procedure . Recent studies have compared not just the rotary files systems in root canal obturation material removal but also the efficacy of reciprocating systems like the waveone and reciproc systems are studied . One study has concluded that both wave one and reciproc when compared were effective but did not completely remove the obturating material from the root canals . These reciprocating systems are no different compared to the rotary systems in gutta - percha removal . One big advantage of reciprocating systems is that it does not extrude apical debris as much as rotary systems do during retreatment procedures . Most of the research concerning retreatment procedures is aimed at establishing the type of system that is more effective in gutta - percha removal, or the type of technique that is faster in the gutta - percha removal . However, in the middle of all this it is important to guide the clinician in employing the type of movement or motion in which the systems are to be used for complete gutta - percha removal . It is most effective if an adaptive motion is employed, by engaging all the sides of the root canal when the retreatment procedure is done, compared to a rotation movement, irrespective of the file system used . It can be concluded that the effective removal of root canal filling material might not be achieved by the use of one system or method . A more effective way of an endodontic retreatment would be the use of both the rotary and hand file systems . The rotary system would help us in achieving the complete removal or filling material form the cervical and middle one third as well as help us in reaching the apical region faster compared to the use of hand files in these areas; the final apical region can be debrided by the use of hand files, thus completing the filling material removal without leaving behind any residual filling materials.
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In recent years, with economic development and social modernization, the number of diabetic patients has been increasing worldwide, including developing countries, posing a global problem in terms of human suffering as well as medical costs [1, 2]. The international diabetes federation (idf) estimated the number of diabetic patients to be 366 million in 2011, and that it will reach 552 million by 2030 unless effective measures are taken . At present, complex interactions between genetic factors and environmental factors in the pathogenesis of diabetes are undergoing extensive study . However, the relationships between genetic and environmental factors are difficult to verify, and direct obtainment of information from humans has intrinsic limitations due to significant ethical restrictions . The use of experimental animal models is essential to resolve these problems, and results of basic research in animal models of diabetes may be useful to clarify the pathogenetic mechanism of human diabetes as well as the causes of complications and in the development of drugs for diabetes . In fact, many animal models of diabetes have contributed to clinical research of diabetes . It is important to develop animal models that correspond to various pathological conditions of human diabetes, and it is urgently necessary to develop models of diabetic complications that can reflect human diabetes, because the number of patients with type 2 diabetes (t2d) is rapidly increasing and progression of complications significantly affects the prognosis of diabetic patients . Although there are many t2d model animals such as goto - kakizaki (gk) rats, zucker diabetic fatty (zdf) rats, and otsuka long - evans tokushima fatty (oletf) rats, and these animal models show diabetic complications, severe diabetic retinopathy has not been observed in the existing models . In these circumstances, spontaneously diabetic torii (sdt) rat (figure 1) has been established as a model of nonobese t2d with three major complications, including ocular complications [46]. This paper provides an overview of the findings from sdt rats such as pathology of diabetes . In 1988, shinohara found five nonobese diabetic rats with polydipsia, polyphagia, polyuria, and sugar urine among approximately 12-month - old elderly male sprague - dawley (sd) rats, which were bred at the laboratory of torii pharmaceutical co., ltd . (305 males and 306 females) after purchase from charles river laboratories japan inc . For long - term studies of spontaneous lesions . These animals were mated with young normal female rats of the same strain to successfully generate diabetic f1, and then attempts were made to preserve the diabetic trait in a closed colony (figure 2(a)). In 1991, some animals in the diabetes - preserved colony developed diabetes at 4 to 5 months of age, leading to sib mating based on positive urine sugar in male rats . In 1997, a new inbred strain of nonobese t2d rats was established and named sdt rat [46]. In the process of strain breeding, the prevalence of diabetes in male rats was 90% or more in the f4 generation and 100% in the f9 and subsequent generations . Diabetes tended to occur earlier in later generations and occurred at approximately 4 months of age in the f7 . This strain of rats was characterized by the development of diabetes only in males since its discovery, but the disease was sporadically observed in females aged 9 months or older in the f7 and subsequent generations . Currently, sdt rat is distributed by clea japan, inc . A clear sex difference is observed in the onset of diabetes in sdt rats (figure 2(b)). While males developed diabetes at approximately 20 weeks of age with a cumulative incidence of 100% at 40 weeks, females developed it at 45 weeks with a cumulative incidence of as low as 33% at 65 weeks . It is suggested that this sex difference may be partly attributed to estrogen, which inhibits the development of diabetes in females . The survival rate at 65 weeks was 92% in males and 97% in females, showing that the rats survive hyperglycemia without insulin treatment (figure 2(c)). The fasting and nonfasting blood glucose levels markedly increased at 20 weeks and thereafter, reaching 700 mg / dl or more at 30 weeks with polyuria characterized by severe sugar urine as well as polydipsia / polyphagia (figure 2(d)). In sdt rats, development of hyperglycemia may be more dependent on decreased insulin secretion than insulin resistance, as shown by the fact that the blood insulin concentration tended to be lower than that in normal sd rats even before the onset of diabetes, and marked hypoinsulinemia developed after the onset of hyperglycemia [8, 9], indicating that this strain of rat is a model of nonobese t2d associated with impaired insulin secretion . Compared with normal sd rats, body weight and body - mass index (bmi) were similar before the onset of diabetes, but decreased with age after the onset (figure 2(e)) [6, 8, 10]. It is clinically known that glucose tolerance decreases before the onset of t2d . In oral glucose tolerance test (ogtt) in sdt rats, glucose tolerance markedly decreased at least 2 months before manifestation of hyperglycemia (around 14 weeks old), and the rate of rise in blood sugar level after glucose load increased with age . In male rats, the severity of impaired glucose tolerance before the onset of diabetes was closely correlated with the age at onset of disease . Impaired glucose tolerance was related to decreased insulin secretory response after glucose load, and decrease in the fasting plasma insulin concentration and loss of insulin secretory response after glucose load were observed after the onset of diabetes (figure 3) [8, 11]. In addition, the insulin secretion level in pancreatic -cells from sdt rats after glucose treatment markedly decreased at 12 weeks of age and thereafter compared with normal sd rats . Likewise, the mrna expression levels for glut2 and glucokinase in the isolated pancreatic islets markedly decreased at 12 weeks and thereafter in sdt rats . In female rats, glucose tolerance also decreased at 25 weeks and thereafter, but insulin was secreted after glucose load, indicating that some factors cause insulin resistance or insulin requirement in the females, unlike in the males . It has also been reported that increased insulin secretion from hypertrophic pancreatic islets delayed the onset of hyperglycemia in high - fat diet - fed sdt rats . As for the biochemical parameters, urine protein, blood urea nitrogen (bun), glycated hemoglobin (hba1c), and triglycerides (tg) markedly increased with the development of hyperlipidemia at 35 weeks of age and thereafter . In male rats, the blood tg concentration after fat load was high with normal tg absorption from the small intestine before the onset of diabetes, suggesting that the tg clearance is already impaired before the onset of disease . It is also suggested that not only the impairment of tg clearance, but also increased tg absorption from the small intestine occurs after the onset of disease . In addition, increased tg absorption may result from the physical increase in tg inflow associated with diabetes - related hyperphagia - induced hypertrophy of the small intestine as well as the increase in enzymes involving in tg absorption in the small intestine [15, 16]. Plasma ghrelin levels, an orexigenic hormone, of sdt rats were significantly higher than those of sd rats at 38 weeks of age . Active ghrelin production and suppression of insulin or leptin may be concerned with diabetic hyperphagia . In female rats as well, free fatty acids and tg were higher at 25 weeks of age before the onset of diabetes, compared with normal sd rats . Sdt rats fed high - sucrose diet showed dyslipidemia and insulin resistance; however, the incidence of hyperglycemia was suppressed . The milder degrees of pancreatic abnormalities in high - sucrose fed sdt rats may be considered as the reason . As for the glucose metabolism - related enzymes in the liver, glucokinase mrna level and glycogen content in the liver decreased in sdt rats at 16 weeks of age, suggesting that glucose metabolism in the liver is already abnormal before the onset of diabetes . After the onset of disease, mrna expression of gluconeogenesis enzymes such as phosphoenolpyruvate carboxykinase (pepck), fructose-1,6-bisphosphatase (fbpase), and glucose-6-phosphatase (g6pase) increased [15, 19]. In sdt rats, the number of pancreatic islets and the area of -cells decreased with almost normal glucose tolerance at 10 weeks of age, compared with normal sd rats of the same age . Around 8 weeks, pancreatic islets with congestion and capillary dilation were sporadically found with those with hemorrhage and edema in the same sections (figures 4(a) and 4(b)). Later, probably accompanying findings such as inflammation and fibrosis in or around the pancreatic islets extended, and fibrosis, hemosiderin deposition and marked decrease in -cells were observed in almost all pancreatic islets at 20 weeks (figures 4(c) and 4(d)). In sdt rats that developed diabetes, atrophy of pancreatic islets occupied by collagenous fibers and virtual disappearance of -cells was observed (figures 4(e) and 4(f)) [5, 6, 8]. These changes in pancreatic islets starting from hemorrhage were observed in female rats around the same time with those in males . Higher sensitivity to streptozotocin (stz), that has selective toxicity to pancreatic -cells, is also suggesting a pancreatic weakness of sdt rats . During the course of the disappearance of -cells, no lymphocyte infiltration was observed, unlike in type 1 diabetes (t1d) models such as nonobese diabetic (nod) mice or biobreeding (bb) rats, but the concentration of interleukin-18 (il-18), an inflammatory cytokine, transiently increased at 9 weeks, resulting in a corresponding increase in interferon gamma (ifn-) and nitric oxide (no) production by spleen cells and peripheral leukocytes, respectively, as well as macrophage infiltration around the pancreatic islet tissue . In sdt rats, the number of white blood cells is increased at 8 weeks . It was immunohistologically found that the il-18 receptor and inducible no synthase (inos) were expressed in pancreatic islet cells . These findings indicate that the development of diabetes in sdt rats may be due to the damage of pancreatic islets resulting from a transient increase in the il-18 concentration through direct effects on the cells and secondary effects via local macrophage infiltration . Pancreas transplantation is generally performed in patients with t1d, but exceptionally in those with t2d, improving insulin sensitivity in both cases . In sdt rats, it is suggested that the elimination of glucose toxicity following pancreas allotransplantation may induce the pancreatic expression of pancreatic and duodenal homeobox 1 (pdx-1), a homeodomain transcription factor, inhibiting the destruction of pancreatic islets and promoting the regeneration of pancreatic islets and -cells [23, 24]. Based on the results of genetic analyses using two control strains, seven quantitative trait loci (qtls) involved in the impairment of glucose tolerance are currently mapped on the rat genome (table 1) [2527]. In a backcross experiment with brown norway (bn) rats, qtls involved in the impairment of glucose tolerance in sdt rats were identified on chromosomes 1, 2, and x, which were named gisdt1, gisdt2, and gisdt3, respectively . It is found that homozygosity or hemizygosity for the sdt allele in each of these qtls markedly increases the risk of hyperglycemia (diabetes), and the interactions between the qtls synergistically worsen glucose intolerance . In an intercross experiment with f344 rats, furthermore, qtls involved in the impairment of glucose tolerance in sdt rats were identified on chromosomes 3, 8, 13, and 14, which were named dmsdt1, dmsdt2, dmsdt3, and dmsdt4, respectively . To evaluate the effects of these qtls on the development of diabetes, test was performed using f2 rats with normal glucose tolerance and those with diabetes, showing that dmsdt1 is the most influential on the development of diabetes . Subsequently, congenic rats were generated by transferring dmsdt1 to f344 rats, and histological analysis was performed, revealing histopathological changes such as inflammation and fibrosis in the pancreas in the congenic rats . These results show that dmsdt1 is the major locus responsible for pancreatic lesions in sdt rats . Of many diabetic ocular complications, retinopathy, cataract, and neovascular glaucoma (hemorrhagic glaucoma) are the most important clinically . Sdt rat is the first diabetic model with all of these complications [5, 6, 2832]. Further progression of the disease was characterized by proliferative retinopathy, with tractional retinal detachment primarily in the optic disc due to fibrovascular membrane resulting from retinal neovascular vessels (figure 5(a)) [5, 6, 2832]. The vascular pathological examination by trypsin digestion method showed a few capillary aneurysms, but revealed capillary narrowing and pericyte loss in sdt rats (figure 5(b)). In fluorescein angiography, abnormal retinal vasodilatation was observed in some animals and may correspond to venous beading in human retinopathy [5, 2832]. In addition, severe fluorescein leakage almost corresponding to the area affected by tractional retinal detachment [2832] (figures 5(c) and 5(d)) was revealed . The prevalence of diabetic retinopathy was 8% at 35 to 50 weeks of age, but increased to approximately 80% at 51 to 60 weeks and 100% at 61 to 82 weeks . At 44 weeks, electroretinogram (erg) revealed the delay and reduction of oscillatory potentials (ops) and a- and b - waves [31, 33], as is the case with human diabetic retinopathy . It is known that not only microangiopathy, but also neurodegeneration occurs in the human diabetic retina . In comparison of changes in the death of neuroretinal cells as well as expression of glial fibrillary acidic protein (gfap: a marker protein for glial cells) and water channel aquaporins (aqps) over time in sdt rats, the aqp expression profile in astrocytes in the nerve fiber layer shifted from aqp-4 to aqp-1 in the retinas of sdt rats at 40 weeks, when the apoptosis of retinal ganglion cells (rgcs) was accelerated . Aqp-0 was predominantly expressed in the bipolar cells of the nondiabetic rat, whereas it was also expressed in the retinal nerve fibers of diabetic rat . The disrupted water transport between astrocytes and retinal nerve fibers may be associated with the apoptosis of rgc induced by diabetes [34, 35]. In sdt rats, the angiotensin ii (aii) concentration in circulating blood was low at 15, 30, and 45 weeks, but the angiotensin - converting enzyme (ace) activity specifically increased in the eye without change in the aortic ace activity at 45 weeks . In addition, continuous treatment with aii resulted in increased retinal expression of the vascular endothelial growth factor (vegf) gene . These findings suggest that specifically increased aii formation in the eye may play an important role in retinal vegf expression in sdt rats . Furthermore, advanced glycation end products (ages) such as carboxymethyllysine (cml) were expressed with vegf in the retina and may be involved in retinopathy in sdt rats . On the other hand, angiogenesis was observed with vegf expression, but it has been reported that retinal neovascularization is not associated with retinal nonperfusion in sdt rats, unlike human diabetic retinopathy . Unlike human diabetic retinopathy, the retinal capillary bed is hardly obstructed in sdt rats, indicating that increased expression of the pigment epithelium - derived factor (pedf) results in the suppression of diabetic retinal vascular disorder and less obstruction of the retinal capillary bed in sdt rats . In human retinopathy, severely advanced retinal ischemia is finally associated with angiogenesis in the iris and anterior chamber angle, presenting with neovascular glaucoma . In sdt rats as well, advanced retinopathy is associated with fibrovascular membrane around the iris and sometimes with anterior chamber hemorrhage . Neovascular membrane around the pupil may cause posterior synechiae and might develop neovascular glaucoma finally . Sdt rat is a first model of iris neovascularization and consequent neovascular glaucoma [5, 6, 28, 29] (figure 5(e)). In male sdt rats, the prevalence of cataract is virtually 100% at 40 weeks of age . Starting with opacity at the posterior pole of the lens, the findings of mature cataract are finally observed (figures 5(f) and 5(g)). Pathological findings include swollen lens fibers, liquefaction, vacuolation, abnormal configuration, and formation of morgagnian droplets as well as partial proliferation of fibroblastoid cells . Advanced cataract is associated with capsular rupture, probably related to swollen lens [5, 6, 2832]. These ocular complications in sdt rats have been shown to be prevented by normalizing blood glucose with insulin treatment or pancreas transplantation and demonstrated to result from the long - term exposure to high blood glucose [31, 40]. Corneal disorder, optic neuropathy, and uveitis are also known as ocular complications in t2d . Though uveitis is not observed in sdt rats, corneal disorder and optic neuropathy are not well investigated . In sdt rats, renal lesions appeared at 24 weeks of age, including the thickening of the glomerular loop and glycogen deposition in the tubular epithelium (armanni - ebstein lesion), dilatation of the renal tubule lumen, and increased hyaline casts . As for the glomerular lesions, slight thickening of the loop was apparent at 24 weeks and consistent with mesangial proliferation as shown by pas, masson's trichrome stain, and type iv collagen immunostaining (figures 6(a)6(f)). Mesangial proliferation intensified with age, and nodular lesions (kimmelstiel - wilson - like nodules) suggestive of more severe glomerular lesions were slightly observed at 68 weeks (figure 6(g)). On the other hand, the renal tubular lesions markedly increased with age, with a severe increase in tubular glycogen deposition at 50 and 68 weeks (figure 6(h)). In addition, urine volume, urine protein, and urine albumin increased with blood glucose at 24 weeks and thereafter, and these changes may be consistent with the development and progression of renal lesions [41, 42]. These renal lesions were also improved by blood glucose control with insulin and thus shown to result from the exposure to high blood glucose [41, 42]. In a study evaluating the involvement of oxidative stress and no in the mechanism for the progression of diabetic nephropathy in sdt rats, the blood asymmetric dimethylarginine (adma) concentration and urinary excretion of oxidative stress markers 8-hydroxydeoxyguanosine (8-ohdg) and nitrogen oxide (nox) increased in sdt rats at 36 weeks, compared with insulin - treated sdt rats and normal sd rats . In addition, renal tissue analysis revealed glomerular hypertrophy and mesangial proliferation, and immunostaining analysis showed that the glomerular 8-ohdg, endothelial no synthase (enos), and nitrotyrosine scores increased . In sdt rats, enos and no increased despite the increase in adma and may thus play an important role in the progression of diabetic nephropathy together with oxidative stress . Metformin, an amp - activated kinase (ampk) activator, decreased renal 8-ohdg levels and subsequent podocyte loss, in spite of the limited effects on hyperglycemia . Both motor nerve and sensory nerve are impaired under in diabetes . In an electrophysiological and morphological study of diabetic peripheral neuropathy (dpn) in sdt rats, the motor nerve conduction velocity (mncv) was not different from that in normal sd rats until 6 months of age, but gradually decreased thereafter to 82% and 76% of that in normal sd rats at 10 and 12 months, respectively (figure 7(a)). Increased nerve sorbitol and fructose contents and decreased myo - inositol contents in sdt rats indicate that the polyol pathway is prominently involved in dpn . Ranirestat, an aldose reductase inhibitor (ari) decreased sciatic nerve sorbitol levels and improved impaired sciatic mncv . In the sural nerve cross - section, no neurologic deficit was observed, but degenerated nerves increased in sdt rats . In morphometry, the myelinated nerve area was not clearly different between the two groups at 6 months, but decreased in sdt rats at 12 months compared with normal sd rats . The number of blood vessels in the nerve sheath was not clearly different; however, occluded / thickened epineurial arterioles were found in sdt rats (figures 7(b)7(e)) [45, 47]. The increased intima possibly results decrease of nerve perfusion and may contribute to development of dpn in sdt rats . In summary, it is shown that sdt rats develop peripheral neuropathy associated with t2d after the onset of disease, including functional / morphological abnormalities of peripheral nerves and vascular lesions . Autonomic nerve is part of the peripheral nervous system and transmits impulses from the central nervous system to peripheral organ systems . In diabetes, autonomic nerve symptoms probably due to diabetic diarrhea are observed in sdt rats . In charcoal propulsion test, gastrointestinal motility increased in sdt rats with higher fecal water content at 28 weeks of age in addition, jejunum and ileum weights and mucosal weight increased, and the lumen diameter and villous height were longer indicating that more nutrients are absorbed with longer villi in diabetes [15, 16, 48]. In a study of voiding dysfunction in sdt rats, voiding pressure, voided volume per micturition, and intermicturition interval tended to increase from 22 weeks to 36 weeks of age compared with normal rats . Immunodeficiency, delayed wound healing, skin ulcer, and osteoporosis are well known . Among these complications, osteoporosis has been reported in sdt rat . In a study of bone lesions in sdt rats with focus on bone density and bone morphometry, bone formation and resorption decreased in sdt rats at 36 weeks of age compared with normal sd rats, but improved in insulin - treated sdt rats . Bone density and strength also decreased in sdt rats compared with normal sd rats (figure 8). Bone lesions in sdt rats were characterized by decreased bone density and low - turnover bone lesions, as seen with t2d primarily due to decreased insulin secretion, and improved with insulin treatment, indicating the deep involvement of diabetic pathology [50, 51]. The study treating carvedilol, a blocker possessing an antioxidant effect, is also suggesting the involvement of oxidative stress on this low - turnover bone disease in sdt rats . Currently, sdt rats are used for the development and application of several diabetic drugs . In addition to insulin [31, 41], sulfonylurea (tolbutamide) and dpp iv inhibitor (jtp-76209), -glucosidase inhibitor (voglibose), sglt inhibitor (phlorizin) [15, 54], and perilla (shiso) tea lowered the blood glucose level of sdt rats . It has been reported that diabetic microangiopathy is caused by increased tissue protein kinase c - beta (pkc-) activity at high blood glucose levels . In sdt rats at 32 weeks of age, abnormal retinal function such as delayed ops in erg were observed . In addition, peripheral and autonomic neuropathies such as decreased caudal mncv, electrocardiographic coefficient of variation of r - r interval (cvr - r), and thermal hypoalgesia were observed . These diabetic complications were improved after 12-week treatment with a pkc- inhibitor jtt-010 . However, histopathological changes including retinal thickening primarily in the optic disc at 68 weeks of age were not improved . Since the tissue pkc activity increased after the onset of diabetes in sdt rats, jtt-010 may have suppressed diabetic neuropathy by inhibiting the pkc- activity . However, the retinal histopathological findings were not affected in sdt rats that developed diabetes along earlier, indicating that factors other than pkc- activation are deeply involved in the progression of ocular complications in sdt rats . Benfotiamine, a transketolase activator that reduces major pathways involved in diabetic microvascular complications (polyol pathway, hexosamine pathway, age pathway, and diacylglycerol - protein kinase c (dag - pkc) pathway) also exhibits effects on peripheral nerve function in sdt rats . In a large - scale clinical study, it was reported that candesartan, an aii type 1 receptor blocker (arb), inhibited the progression of retinopathy in type 2 diabetic patients . In an efficacy study of an telmisartan for the progression of ocular lesions in sdt rats, the blood glucose level was not changed, but blood pressure was decreased by telmisartan . Under these conditions, delayed ops and a - wave in erg were prevented by telmisartan . In fluorescein fundus angiography, fluorescein leakage in sdt rats was decreased by telmisartan, suggesting that the arb may inhibit the development of proliferative retinopathy in sdt rats . It has also been reported that arbs (candesartan and olmesartan) improved coronary angiogenesis, cardiomyocyte fibrosis, and hypertrophy associated with the progression of diabetes in sdt rats [59, 60]. In addition, candesartan decreased the pentosidine, a biomarker for age, content in the lens / vitreous body in sdt rats at 44 weeks of age, and immunohistologically, it inhibited the accumulation of pentosidine in the retinal vascular wall and decreased retinal vegf mrna expression . These findings indicate that arbs can inhibit the development of proliferative diabetic retinopathy by inhibiting age formation . Furthermore, it has been reported that cataract and retinopathy in sdt rats were prevented by ari fidarestat and ranirestat, age inhibitor aminoguanidine, and 1/ blocker nipradilol . With application of gene therapy, the soluble vegf receptor (sflt-1) gene was introduced into the retina in sdt rats to evaluate the preventive effect of sflt-1 expressed in the retina against diabetic retinopathy . At 57 weeks of age, fluorescein fundus angiography revealed that the development of retinopathy was inhibited in the retina engineered to express sflt-1 using an adeno - associated virus (aav) vector as compared with the contralateral nave retina . Since the local introduction of sflt-1 gene in the retina with the use of an aav vector is effective in preventing the development of retinopathy in sdt rats, gene therapy for diabetic retinopathy with antiangiogenic factors will be a promising therapeutic option for human patients [65, 66]. Proliferative retinopathy, especially, resulting from retinal neovascular vessels is a unique characteristic of this model . In addition, diabetic neuropathy (e.g., mesangial proliferation, nodular lesion) and diabetic peripheral / autonomic nephropathy (e.g., decreased ncvs, hypoalgesia, diarrhea, and voiding dysfunction) seem to be caused by postprandial long - term hyperglycemia in sdt rats . Bone disorder such as decreased bone density and low - turnover bone lesions are also observed . At younger age, glucose intolerance, caused by pancreatic islet lesions with inflammatory cell infiltration and fibroblasts, is also a characteristic of this animal . Taking advantage of these features, sdt rat has been used for evaluating antidiabetic drugs and drugs / gene therapy for diabetic complications . Although there are insurmountable discrepancies between human and rodents, sdt rat seems to be a better animal model of diabetes than other models . In conclusion, findings indicate that the sdt rats should be a potential t2d model for studies on the pathogenesis and treatment of diabetes and its complications.
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Alopecias can be broadly classified into non - scarring or non - cicatricial and scarring or cicatricial forms . Pathologically, a scar constitutes the end point of reparative fibrosis with permanent destruction of the preexisting tissue.1 scarring alopecias are further subdivided into primary and secondary types . In primary scarring alopecias, the hair follicle is the primary target of destruction, which is microscopically evident as preferential destruction of follicular epithelium and/or its associated advential dermis with relative sparing of the reticular dermis.2 this group includes the following clinical entities: chronic cutaneous lupus erythematosus (ccle), lichen planopilaris (lpp), pseudopelade of brocq (pb), folliculitis decalvans (fd), dissecting cellulitis / folliculitis (df). If the follicular destruction is secondary to the scarring and occurs outside the follicular unit, e.g., the reticular dermis, epidermis, or sub cutis, it can eventually impinge upon and even eradicate the whole follicle . The term secondary scarring alopecia implies that follicular destruction is not the primary pathologic event . Exogenous factors, such as burns, and endogenous infiltrative and inflammatory diseases, such as sarcoidosis, pemphigus vulgaris and reticular dermal sclerosis, can result in secondary alopecias . In 2001, a group of hair clinicians, pathologists and researchers, under the rubric of the north american hair research society (nahrs),3 issued a consensus opinion on the classification of primary cicatricial alopecias . The proposed classification subdivides primary scarring alopecias on the basis of the predominant type of inflammatory cell component, an approach that had already published1,4,57 and was further refined by the workshop . In addition to the lymphocyte- and neutrophil - associated subgroups, a mixed and nonspecific group was differentiated and newly defined (table 1). In the late phase of cicatricial alopecias, a histopathologic diagnosis is more difficult because the main criteria of classification, i.e., the type of inflammatory infiltrate, cannot always be evaluated . In these cases, additional criteria, such as the evaluation of the perifollicular elastic sheet and the fibrosis, the goal of this study was to review clinical and histopathologic findings of 38 patients diagnosed with late, pauci or non - inflammatory phase of cicatricial alopecias at the dermatologic department of the hospital das clinicas, so paulo university medical school, over a six - year period in order to classify them by sub - types according to nahrs and evaluate the dermal elastic system and thickness of the epidermal basement membrane, using weigert and pas stains, respectively . Biopsy specimens obtained using 5-mm punches from patients seen between 2000 and 2005 at the dermatologic department of hospital das clinicas, so paulo university medical school with cicatricial alopecias in the late, pauci or non - inflammatory phase were reevaluted . Essential criteria included histopathologic diagnosis of cicatricial alopecia and accessibility of the patient s clinical records . Slides stained with hematoxylin and eosin, pas and stain for elastic tissues (weigert with previous oxidation by peracetic acid) were reviewed, evaluating the presence or absence of criteria listed in the table 2 . A total of 38 cases with a histopathologic diagnosis of cicatricial alopecia were included in the study . These cases had been previously diagnosed as follows: eight cases of ccle, two cases of lpp, 13 cases of pb, two cases of fd, one case of scleroderma and 13 cases of non - specific cicatricial alopecia . Twelve of 13 cases (92.3%) that were initially classified as non - specific cicatricial alopecias could be specifically reclassified . Chronic cutaneous lupus erythematosus was diagnosed in 17 cases, representing 43.6% of the total number of scarring alopecias . The female to male ratio was 4.7: 1 (14 females and three males). The age at onset ranged from 29 to 75 years, with a mean age of 46.5 years . Characteristic histopathologic findings of the late phase included hyperkeratosis, horn plugs, atrophy of the malpighian layer, slight vacuolar degeneration of the basal layer, and fibrous tract replacing the follicles . A thickened basal membrane could be seen in 58.8% of the cases on pas stained sections, and an incomplete elastic sheet occurred around the fibrous tracts . In all cases on weigert- stained sections . Elastolytic foci were also found in areas of fibrosis outside the perifollicular zone (figure 1d f). This category of cicatricial alopecia represented 10.2% of the total number of biopsies, totaling four cases . In one case, there was a lichenoid infiltrate around the infundibuloisthmic segment of one follicle in addition to fibrous tracts replacing follicles . In all the others, absence of inflammation, absence of sebaceous epithelium, and atrophy of the bulge area occurred, resulting in hourglass figures . Fibrous tracts replacing the follicle, with or without the presence of colloid bodies, were also observed . The basal membrane was not thickened on pas - stained sections, and the perifollicular elastic sheet was partially destroyed, as observed with the weigert stain (figure 1g pseudopelade of brocq was diagnosed in 12 cases, representing 30.8% of the total number of biopsies . Age of onset ranged from 23 to 69 years, with a mean age of 54.4 years . Characteristic histopathologic findings were the absence of criteria seen in ccle and lpp and preservation of the elastic sheet around the follicles (figure 1a c). Folliculitis decalvans was diagnosed in three cases, representing 7.7% of the total number of biopsies . The histopathologic hallmark was the presence of superficial suppurative folliculitis and fibrosis replacing the follicle and the perifollicular area, with elastolysis visible with the weigert stain (figure 2a c). This category of cicatricial alopecia represented 2.6% of the total number of cicatrizing biopsies, totaling one case in a female 19 year - old patient . In this case, the inflammation was slight, and there was an extensive and deeper dermal fibrosis extending to the hypodermis, with elastolysis (figure 2d f). One case of non - specific alopecia was diagnosed due to the destruction of the follicles being the only histopathologic finding . This category of cicatricial alopecia represented 10.2% of the total number of biopsies, totaling four cases . In one case, there was a lichenoid infiltrate around the infundibuloisthmic segment of one follicle in addition to fibrous tracts replacing follicles . In all the others, absence of inflammation, absence of sebaceous epithelium, and atrophy of the bulge area occurred, resulting in hourglass figures . Fibrous tracts replacing the follicle, with or without the presence of colloid bodies, were also observed . The basal membrane was not thickened on pas - stained sections, and the perifollicular elastic sheet was partially destroyed, as observed with the weigert stain (figure 1g pseudopelade of brocq was diagnosed in 12 cases, representing 30.8% of the total number of biopsies . Age of onset ranged from 23 to 69 years, with a mean age of 54.4 years . Characteristic histopathologic findings were the absence of criteria seen in ccle and lpp and preservation of the elastic sheet around the follicles (figure 1a c). Folliculitis decalvans was diagnosed in three cases, representing 7.7% of the total number of biopsies . The histopathologic hallmark was the presence of superficial suppurative folliculitis and fibrosis replacing the follicle and the perifollicular area, with elastolysis visible with the weigert stain (figure 2a c). This category of cicatricial alopecia represented 2.6% of the total number of cicatrizing biopsies, totaling one case in a female 19 year - old patient . In this case, the inflammation was slight, and there was an extensive and deeper dermal fibrosis extending to the hypodermis, with elastolysis (figure 2d f). One case of non - specific alopecia was diagnosed due to the destruction of the follicles being the only histopathologic finding . Considering the group of primary lymphocytic cicatricial alopecias in the inflammatory phase, a differential diagnostic consideration for ccle is lpp . The superficial and deep perivascular and periecrine patterns of inflammation further aid in differentiating ccle from lpp . In the late pauci or non - inflammatory phase, as observed in our cases of ccle, interfollicular epidermis alterations were evident (hyperkeratosis and atrophy), in an area usually spared in lpp cases . Commonly, in the dermis in lpp, the only disturbance was the fibrous tract replacing the follicle, while in ccle, there was frequent extra follicular fibrosis and elastolysis; even in this late phase, the basement membrane was thickened in 58.8% of cases . Ccle is differentiated from pb by the presence of predominately vacuolar interface changes at the level of follicular infundibulum in the former and lack of interface alterations in the latter.1 in our cases, the perifollicular elastic sheet was preserved in pb cases, while it was partially destroyed in lpp and ccle, confirming the previous findings of pinkus et al.7 some authors believe that pb is not a distinct clinical - pathologic entity but a variant of certain primary cicatricial alopecias or, alternatively, a form of end - stage alopecia caused by other scarring alopecias, such as ccle, lpp and fd.79 according to stephen (1993)1 and other authors, with whom we agree, pb does indeed have sufficient distinct pathologic features to merit a separate classification.5,1012 klaus and wilma (2006)1 believe that the close clinicopathlogic correlation in the histologic absence of significant follicular plugging, as well as the use of elastic fiber stain in addition to evaluation of conventional he - stained sections, should enable the differentiation of classic pb from late stage lesions of lpp and ccle in most cases and further justify the classification of the condition as an entity sui generis . In relation to the neutrophilic cicatricial alopecias, it is remarkable that even in the late phases of this disorder, it seems that the inflammatory process persists in our cases,, and a neutrophilic folliculitis can frequently be seen, which helps make the correct histopathologic diagnosis . We could also see that the fibrotic process that is secondary to this neutrophilic folliculitis was more intense and proceeded by a granulation tissue, as is expected after a suppurative process . Sinus tracts are the histopathologic hallmark of dissecting cellulites and are not seen in either folliculitis decalvans or acne keloidalis and were not observed in our late phase cases . Bacterial or fungal folliculitis (kerion and favus) may have to be excluded in the group of neutrophilic cicatricial alopecias, with the use of special stains . Acne keloidalis cases were not seen in our series because they are histopathologically reported in our laboratory as a superficial and deep suppurative and giant cellular granulomatous folliculitis with extensive fibrosis compatible with acne keloidalis, not as a cicatricial alopecia . Besides, the clinical aspect of this disorder is so typical that histopathologic exam is seldom required . A scalp biopsy is mandatory in all cases of cicatricial alopecias, representing the clinically inflammatory area . If there is no evidence of inflammation, a biopsy at the border of the cicatricial zone should be performed . A precise diagnosis is possible, even in the late, pauci or non - inflammatory phase of cicatricial alopecias if a systematic evaluation of a constellation of criteria is employed, using routine he, pas and a stain for elastic tissue . In our cases, this method allowed a precise diagnosis in 97.4% of cases, even in those initially considered non - specific cicatricial alopecia.
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Whether signals mediated via growth factor receptors (gfrs) might influence lineage fate in normal multipotent progenitors (mpps) and stem cells remains unclear and disputed . New insights into this process have recently been gained through studies in vitro (rieger et al ., 2009); however, whether gfr signaling instructs lineage specification in vivo remains a key unresolved issue (enver and jacobsen, 2009). Studying the impact of gain - of - function mutations is an alternative approach to determine whether and how gfrs might instruct lineage fate in vivo . Indeed, activating mutations of gfrs and downstream signaling pathways are common events in cancer, particularly in hematopoietic malignancies such as acute myeloid leukemia (aml), and often show striking associations with distinct clinical and cell - lineage phenotypes (croce, 2008). In some cases, this might simply reflect the fact that such mutations primarily target a specific cell lineage . However, more intriguingly, if a mutation targets a primitive multipotent cell, it might instruct lineage - fate decisions . As recent investigations of human aml have suggested that the propagating cell might frequently represent the counterpart of normal mpps (goardon et al ., 2011), it is possible but still unclear to what degree gfr signaling mutations might also influence lineage specification in multipotent cells . A good example of aberrant gfr signaling associated with specific leukemia phenotypes relates to the fms - like tyrosine kinase 3 receptor (flt3), which is expressed in the majority of cases of aml and acute lymphoblastic leukemia (all) (stirewalt and radich, 2003). Constitutively activating internal tandem duplications (itds) within the juxtamembrane domain of flt3 occur in 25% of cases of aml, conferring an adverse prognosis (stirewalt and radich, 2003). However, despite the high frequency of flt3 expression in all and the key role of flt3 in early lymphoid development (sitnicka et al ., 2002), including high - level flt3 expression in lymphoid - primed mpps (lmpps) with combined lymphoid and myeloid potential (adolfsson et al ., 2005), flt3-itds are rare in cases of all, occurring in <1% in larger series (leow et al ., 2011). As is the case for most leukemic mutations, it is unclear how often flt3-itds are a true initiating event in myeloid malignancies . Indeed, a number of lines of evidence support the notion that flt3-itds are often acquired secondarily to an initiating clonogenic event (gale et al ., 2008; jan et al ., 2012), although when involved in chronic myelomonocytic leukemia (cmml), they may indeed be the initiating mutation (lee et al ., 2007) nevertheless, in line with the normal expression pattern of flt3 (adolfsson et al ., 2005), results from studies in patients are compatible with the notion that flt3-itds occur within the human mpp compartment (levis et al ., 2005) and are an essential driver mutation within the founding leukemic clone (ding et al ., 2012; smith et al ., thus, even though flt3-itds might occur secondarily to an initiating clonogenic event in many cases, these mutations occur in multipotent cells (levis et al ., 2005) and flt3-itd gfr signaling appears to be an essential requirement for leukemia propagation (smith et al ., 2012). It is possible, therefore, that flt3-itds may act to dictate the lineage fate and phenotype of the resulting leukemia . Compatible with a role of flt3-itd signaling in lineage determination, two different knockin mouse models of flt3-itd have been reported to develop a myeloproliferative phenotype exclusively (lee et al . . However, although it was recently suggested that flt3-itds deplete hematopoietic stem cells (hscs) (chu et al ., 2012), the key progenitor population that propagates flt3-itd - induced myeloid disease, as well as the cellular and molecular bases of their myeloid lineage bias, remains unclear . Using a mouse knockin model of the flt3-itd mutation, we investigated the cellular and molecular mechanisms by which constitutive gfr signaling might subvert lineage specification in mpps and alter the cell fate of early lymphoid progenitors, in order to explain the myeloid bias of the resulting leukemias . To definitively determine whether physiologically expressed flt3-itd impacts the establishment of myeloid versus lymphoid leukemia development, we crossed flt3 with runx1 (growney et al ., 2005) and mx1-cre mice to induce runx1 deletion in mpps . Importantly, runx1 loss - of - function mutation is associated with both lymphoid and myeloid leukemia (grossmann et al ., 2011; schnittger et al ., unexpectedly, even without poly i: c induction, flt3runx1mx1-cre mice developed a high - penetrance, short - latency acute leukemia (figure 1a) characterized by marked leukocytosis, anemia, and thrombocytopenia (figures s1a s1c) and hepatosplenomegaly . Peripheral blood (pb) and bone marrow (bm) morphology resembled aml in all diseased mice (figures 1b1d), and myeloid lineage was confirmed by flow cytometry (figures 1e and s1d). Leukemias showed deletion of the runx1 dna - binding domain (figure s1e). The uniform myeloid - lineage leukemias in flt3runx1mx1-cre mice demonstrate a key role for flt3-itd signaling in introducing a myeloid - lineage bias during leukemogenesis . As shown previously (lee et al ., 2007), the multipotent linsca1c - kit (lsk) compartment was expanded in flt3 mice (figure 2a). We applied cd150 and cd48 to determine the nature of the expanded cells within the lsk compartment (kiel et al ., 2005), as flt3-itd is not detectable at the cell surface (figure s2a). Notably, the expansion of lsks was wholly attributable to a marked expansion of lskcd15048 mpps in 8- to 10-week - old flt3 mice (figures 2b and 2c), in line with the recent observation that flt3-itd may suppress the hsc compartment (chu et al ., 2012). Heterozygous (flt3) mice had an intermediate phenotype between flt3 and flt3 mice (figures s2b and s2c). High - level flt3 expression in the normal lsk compartment defines lmpps that lack self - renewal and megakaryocytic (mk) and erythroid (e) potential but sustain lymphomyeloid capability (adolfsson et al ., 2005), a progenitor that is also implicated in human aml (goardon et al ., 2011). Because flt3 lmpps reside almost exclusively in the lskcd15048 compartment (figure s2d), we explored whether the expanded lskcd15048 cells in flt3-itd mice had lmpp - like characteristics . As expected for lmpps (mnsson et al ., 2007), hsc- and mke - affiliated gene expression was downregulated in flt3 lskcd15048 mpps (figures 2d, 2e, and s2e), and in line with the molecular data they possessed little or no mk potential in vitro (figure 2f). In contrast, myeloid - affiliated gene expression was upregulated in flt3 lskcd15048 cells (figure 2 g), paralleled by high granulocyte - macrophage (gm) potential in vitro (figure 2h). Early lymphoid transcriptional programs were downregulated in both flt3 and flt3 lskcd15048 mpps (figures 2i and 2j), paralleled by severely reduced b cell potential in vitro (figure 2k). Importantly, the lymphoid transcriptional program was already suppressed in flt3 lskcd15048 mpps in embryonic day 15 (e15) fetal liver (fl) (figure s2f), at which time the phenotype and number of lskcd15048 mpps were unaffected (figure s2 g). In keeping with this suppression of early lymphoid programs in fetal mpps, the number of b220cd19 b cells was suppressed in flt3-itd e15 fl (figure s2h), preceding the emergence of a myeloproliferative phenotype at this early stage of ontogeny (figure s2i). This supports the notion that lymphoid suppression and myeloid bias in mpps occurs as a cell - intrinsic and direct consequence of flt3-itd signaling . Because flt3-itds in humans often occur secondarily to an initiating clonogenic event (jan et al ., 2012), it is possible that in such cases it is the other genetic events and not the flt3-itd that influence the leukemic phenotype . In order to address this issue, we used vav - cre mediated recombination to examine the impact of runx1 loss of function on lineage priming in mpps in the absence of flt3-itd . The data obtained demonstrate a significant upregulation of il7r and rag1 expression in mpps with no significant impact on sigh expression (figure s2j). This finding is in keeping with the high incidence of lymphoid malignancies in mouse models of runx1 mutation (jacob et al ., 2010; kundu et al ., 2005; putz et al ., 2006) and contrasts markedly with the suppression of lymphoid programs caused by flt3-itds in mpps . This supports the notion that it is gfr signaling through flt3-itd that instructs the uniform myeloid phenotype of leukemias resulting from the collaboration between runx1 deletion and flt3-itd in lmpps . Because flt3-itd compromised lymphoid - transcriptional priming in lmpps, which have been implicated as critical thymus - seeding progenitors (luc et al ., 2012), we next investigated whether t lymphopoiesis might be suppressed in flt3-itd mice . Thymic cellularity was found to be progressively reduced, and the earliest thymic progenitors (double - negative 1 kit+ [dn1kit]) were almost completely lost in flt3 mice (figures 3a3c). Also, subsequent stages of dn2kit and dn3 thymocyte progenitors were severely reduced (figures s3a and s3b). Of relevance, gene and protein expression for the chemokine receptor ccr9, which is critical for migration of thymus - seeding progenitors from the bm to the thymus (schwarz et al ., 2007), was suppressed in flt3 lskcd15048 mpps (figures 3d, s3c, and s3d). Moreover, dn1 cells showed large reductions in expression of notch1 and its targets (hes1 and hes5; figure s3e), which are critical for early t cell development . Although expression of some early lymphoid genes (il2ra, il7r, and gata3) was maintained, myeloid genes (cebpa, sfpi1, csf1r, csf2r, and csf3r) were highly upregulated in dn1 thymic progenitors (figure s3e). In keeping with this myeloid bias, flt3 dn1 progenitors showed low t cell potential in vitro (figure 3e). Previous studies demonstrated a reduction of mature b cells in flt3-itd mice (lee et al . We investigated whether this might reflect flt3-itd - induced perturbation at the earliest b cell commitment stages, and in support of this found a notable 11-fold expansion of lincd19cd24aa4.1cd43b220 pre - pro - b cells in flt3 mice (figures 3f and 3 g), whereas pro - b cells, the next stage in b cell development, were severely reduced (figures 3h and s3f). Because pre - pro - b cells are known to sustain low - level myeloid potential (rumfelt et al ., 2006), we next investigated lymphoid and myeloid gene expression in flt3 pre - pro - b cells . Key early lymphoid genes (cd79a, il7r, sigh, ebf1, pax5, rag1, and rag2) were all suppressed (figure 3i), whereas myeloid - affiliated genes (cebpa, cebpb, sfpi1, csf1r, and csf2r) were upregulated in flt3 pre - pro - b cells (figure 3j). In agreement with this, flt3 pre - pro - b cells had severely reduced b cell potential (figure 3k). These findings demonstrate that flt3-itds markedly impair the earliest stage of both t and b lymphopoiesis and upregulate myeloid gene expression in the earliest b and t lymphoid progenitors, in agreement with the notion that myeloid bias initiates in multipotent lmpps . To further explore the mechanistic basis of flt3-itd - induced myeloid bias of early lymphoid progenitors, we examined pu1 protein expression using a pu1-yfp reporter line . Whereas in wild - type (wt) mice lin cells showed a bimodal distribution between pu1 and pu1 cells, almost all lin cells in flt3 mice were pu1 (figure 4a). Flt3 mice showed a modest increase in pu1 expression in lskcd15048 mpps (figure 4b) and markedly enhanced levels of pu1-yfp in pre - pro - b cells (figure 4c). In keeping with the observed increased expression of pu1, gene set enrichment analysis (gsea) demonstrated upregulation of pu1 target genes (steidl et al . Furthermore, gsea also demonstrated a similar upregulation of pu1 target genes in flt3-itd mutated lmpp - like leukemia stem cells in human aml (goardon et al . Because transition from pre - pro - b cells to subsequent stages of b cell development is associated with downregulation of pu1 expression (figure s4b), we attempted to rescue the suppressed b cell phenotype in flt3-itd mice through generation of flt3 pu1 mice . Notably, whereas pu1 haploinsufficiency did not increase the number of b cells or their progenitors on a wt background, it resulted in a 10.3-fold increase in b cells in flt3 mice (figure 4e). Furthermore, pu1 haploinsufficiency led to a 2-fold reduction in the expanded pre - pro - b cell population in flt3-itd mice (figure 4f) together with a 13.5-fold rescue of the suppression of pro - b cells (figure 4 g). Strikingly, in the presence of pu1 haploinsufficiency, the expansion of mpps was also restored to wt levels (figure 4h) together with a significant amelioration of the myeloproliferative phenotype in flt3 mice (figure 4i). Stat3 is aberrantly activated by flt3-itd signaling (schmidt - arras et al ., 2009) and pu1 is a direct target gene of stat3 (hegde et al ., 2009). In line with this, flt3 bm showed aberrant activation of stat3 (figure s4c), and mpps and pre - pro - b cells showed significantly increased expression of stat3 target genes (figures 4j4l). Collectively, these data suggest that pu1 upregulation is a key mediator of the flt3-itd - induced myeloid bias and progenitor phenotype . In the studies presented here, we explored whether and how gfr signaling might influence lineage specification in vivo . To that end, we used a knockin model of flt3-itd to investigate whether oncogenic mutations that result in constitutive gfr signaling influence the lineage fate of mpps in vivo . In order to first establish the physiological relevance of flt3-itd as an aml - inducing mutation, we developed a mouse model in which flt3-itd was coexpressed with an inducible deletion of the dna - binding domain of runx1 (growney et al ., 2005) in mpps . Importantly, although runx1 loss - of - function mutations are found in patients with both all and aml, only the latter is often found in association with flt3-itds (grossmann et al . Deletion of the dna - binding domain of runx1 during adult murine hematopoiesis results in mild myeloproliferation (growney et al ., 2005) or lymphoblastic leukemias / lymphomas (jacob et al ., 2010; kundu et al ., 2005; putz et al .,, we demonstrate that the combination of runx1 mutation with flt3-itds results in aggressive leukemia with 100% penetrance and a uniform myeloid phenotype . In addition to demonstrating the functional relevance for flt3-itd - induced aml, this aml model has a number of important features . First, in contrast to many other mutation collaboration models, both mutations are targeted to the physiologically relevant endogenous genetic loci . Second, in isolation each mutation results in only a modest phenotype, but in combination a minor clone of deleted cells rapidly expands and becomes clonally dominant, paralleling somatic mutations during leukemogenesis . Third, the evolution of leukemia is rapid, supporting the notion that it occurs without the requirement for additional genetic events . Finally, the model recapitulates a particularly poor prognostic form of aml with a major unmet need for novel therapeutic approaches and thus provides a powerful model for future studies of the cellular and molecular mechanistic bases for collaboration of these mutations in aml . The most primitive progenitor population that was expanded by flt3-itds consisted of lmpp - like cells, demonstrating upregulation of the myeloid program, whereas the transcriptional expression of lymphoid - affiliated genes was markedly reduced in both heterozygous and homozygous flt3-itd mice . This myeloid transcriptional bias and suppression of lymphoid transcripts was also present during early b and t cell development and, importantly, was already present in flt3-itd fl mpps, preceding the expansion of mpps and development of a myeloproliferative phenotype, supporting the notion that it is a direct consequence of flt3-itd signaling in lmpps . The severe suppression of the earliest thymic progenitors in flt3-itd mice and concomitant lack of ccr9 upregulation in lmpps, which have been implicated as key thymus - seeding progenitors (luc et al ., 2012), further suggests that thymic seeding might also be impaired by flt3-itds . These findings demonstrate that the myeloid propensity of flt3-itds results from flt3-itd introducing a myeloid bias in multipotent lymphomyeloid progenitors as well as in the earliest b and t lymphoid progenitors . Pu1 is a dosage - sensitive regulator of myeloid - lymphoid cell - fate decisions that promotes myeloid differentiation when overexpressed (dekoter and singh, 2000). Using a pu1-yfp reporter, we demonstrated that flt3-itds upregulate pu1 expression in mpps and the earliest b lymphoid progenitors, paralleled by increased expression of pu1 target genes in mpps . Furthermore, we confirmed the relevance of these findings for human aml by demonstrating upregulation of pu1 target genes in flt3-itd mutated lmpp - like human aml stem cells (goardon et al ., 2011). In agreement with a key mechanistic role for pu1 overexpression, flt3-itd - induced suppression of b cell development, as well as the flt3-itd - associated progenitor phenotype, constitutive flt3-itd signaling is distinct from wt flt3 signaling due to abnormal anchoring of flt3-itd in the endoplasmic reticulum, with markedly reduced surface flt3 expression resulting in aberrant stat3 activation, as confirmed in our study (schmidt - arras et al ., 2009). Because pu1 is a direct target gene of stat3 (hegde et al ., 2009) and stat3 target genes were upregulated in flt3-itd lskcd15048 mpps and pre - pro - b cells, this provides a putative direct link between aberrant constitutive flt3-itd signaling and pu1 overexpression as a cause of aberrant myeloid bias . Several lines of evidence indicate that flt3-itds frequently arise early in the transformation process in mpps (levis et al ., 2005) and as a driver mutation in the founding leukemic clone (ding et al ., 2012; smith et al ., 2012), and thus may influence the lineage of resulting leukemias . Together with our findings, this is consistent with a model in which lmpps, which normally express high levels of flt3 (adolfsson et al ., 2005), acquire flt3-itd mutations that confer both a strong clonal advantage and a marked myeloid bias . This results in myeloid expansion and suppression of early lymphoid development, strongly supporting a fundamental role for flt3-itds in promoting myeloid lineage leukemia development at the mpp level . Our study also further highlights lmpps as a key target population in aml, as also supported by recent findings in human aml (goardon et al ., 2011). Transformation to an aggressive leukemia exclusively of a myeloid phenotype by introduction of runx1 mutation demonstrates the functional relevance of this flt3-itd - induced myeloid bias and clonal dominance, providing insights into the process by which oncogenic mutations might determine the lineage fate of the resulting leukemias at the precommitment stage . Our findings are also of considerable relevance for normal hematopoiesis, as it remains disputed whether key cytokine receptor signaling pathways mediate critical in vivo functions in blood lineage development through purely permissive rather than instructive actions (enver and jacobsen, 2009). Thus, although flt3-itd elicits aberrant signaling, our findings clearly provide support for the notion that cytokine receptors are also capable of eliciting lineage - instructive signaling in mpps in vivo . All animals used were bred and maintained in accordance with regulations of the uk home office . Bm samples from aml patients were obtained with informed consent and the approval of oxford ethics committee b (protocols 06/q1606/110 and 05/mre07/74). Details of the antibody staining panels and protocols are provided in the extended experimental procedures . Antigens and antibodies used for identification of specific cell populations are shown in tables s1 and s2 . Cells were lysed in 1% np40 buffer and analyzed by western blot as previously described (pecquet et al ., 2010) with antibodies against pstat3 (cell signaling) and beta - actin (sigma). Evaluation of megakaryocytic, gm, and lymphoid potentials was carried out as previously described (mnsson et al ., 2007) and detailed in the extended experimental procedures . Multiplex quantitative pcr was performed as previously described (kharazi et al ., 2011) and detailed in the extended experimental procedures . Analysis by affymetrix mouse genome 430 2.0 arrays was carried out at the stanford protein and nucleic acid facility as described previously (mnsson et al ., 2007) and in the extended experimental procedures . Extended experimental proceduresquantification of hematologic indiceshemoglobin, wbc, and platelet count per milliliter of blood were quantified on a sysmex kx-21n 3-part differential hematology analyzer.animalsflt3-itd knockin mice on c57bl/6 background have been previously described (kharazi et al . Homozygous (flt3) and heterozygous (flt3) flt3-itd mice were studied as specified . Runx1 mice have been previously described (grundler et al ., 2005) and were on a 129s1 x cba background . Vavcre mice have been previously described (de boer et al ., 2003) and were on a c57bl/6 background . Mx1-cre mice were on c57bl/6 background and have been previously described (khn et al . Mice carrying a germline deletion of pu1 (pu1) were derived by crossing a previously reported pu1 line (iwasaki et al ., 2005) on a c57bl/6 129s1 x cba background with a constitutive cmv - cre recombinase, on a c57bl/6 background . Pu1-yfp mice on a c57bl/6 background have been previously described (kirstetter et al ., 2006). Mice were analyzed with littermate controls of the appropriate genotype when available.flow cytometry analysis and facs purificationantibodies and viability dyes used for flow cytometry analysis are shown in table s1 . Combinations of antibodies used for identification and purification of indicated populations are shown in table s2.all antibodies were used at predetermined (titrated) optimized concentrations . Cell acquisition and analysis were performed on a 4-laser lsrii (becton dickinson, san jose, ca) using flowjo software (treestar, ashland, or). Cells used in cell sorting experiments were either un - enriched or enriched for cd117 with macs cell separation (miltenyi biotec) followed by fc - block incubation and staining with anti - mouse antibodies . Fluorescence minus - one controls as well as negative populations were used as gate - setting controls . Gates were set using a combination of fluorescence minus one controls and also populations that are known to be negative for the antigen . For example, lskcd150cd48 cells, which are known to not express flt3, were used to set the gate for surface flt3 expression on lskcd150cd48 cells.microarray analysisglobal gene expression analysis was performed on lskcd150cd48 cells from flt3 and flt3 (4 mice of each genotype) as previously reported (luc et al ., 2012). Two thousand cells were sorted directly into trizol (invitrogen) and the rna extraction carried out as per manufacturer s instructions and previously as described (luc et al ., 2012). Using the same total amount of input rna, samples were amplified using the nugen kit wt - ovation pico rna amplifications system followed by the wt ovation cdna biotin module v2 for cdna labeling (nugen) and fragmentation and finally hybridized to affymetrix mouse genome 430 2.0 arrays using standard protocols (affymetrix) at the stanford protein and nucleic acid facility . These data will be available through geo accession number gse35805 (http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?token=dfcvjocouaoccds&acc=gse35805).microarray data were normalized using the robust multi - array averages (rma)(irizarry et al ., 2003) method in biocondcutor / r and analyzed using gene set enrichment analysis (gsea) (subramanian et al ., 2005). Erythroid, myeloid (chambers et al ., 2007), pu1 (steidl et al ., 2006), and stat3 (alvarez et al ., 2005) target gene lists have been previously described . A signature for early b cell commitment was generated using data from the immunological genome project (heng et al ., 2008) (http://www.immgen.org) by comparing the gene expression profile of long term reconstituting stem cells with that of pre - pro - b cells using the limma package (smyth, 2004) in r; genes which were greater than 2-fold upregulated (limma b statistic> 3) were used for the b cell commitment signature . Gsea analysis of lmpp - like human aml stem cells, stratified according to the presence of flt3-itd mutation, was carried out using previously described data set (goardon et al ., 2011), using human equivalents of the above murine pu1 target genes (steidl et al ., 2006).multiplex quantitative real - time pcr analysismultiplex quantitative pcr was performed as previously described (kharazi et al ., 2011). For each cell population, two to four biological replicates (different mice) were prepared, and two to three separately sorted cell samples from each mouse were cell sorted and analyzed . For lskcd150cd48 cells, lskflt3 cells, and pre - pro - b cells, 100 cells per replicate were analyzed . For dn1 cells, 20 cells per replicate were analyzed as cell numbers of this population were highly limited in flt3 mice . Details of the taqman assays used are shown in table s3.for cdna synthesis and preamplification of target genes cellsdirect one - step qrt - pcr kit (invitrogen) was used . Cells were sorted directly into pcr plates or 0.2 ml pcr tubes containing 2.5 l gene specific 0.2x taqman gene expression assays (applied biosystems), 5 l of cellsdirect 2x reaction mix (invitrogen), 1.2 l cellsdirect rt / taq mix, 1.2 l te buffer and 0.1 l superase - in rnase inhibitor (ambion). Conditions for reverse transcription and target gene amplification were: 15 min at 50c; 2 min at 95c; 22 cycles of 95c for 15 s and 60c for 4 min . Preamplified products were diluted 1:5 in te buffer and analyzed on dynamic array (fluidigm) using the following pcr cycling condition: 95c for 10 min; 40 cycles of 95c for 15 s and 60c for 60 s. data was analyzed using the ct method; results were normalized to hprt expression and expressed as mean expression level relative to hprt.in vitro assays for megakaryocyte, gm, and lymphoid potentialsevaluation of megakaryocytic and gm potentials was carried out as previously described (luc et al ., 2008). For evaluation of megakaryocytic potential, 150 cells were sorted into 3 ml of x - vivo 15 (lonza) supplemented with 0.5% detoxified bovine serum albumin (bsa; stemcell technologies inc), 10% fetal calf serum (fcs), 2-mercaptoethanol (2-me; 10 m; sigma - aldrich co), 1% penicillin / streptomycin (paa) and 50 ng / ml murine stem cell factor (mscf; peprotech, rocky hill, nj), 50 ng / ml human fms - like tyrosine kinase 3 ligand (hfl; immunex, seattle, wa), 50 ng / ml human thrombopoietin (hthpo; peptrotech), 5 u / ml human erythropoietin (hepo; boehringer mannheim, mannheim, germany) and 20 ng / ml murine interleukin 3 (mil-3; peprotech). For evaluation of gm potential, cells were sorted into media as above but with the following cytokines: mscf, hfl, hthpo, hg - csf (neopogen, amgen, thousand oaks, ca) (all 25 ng / ml), mgm - csf (immunex, seattle, wa) and mil-3 (both 20 ng / ml). The cell suspension was thoroughly mixed and 20 l was pipetted into each well of two 60-well plates per replicate (nunc minitrays catalog number: 163118, nunc a / s, roskilde, denmark). Wells were scored for clonal growth and/or frequency of wells with megakaryocytes with an inverted light microscope after 8 days of culture . The reliability of this approach to score cultures for megakaryocytic potential has been validated by morphological validation of giemsa stained cytospins of the cultures (mnsson et al ., 2007). Percentage of cloning efficiency was calculated according to the poisson distribution which predicts that 63% of wells should contain 1 or more cells following manual plating (76 of 120 wells).b cell and t cell potential was evaluated by sorting 10 mpps, dn1 or pre - pro - b cells onto approximately 80% confluent monolayers of op9 (b cell potential) and op9dl1 (t cell potential) stromal cells, as previously described (adolfsson et al ., 2005; luc et al ., 2012). Clones were identified and picked at 3 to 4 weeks (depending on clonal size), and subsequently analyzed by facs for the presence of b cell (defined as b220cd19) or t cell (cd4/8 double positive) committed progeny.statistical analysisthe statistical significance of differences between samples was determined using the 2-tailed t test . Hemoglobin, wbc, and platelet count per milliliter of blood were quantified on a sysmex kx-21n 3-part differential hematology analyzer . Flt3-itd knockin mice on c57bl/6 background have been previously described (kharazi et al ., 2011; homozygous (flt3) and heterozygous (flt3) flt3-itd mice were studied as specified . Runx1 mice have been previously described (grundler et al ., 2005) and were on a 129s1 x cba background . Vavcre mice have been previously described (de boer et al ., 2003) and were on a c57bl/6 background . Mx1-cre mice were on c57bl/6 background and have been previously described (khn et al ., 1995). Mice carrying a germline deletion of pu1 (pu1) were derived by crossing a previously reported pu1 line (iwasaki et al ., 2005) on a c57bl/6 129s1 x cba background with a constitutive cmv - cre recombinase, on a c57bl/6 background . Pu1-yfp mice on a c57bl/6 background have been previously described (kirstetter et al ., 2006). Combinations of antibodies used for identification and purification of indicated populations are shown in table s2 . All antibodies were used at predetermined (titrated) optimized concentrations . Cell acquisition and analysis were performed on a 4-laser lsrii (becton dickinson, san jose, ca) using flowjo software (treestar, ashland, or). Cells used in cell sorting experiments were either un - enriched or enriched for cd117 with macs cell separation (miltenyi biotec) followed by fc - block incubation and staining with anti - mouse antibodies . Fluorescence minus - one controls as well as negative populations were used as gate - setting controls . Gates were set using a combination of fluorescence minus one controls and also populations that are known to be negative for the antigen . For example, lskcd150cd48 cells, which are known to not express flt3, were used to set the gate for surface flt3 expression on lskcd150cd48 cells . Global gene expression analysis was performed on lskcd150cd48 cells from flt3 and flt3 (4 mice of each genotype) as previously reported (luc et al ., 2012). Two thousand cells were sorted directly into trizol (invitrogen) and the rna extraction carried out as per manufacturer s instructions and previously as described (luc et al ., 2012). Using the same total amount of input rna, samples were amplified using the nugen kit wt - ovation pico rna amplifications system followed by the wt ovation cdna biotin module v2 for cdna labeling (nugen) and fragmentation and finally hybridized to affymetrix mouse genome 430 2.0 arrays using standard protocols (affymetrix) at the stanford protein and nucleic acid facility . Microarray data were normalized using the robust multi - array averages (rma)(irizarry et al ., 2003) method in biocondcutor / r and analyzed using gene set enrichment analysis (gsea) (subramanian et al ., 2005)., pu1 (steidl et al ., 2006), and stat3 (alvarez et al ., 2005) target gene lists have been previously described . A signature for early b cell commitment was generated using data from the immunological genome project (heng et al ., 2008) (http://www.immgen.org) by comparing the gene expression profile of long term reconstituting stem cells with that of pre - pro - b cells using the limma package (smyth, 2004) in r; genes which were greater than 2-fold upregulated (limma b statistic> gsea analysis of lmpp - like human aml stem cells, stratified according to the presence of flt3-itd mutation, was carried out using previously described data set (goardon et al ., 2011), using human equivalents of the above murine pu1 target genes (steidl et al ., 2006). Multiplex quantitative pcr was performed as previously described (kharazi et al ., 2011). For each cell population, two to four biological replicates (different mice) were prepared, and two to three separately sorted cell samples from each mouse were cell sorted and analyzed . For lskcd150cd48 cells, lskflt3 cells, and pre - pro - b cells, 100 cells per replicate were analyzed . For dn1 cells, 20 cells per replicate were analyzed as cell numbers of this population were highly limited in flt3 mice . For cdna synthesis and preamplification of target genes cellsdirect one - step qrt - pcr kit (invitrogen) was used . Cells were sorted directly into pcr plates or 0.2 ml pcr tubes containing 2.5 l gene specific 0.2x taqman gene expression assays (applied biosystems), 5 l of cellsdirect 2x reaction mix (invitrogen), 1.2 l cellsdirect rt / taq mix, 1.2 l te buffer and 0.1 l superase - in rnase inhibitor (ambion). Conditions for reverse transcription and target gene amplification were: 15 min at 50c; 2 min at 95c; 22 cycles of 95c for 15 s and 60c for 4 min . Preamplified products were diluted 1:5 in te buffer and analyzed on dynamic array (fluidigm) using the following pcr cycling condition: 95c for 10 min; 40 cycles of 95c for 15 s and 60c for 60 s. data was analyzed using the ct method; results were normalized to hprt expression and expressed as mean expression level relative to hprt . Evaluation of megakaryocytic and gm potentials was carried out as previously described (luc et al ., 2008). For evaluation of megakaryocytic potential, 150 cells were sorted into 3 ml of x - vivo 15 (lonza) supplemented with 0.5% detoxified bovine serum albumin (bsa; stemcell technologies inc), 10% fetal calf serum (fcs), 2-mercaptoethanol (2-me; 10 m; sigma - aldrich co), 1% penicillin / streptomycin (paa) and 50 ng / ml murine stem cell factor (mscf; peprotech, rocky hill, nj), 50 ng / ml human fms - like tyrosine kinase 3 ligand (hfl; immunex, seattle, wa), 50 ng / ml human thrombopoietin (hthpo; peptrotech), 5 u / ml human erythropoietin (hepo; boehringer mannheim, mannheim, germany) and 20 ng / ml murine interleukin 3 (mil-3; peprotech). For evaluation of gm potential, cells were sorted into media as above but with the following cytokines: mscf, hfl, hthpo, hg - csf (neopogen, amgen, thousand oaks, ca) (all 25 ng / ml), mgm - csf (immunex, seattle, wa) and mil-3 (both 20 ng / ml). The cell suspension was thoroughly mixed and 20 l was pipetted into each well of two 60-well plates per replicate (nunc minitrays catalog number: 163118, nunc a / s, roskilde, denmark). Wells were scored for clonal growth and/or frequency of wells with megakaryocytes with an inverted light microscope after 8 days of culture . The reliability of this approach to score cultures for megakaryocytic potential has been validated by morphological validation of giemsa stained cytospins of the cultures (mnsson et al ., 2007). Percentage of cloning efficiency was calculated according to the poisson distribution which predicts that 63% of wells should contain 1 or more cells following manual plating (76 of 120 wells). B cell and t cell potential was evaluated by sorting 10 mpps, dn1 or pre - pro - b cells onto approximately 80% confluent monolayers of op9 (b cell potential) and op9dl1 (t cell potential) stromal cells, as previously described (adolfsson et al ., 2005; clones were identified and picked at 3 to 4 weeks (depending on clonal size), and subsequently analyzed by facs for the presence of b cell (defined as b220cd19) or t cell (cd4/8 double positive) committed progeny.
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In a recent breast cancer study conducted by the clinical proteomic tumor analysis consortium (cptac), extensive global- and phospho- protein profiling were obtained for a subset of breast cancer samples that have been extensively characterized in the the cancer genome atlas (tcga). This is so far the first attempt to study protein activities in breast cancer samples using sophisticated protein experiments on a large scale . Then, by leveraging genomic analytical outputs from tcga on these samples, we have the unique opportunity to characterize and compare gene coexpression networks and protein coexpression networks based on the same set of samples . This is of great interest because knowledge about the common and different coexpression patterns among rnas and proteins could improve our understanding of complicated gene regulatory mechanisms in tumor samples and could also facilitate the detection of important disease genes and therapeutical targets . In the past decade, numerous statistical and machine learning methods have been proposed to construct gene gene regulatory networks and protein protein regulatory networks, including coexpression network methods, bayesian network methods, and gaussian graphical models . Review of these and other methods are available in hecker et al . And lee et al . ; however, all of these proposed models are designed to construct one network at a time . Presumably, we can use rna expression data and protein expression data to construct two networks separately and then compare, but such an approach is certainly less optimal, as gene expressions and protein expressions in one tumor sample are closely related . On one hand, protein levels are regulated by their rna levels, so we expect the two coexpression networks share common structures . On the other hand, protein activities are subject to a large amount of post - transcriptional modifications, and thus we also expect to observe unique interaction patterns in each network . This motivates us to perform joint learning of both rna - seq and proteomic networks, so we are able to borrow information across different data sets and better capture the common correlation structure, which shall then improve the overall accuracy of network estimation . Some of them have been specifically designed to estimate time - varying graphical models in the context of time - series data . Proposed likelihood - based methods for the joint estimation of multiple related gaussian graphical models . Their approaches rely on two different regularization schemes penalizing differences across partial correlation structures of different classes . While both papers successfully demonstrate the advantage of jointly modeling multiple networks over estimating individual networks separately, the performance of the methods heavily depends on the multivariate gaussian assumptions of gene expression distributions, which may not hold in real biological systems . Recently, random - forest - based methods have been utilized for the construction of grn . Random forest is a decision - tree - based nonparametric method for building powerful prediction models . Its ensemble structure combined with its nonparametric nature delivers excellent performance even when the sample size is moderate . The key idea is to model the expression of each target gene as a function of the expression of all other genes via random forest . The superior performance of genie3 was demonstrated in both dream 4 in - silico multifactorial challenge and dream 5 network inference challenge . In both challenges,, genie3 was further modified to estimate grn based on time - series data . Proposed irafnet, another random - forest - based method that combines different types of heterogeneous data, such as protein protein interactions, transcription factor - dna binding, and gene knock - down to estimate grn . While these random - forest - based methods greatly advance the field of grn construction, none of them is designed to estimate simultaneously multiple related networks . We propose jrf (joint random forest), a new algorithm for the simultaneous estimation of protein coexpression and gene coexpression networks . The key idea is to borrow information across different data by forcing the class - specific tree ensembles to use the same genes for the splitting rules . In this way, regulatory relationships playing important roles in multiple classes will be detected with better power . After testing the performance of our algorithm on several in silico experiments and comparing jrf with different methods including genie3 the joint graphical lasso (jgl), we applied jrf for the simultaneous construction of gene coexpression networks and protein coexpression networks . For our analysis, we considered gene expression from tcga and protein expression from cptac data for 62 breast cancer patients . We derived the two networks and identified protein - specific hub genes and gene modules, showing the unique contribution of proteomic data to breast cancer research . Random forest is a nonparametric algorithm that models a response variable via a collection of decision trees . Specifically, each decision tree is constructed based on a random subset of training samples, and the splitting variable at each node of a decision tree is chosen from a randomly sampled subset of predictors upon maximizing a certain utility function (e.g., decrease in node impurity). Recently, huynh - thu et al . Introduced genie3, a random - forest - based model for inferring gene regulatory networks (grns). In genie3, first, for each target gene k, its expression is modeled as a function of the expression of all other genes via random forest . Then, regulatory event (j k) is measured by ijk, the importance score of gene j in the random forest model of gene k. specifically, the importance score ijk is defined as the summation of node impurities across all nodes that utilize predictor j for the splitting rule divided by the total number of trees in the random forest model of gene k. we start with some notations . For each class g {1,..., g}, denote the gene expression data for p genes and ng individuals as xngpg, and the ith observation of gene j under class g as xijg . An overview of jrf is shown in figure 1, where, for simplicity, we consider only two classes . The key step of jrf is to construct g random forest models simultaneously using data from g classes when predicting the expression of a target gene k based on the expression of all other genes . We propose to use the same predictor variables for splitting rules in different trees corresponding to different classes . The goal is to borrow information across different classes, so that regulatory relationships can be better detected if there are coherent signals across different classes . Specifically, when we grow g decision trees in parallel for g classes, at one node, we decide the splitting variable based on the following procedure: (1) randomly sample a set containing n genes from the entire set of genes except gene k. (2) for each class, the best splitting rule based on each candidate predictor in set is derived . Let be the subset of samples allocated to node under class g. the splitting rule based on the jth predictor is of the formwhere and are the subsets of samples in class g allocated to the left and right children of node with a splitting rule based on the jth predictor . For each predictor in set, the best threshold agj*() is derived as followswhereand with x representing the mean of set . In other words, cgj is the decrease in node impurity after splitting node in the gth tree according to gene j. (3) finally, gene g * is chosen as the splitting variable of node for all classes based on the following criteria: because jrf utilizes the same splitting variable for corresponding nodes across all classes, predictors associated with gene k in multiple classes are more likely to be chosen for the splitting rules . For each step in the tree construction, (13) only apply to classes for which is not a final node . As in the original random forest model, each tree grows until either the total number of observations allocated to the final leaves falls below a certain prespecified threshold or the maximum number of possible nodes is reached . It is worth mentioning that when the number of classes is one, jrf reduces to genie3, the original random forest model for network inference . It is worth noting that to implement jrf, data sets need to be standardized to mean zero and unit variance . Importance scores depend on the scale of the data and therefore variables (genes) need to be standardized before the random forest models are fitted.figure 1jrf schematic . For simplicity, let us assume that there are only two classes and that each data contains the same number of samples . For each target gene, gk, we run random forest for each class and the two tree ensembles are forced to share some structure as follows . First, for each node, the same set of genes s is randomly sampled from the entire set of genes (a). Then, the same variable in set s is chosen for the splitting rule . This is achieved by deriving the splitting rule for any gene contained in set s (b) and choosing the best variable to maximize the sum of decrease in node impurity over different classes (c). Let us assume that there are only two classes and that each data contains the same number of samples . For each target gene, gk, we run random forest for each class and the two tree ensembles are forced to share some structure as follows . First, for each node, the same set of genes s is randomly sampled from the entire set of genes (a). Then, the same variable in set s is chosen for the splitting rule . This is achieved by deriving the splitting rule for any gene contained in set s (b) and choosing the best variable to maximize the sum of decrease in node impurity over different classes (c). On the basis of the previously described procedure, jrf constructs g random forest models simultaneously for each target gene k and returns a ranking of gene gene interactions based on importance scores . To assess the performance of jrf in predicting the true interactions, receiver operating characteristic curves (roc) and precision - recall curves can be computed by setting different thresholds on importance scores . In this paper, jrf is compared with jgl, genie3-sep, and genie3-comb on several in silico experiments . Genie3-sep is genie3 used to estimate networks based on data from g classes separately, while genie3-comb is genie3 used to estimate a unique network based on the union of data from all classes . All random - forest - based algorithms (jrf, genie3-comb, and genie3-sep) provide a ranking of regulatory relationships based on importance scores, and roc curves were computed by setting different thresholds on these scores . Instead, for jgl, roc curves were constructed by considering different values for the two parameters controlling the level of sparsity (as shown by figure s1 in the supporting information). Another approach to evaluate the performance of jrf is choosing a proper cutoff value for importance scores using permutation techniques . Let ijkg be the importance score associated with the regulatory event (j k) in the gth class - specific tree ensemble . Specifically, this importance score is defined as where t is the number of trees and is the set of nodes which utilize gene j for the splitting rule in the tree ensemble used to predict gene k based on the gth class - specific data . Because in this paper we are interested in estimating undirected networks, we derive importance score ij kg for every edge (j k) as the average between importance scores ikjg and ijkg, and the final undirected networks can therefore be derived by applying a cutoff on the edge importance scores . To derive a proper cutoff value for importance scores, we utilize the following permutation - based procedure: (a) for b {1,, b}, with b being the number of permutations: (a.1) for any target gene k, we first permute its sample order within each class and fit g random forest models via jrf to predict the expression of gene k based on the expression of all other genes in g classes, respectively . (a.2) repeat (a.1) for all genes and compute the final importance scores for each edge in each class, which are denoted as {ippg(b)}g=1 g . (b) for each threshold, we computewhere 1() is the indicator function, equal to one if event occurs and zero otherwise . F() can serve as an approximation of the false discovery rate (fdr). In the following numerical studies, we use 0 = min{: f() 0.001} and declare an edge between j and k in class g if ij the computational complexity of jrf is o(ptng=1glog(ng)ng), where p is the number of genes, t is the number of trees in each forest, n is the number of variables sampled at each node, g is the number of classes, and ng is the number of samples in each class . This complexity is in the same order as the complexity of applying genie3 to estimate g networks for g classes separately . In the contrast, the time complexity of ggm - based approaches, such as jgl, often has the order of p. so when p is large, jrf and genie3 enjoy better computational efficiency, which is supported by our numerical investigations (see supporting information). In practice, the number of trees (t) and the number of potential regulators to be sampled at each node (n) are user - specified parameters . T is usually chosen sufficiently large because the tree ensemble provides more accurate results as t increases . In our numerical studies, we reported results for t = 1000 . We also evaluated larger t values, such as 2000 and 5000, but observed similar results (data not shown). For n, a common choice is where (p 1) is the number of predictors for each target gene . In general, large value of n results in predictions with high bias; while low value results in predictions with high variance . We implemented an r package jrf using c routines wrapped with an r interface . Similarly to other tree - based algorithms for network construction, jrf can be easily parallelized because the estimation process consists of p - independent subproblems . A comparison between jrf, jgl, genie3-sep, and genie3-comb in terms of computational time is presented in table s1 . For data generation, we first considered two networks containing 500 nodes connected by 498 and 249 edges, respectively . In particular, network 1 is a network with two disjoint components and network 2 consists of only the first component of network 1 . In this example, we focused on two network topologies: power law and star topology (figure s2 in supporting information). For each network topology, we simulated 20 replicates involving n = 50 samples from a gaussian graphical model . Network structures and covariance matrices were simulated in the same way as danaher et al . (2014) (further details can be found in the supporting information). Besides this example, we also consider a series of other different simulation settings, including (1) two non - nested networks; (2) five non - nested networks; (3) two nonrelated networks; and (4) data from non - gaussian distributions . The expressions of 5864 proteins for 62 breast cancer tumors (proteome-ratio-norm-nona-normal.gct(04-jun-2014 version)) were downloaded from the cptac data coordinating center (http://proteomics.cancer.gov/programs/cptacnetwork) sponsored by the national cancer institute . For three samples having technical duplicates we further standardized protein expression so that each sample had median 0 and median absolute deviation (mad) 1 . Finally, we excluded proteins with interquartile range <70% quantile . Level - three rnaseq data of breast tumor samples were obtained from the tcga web site (http://tcga-data.nci.nih.gov/tcga/). First, we log - transformed the data and then replaced all missing values with 0 . Then, we standardized each sample to have median 0 and mad 1 . We focused on the 62 samples contained in the proteomic data and excluded genes with more than 10% missing values . Then, we selected the top 10% genes with largest interquartile range across samples . As final subset, we considered 528 genes obtained by overlapping the set of 1464 selected genes and the set of 1759 selected proteins . For each data in this section, jrf was compared with jgl, genie3-sep, and genie3-comb on several in - silico experiments . In all implementations, we used t = 1000 and . We evaluated the performances of different algorithms based on different network structures, sample sizes, and number of genes . Figure 2 shows the average roc curves of each method over 20 replicates for network 1 and network 2 separately . Jrf outperforms all other methods under all different settings, while genie3-sep is the second best performer . Figure s3 shows the boxplot of the area under the precision - recall curve (aupr) for jrf, genie3-sep, and genie3-comb . The advantage of jrf over genie3-sep is more evident for network 2, which contains only shared edges . This is expected because jrf should be able to detect common edges with better power . Mean of roc curves for network 1 (first column) and network 2 (second column) over 20 replicates for jrf (red), genie3-comb (green), genie3-sep (blue), and jgl (black). For each replicate, we sampled 50 samples from a gaussian graphical model based on the power law topology (first row) and star topology (second row). To further assess the performance of the proposed model, we included more simulation scenarios in the supporting information . In particular, in supporting information section 1.3, we considered the case of non - nested networks sharing some structure, while, in section 1.4, we considered the case where five different networks were estimated simultaneously . As shown by figures s4 and s5, jrf outperformed competitors under all of these simulation scenarios . In addition, it would be interesting to evaluate the performance of jrf when class - specific networks do not share any structure . For this purpose, we simulated two different networks with no common edge and applied different methods on the corresponding data (supporting information section 1.5). As shown in figure s6, performance of jrf is very comparable to that of genie3-sep on this simulation example . This suggests that jrf is sufficiently robust and works in the absence of common structure . Moreover, to assess the performance of jrf in the presence of nonlinearities in the data, we further generated data examples using genenetweaver, an open - source software for the generation of in - silico data from a set of ordinary and stochastic differential equations (supporting information section 1.6). As shown in the supplemental figure s7 and s8, jrf again outperformed other algorithms in terms of both area under the roc curve (auc) and area under precision - recall curve (aupr). Finally, figure s9 shows results for different algorithms in estimating networks with 1000 nodes (further details can be found in supporting information section 1.7), in which we can see that jrf outperformed competitors in terms of both auc and aupr . It is worth mentioning that the increased dimensionality did not negatively affect results, suggesting that jrf can handle problem with high dimensionality . In this section, we assess the performance of jrf and other algorithms on data sets involving different number of samples . Figure 3 shows boxplots of auc over 20 replicates resulting from jrf, genie3-sep, and genie3-comb for different sample sizes, that is, n = 50, 100, 200 . The aucs of jrf are significantly larger than that of genie3-sep based on 20 replicates under all sample sizes . As shown in the supplemental figure s10, jrf outperforms competitors also in terms of aupr for different samples sizes . As shown, the smaller the sample size the bigger the improvement of jrf over genie3-sep is . This result is expected because as the sample size increases genie3-sep should have sufficient information to estimate the networks separately . Boxplot of auc over 20 replicates for jrf (red), genie3-comb (green), and genie3-sep (blue) for different sample sizes, that is, n = 50, 100, and 200 . Data: samples simulated from a gaussian graphical model on a power law topology . In this section, we compare jrf and genie3-sep regarding their abilities to estimate class - specific edges . We considered the 20 replicate data sets (n = 100), which was simulated based on the power law network topology in the previous section . For both jrf and genie3-sep, edges were declared using the permutation procedure illustrated above with b = 500 and an fdr threshold of 0.001 . Table 1a shows the performance of jrf and genie3-sep in estimating network 1 and network 2 . For each network, we reported the minimum and the maximum value of true positive rate (tpr), false - positive rate (fpr), and false discovery rate (fdr) across 20 replicates . In particular, these quantities are defined as tpr = tp/(tp + fn), fpr = fp/(fp + tn), and fdr = fp/(fp + tp), where tp is the number of true positives, fp is the number of false positives, tn is the number of true negatives, and fn is the number of false negatives . For both networks, jrf achieved better power (tpr) than genie3-sep, while the resulting fprs were quite comparable . Table 1b shows a comparison of the two algorithms regarding the detection of class - specific (differential) edges . Again, we computed the rate of true differential edges and false differential edges and showed the minimum and the maximum value of these quantities over 20 replicates . While the tprs for detecting differential edges were similar for the two methods, the fprs of genie3-sep were significantly larger than that of jrf . In addition, jrf leads to substantially lower fdrs for detecting differential edges than genie3-sep . For each quantity (tpr, fpr, and fdr) we show the minimum value (min) and the maximum value (max) across 20 replicates . Note that the true discovery rate (tdr) is defined as tdr = (1 fdr). We applied jrf to construct coexpression networks based on the cptac global proteomics data set and tcga rnaseq data of 62 breast cancer samples . For simplicity, we will refer to the coexpression networks constructed by jrf from global proteomics data and rnaseq data as protein - network and rna - network . At fdr cutoff of 0.001, we detected 687 common edges shared by both networks, 502 edges unique to protein - network, and 382 edges unique to rna - network . The topologies of the two networks are shown in figure 4, while the full list of interactions can be found in additional file 1 in the supporting information . Interestingly, a few network modules in protein - network have a high percentage of edges unique to protein - network . Figure 5 highlights genes whose degrees (i.e., total number of edges connecting to one gene) in protein - network are much higher than that in rna - network . Three out of the top five genes with more protein - unique edges, c3, cfb, and mpo, are related to immune response functions . It is well known that immune response plays a major role in the patient response to chemotherapy treatments . Some of these genes have been shown to be predictive of survival outcomes of breast cancer patients . For example, c3 and cp are predictive of chemoresistance in breast cancer patients, and high activity of mpo genotypes can enhance efficacy of chemotherapy for early - stage breast cancer . . Genes belonging to the mmp family have been linked to cancer progression for their ability to degrade the extracellular matrix and may be implicated in the formation of metastasis . Network based on proteomic data (a) and rnaseq data (b) resulting from jrf . Green edges (687) are shared between proteomic and rnaseq data, red edges (502) are unique to proteomic data, while blue edges are unique to rnaseq data (382). Only names of genes with at least ten connecting edges are shown in the plot . For both networks, the full list of interactions can be found in additional file 1 . Number of connecting edges for top connected genes in the network based on proteomic data . For each gene, the green bar corresponds to the number of connecting edges shared between proteomic and rnaseq data; the red bar indicates the number of protein - specific edges; while the blue bar indicates the number of rna - specific edges . For each gene, we list its biological functions . We then focused on network modules unique to protein - network . We derived network modules based on edge betweenness using function edge.betweenness.community available in the r package igraph . In figure 6, we show the protein - network with gene modules enriched of at least one go category . The full list of genes contained in each module can be found in additional file 1 . Figure 6 shows, for each module, the most enriched go category and the corresponding benjamini adjusted p - value . Two interesting protein - specific modules (with more protein - specific edges than shared edges) are c1 and c2 . Fibroblast growth factor receptor activity, as shown in figure 7, genes are more tightly correlated in the proteomic data compared with the rnaseq data . Various studies have investigated the role of the fgfr pathway as a predictive marker for breast cancer . As shown in figure 7, fgfr2 and fgfr3 cerliani et al . Reported a strong correlation between fgfr2 and fgfr3 based on protein expression mentioning that there is no previous evidence of correlation between these two proteins for breast cancer patients . In addition, the interaction between fgfr2 and fgfr3 is contained in the string database (see table s2). Similarly to module c1, genes in module c2 are higher correlated in the proteomic data compared with the rnaseq data (figure 7). This cluster contains genes such as apob, c3, orm1, and cp that have been recently shown to be predictive of chemoresistance in breast cancer patients . Another important gene in this module with a high number of connecting edges is apoa1 (the fifth highest connected node according to figure 5). This gene has been recently identified as a potential target for disease progression using whole - genome trancriptomic and whole proteomic . As shown in table s2, 11 of the relationships in figure 7 are contained in the string database . These results suggest that proteomic data can provide complementary information to genomics data and help to reveal important biological mechanisms ., we show the most enriched go category with corresponding benjamini adjusted p - value (p). In particular, only modules reporting a significant enriched benjamini adjusted p - value (p 0.01) are highlighted . Heatmap of the absolute value of correlation between genes based on proteomic and rnaseq data for module c1 (a) and c2 (b) in figure 6 . For each module, we include a pie plot showing the number of protein - specific interactions (red) and interactions shared across proteomic and rnaseq data (green). Because we are interested in assessing the utility of a joint learning, jrf was compared with the standard random - forest algorithm that constructs the two networks separately . For both methodologies, we derived undirected networks using the same fdr cutoff (0.001). The protein - network resulting from genie3-sep can be found in figure s11 in the supporting information . As shown, 57% of the edges are shared across protein- and rna - networks under jrf, while only 26% are shared under genie3-sep . This finding can be explained by the fact that a joint learning can facilitate the detection of common edges . In this section, we validate our findings using go categories . In particular, we consider 596 go terms containing fewer than 200 genes (the list of go terms can be found in tables s3s6 in the supporting information). Figure 8a shows the number of edges contained in at least one go term for different fdr cutoff for both protein - networks . First, we constructed a network based on go terms where an edge (a b) was drawn between every pair of genes (a, b) sharing at least one go term . Then, a contingency table was constructed considering variables network based on go terms and network based on proteomic data with categories number of edges contained and number of edges non - contained, and a fisher s exact test was performed to derive enrichment p - values . Figure 8a, b shows that jrf results in a network more overlapping with go terms than the standard random - forest algorithm genie3-sep . We validate protein - networks in figure 4 using go terms and knockout signatures . The first row contains network validation based on go terms . For our analysis, we considered 596 go terms containing fewer than 200 genes . (a) number of edges contained in at least one go term for different fdr cutoff . The second row contains validation based on esr1 (c) and gata3 (d) signatures . For a particular neighborhood size of either esr1 or gata3, we show the total number of knockout signatures contained under jrf () and genie3-sep (). To further validate our findings, we overlapped both networks with esr1 and gata3 knock - down signatures . We identified sirna knock - down signatures of key transcription factors (tfs) of breast carcinoma in mcf7 cells1 by accessing gene expression data available under gse31912 in gene expression omnibus (further details about these signatures can be found in section 2 of supporting information). For each network, a neighborhood was defined by defining a threshold k for the number of edges connecting a particular gene to the target gene of interest (either esr1 or gata3). For each network in figure 8, we considered different values of k and counted the number of knockout signatures contained in the neighborhood . Figure 8c, d shows the total number of signatures contained in k - size neighborhoods for esr1 and gata3, respectively . As shown, also in this case, jrf results in a more enriched network . Figure s12 contains the same comparison for rna networks . In this paper, we developed jrf, a random - forest - based algorithm for the simultaneous construction of gene coexpression and protein coexpression networks . Jrf is designed to borrow information across different expression data by selecting the same set of predictors (genes) as splitting variables in the random forest model corresponding to each data . In this way, jrf is able to detect common relationships (edges) with better power and detect differential edges - specific to individual data with fewer false positives . Because in this study we were mainly interested in assessing the unique contribution of proteomic data to breast cancer research, we focused our attention on protein - specific hub genes and modules . We identified two interesting protein - specific modules containing potential targets for breast cancer treatment . In addition, we compared our algorithm to the original random - forest algorithm, which constructs the two networks separately, and showed that our algorithm leads to networks more overlapping with available knockout signatures and existing go terms . The current version of jrf requires the feature (protein) space to be the same across different classes . Extension of jrf for handling data involving different sets of variables remains as future work . This problem arises in the protein domain because protein abundance can be measured for different phosphorylation - sites that map to a unique protein . As future work, we will design a model able to jointly estimate networks from phosphorylation - site abundance and rnaseq data, simultaneously . Besides estimating gene - regulatory network and protein - regulatory network, as shown in the paper, jrf can be used in many other applications . For example, we can apply jrf to estimate grns for the three breast cancer subcategories (luminal, basal, and her2), grn for different tissues, and grn for cancer and normal tissues . Another interesting direction would be assessing the association between mirnas - genes and mirnas - proteins simultaneously . It is well known that the downregulation of some mirnas may play an important role in the progression of cancer . Therefore, a better understanding of the interactions between mirna, mrna, and protein expression is crucial to cast light on the potential disease mechanisms of cancer . In this context, jrf can be easily utilized to jointly detect which mirnas regulate genes and proteins . Jrf can be easily parallelized because the computation can be divided into p - independent subproblems . On the contrary, for many existing methods such as bayesian networks and ggm - based algorithms, the merit of jrf depends on the assumption that networks of different classes share some common structures . To assess the performance of jrf in the absence of common structure, we conducted numerical studies to investigate such cases and concluded that jrf is sufficiently flexible to guarantee good performance even when nonrelated networks are considered . In jrf, we propose a permutation - based procedure to derive proper cutoffs on importance scores for selecting confident edges . Specifically, we introduce f() as an approximation of the false discovery rate (fdr) of the selected edge set based on cutoff; however, in the numerical studies, we observe that f() tends to underestimate fdr . New methods to obtain more accurate estimate of fdr for jrf warrant future research.
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Maintenance of low serum urate (sua) levels is important for the management of gout.1 achieving the recommended sua levels of less than 6.0 mg / dl2,3 is difficult in elderly (65 years of age and older) patients with renal impairment . Allopurinol has been used as a first - line drug for the treatment of hyperuricemia.3 adverse reactions such as hepatic disorder, hypersensitivity vasculitis, and bone marrow depression have been reported.48 moreover, the dose of allopurinol requires reduction according to the degree of renal impairment.9,10 febuxostat is a nonpurine selective inhibitor of xanthine oxidase that forms a very stable interaction with both the oxidized and reduced forms of the enzyme.11 becker et al reported superior efficacy of febuxostat compared with that of allopurinol in diabetic gout patients.12 however, the efficacy of febuxostat was not superior to that of allopurinol in the febuxostat versus allopurinol controlled trial,6 and there was no significant difference between the two drugs with respect to the rate of adverse events (aes).6,13,14 febuxostat has been shown to be efficacious in elderly patients;13 however, the efficacy and safety of febuxostat in elderly female patients with hyperuricemia remain unclear . To promote proper use of febuxostat elderly japanese patients (65 years of age or older) who were treated with febuxostat at fujita health university hospital from january 2012 to december 2013 were included in the study . Patients treated with hemodialysis or peritoneal dialysis, those treated with anticancer or immunosuppressive therapies, those with baseline sua levels of less than 7.0 mg / dl, and those for whom sua levels were not measured within 16 weeks after starting febuxostat therapy were excluded . This retrospective cohort study used information from the electronic medical records of fujita health university hospital . These values were calculated using the modified isotope dilution mass spectrometry - traceable modification of diet in renal disease study equation.15 the treatment goal was defined as achievement of sua levels of 6.0 mg / dl or less16 within 16 weeks; this was the primary end point in the present study . Aes of febuxostat were defined as more than twofold increases in common terminology criteria for adverse events scores compared with baseline . We focused on aes commonly observed at high frequency when using xanthine oxidase inhibitors.68,17 among them, leukopenia hepatic disorder, thrombocytopenia, and anemia were selected for use as secondary end points in the present study . Continuous data are presented as the mean (range), and nominal data are presented as percentages . Continuous and nominal data were analyzed with student s t - test and chi - square test, respectively . The mean time to achievement of the treatment goal was measured from the initiation of febuxostat treatment until the first observation of sua levels of 6.0 mg / dl or less . Time - to - event curves were plotted using the kaplan meier method, and comparisons among groups were performed with the log - rank test . In these tests, spss version 22.0 software (ibm corporation, armonk, ny, usa) was used for statistical analysis . Elderly japanese patients (65 years of age or older) who were treated with febuxostat at fujita health university hospital from january 2012 to december 2013 were included in the study . Patients treated with hemodialysis or peritoneal dialysis, those treated with anticancer or immunosuppressive therapies, those with baseline sua levels of less than 7.0 mg / dl, and those for whom sua levels were not measured within 16 weeks after starting febuxostat therapy were excluded . This retrospective cohort study used information from the electronic medical records of fujita health university hospital . These values were calculated using the modified isotope dilution mass spectrometry - traceable modification of diet in renal disease study equation.15 the treatment goal was defined as achievement of sua levels of 6.0 mg / dl or less16 within 16 weeks; this was the primary end point in the present study . Aes of febuxostat were defined as more than twofold increases in common terminology criteria for adverse events scores compared with baseline . We focused on aes commonly observed at high frequency when using xanthine oxidase inhibitors.68,17 among them, leukopenia hepatic disorder, thrombocytopenia, and anemia were selected for use as secondary end points in the present study . Continuous data are presented as the mean (range), and nominal data are presented as percentages . Continuous and nominal data were analyzed with student s t - test and chi - square test, respectively . The mean time to achievement of the treatment goal was measured from the initiation of febuxostat treatment until the first observation of sua levels of 6.0 mg / dl or less . Time - to - event curves were plotted using the kaplan meier method, and comparisons among groups were performed with the log - rank test . In these tests, spss version 22.0 software (ibm corporation, armonk, ny, usa) was used for statistical analysis . We evaluated 82 patients treated with febuxostat during the observation period and classified them into male (n=53) and female (n=29) groups . The mean body weight and body surface area of the female group were significantly lower than those of the male group . Baseline renal function was lower in the female group, whereas liver function and sua levels were similar between the 2 groups . Morbidity rates for hypertension and diabetes mellitus were not significantly different between the 2 groups . The results of mean time to achievement of the treatment goal are presented in figure 1 . The mean time in the female group (53 days; 95% confidence interval, 3968 days) was significantly shorter than that in the male group (71 days; 95% confidence interval: 6083 days). Aes after treatment with febuxostat are indicated in table 2, and the grades of aes are shown in table 3 . There were no significant differences in all terms between the 2 groups . The dose of febuxostat commonly prescribed in japan (1020 mg / day) is lower than that prescribed in other countries, and there was a time lag preceding the drug s introduction in japan . Moreover, the majority of patients with hyperuricemia are male . Thus, clinical evidence of the suitability of febuxostat for elderly female patients in japan is less than that accumulated in many other countries . To increase the usefulness of febuxostat, we evaluated the efficacy and safety of febuxostat in elderly female patients in japan . Although achievement of the treatment goal was difficult in patients with renal impairment, more than 70% of the female patients achieved sua levels of 6.0 mg / dl or less within 16 weeks . Furthermore, the mean time to achievement of the treatment goal was shorter in female patients than in male patients . Khosravan et al concluded that neither age nor sex has a clinically significant effect on the pharmacokinetics, pharmacodynamics, or safety of febuxostat.18 their results are in conflict with those of the present study . Khosravan et al investigated these parameters in healthy subjects and assessed the parameters after 7 days,18 whereas the present study focused on elderly patients using low doses of febuxostat and observed the parameters for 16 weeks . Therefore, their subjects, doses, and duration of evaluation were different from those of the present study . Moreover, renal function and body surface area levels in female patients in the present study were low; thus, the blood concentrations of febuxostat are expected to have been higher than those observed in the previous study . Some previous reports have suggested that the efficacy of febuxostat13,19,20 increases in a dose - dependent manner, whereas its safety in elderly patients is not affected by high doses.13 although the blood concentration of febuxostat may be increased in female patients, the incidence of aes in female patients was similar to that in male patients . These results suggest that a low dose of febuxostat is safe and efficacious in elderly female patients . Therefore, larger, prospective, multicenter studies (eg, febuxostat versus allopurinol controlled trial) will be needed to further validate our results . Further studies that focus on the pharmacodynamics of febuxostat in elderly patients will be needed . In the present study, we demonstrated that the efficacy of febuxostat in elderly female patients is superior to that in elderly male patients, whereas the safety is equivalent.
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At this institution, a retrospective review was performed and 36 consecutive patients were identified with decompensated cirrhosis with refractory or large - volume ascites where indwelling peritoneal catheter placement was utilized . Each patient had biopsy - proven cirrhosis, and an etiology was determined for each patient . Records reviewed included complete blood count, comprehensive metabolic panel, coagulation panel, viral serologies, and biopsy reports . All patients were up to date with alpha - fetoprotein and abdominal ultrasound testing for hepatocellular carcinoma with three patients undergoing active therapy . A pigtail catheter was utilized and placed into the peritoneal cavity via the seldinger technique under sterile procedure (10, 12). Prior to placement, the patient was evaluated with both shifting dullness and abdominal ultrasound to document large - volume ascites . A site was determined with shifting dullness or abdominal ultrasound, whichever was more appropriate . After site determination, a surgical scrub was utilized to prep the surface and a sterile drape was placed overlying the area . Under sterile technique, the skin was anesthetized with 510 ml of 1% xylocaine solution . Free fluid was aspirated with the small 22-gauge needle to assure adequate placement and positioning . A needle was then introduced into the peritoneal space through the anesthetized tract . The pigtail catheter a locking device was then placed on the catheter . The drain was then sutured in using a 2 - 0 prolene . Ascites was removed at a rate of 3 l every 8 h. if 3 l was achieved, the nursing staff would utilize the locking device to halt additional fluid output until an 8-h shift had passed before again allowing gravity drainage to take place . Albumin (25%, 50 g) was infused if 3 l was removed in the 8-h shift . Daily ascitic fluid cell counts were performed in addition to daily laboratory profiles with complete blood count (cbc) and comprehensive metabolic panel (cmp). All patients signed an informed written consent for paracentesis with indwelling peritoneal drainage for up to 72 h. in addition, this retrospective study was approved by the institutional review board at university of illinois college of medicine at peoria . Analysis was conducted with sas 9.4 (by sas institute inc, cary, nc, usa). Descriptive results were reported as meanstandard deviation for continuous variable, and percentage for categorical variable . Generalized estimating equations were used to estimate and compare clinical characteristics at different time points . A two - tailed p value was calculated for all tests, and p0.05 is considered as being statistically significant . The values of serum creatinine improvement, albumin infused, plasma white blood cell count, ascitic fluid white blood cell count, and blood urea nitrogen (bun) are given in table 2 . The mean ascitic fluid removed on day 1, 2, and 3 was 6.60, 5.72, and 4.21 l respectively . The amount of albumin given directly correlated with the amount of fluid removed with 80.76, 68.26, and 60.0 g being infused on day 1, 2, and 3, respectively, correlating to 12 the mean weight on day 1 was 86.5 kg (range: 75.997.0 kg), and on day 3, it was 77.8 kg (range: 66.888.8 kg). Serum creatinine declined statistically from 1.37 on day 1 to 1.21 on day 3 (p<0.05). Predicted values (mean, 95%ci) of clinical characteristics based on generalized estimation equations p<0.05 compared to the baseline (day 1). Follow - up within 26 weeks . At a follow - up at a mean time of 17 days, serum bun values were not affected significantly throughout with mean values of 31.1, 31, 29, and 25.5 mg / dl on day 1, 2, 3, and follow - up, respectively . No patient required a repeat paracentesis or indwelling peritoneal drain for at least 2 weeks after the initial drain placement . No patient was noted to develop any clinical signs or laboratory evidence of infection or secondary bacterial peritonitis . The ascitic fluid white blood cell count trended upwards from 126 on day 1 to 234 on day 3 . None of the patients met the criteria for secondary bacterial peritonitis of neutrophil count> 250 cells/l . The most common complaints encountered were pain at insertion site in seven patients and minor bleeding in two patients as represented in table 3 . Paracentesis is known to reduce short - term mortality and improve outcomes in patients with cirrhosis . A nationwide analysis showed in - hospital mortality reported to be lower in decompensated cirrhosis patients who underwent paracentesis within 24 h of admission (5.7% vs 8.1%; p=0.49) (13). Traditionally, large - volume paracentesis with colloid infusion has been shown to be more effective than diuretics alone in managing ascites in hospitalized patients (5). This often results in large - volume shifts with development of intravascular volume depletion and renal insufficiency ., we attempted to demonstrate the efficacy and safety of indwelling peritoneal catheter placement for refractory and large - volume ascites, especially its effect on renal function . Patients with refractory ascites often have contraindications to the use of diuretics due to their concomitant renal dysfunction, fostering electrolyte imbalance and precipitating hepatic encephalopathy (14). While previous studies have documented its safety, its role in maintaining renal function has not been reported . It has been shown that increased intra - abdominal pressure can cause acute renal failure in humans and animals (15, 16). Removal of this pressure resulted in immediate improvement in renal function and resolution of anuria (1619). In our study, a mean of 16.53 l of fluid was removed over a period of 72 h. this corresponded with a mean drop of 8.7 kg body weight between day 1 and day 3 . The removal of this volume of fluid from the patient alone could be responsible for causing a significant improvement in renal perfusion and subsequent renal function seen in our patient population, secondary to reduced intraperitoneal pressures . Large - volume paracentesis supported with albumin infusion has been shown to decrease the risk of post - paracentesis related circulatory dysfunction (ppcd). It has been reported that the use of albumin was associated with a significantly reduced risk of ppcd (reducing odds by 66%) and reduction in morbidity and mortality by 36% . While this reduction in ppcd is mainly believed to be due to albumin, the slow gravity - dependent removal of large volume of fluid could play a further role in preventing this complication (20). Moreover, slow gravidity - dependent removal of ascites along with albumin infusion not only helps in effective removal of ascitic fluid but also facilitates fluid movement from the interstitium into the vascular space due to the increased oncotic effect of albumin in the plasma . This also plays a role in preventing the rapid re - accumulation of fluid as well as improved renal perfusion (10). In this study g on days 1, 2, and 3, respectively, with albumin infusion directly correlating with the amount of fluid evacuated . It has already been demonstrated that albumin infusion helps in the resolution of hepatorenal syndrome (21). We believe that this slow gravity - dependent removal of ascites along with albumin infusion could have a further beneficial role in preventing renal dysfunction and subsequent development of hepatorenal syndrome, but more prospective studies are needed to validate such findings . Overall there is a paucity of literature regarding the efficacy and side effects of an indwelling catheter in the management of non - malignant refractory ascites 1012 . In addition, concerns have been raised regarding the risk of bacterial peritonitis with the use of indwelling peritoneal catheters (11, 12, 15). Recently, a retrospective observational study reported the risk of secondary bacterial peritonitis as high as 10% in patients with indwelling peritoneal catheters . This was based solely of neutrophilic criterion of fluid analysis and did not correlate with cultures or known clinical decompensation after drain removal (11, 22). In our study, none of our 36 patients developed bacterial peritonitis in the 72-h time frame of indwelling peritoneal catheter . Our ascitic fluid white blood cell count trended from a mean value of 126/mm on day 1 to 234/mm on day 3 . This trend likely represents several pathophysiologic processes occurring in patients with an indwelling peritoneal catheter . The first is a concentration of the ascitic fluid volume within the peritoneal cavity, thereby increasing all cellular material per unit of measurement . The second is mild abdominal wall irritation from the drain catheter with a resulting mild inflammatory response . In addition, over a 4-week follow - up period, no patients presented to the hospital with clinical findings or laboratory evaluation consistent with bacterial peritonitis . The most common side effects encountered in our study were pain at insertion site in seven patients (19.4%) and minor bleeding from the drain site in two patients (5.6%). Both cases of minor bleeding responded to local pressure for 10 min to the insertion site with no recurrence . The mortality also did not seem to be affected by drain placement with average survival being around 22 months after drain placement varying in different patients according to the severity of their respective meld scores (11). Our results are consistent with a previous study done by van thiel and colleagues . In their study the duration of indwelling drain placement was up to a maximum of 72 h similar to our study . The most common complaint accompanied in their study was abdominal wall discomfort in 63% of the patients and abdominal wall hematoma in 5% of the patients (10, 22). Another study by nadir and colleagues showed the risk of spontaneous bacterial peritonitis (sbp) to be more only in those patients in whom the drain was left in for more than 3 days (12). In addition, previous studies have shown increased risk of concurrent proton pump inhibitor (ppi) therapy and development of sbp (23, 24). This risk is greater with a ppi therapy (n=3,815; or 3.15, 95% ci 2.094.74) as compared with those on h2 therapy (n=562; or 1.71, 95% ci 0.973.01) (25). In our study 30 (~75%) patients were on some form of ppis while the indwelling catheter was placed in and none had clinical sbp, thus suggesting that ppis did not enhance the risk of developing sbp . In conclusion, continuous paracentesis with an indwelling peritoneal catheter represents an important modality in the evacuation of ascites in selected patients . This technique when used for less than 72 h and with albumin infusion can be a safe and effective means to manage patients in the hospital with large - volume tense ascites . Sw, za, and kr collected the data; rj analyzed the data; and dm and sd reviewed the literature and made critical revisions related to the content of the article . The authors have not received any funding or benefits from industry or elsewhere to conduct this study.
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According to the international diabetes federation, the worldwide prevalence of adults with diabetes mellitus (dm) is 8.3%, accounting for approximately 382 million people.1 in the people s republic of china, the prevalence of dm in adults aged 20 years or older has increased to 9.7%, accounting for 92.4 million adults with dm.2 with regard to the association between family history risk categories and prevalence of dm in chinese adults aged 20 years or older and living in the people s republic of china, the prevalence has reached 32.7%, 20.1%, and 8.4%, representing at least two generations and one generation of first - degree relatives with dm, and no first - degree relatives with dm, respectively.3 with the same tendency, the prevalence of type 2 dm has also increased sharply among chinese immigrants in the usa, so a relevant and interesting conceptual model of type 2 dm self - management was designed and developed by a group of researchers from the people s republic of china and the usa for this specific group of individuals . This model has seven interrelated factors, ie, sociodemographic characteristics, behavioral and psychological characteristics, social support, linguistic barriers, cultural characteristics, type 2 dm self - management behaviors, and diabetes - related health outcomes (eg, health - related quality of life [hrqol], hba1c, blood pressure, and other cardiovascular risk factors), and postulates that the first five components have an influence on type 2 dm self - management, and that all factors except for the latter predict health outcomes.4 in portugal, the prevalence of dm in people aged between 20 and 79 years has reached a worrying rate of 11.7%.5 hrqol aggregates the individual s subjective perception of physical, emotional, and social well - being,69 includes a cognitive and emotional component,9 and is becoming increasingly important for those providing health care for diabetic patients.10,11 concerning the relationship between age, sex, body mass index (bmi), different treatment regimens of type 1 and type 2 patients, disease duration, and hrqol in patients with dm, the studies have not been consistent, so it is important to evaluate the relationships between these factors and hrqol . To our knowledge, the research carried out in portugal that evaluated the effect of certain demographic and clinical variables on hrqol in patients with dm did not make the adjustment of hrqol scores for age, despite the fact that they can mediate some of these relationships . Most studies have found that older age is related to worse hrqol in dm patients.1225 however, one study26 reported that older age of diabetic patients was related to better hrqol, whereas fal et al7 did not find a relationship between age and hrqol . Other studies2729 found that older age in diabetic patients was related to worse and better hrqol in different dimensions . Nevertheless, some studies7,19,20,25,33 did not find any relationship between sex and hrqol in diabetic patients . Concerning the relationship between bmi and hrqol in dm, most of the studies have shown that patients with higher bmi had worse hrqol.16,20,21,23,28,34,35 however, mier et al36 did not find a relationship between bmi and hrqol in type 2 diabetic patients . As far as we know, there are no studies conducted in portugal that have evaluated the relationship between hrqol and bmi in individuals with dm . According to two meta - analyses of randomized controlled trials that studied the effect of weight loss on hrqol, positive clinical changes in bmi do not always produce a gain in hrqol.37,38 with regard to the relationship between type of dm and hrqol, some studies22,39,40 found that type 2 dm was related to worse hrqol than type 1 dm, while other authors30,41 found no relationship between type of dm and hrqol . Trief et al27 found that type 2 dm was related to poor and better hrqol in the different domains than type 1 dm . Another study33 found that patients with type 2 dm reported better hrqol in one dimension of the short - form 36 than patients with type 1 dm, whereas in other dimensions of this generic tool assessing hrqol, the two groups of patients did not differ . Results of research on the relationship between treatment regimens in type 2 diabetic patients and hrqol have found that insulin therapy was related to lower hrqol.16,18,19,28,34,36,42 nevertheless, two studies17,26 reported an absence of a relationship between type of therapeutic regimen in type 2 dm and hrqol, while two studies7,33 found that patients with type 2 diabetes treated with insulin had poor and better hrqol in the different domains than those receiving oral hypoglycemic agents . Concerning the relationship between treatment regimens in type 1 dm and hrqol, one study22 found that type 1 patients on a conventional insulin regimen reported worse hrqol than those on an intensive insulin regimen . However, other study43 reported no relationship between treatment regimen in type 1 dm and hrqol . Finally, some studies looking at the relationship between duration of dm and hrqol have shown that a longer duration of the disease was related to lower hrqol;20,22,23,32,36 however, others7,12,16,26,33 found no relationship between diabetes duration and hrqol, and one43 found that an increased duration of dm was related to better hrqol . In summary, studies that have investigated the relationship between demographic and medical variables have used a wide range of methods6 and questionnaires44 to assess hrqol, and have covered different aspects of hrqol . These differences may contribute to the discrepancy between study results, and undermines the discussion of our present results . Given the discrepancies found in the literature, and the lack of studies assessing hrqol in portuguese diabetic patients, the aim of this cross - sectional study was to assess the relationship between hrqol and age, sex, bmi, type of dm and treatment regimens, and duration of dm . A consecutive sample of 124 patients from the outpatient department of endocrinology, diabetes and metabolism, centro hospitalar so joo, porto, portugal, was recruited . Patients were included in the study if they had a diagnosis of type 1 or type 2 dm, were at least 18 years old, and were not currently pregnant . The protocol for the study was approved by the ethics committee for health at the centro hospitalar so joo, and informed consent was obtained from all participants before participation in the study . Bmi was expressed as weight (in kilograms) divided by the square of height (in meters), and categorized according to the world health organization45 as normal weight (one or two daily administrations of insulin) or intensive treatment (at least three daily administrations of insulin). Type 2 dm patients were classified as on oral hypoglycemic agents or insulin therapy (the latter including patients only on insulin therapy or on oral hypoglycemic agents plus insulin therapy). Less than 10 years, 1019 years, or 20 years or more . General hrqol was assessed by the portuguese version of the medical outcomes study 36-item short - form health survey (sf-36).46,47 the eight domains used to assess hrqol in this analysis were: physical functioning, role - physical, bodily pain, general health, vitality, social functioning, role - emotional, and mental health . Raw dimension scores were transformed to scales ranging from 0 to 100, in which higher scores indicate higher hrqol . Given the relationship between age and hrqol, we adjusted our sf-36 results to the age of the participants; therefore, all other variables were related to the residuals of simple linear regression models having each dimension as a dependent variable and age as the independent variable . All questionnaires were administered by one of the researchers . Statistical package for the social sciences version 20.0 for windows software (ibm corporation, armonk, ny, usa) was used for the analysis . Descriptive statistical analysis included calculation of means and standard deviations for cardinal variables, and frequencies for ordinal and nominal variables . Independent samples t - tests and one - way analysis of variance were used to compare means of independent samples; when significant differences were observed using analysis of variance, we performed multiple comparisons for observed means using the bonferroni correction . The degree of association between pairs of variables was measured by pearson s (r) or spearman s (rs) correlation coefficients . Most patients were male, had type 2 dm, had had the disease for 10 years or more, and were overweight or obese . The average values obtained by patients in the eight dimensions of the sf-36 are shown in table 3, as well the relationship between these scores and age . There was an association between age and six of the eight dimensions of the sf-36: increased age was associated with worse hrqol in terms of physical functioning, role - physical, bodily pain, vitality, social functioning, and role - emotional . The age - adjusted scores of hrqol by sex are shown in table 4, bmi class in table 5, and type of dm and treatment regimens in table 6 . Diabetic women had worse hrqol in terms of physical functioning, general health, vitality, social functioning, role - emotional, and mental health than men, and evidenced a tendency in the same direction in terms of role - physical and bodily pain . Obese patients had lower hrqol in terms of physical functioning when compared with normal and overweight patients, and reported worse vitality than normal weight patients . Type 2 patients on insulin therapy had worse physical functioning and vitality than type 2 patients only on oral hypoglycemic agents . Globally, type 2 patients taking insulin tended to show poorer hrqol than patients in other treatment regimens, except in terms of role - physical . Intensively treated type 1 patients tended to have better hrqol than patients on other treatment regimens, except in terms of social functioning and role - emotional . An increased duration of dm (measured according to the categories presented in table 1) was associated with lower hrqol in terms of physical functioning (rs = 0.271, p=0.002), role - physical (rs = 0.209, p=0.020), general health (rs = 0.372, p<0.001), vitality (rs = 0.312, p<0.001), role - emotional (rs = 0.224, p=0.012), and mental health (rs = 0.197, p=0.028). In terms of social functioning, there was a tendency in the same direction; however, it did not reach statistical significance (rs = 0.160, p=0.077). Regarding bodily pain dimension, it seemed to exist no relationship with duration of disease (rs = 0.045, p=0.621). Demographic and clinical variables in patients with dm were related to hrqol, regardless of age . Globally, we highlight physical functioning and vitality as the dimensions more related to these variables . The adjustment of sf-36 scores for age may obscure relationships between several clinical variables and hrqol dimensions that strongly depend on a concomitant relationship with age . Therefore, absolute scores on the sf-36 and their relationship with clinical variables may have clinical usefulness, but to analyze their direct effect on hrqol, which was our intention, it is recommended that values are adjusted for age . Our results showed that being female was related to worse hrqol in terms of physical functioning, general health, vitality, social functioning, role - emotional, and mental health, and a tendency in the same direction in terms of role - physical and bodily pain . These findings are partially in agreement with those of kleefstra et al, neves et al, and papadopoulos et al,2123 who found that diabetic females reported, respectively: lower hrqol in all dimensions of sf-36 with the exception of the general health dimension; in physical functioning, bodily pain, vitality, and mental health of sf-36; and in all dimensions of the aforementioned instrument . Different explanations for the worse hrqol of women with dm have been presented: females have an increased risk of developing cardiovascular disease,48 being hypertensive, obese, having more symptoms related to hyperglycemia,49 presenting higher prevalence rates of depression,31,50 anxiety, having less energy,31 and presenting greater difficulty in doing physical exercise,50 and this factor may lead to worse hrqol, particularly in some dimensions . An alternative explanation may be the fact that women find themselves overburdened with several competing demands, such as their commitments toward their closest relatives, as well as with their jobs and household activities, whereas men are more concentrated in their paid jobs, which may contribute to greater physical and psychological overload.51 these above - mentioned sex patterns with regard to work roles were observed across countries . It is worth noticing that in our study women showed the longest duration of dm and a higher prevalence of obesity, which may act as confounders contributing to the worse hrqol . On the other hand, the relationships between age and some clinical variables, namely the type of dm and bmi, highlight the relevance of age - adjustment to the hrqol scores . Although mier et al36 reported no relationship between obesity in type 2 dm and hrqol, we found that obese patients assessed their hrqol as being worse than normal and overweight individuals in terms of physical functioning . It was previously shown that an increasing bmi in type 2 dm was related to lower physical functioning,20,21,23,34 as well as in worse hrqol in other dimensions of the sf-36: role - physical,20,21 bodily pain,21 general health,34 social functioning,21,34 and role - emotional.20 we also emphasize that obese patients had lower vitality than their normal weight counterparts . However, as far as we know, we found no study reporting that obese diabetic patients had worse hrqol in this particular dimension in comparison with normal and overweight ones . A possible explanation for the worse physical functioning and vitality in obese patients may be related to the coexistence of hypertension or other chronic morbidities in this group of patients . While wexler et al and caldwell et al17,26 did not observe any relationship between treatment regimens in type 2 dm and hrqol, we observed that insulin therapy in type 2 dm was related to lower scores on the physical functioning dimension compared with those who were on oral hypoglycemic agents, in accordance with previous research.18,19,34 moreover, johnson et al19 reported that insulin use in type 2 dm was related to worse hrqol in terms of role - physical, general health, and social functioning . Interestingly, fal et al7 observed that patients with type 2 diabetes on insulin therapy had worse hrqol in the somatic and environmental domains and better hrqol in the psychological domain than those receiving oral hypoglycemic agents . Additionally, we found that type 2 diabetic patients treated with insulin showed poorer vitality than type 2 patients not treated with insulin therapy . Nevertheless, to our knowledge, there are no studies reporting that insulin use is related to worse hrqol for this specific dimension in type 2 diabetic patients . Different explanations have been put forward by various authors to explain the lower hrqol in patients requiring insulin to achieve better metabolic control: the side effects of the medication and its influence on scheduling and organizing their daily activities,7 the fear of weight gain, and concerns about the impact of the insulin regimen on their social environment.52 alternative explanations for these findings in such patients may be the longer duration of the disease, a sense of frustration after failure of oral medications, or the onset of diabetes - related complications and subsequent necessity to start insulin . Patients commonly held erroneous and irrational beliefs about the relationship of the need of insulin therapy and the development of chronic complications of dm . These hypotheses should be tested in future research, on the one hand using larger samples allowing multivariate analysis, and on the other hand by qualitative studies focusing on the personal representations of different features of treatment regimens . In our study, there were no differences on any of the hrqol dimensions between type 1 patients on a conventional regimen and those on an intensive insulin regimen, which is consistent with the results obtained by eiser et al43 but different from the findings of neves et al,22 who found better hrqol in terms of physical functioning and vitality in type 1 patients on an intensive insulin regimen . Eventually, the absence of differences in terms of hrqol between insulin treatment regimens in type 1 dm may be related to the small number of patients interviewed . Some studies did not find a relationship between duration of diabetes and hrqol in type 2 dm7,12,16,26,33 or in type 1 dm,33 and another study43 reported better hrqol in terms of diabetes satisfaction with increasing duration of the disease in young adults with type 1 dm . In contrast, across both type 1 and type 2 patient groups, we found a negative association between duration of diabetes and hrqol in terms of physical functioning, role - physical, general health, vitality, role - emotional and mental health, and a tendency in the same direction in terms of social functioning . These results, on one hand, are partially overlapping with the findings of neves et al,22 since in that study an increased duration of dm was related to lower hrqol in all dimensions of the sf-36, but on the other hand partially agree with the findings of kalda et al, papadopoulos et al, and mier et al,20,23,36 who found that a longer duration of diabetes was related to worse general health, vitality, social functioning,20,23 bodily pain,23 mental health,20 and physical component.36 possible explanations for this reduction in hrqol for diabetic patients with a longer duration of disease may be related to the sharp decrease of energy, vitality, and social relationships, the development of long - term complications of dm in the later stages of the disease, and consequently to the intensification and complexity of the treatment regimen.20 it is worth noticing that, by comparing age - adjusted hrqol scores, the relationship found between duration of diabetes and hrqol is not due to the older age of patients, but to the longer duration of dm . The difference in the results between this study and others may be explained, on the one hand, by the different generic tools used to assess hrqol, which do not measure exactly the same dimensions, and by the fact that some studies have evaluated only type 1 or type 2 diabetic patients, whereas in the present study both types of patients were included . Given the frequent age difference between patients with type 1 diabetes and those with type 2 diabetes, and due to the fact that not all studies make the age - adjustment of the values of the generic instruments to assess hrqol for this variable, we highlight the implications for clinical practice when hrqol is evaluated considering age - adjusted values . Several limitations of this study should be considered when interpreting its results . Further, most of the patients interviewed were of low sociocultural status, which may have limited their understanding of the questions in the instrument used to assess hrqol . Generalization of our findings may be limited because our sample was collected in a central public hospital and might not be representative of the diabetic population in other care centers or in primary care . Further investigations are warranted to adjust the dimensions of hrqol to other variables (eg, sex, bmi, type of dm and treatment regimens, and disease duration) to explore the relationships between hrqol and the demographic and clinical variables considered, we think may explain some of these relationships . As previously stated, the use of qualitative methodology could be useful to obtain information complementary to that achieved by quantitative studies, allowing more accurate interpretation of some results . Finally, participants self - reported duration of diabetes, and height and weight values when calculating bmi might not be as reliable as measurements taken from official medical records . One of the main points of our results is the relationship found between the vitality dimension of the sf-36 and obesity and insulin use in patients with type 2 dm . To our knowledge, there are no studies providing such data (ie, reporting the negative impact of these two factors on this specific dimension of hrqol), which seems worthy of exploring further . Apart from the reasons previously stated, adjusting hrqol values for age is important, since type 1 patients are significantly younger than those with type 2 diabetes, and our analysis allowed us to analyze and compare these two groups without the confounding effect of age . Our findings suggest that being female, obese, having type 2 dm and taking insulin, and having diabetes for a longer period of time are characteristics associated with worse age - adjusted hrqol in patients with dm . Globally, physical functioning and vitality were the dimensions more related to these demographic and clinical variables.
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Among menstrual disorders, menorrhagia, dysmenorrhea, and abnormal cycles are highly prevalent and are considered as disabling conditions among women at reproductive ages . Dysmenorrhea is defined as painful cramps in lower abdomen during menstruation without any evidence of pelvic pathologic lesions . This condition occurs right before or during menstruation without any describable macroscopic pelvic pathology . In general, 50% of women suffer from dysmenorrhea and 10% experience this condition for 13 days a month . One study reported the prevalence of dysmenorrhea to be over 75% . In another study in sweden, more than 72% of 19-year - old women suffered from this condition (34%, 23%, and 15% of the participants had mild, moderate, and severe symptoms, respectively). Dysmenorrhea can reduce efficiency and production due to daily stresses and socioeconomic losses . In the us, at least 600 million hours and 2 billion dollars are lost due to dysmenorrhea every year . In addition, dysmenorrhea affects young girls' opinion about femininity and method of childbirth . For the treatment and control of dysmenorrhea, there are many ways: common drug interventions for reduction of pain include nonsteroidal anti - inflammatory drugs (nsaids) and oral contraceptive pills . Despite their usefulness, their side effects include diarrhea, stomachache, nausea, renal complications, hepatic complications, blood problems, edema, sleepiness, indigestion, and fatigue . Other treatment methods also include supplementary vegetarian diet, vitamins, minerals, nerve block, spinal manipulation, magneto - inertial fusion, percutaneous electrical nerve stimulation, acupressure, acupuncture, rest, muscle relaxation, reflexology, hand pressure, heat, aromatherapy, and physical therapy . Since the acupressure is a new therapy, the side effects of drug therapies are not readily acceptable . Hence, but the rationales for using complementary and alternative medicine have been widely used for many years, and they are accepted by many people and doctors . Approximately 38.2 million adult people in america have used complementary medicine (herbs and supplements) in 2002 . Moreover, more than half of them said that complementary medicine was important to their health and well - being, besides the points examined in this study . In reviewing the past 30 years, no article has been found . Hence, the acupressure points in this research are entirely new . According to the gate control theory of pain, cutaneous stimulation through massaging, needling, and scratching can stimulate large fiber nerves that transfer nervous impulses to the spinal cord . In case this stimulation is continued, it can close pain gates, eventually reducing the feeling of pain . Evidence has shown acupressure as a noninvasive, inexpensive, safe, and simple method for reduction of labor pain . In the study by wang et al ., (2010) acupressure was applied at sanyinjiao (sp-6) acupoint for 20 min to assess its effect on menstrual pain and distress in young women suffering from dysmenorrhea . The results revealed a significant decrease in pain in acupressure and self - care training groups based on short - form mcgill pain questionnaire . In another study in iran, acupressure at sp-6 acupoint significantly reduced the intensity of dysmenorrhea in the intervention group compared to the control group 30 min and 1, 2, and 3 h after the intervention (p <0.05). In the present study, our aim is to perform acupressure at guan yuan (rn-4) and qu gu (rn-2) acupoints located on the abdominal meridian between the navel and the symphysis pubis and compare its effect to that of using ibuprofen (control group). Rn-2 acupoint is also located on the midline, 5 cuns below the navel, 1 cun between the two grooves of the interphalangeal joints of the massager s middle finger . The rationale for using self - care education to increase awareness of one s own body and the adoption of appropriate health behavior and lifestyle modification such as diet and exercise will lead to the improvement of dysmenorrheal . Considering the limited interventions in this regard and importance of complementary medicine, the present study aims to compare self - care behaviors training, acupressure at rn-4 and rn-2 acupoints, and ibuprofen consumption . The present randomized clinical trial was conducted on 120 nonmedical students (three groups of 40) living in dormitories of shiraz university shiraz, iran . The sample size was determined based on the study by kashefi et al . And considering the loss rate of 10% . Of course, the initial sample comprised 157 patients with primary dysmenorrhea, of which 37 patients were excluded from the trial due to lack of inclusion criteria [figure 1]. Consort diagram of sampling at first, five dormitories of shiraz university were selected randomly . Then, one, two, and two dormitories were randomly considered as control, self - care behaviors training, and acupressure groups, respectively . The inclusion criteria of the study were being single, being below 25 years old, having primary dysmenorrhea with pain intensity of four and above based on visual analog scale (vas), having regular menstrual cycles (2135 days), having experienced dysmenorrhea in the recent six menstrual cycles (self - report), not having used oral contraceptive pills, and treatment interventions (nsaids, antiprogestins, and supplements) since 2 months before the study, not suffering from physical (vertebral fracture, herniated disk, acute inflammation, deep vein thrombosis, and gynecological diseases) and mental disorders, not consuming special medications such as benzodiazepines and antidepressants, and not having experienced stress due to parents' divorce or loss of first - degree relatives during the recent 6 months . On the other hand, the exclusion criteria of the study were having symptoms such as burn, itch, and abnormal vaginal discharges, experiencing stressors (parents' divorce, loss of first - degree relatives, etc .) During the study, and not being willing to participate in the study . All interventions and inclusion criteria carried out by the researcher, and research was done in student dormitories . Before the intervention, intensity of pain was determined in all the three groups . It should also be noted that researcher s performance of acupressure was confirmed by a specialist . In doing so, pressure was repeatedly applied with the thumb and was controlled by a scale to equalize its amount in each application . First group, in the acupressure group, pressure was applied on the abdominal meridian using the thumb for 20 min (15 s pressure, 15 s rest) over the 1 2 days of menstruation for two cycles . Pain intensity was assessed h, 1 h, and 2 h after application of pressure . It should be mentioned that pain intensity was assessed by the researcher s assistant to avoid bias . The method of compression in acupressure groups was done after obtaining confirmation from a trained researcher (i.e., from a specialist in physical medicine and rehabilitation). After confirmation from the researcher, the intervention was conducted . In the second group, self - care behaviors were conducted in two parts: teaching theoretical base of using lecture, discussion, and experience sharing was performed through four sessions (one - 6090 min session a week). The educational content included anatomy and physiology of the genital system, nutrition during the menstrual cycle (based on books, articles, and a nutritionist s advice, emphasis was put on the resources that were effective in dysmenorrhea), the second part was practical training and includes isometric exercises during the menstrual cycle . It should be noted that isometric exercises were educated to researcher by a master of physical education, and researcher was trained to girls in the gymnasium dormitory . Isometric exercise duration was 8 weeks, in the first 4 weeks was conducted by the researcher . In the second 4 weeks with an appropriate images of playing sports the girls were given regarding how exercise . And the girls without researcher continued (a total of 8 weeks). To ensure the exercise was done in the dormitory to follow up, as well as phone calls, for continued exercise classes were encouraged after the 1 4 weeks . Food recommendations and other educational content have been on the books and articles . In group three, the control group received the routine treatment for dysmenorrhea, i.e., taking ibuprofen 400 mg three times a day for three cycles . Of course, they were provided with the educational pamphlet at the end of the study . Questionnaires were completed in the student dormitories and after the intervention; to avoid sampling bias in research, pain assessment was conducted by a research assistant . In this study, intensity of pain was assessed using mcgill pain questionnaire developed by melzack, which has been introduced as a reliable instrument for the assessment of pain intensity . However, since this questionnaire takes a long time to be completed, its short form was designed and has been frequently used in persian studies . The short form of mcgill pain questionnaire consists of 15 sensory (11 questions) and emotional (4 questions) items, and the patients determine the quality of their pain by selecting one of the options of none, mild, average, and severe . The validity and reliability of this scale in the current research were based on the study performed by adelmanesh et al . That reported cronbach s alpha coefficients of 0.951, 0.832, and 0.840 for sensory, emotional, and total scores, respectively . Wallis test was used to compare the three study groups . Besides, paired t - test was employed to compare the means before and after the intervention . Finally, qualitative variables were compared using chi - square test . Besides, written informed consents for taking part in the study were obtained from all the participants . Hence, thesis number: 92 - 6900, irct: 2013071013940n1 and ethic number: 1392 - 12 - 11 - 6900 . The present randomized clinical trial was conducted on 120 nonmedical students (three groups of 40) living in dormitories of shiraz university shiraz, iran . The sample size was determined based on the study by kashefi et al . And considering the loss rate of 10% . Of course, the initial sample comprised 157 patients with primary dysmenorrhea, of which 37 patients were excluded from the trial due to lack of inclusion criteria [figure 1]. Consort diagram of sampling at first, five dormitories of shiraz university were selected randomly . Then, one, two, and two dormitories were randomly considered as control, self - care behaviors training, and acupressure groups, respectively . The inclusion criteria of the study were being single, being below 25 years old, having primary dysmenorrhea with pain intensity of four and above based on visual analog scale (vas), having regular menstrual cycles (2135 days), having experienced dysmenorrhea in the recent six menstrual cycles (self - report), not having used oral contraceptive pills, and treatment interventions (nsaids, antiprogestins, and supplements) since 2 months before the study, not suffering from physical (vertebral fracture, herniated disk, acute inflammation, deep vein thrombosis, and gynecological diseases) and mental disorders, not consuming special medications such as benzodiazepines and antidepressants, and not having experienced stress due to parents' divorce or loss of first - degree relatives during the recent 6 months . On the other hand, the exclusion criteria of the study were having symptoms such as burn, itch, and abnormal vaginal discharges, experiencing stressors (parents' divorce, loss of first - degree relatives, etc .) During the study, and not being willing to participate in the study . All interventions and inclusion criteria carried out by the researcher, and research was done in student dormitories . Before the intervention, intensity of pain was determined in all the three groups . It should also be noted that researcher s performance of acupressure was confirmed by a specialist . In doing so, pressure was repeatedly applied with the thumb and was controlled by a scale to equalize its amount in each application . First group, in the acupressure group, pressure was applied on the abdominal meridian using the thumb for 20 min (15 s pressure, 15 s rest) over the 1 2 days of menstruation for two cycles . Pain intensity was assessed h, 1 h, and 2 h after application of pressure . It should be mentioned that pain intensity was assessed by the researcher s assistant to avoid bias . The method of compression in acupressure groups was done after obtaining confirmation from a trained researcher (i.e., from a specialist in physical medicine and rehabilitation). After confirmation from the researcher, the intervention was conducted . In the second group, self - care behaviors were conducted in two parts: teaching theoretical base of using lecture, discussion, and experience sharing was performed through four sessions (one - 6090 min session a week). The educational content included anatomy and physiology of the genital system, nutrition during the menstrual cycle (based on books, articles, and a nutritionist s advice, emphasis was put on the resources that were effective in dysmenorrhea), the second part was practical training and includes isometric exercises during the menstrual cycle . It should be noted that isometric exercises were educated to researcher by a master of physical education, and researcher was trained to girls in the gymnasium dormitory . Isometric exercise duration was 8 weeks, in the first 4 weeks was conducted by the researcher . In the second 4 weeks with an appropriate images of playing sports the girls were given regarding how exercise . And the girls without researcher continued (a total of 8 weeks). To ensure the exercise was done in the dormitory to follow up, as well as phone calls, for continued exercise classes were encouraged after the 1 4 weeks . Food recommendations and other educational content have been on the books and articles . In group three, the control group received the routine treatment for dysmenorrhea, i.e., taking ibuprofen 400 mg three times a day for three cycles . Of course, they were provided with the educational pamphlet at the end of the study . Questionnaires were completed in the student dormitories and after the intervention; to avoid sampling bias in research, pain assessment was conducted by a research assistant . In this study, intensity of pain was assessed using mcgill pain questionnaire developed by melzack, which has been introduced as a reliable instrument for the assessment of pain intensity . However, since this questionnaire takes a long time to be completed, its short form was designed and has been frequently used in persian studies . The short form of mcgill pain questionnaire consists of 15 sensory (11 questions) and emotional (4 questions) items, and the patients determine the quality of their pain by selecting one of the options of none, mild, average, and severe . The validity and reliability of this scale in the current research that reported cronbach s alpha coefficients of 0.951, 0.832, and 0.840 for sensory, emotional, and total scores, respectively . Wallis test was used to compare the three study groups . Besides, paired t - test was employed to compare the means before and after the intervention . Finally, qualitative variables were compared using chi - square test . This study was approved by the ethics committee of shiraz university of medical sciences . Besides, written informed consents for taking part in the study were obtained from all the participants . Hence, thesis number: 92 - 6900, irct: 2013071013940n1 and ethic number: 1392 - 12 - 11 - 6900 . The mean age of the participants was 20.02 1.44, 20.12 1.50, and 20.37 1.54 years in the acupressure, training, and control groups, respectively (p = 0.569). Besides, the mean time of beginning of pain after menarche was 1.80 0.82 years in the acupressure group, 1.90 0.77 years in the training group, and 1.87 0.79 years in the control group (p = 0.844). According to the results of kruskal wallis test, the three groups were homogeneous regarding these two variables [table 1]. Comparison of the age of in three groups (acupressure - education of self - care behaviors and control) in the training group, the mean intensity of pain was 16.90 7.43 before the intervention and 10.56 5.71 after the intervention, and the results of paired t - test showed that the difference was statistically significant (p <0.001). In the acupressure group also, the mean intensity of pain was 20.25 6.93 before the intervention, and the results showed a significant difference in this regard before and 1 and 2 months after the intervention (p <0.001) [table 2]. Comparison of the intensity of pain after and before the acupressure and educational groups based on the mcgill questionnaire in the control group, the mean intensity of pain was 31.77 6.36 before the intervention, 23.52 5.55 1 month after the intervention (p <0.001), 9 5.99 2 months after the intervention (p <0.001), and 14.57 6.40 3 months after that (p comparison of the intensity of pain after and before the acupressure and educational and control groups based on the mcgill questionnaire based on mcgill pain questionnaire, the mean intensity of pain was 10.65 5.71 in the training group, 19 5.41 in the control group, and 14.40 6.87 in the acupressure group after the intervention . Wallis test, showed that these differences were statistically significant, and both interventions were more effective compared to the control group [table 4]. Comparison of the difference mean scores severity of pain of in three groups (acupressure - education of self - care behaviors and control of the mcgill questionnaire according to the present study results, discussion is on three main axes: in our study, the mean intensity of pain significantly decreased in the self - care training group . Chang and chuang conducted a study to introduce the effective factors in dysmenorrhea . In that study, the participants with moderate dysmenorrhea received self - care behaviors training, and the results showed that training self - care behaviors at schools were one of the effective methods in reduction of dysmenorrhea . Chen et al . Also carried out a research on self - care behaviors using herbal medicines and complementary medicine in primary dysmenorrhea among taiwanese adolescents . The results of the study by chen et al . Revealed the necessity to educate individuals regarding the effect of food diet and effective herbal medications on the treatment of dysmenorrhea . In that study, the participants mentioned the reduction of physical activities as a self - care behavior, but the researchers modified this opinion and trained individuals regarding aerobic exercises as a self - care behavior to decrease their pain ., who investigated the effect of heavy exercises on the treatment of dysmenorrhea using mcgill pain questionnaire . The results of that study demonstrated that exercising was effective in reduction of pain without any side effects . In contrast, shafaie - sehati et al . Found no significant difference between athletic and nonathletic groups with respect to the frequency of dysmenorrhea . In the present study, self - care behaviors training resulted in a reduction of pain and exercising was among the bases of this training . Exercising enters the body in the state of hypoxia where beta - endorphin levels increase . Therefore, hypoxia and acidosis stimulate production of beta - endorphins, and the relationship between breathing and beta - endorphin production can be associated with the effect of acidosis on the two through different mechanisms . Reduction of pain after self - care training can also be attributed to the control of prostaglandins . In fact, aerobic exercises empty the uterus from waste materials and prostaglandins, which are the main cause of pain, eventually reducing the duration of pain through the menstrual cycle . In another survey exercise after 6 weeks is created flexibility in the hamstring, back and hip muscles, and reduces pain . The current study at the second part findings showed a significant difference in the acupressure group s pain intensity before and after the intervention . Considering the limited number of studies conducted on rn-4 and rn-2 acupoints, the results of performance of acupressure at other points have been discussed in this section . In the study by yeh et al ., pressure was applied for 1 min four times a day for 2 days, which is different from our study . The results showed a significant difference in each group s pain intensity before and after the intervention . In the present study, however, the study groups' pain intensity was significantly different only based on mcgill pain questionnaire . Other researches have also revealed the effectiveness of acupressure in treatment of dysmenorrhea and relaxation . In a review study about efficacy of self - acupressure showed several studies (eight rcts and two quasi - rcts) have a positive effect and effective on primary dysmenorrhea . In another study concluded that enough evidence not exists for the effectiveness of acupressure or acupuncture and rct larger studies are needed . The analgesic effect of acupressure can be due to release of endorphins and glucocorticoids that block pain gates . Acupressure, in fact, refers to the touch technique for balancing human body s energy flow or qi . This method leads to the release of various neurotransmitters, which cut the nervous signals transferred by the nervous system . It also inhibits secretion of prostaglandins, reduces stimulation of cerebral cortex, and regulates secretion of endocrine hormones . Based on mcgill pain questionnaire scores, the control group s pain intensity scores were significantly different before and after the intervention . Similarly, the results of the studies by zafari and witt et al . Indicated the effectiveness of ibuprofen . However, the results obtained by salmalian were on the contrary to those of the present study . In general, nsaids are the first - line treatment, but their side effects, including gastrointestinal problems, make patients choose alternative therapies . Overall, the results of the current study showed a significant difference among the three groups regarding the intensity of pain, and that training and acupressure were more effective compared to consumption of ibuprofen . In this study, new and effective acupoints were employed, whereas the previous studies had not used such a combination of effective points . One of the limitations of our study was the difficulty in obtaining the required permissions from dormitories and performing the procedures privately . Hence, probable physiological tolerance of pain in each people is different, so feel pain is different, as well as contrast to the reactions and feelings of loss pain after pain relief methods will be different, which is beyond the control of the researcher . Another limitation was to assess the severity of pain after intervention . In order to solve the problem and avoid bias in sampling, finally, due to the different time periods, research conducted at the same time for all units is not possible . Future studies are recommended to compare the effects of acupressure at other acupoints and common chemical and herbal drugs on primary dysmenorrhea in larger populations . Suggestions for future research: (1) comparison the effect of other point of acupressure on severity of primary dysmenorrhea with the rn-2, rn-4 points . (2) the application of educational models such as basnef models on knowledge and attitudes of people with primary dysmenorrhea . According to the current study results, the most effective method in reduction of dysmenorrhea was self - care behaviors training followed by acupressure at rn-4 and rn-2 acupoints and consumption of ibuprofen . Considering the side effects of consuming ibuprofen drug, training and acupressure as nonpharmacological, easy, safe, and inexpensive methods can be incorporated into the curricula of girls' high schools . Considering the effectiveness of acupressure in reducing dysmenorrheal, obstetricians, gynecologists, and midwives have recommended acupressure not only in the treatment of dysmenorrhea but also in reducing labor pain and pain control . The effective nondrug method that is the responsibility of the midwifery, and in clinics and delivery room applications and this study is funded by the shiraz university of medical sciences, shiraz, iran . This study is funded by the shiraz university of medical sciences, shiraz, iran . All authors contributed to the conception of the work, conducting the study, revising the draft, approval of the final version of the manuscript, and agreed for all aspects of the work.
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Stroke commonly causes loss of motor function due to weakening of upper / lower extremity muscles1 . According to ryerson2, use the affected upper extremity decreases because of the patient s dependency on the unaffected upper extremity for normal functions, which results in problems such as learned disuse, asymmetric postural patterns, contractures, and aggravated functional restrictions involving the affected upper extremity . Therefore, to improve functions of the affected upper extremity in stroke patients, measures that maximize opportunities to use the affectedupper extremity are necessary . Bilateral activities have been discussed as measures to improve the body symmetry and to reduce abnormal muscle tone3, thereby promoting voluntary movement of the affected upper extremity4 . Thus far, bilateral upper extremity coordination movements have been applied in the form of bilateral single exercises utilizing tasks such as figure imitation5, robot arm upper extremity mechanisms6 and functional stretching7, and bilateral complex exercises combined with the principle of motor learning, such as rhythmic acoustic8, unaffected extremity weight addition9, and active neuromuscular electrical stimulation in stroke patients10 . Most previous studies have reported the positive effects of these exercises on motor function recovery in stroke patients . Whitall et al.8 reported that when chronic hemiplegia patients underwent bilateral training to push and pull upper extremity apparatuses, including acoustic signals, their upper extremity functions were improved . In a study conducted by summer et al.7 with 12 chronic stroke patients, the movement time of the patients affected side upper extremity decreased and upper extremity functions showed more improvement when they performed tasks to hold objects by stretching both hands simultaneously then when they performed tasks to hold objects by stretching only one hand . However, lee su - jin11 advised that the tasks in those studies involved mostly gross motor exercises of the upper extremity and simple repetitive training tasks, and that they were not sufficient to improve patients grasping ability to hold objects with various sizes, shapes, and weights using the shoulder, elbow, wrist, and fingers . Although studies that applied diverse functional training tasks including activities of daily living to symmetric bilateral upper extremity exercises have been conducted recently12, 13, these studies are still quite insufficient, and the effects of these new functional tasks on the recovery of upper extremity functions should be compared with those of existing bilateral upper extremity exercises . Therefore, the purpose of this study was to compare the effects of symmetric bilateral upper extremity exercises using diverse functional training tasks with the effects of repetitive bilateral upper extremity exercises including acoustic signals on the recovery of upper extremity function and daily living activities in stroke paitents . Forty patients diagnosed with hemiplegia resulting from stroke by a rehabilitation doctor in the n hospital located in daegu, south korea participated in the study . They were randomly and equally assigned to a task - oriented bilateral arm training group (tbg: 11 males, 9 females) and a repetitive bilateral arm training group (rbg: 13 males, 7 females). The meansd age, height, and weight of the patients in the tbg were 51.1514.81 years, 163.458.53 cm, and 62.079.79 kg, respectively . Eleven of the 20 patients in the tbg group had right hemiplegia, and 9 had left hemiplegia . The meansd age, height, and weight of the patients in the rbg were 48.6512.81 years, 164.108.11 cm, and 63.809.26 kg, respectively . Eight of the 20 patients had right hemiplegia, and 12 had left hemiplegia . The inclusion criteria were as follows: no visual field defect, no abnormality in the vestibular organs, no orthopedic disease, an unrestricted range of motion, ability to understand and perform the exercises as instructed by the researcher, a score of 24 or higher on the mini - mental state examination - korean version, and upper extremity paresis with ability to extend the affected wrist and fingers to at least 15 degrees . This study was approved by the university institutional review board and all the subjects understood the purpose of this study and provided their written informed consent prior to their participation in the study, in accordance with the ethical standards of the declaration of helsinki . The tbg bilateral training with 5 functional tasks such as towel sweep on the table, staking cups, positioning the cup upright, carrying a wooded block, and drinking water . Both groups trained for 5 minutes and had a rest break of 1 minute . Both groups performed their exercises for 30 minutes each day, 5 times per week . Measurements were collected twice, before and after the intervention, which lasted for 12 weeks . The main outcome measurements were box and block test (bbt), jebsen - taylor test (jbt), and modified barthel index (mbi) to determine upper limb function and adl ability . After the general characteristics of the subjects were determined, the paired t - test was used to compare the variations in bbt, jtt, and mbi between pre- and post - intervention within each group . The significance of the difference between the two groups was investigated using the independent t - test . Both groups demonstrated significant improvement on the bbt, jtt, and mbi after the intervention (p<0.05) (table 1table 1.comparison of the bbt, jtt, and mbi between pre- and post- intervention of each grouptbgrbgpre - interventionpost - interventionpre - interventionpost - interventionbbt29.40 11.347.7 7.932.8 11.444.1 11.5*jtt45.3 23.361.1 23.155.8 19.564.9 19.9*mbi51.1 17.371.1 19.050.9 20.762.2 21.6*meansd . * significant difference from pre - test at p<0.05 . Tbg: task oriented bilateral arm training group; rbg: repetitive bilateral arm training group; bbt: box and block test; jtt: jebson - taylor test; mbi: modified barthel index). The differences in the bbt, jtt, mbi between the 2 groups were compared and were found to be significant (p<0.05) (table 1). Tbg: task oriented bilateral arm training group; rbg: repetitive bilateral arm training group; bbt: box and block test; jtt: jebson - taylor test; mbi: modified barthel index this study was conducted to examine the effects of symmetric bilateral upper extremity exercises applied with diverse functional training in comparison with repetitive bilateral upper extremity exercises including acoustic signals on the recovery of upper extremity functions and daily living activities in stroke patients . According to the results of this study, both the tbg and rbg showed significant effects on the recovery of upper extremity motor functions and daily living activities . After implementing the exercise programs, both groups showed significant improvement in the bbt, jtt, and mbi scores . In a study conducted by lin et al.12 on chronic stroke patients who had had the disease for at least 6 months, the group that performed bilateral upper extremity exercises showed further improvement in spatial - temporal control of the affected upper extremity and fugel - meyer assessment scores than the control group . In a study conducted by cauraugh et al.9, 2 complex bilateral upper extremity exercise groups were able to move more blocks than the control group in the bbt . In a study conducted by brunner et al.13 with sub - acute stroke patients that compared the effects of modified constraint induced movement therapy (mcimt) with those of bilateral upper extremity training, results indicated that bilateral upper extremity training was as effective as mcimt in the improvement of upper extremity motor functions . According to researchers most sub - acute stroke patients were not required to wear a restraining band during training of the affected upper extremity . Similar to the above - mentioned previous studies, the results of this study indicated that upper extremity functions and daily living activity functions were improved after performing bilateral upper extremity exercises, which supports the opinion that when both hands move simultaneously, instead of acting as separate units, they act as a unit, which is perceived as a coordinated unit by the brain14 . The study also found that when homologous muscle groups on the left and right sides are activated simultaneously by symmetrical bilateral exercises, the neural networks in the 2 cerebral hemispheres that are involved in upper extremity movement on both sides are similarly activated15 . In this study, in comparison with the group that performed repetitive bilateral upper extremity exercises, the group that underwent task - oriented bilateral upper extremity exercise showed significant improvement in the bbt, jtt, and mbi scores . Given this result, functional task training related to movement frequently used in daily life is considered to have more positive effects on the recovery of upper extremity motor functions in stroke patients than repetitive training using simple movement . In a study conducted by wu et al.16 on the performance of stretching motions in chronic stroke patients, kinematically better performance was shown when actual objects used in daily life were used when no such objects were available . In a study conducted by thielman et al.17, in the case of subjects with low functional levels, coordination between the shoulder and elbow joints was improved when they underwent task - related training than when they performed gradual resistive exercises . Senesac et al.18 conducted a study on whether bilateral arm training with rhythmic auditory cueing (batrac) helped the recovery of motor functions the affected upper extremity and whether the effects of the recovery of motor functions would be transmitted to the performance of new tasks . The subjects underwent batrac for a total of 8 times in 2 weeks, and the effects of the training were evaluated using similar tasks and new tasks . Although the maximum speed increased in both tasks, the training did not affect exercise time, speed up areas, or acceleration . With regard to such results, the researchers presented a stated opinion that if the repetitive bilateral training was composed of significant movement related to the patients daily living activities, the training should have shown positive effects on the recovery of the patients motor functions . In this study, only the functional aspect of upper extremity exercises was evaluated, and the performance of the exercises was not evaluated kinematically . In future studies, the functional aspect of upper extremity exercises and the kinematic and qualitative aspects of the exercises as well as the recovery in terms of neurophysiology should be evaluated . Based on the results of this study, it can be seen that bilateral upper extremity exercises applied with functional tasks are more effective in improving upper extremity functions and daily living activities in stroke patients compared to simple, repetitive bilateral upper extremity exercises . Therefore, performing symmetrical bilateral upper extremity exercises which utilize treatment tools of different sizes and weights and movements involved in daily living activities, can be used as an effective therapeutic intervention method in the recovery of upper extremity functions and daily living in stroke patients.
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Several dozen autoantigens related to type 1 diabetes have been described engendering interest in developing an immunotherapeutic treatment . Phase iii clinical trials in newly diagnosed patients using noncomplement fixing anti - cd3 ab failed to achieve primary endpoints . Together with heightened risks to patients receiving any type of systemic immunosuppression, this setback for nonspecific immunological control of disease progression in patients emphasizes the impetus to explore optimization of antigen - specific immunotherapy for prevention and treatment of type 1 diabetes [35]. Experimental immunotherapies that target individual antigens have been tested but which have also proven disappointing; phase iii gad vaccination trials failed [7, 8] and preclinical studies targeting hsp60 (p277) showed some disease protection but a late stage clinical trial study was retracted . More promisingly, preclinical and phase i / ii trials using tolerogenic dna plasmids encoding proinsulin showed encouraging improvement in subject c - peptide levels . Since cd8 t cells play a causal role in diabetogenesis an important goal in development of an effective immunotherapy is identification of specific epitopes that elicit diabetogenic cd8 t cells . A substantial number of candidate mhc class i epitopes have been identified in both the nod mouse and patients including multiple peptides derived from cell antigens that elicit robust ab response in patients as well as other candidate antigens not exclusively expressed in cells [15, 16]. Humoral responses are successfully used clinically to forecast disease susceptibility [13, 17], and since adaptive b cell response usually requires t cell help, the presence of reactive ab in patients implies the involvement of cd4 t cells . Study of the nod model has revealed a temporal pattern in development of t cell responses: circulating or islet - infiltrating cd8 t cells reactive to certain antigens are detected at different ages and disease status . Attempts to induce antigen - specific tolerance in diabetogenic cd8 t cells aim to develop therapies to treat patients such that cell function recovers, which might be replaced via endogenous stem cells or exogenous allograft transplants . In spite of abundant representation in young prediabetic mice, t cells specific for glutamic acid decarboxylase 65 (gad65) or islet - specific glucose-6-phosphate catalytic subunit - related protein (igrp) appear to not be dominant in type 1 diabetes initiation since mice tolerant to those antigens develop disease [18, 19]. However, induction of tolerance to insulin or proinsulin in young mice prevents development of both t cells reactive with other cell antigens and disease progression therein implicating immune response to insulin as the major initiating event in type 1 diabetes . Since noninsulin antigens likely contribute to development or expansion of clinical disease and thus represent candidate therapeutic targets for tolerance induction, we have sought and identified cd8 epitopes that may contribute to optimal tolerization in type 1 diabetes . Mice . Nod / shiltj female mice (the jackson laboratory, bar harbor, me) were housed five per cage in a pathogen - free barrier facility and were maintained on a 12 hr light / dark cycle (7 am to 7 pm) with ad libitum access to autoclaved food and water . Experiments involving animals were conducted with the approval of the new york university school of medicine iacuc (protocol #150219). Peptides . Purity of peptides (lifetein, south plainfield, nj) used in proliferation and ifn- assays was> 85% and that used in tolerance assays was 99% . Peptides were solubilized in sterile pbs and 0.1 mg / ml of each peptide was analyzed for lps (lonza, allendale, nj). Any peptide having> 0.1 eu / mg lps was treated to remove endotoxin (thermo scientific, waltham, ma) until samples were <0.1 eu / mg (1 eu = 0.1 ng endotoxin). The naturally presented epitope is expected to be within the top 2% of peptides predicted assuming 80% reliability . For the rankpep analysis binding thresholds for the candidate epitopes are as follows: igrp> 17.9% for d and> 19.2% for k, dh> 17.9 for d and> 14.5% for k, insulin> 17.9% for d and> 14.5% k, gad65> 17.9% for d and> 14.5 for k, icap69> 17.9% for d and> 14.5% for k, znt8> 17.9% for d and> 14.5% for k. the iedb analysis (http://www.iedb.org/) was performed using settings: default prediction method, the immunoproteasome cleavage prediction type, and the tap transport prediction . A known mhc class i k binding peptide was synthesized and used as control [influenza np147155 (tyqrtralv)]. There were a total of 69 peptides synthesized including previously characterized epitopes in insulin(3947 v), igrp(206214), igrp(251259), gad65(178186), and gad65(546554) intended to serve as positive controls . The insulin epitope was prepared with a g> v substitution in the last position since g binds poorly to the pocket f . Antibodies and flow cytometry . Antibodies (anti - k (sf1 - 1.1.1), anti - d (28 - 14 - 8), and anti - ifn (r4 - 6a2)) were purchased from ebioscience (san diego, ca) and used as described previously [25, 26]. Analyses were performed on a facs calibur flow cytometer (becton - dickinson, palo alto, ca). Supernatants of 2 10 cells cultured for 48 h with 10 m peptide (from 2 - 3 pooled mice pln) were assayed by elisa using 3,3,5,5-tetramethylbenzidine as substrate (ebioscience, #88 - 8314 - 88, minimum sensitivity = 0.7 pg / ml). Single - cell suspensions were prepared and analyzed in quadruplicate wells using 96-well plates by measurement of [h]-thymidine incorporation after stimulation with peptides as indicated or as positive control plate - bound anti - tcr (h57 - 597) using 2 10 ln cells / well . Stimulation with anti - tcr gave incorporation typically> 1020 times greater than nonpulsed wells . Incorporation of thymidine into nonpulsed wells varied in experiments and ranged from ~200 to ~900 cpm . Incorporation of thymidine into wells pulsed with control flu np147155 peptide varied in experiments and ranged from ~400 to ~1,100 cpm . Thus, the average thymidine incorporation at a given peptide concentration was divided by average incorporation into wells pulsed with flu np147155 and shown as error bars represent standard error of the four wells at each peptide concentration tested for a representative assay . Rma - s cells expressing both d and k [2830] (from m. bevan, univ . Washington, seattle, wa) were maintained in complete rpmi-1640 medium containing g418 (biowhittaker, walkersville, md). Cells were cultured at 26 overnight, pulsed with candidate peptide (1010 m) for 60 min at 26, incubated at 37 for 4 h, and assayed by flow cytometry for cell surface expression of d and k. assays were performed more than three times for each peptide . Fluorochrome - conjugated tetramers were prepared by the nih tetramer core facility at emory university (atlanta, ga). Cells were stained with apc - conjugated anti - cd8 simultaneously with alexa 488-conjugated tetramer, or pe - conjugated cd8 and apc - conjugated tetramer (1: 1001: 200 dilution). Flow cytometry data was analyzed using flowjo (tree star, ashland, or). The plasmid pbht-568 expresses the entire murine proinsulin (lacking the signal sequence) and thus contains the dominant epitope residues 3947 . The proinsulin sequence was removed and full - length cdnas encoding znt8 and encoding separately dh (both from open biosystems) were cloned into . Plasmids were purified (endofree giga kit #12391, qiagen corp, valencia, ca) followed by removal of residual endotoxin as for synthetic peptides . High zone tolerance . Mice were injected at weeks 4, 6, 8, 11, and 14 . 2 mg was mixed with 0.1 mg peptide in a final volume of 0.2 ml sterile pbs and injected i.p . This experiment was conducted twice using 10 mice per group and the data were combined for statistical analysis . Dna plasmid tolerance . Four - week - old mice were injected weekly with 0.025 mg of individual plasmids dissolved in pbs + 0.9 mm cacl2 into each quadriceps in 0.1 ml (0.05 mg plasmid / injection / mouse) for a total of 6 injections . This experiment was conducted twice using 10 mice per group and the data were combined for statistical analysis . Pancreata were fixed overnight in 10% buffered formalin before embedding in paraffin and processed for h / e staining . 10-micron sections were made of the whole organ and slides were scored in a blinded manner for both the number of islets per pancreas and the extent of islet infiltration (0%, 125%, 2650%, 5175%, and> tail vein blood glucose was tested using onetouch ultra test strips and monitor (lifescan, shelton, ct). The log - rank test (mantel - cox) was used for statistical analysis of diabetes incidence at 30 weeks (graphpad software, san diego, ca). The literature was reviewed for proteins that induce humoral immunity in human type 1 diabetes; six proteins were selected, five for which candidate epitopes have been previously identified and one, dh, which has been reported as being present in islets [3234] but has not previously been implicated in type 1 diabetes . Candidates were analyzed by predictive algorithms for mhc class i - restricted epitopes (9 amino acids, using iedb, syfpeithi, and rankpep), and predicted epitopes were synthesized (table 1) and tested for the ability to stimulate in vitro proliferation of total splenocytes obtained from mice of different ages, 3 or 13 weeks, or overtly diabetic (having blood glucose> 250 mg / dl). Most candidate epitopes failed to demonstrate splenocyte proliferation and the level of stimulation was in general modest, possibly reflecting low abundance of antigen - specific cells in spleen (data not shown). Peptides were then tested for stimulation of pancreatic ln cells (figure 1(a)). Surprisingly the majority of candidate epitopes did not reproducibly stimulate ln proliferation, even peptides that were scored highly by predictive algorithms . However, several previously described epitopes were highly stimulatory: proinsulin3947, igrp206214, and two epitopes from gad65 . In addition, a candidate peptide of icap69, two znt8 peptides, and a peptide from dh each stimulated robust ln cell proliferation, predominately in older prediabetic and overtly diabetic mice . We next tested candidate peptides for the ability to stimulate secretion of ifn from pln (figure 1(b)). Several peptides stimulated ifn secretion, usually from cells of mice of the same age that scored in the proliferation assay except for insulin3947 where ln cells from very young mice also responded . Interestingly, in spite of the presumed greater sensitivity of the cytokine production assay, several peptides that stimulated robust ln cell proliferation failed to induce ifn expression: gad65178186, icap697886, and znt8158166 . This could be due to several factors including cell intrinsic lack of ifn expression by those clones, but no peptide stimulated ifn secretion that did not also stimulate ln cell proliferation . Mhc class i stabilization assay . In order to assess the ability of candidate peptides to bind to mhc class i, rma - s stabilization assays were performed and these data are summarized in figure 1(c). The mhc stabilization data was in general concordant with the ifn production assay in that, with the exception of znt8158166 and to a lesser extent icap697886, candidate epitopes that stimulated ifn production also strongly stabilized mhc class i. collectively considered the three types of assays indicate the existence of cd8 t cells that recognize the predicted cognate epitopes . Mhc class i tetramers of the appropriate allele were prepared and nod splenocytes were assessed by flow cytometry after costaining with anti - cd8 (summarized in figure 1(d)). These analyses were in general concordant with the ifn assay in that tetramer - reactive t cells were detected in mice of certain ages coincident with identification of functional in vitro t cell activity using cognate peptides . Induction of epitope - specific tolerance . Using 3 different protocols, we asked if the predicted novel t cell epitopes could be used to influence development of elevated blood glucose . In the first approach, purified znt8 and dh peptides were injected at 2 - 3-week intervals beginning at 4 weeks of age under noninflammatory conditions (figure 2(a)). In control mice receiving buffer injection, diabetes developed with kinetics and penetrance typical in our mouse facility: ~85% of females had elevated blood glucose by ~30 weeks of age . However disease development was significantly prevented by treatment with znt8282290, znt8158166, or dh233241 implying that these epitopes may be tolerogenic . To confirm the tolerizing potential of these epitopes, in the second approach, znt8 candidate epitopes were mixed with a stimulator of tlr2 (zymosan) that has been shown to induce immunological tolerance . In this experiment (figure 2(b)) the stringency of the tolerizing protocol was increased and we began treatment on older mice that developed elevated blood glucose (~170 mg / dl). In addition, epitopes from previously described antigens igrp, gad65, and insulin were separately tested (and control mice were injected with zymosan plus an irrelevant k binding peptide, flu np147155). Control mice receiving zymosan plus flu np147155 peptide developed disease with slightly slower kinetics than mice receiving only buffer but ultimately the same percentage became diabetic . Although fewer mice treated with znt8282290 or znt8158166 developed disease compared to controls, the results were not significant . The same was noted for mice receiving igrp206214, gad65546554, or insulin3947 peptides leading to the conclusion that coadministration of tlr2 agonists together with these candidate peptide epitopes did not enhance tolerance induction . The third tolerizing protocol used injection of dna plasmids encoding antigens containing candidate epitopes [11, 12, 35]. Normoglycemic mice were injected with plasmids encoding either dh or znt8, proinsulin, the empty plasmid (as negative control), or combinations of these plasmids (figures 2(c) and 2(d)). As was observed for mice receiving admixture of zymosan and peptides, injection of individual znt8 or dh plasmids reduced disease incidence but not to statistical significance . However, diabetes prevention in mice receiving the proinsulin plasmid was statistically significant (figure 2(c)). Mice receiving combinations of plasmids were protected to a greater extent than any single plasmid, including proinsulin alone (figure 2(d)), although comparison of combinations of plasmids to insulin plasmid alone was not statistically significant . Combination of targets was also better at impacting disease compared to single plasmid treatment seen when the data was analyzed for median age of disease development (figure 3) where each dual combination of plasmid was superior to that of insulin alone, and targeting both znt8 and dh in combination with insulin was the most successful . In order to characterize the effect of dna vaccination on islet inflammation, pancreases in mice receiving dna plasmids that did not show elevated blood glucose at the completion of the experiment were analyzed for extent of lymphocytic infiltration (figure 4). Reflecting the tolerization data (figure 2), combinations of plasmids dramatically and significantly reduced the number of islets having severe infiltration (defined as> 50%). Interestingly, the number of islets having no insulitis did not decrease, even compared to the control group, and the number of islets having demonstrable insulitis (2650% of islet area infiltrated) increased in all groups of treated mice . Immunotherapeutic reversal or downregulation of the diabetogenic adaptive autoimmune response has been pursued as a way to prevent or reverse type 1 diabetes . Since there are more than two dozen non - mhc - linked genes associated with type 1 diabetes, each of which may contribute to disease development and/or maintenance, monotherapy that targets any given gene seems unlikely to succeed . Adding more complexity to this task is the considerable antigenic variety revealed by the large number of cell proteins that elicit an immune response many of which are candidates for antigen - specific tolerance induction [3, 38] nevertheless, although most experimental immunotherapy trials have been unsuccessful at inducing long - term restoration of regulated insulin secretion, several showed at least partial effects in that the loss of c - peptide is reduced implying enhanced cell function and a reduced rate of disease progression [11, 12, 39]. In the nod model where experimental design can be more carefully controlled, several approaches to tolerance induction have showed that tolerance to gad65 and igrp peptide epitopes can be induced in prediabetic mice, but which has little or minimal effect on disease development [18, 23]. Those observations illustrate that although gad65 and igrp are potent autoantigens and immune response to those proteins may significantly contribute to disease maintenance, they do not provide dominant epitopes in the initiation of disease . Preclinical tolerizing therapy protocols in the nod model have showed that insulin is a primary autoantigen and initiates type 1 diabetes . This was convincingly demonstrated when a key residue in the insulin b923 epitope was replaced; disease is prevented . Even though anti - insulin t cells are produced very early in disease, t cells having other, noninsulin specificities accumulate in islets as nod mice age . This occurs before overt disease develops leading to the notion that pathogenic t cells having specificities for other antigens contribute in a substantial way to disease . This probably underlies the observation in an experimental trial that, even with successful reduction of insulin - specific t cells following tolerization, insulin independence was not achievable (in addition, results from one phase iii trial that targeted a hsp peptide (diapep277) that was supported by encouraging early stage data was subsequently retracted). Considered collectively these data suggest that an unknown number of cell - reactive t cells likely need to be simultaneously rendered inactive in order to reduce or eliminate islet inflammation and destruction of function . Based upon data achieved by screening candidate peptides with patient peripheral blood lymphocytes that identified dozens of peptides having stimulatory activity for example, for the antigen znt8 enee and colleagues have identified nine candidate epitopes although the existence of a given t cell with islet reactivity, even prior to development of type 1 diabetes, does not establish if that t cell is causally associated with disease, evidence which needs to be achieved before consideration as part of an immunotherapeutic protocol . Conclusive proof for a role of any given peptide in development of diabetes requires being able to influence the incidence or progression of disease by gene deletion or epitope - specific induction of tolerance . Our results that identify new cell diabetogenic epitopes illustrate this point and argue in favor of continued effort at antigen identification . Our approach to identification of diabetogenic epitopes focuses on candidate peptides that have high hla binding affinity (which are then screened for biological activity) which appears to be valid in the sense that several novel diabetogenic epitopes have been identified . However, it remains possible that low affinity candidate epitopes that were not strong binding peptides may nonetheless be associated with disease, but corresponding synthetic peptides were not tested for tolerizing potential in vivo because of exclusion in preliminary experiments . Screening all candidate peptides using in vivo tolerizing experiments, and in multiple combinations (which appears a likely requirement for successful therapy of patients), is a definitive, but challenging and unfortunately perhaps untenable, approach for epitope identification . Those candidate epitopes that may be excluded from further consideration on the basis of low responses in peptide - based in vitro assays are contained within the cdna plasmids used for combinatorial in vivo tolerizing experiments and thus may contribute to the biological effects of plasmid - based therapy . A major future goal for this experimental approach is to determine if plasmid vaccination using any given diabetogenic antigen results in development of multiple t cells having different specificities and determination if those t cells contribute to tolerance induction . The mechanism of tolerance induced by the znt8282290 and dh233241 epitopes is currently unknown but, since tetramer - reactive cells were detected in tolerant normoglycemic mice at> 30 weeks of age (dns), likely does not involve clonal deletion as has been suggested for plasmid - based targeting of proinsulin - reactive t cells in a recent human trial . In addition, total numbers of pln treg are not changed in treated mice (dns). Possibilities on how tolerance is developed include activation of antigen - specific treg that suppress the diabetogenic phenotype, functional anergy, development of cryptic (subdominant) low affinity t cells that block the effector phase of diabetogenic clones, or deviation of cytokine expression in the cognate t cell.
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